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Sample records for shows abnormal phosphorylation

  1. Abnormal serine phosphorylation of insulin receptor substrate 1 is associated with tau pathology in Alzheimer's disease and tauopathies

    PubMed Central

    Yarchoan, Mark; Toledo, Jon B.; Lee, Edward B.; Arvanitakis, Zoe; Kazi, Hala; Han, Li-Ying; Louneva, Natalia; Lee, Virginia M.-Y.; Kim, Sangwon F.; Trojanowski, John Q.; Arnold, Steven E.

    2015-01-01

    Neuronal insulin signaling abnormalities have been associated with Alzheimer's disease (AD). However, the specificity of this association and its underlying mechanisms have been unclear. This study investigated the expression of abnormal serine phosphorylation of insulin receptor substrate 1 (IRS1) in 157 human brain autopsy cases that included AD, tauopathies, α-synucleinopathies, TDP-43 proteinopathies, and normal aging. IRS1-pS616, IRS1-pS312 and downstream target Akt-pS473 measures were most elevated in AD but were also significantly increased in the tauopathies: Pick's disease, corticobasal degeneration and progressive supranuclear palsy. Double immunofluorescence labeling showed frequent co-expression of IRS1-pS616 with pathologic tau in neurons and dystrophic neurites. To further investigate an association between tau and abnormal serine phosphorylation of IRS1, we examined the presence of abnormal IRS1-pS616 expression in pathological tau-expressing transgenic mice and demonstrated that abnormal IRS1-pS616 frequently co-localizes in tangle-bearing neurons. Conversely, we observed increased levels of hyperphosphorylated tau in the high-fat diet-fed mouse, a model of insulin resistance. These results provide confirmation and specificity that abnormal phosphorylation of IRS1 is a pathological feature of AD and other tauopathies, and provide support for an association between insulin resistance and abnormal tau as well as amyloid-β. PMID:25107476

  2. Tau in Alzheimer's disease and Down's syndrome is insoluble and abnormally phosphorylated.

    PubMed Central

    Hanger, D P; Brion, J P; Gallo, J M; Cairns, N J; Luthert, P J; Anderton, B H

    1991-01-01

    Some investigators have described the presence in Alzheimer's disease brain extracts of several abnormal forms of the microtubule-associated protein tau, based on their unusual mobility in SDS/PAGE. It has been proposed that these abnormal forms of tau may be the result of aberrant tau phosphorylation. In this study we show that tau in extracts of Alzheimer's disease brain can be separated into two fractions based upon its solubility (100,000 g x 1 h supernatant) in non-denaturing conditions (100 mM-Mes, pH 6.5, 0.5 mM-MgCl2, 1 mM-EGTA and 1 M-NaCl). The tau isoforms with decreased mobility in SDS/PAGE are predominantly in an insoluble fraction, whereas the soluble tau is indistinguishable by its mobility in SDS/PAGE from tau in soluble extracts of control brain. Insoluble tau displaying abnormal mobility on SDS/PAGE was only found in Alzheimer and adult Down's syndrome brains and was absent from the brains of age-matched controls and from foetal and infant Down's syndrome brains. There was a good correlation between the presence of insoluble tau in brain extracts and the abundance of neurofibrillary tangles and senile neuritic plaques. The monoclonal antibody Tau. 1 stained insoluble tau on Western blots only after treatment of the nitrocellulose transfers with alkaline phosphatase, implying that this insoluble tau is in a particular state of phosphorylation. We conclude that, in Alzheimer's disease, a fraction of tau has a modified phosphorylation state and a decreased solubility; these modifications may precede formation of the neurofibrillary tangles characteristic of Alzheimer's disease and Down's syndrome in adults. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:1826835

  3. LRRK2 phosphorylation level correlates with abnormal motor behaviour in an experimental model of levodopa-induced dyskinesias.

    PubMed

    Stanic, Jennifer; Mellone, Manuela; Cirnaru, Maria Daniela; Perez-Carrion, Maria; Zianni, Elisa; Di Luca, Monica; Gardoni, Fabrizio; Piccoli, Giovanni

    2016-01-01

    Levodopa (L-DOPA)-induced dyskinesias (LIDs) represent the major side effect in Parkinson's disease (PD) therapy. Leucine-rich repeat kinase 2 (LRRK2) mutations account for up to 13 % of familial cases of PD. LRRK2 N-terminal domain encompasses several serine residues that undergo phosphorylation influencing LRRK2 function. This work aims at investigating whether LRRK2 phosphorylation/function may be involved in the molecular pathways downstream D1 dopamine receptor leading to LIDs. Here we show that LRRK2 phosphorylation level at serine 935 correlates with LIDs induction and that inhibition of LRRK2 induces a significant increase in the dyskinetic score in L-DOPA treated parkinsonian animals. Our findings support a close link between LRKK2 functional state and L-DOPA-induced abnormal motor behaviour and highlight that LRRK2 phosphorylation level may be implicated in LIDs, calling for novel therapeutic strategies. PMID:27169991

  4. HS3ST2 expression is critical for the abnormal phosphorylation of tau in Alzheimer's disease-related tau pathology.

    PubMed

    Sepulveda-Diaz, Julia Elisa; Alavi Naini, Seyedeh Maryam; Huynh, Minh Bao; Ouidja, Mohand Ouidir; Yanicostas, Constantin; Chantepie, Sandrine; Villares, Joao; Lamari, Foudil; Jospin, Estelle; van Kuppevelt, Toin H; Mensah-Nyagan, Ayikoe Guy; Raisman-Vozari, Rita; Soussi-Yanicostas, Nadia; Papy-Garcia, Dulce

    2015-05-01

    Heparan sulphate (glucosamine) 3-O-sulphotransferase 2 (HS3ST2, also known as 3OST2) is an enzyme predominantly expressed in neurons wherein it generates rare 3-O-sulphated domains of unknown functions in heparan sulphates. In Alzheimer's disease, heparan sulphates accumulate at the intracellular level in disease neurons where they co-localize with the neurofibrillary pathology, while they persist at the neuronal cell membrane in normal brain. However, it is unknown whether HS3ST2 and its 3-O-sulphated heparan sulphate products are involved in the mechanisms leading to the abnormal phosphorylation of tau in Alzheimer's disease and related tauopathies. Here, we first measured the transcript levels of all human heparan sulphate sulphotransferases in hippocampus of Alzheimer's disease (n = 8; 76.8 ± 3.5 years old) and found increased expression of HS3ST2 (P < 0.001) compared with control brain (n = 8; 67.8 ± 2.9 years old). Then, to investigate whether the membrane-associated 3-O-sulphated heparan sulphates translocate to the intracellular level under pathological conditions, we used two cell models of tauopathy in neuro-differentiated SH-SY5Y cells: a tau mutation-dependent model in cells expressing human tau carrying the P301L mutation hTau(P301L), and a tau mutation-independent model in where tau hyperphosphorylation is induced by oxidative stress. Confocal microscopy, fluorescence resonance energy transfer, and western blot analyses showed that 3-O-sulphated heparan sulphates can be internalized into cells where they interact with tau, promoting its abnormal phosphorylation, but not that of p38 or NF-κB p65. We showed, in vitro, that the 3-O-sulphated heparan sulphates bind to tau, but not to GSK3B, protein kinase A or protein phosphatase 2, inducing its abnormal phosphorylation. Finally, we demonstrated in a zebrafish model of tauopathy expressing the hTau(P301L), that inhibiting hs3st2 (also known as 3ost2) expression results in a strong inhibition of the

  5. Abnormal Phosphorylation of the Microtubule-Associated Protein τ (Tau) in Alzheimer Cytoskeletal Pathology

    NASA Astrophysics Data System (ADS)

    Grundke-Iqbal, Inge; Iqbal, Khalid; Tung, Yunn-Chyn; Quinlan, Maureen; Wisniewski, Henryk M.; Binder, Lester I.

    1986-07-01

    A monoclonal antibody to the microtubule-associated protein τ (tau) labeled some neurofibrillary tangles and plaque neurites, the two major locations of paired-helical filaments (PHF), in Alzheimer disease brain. The antibody also labeled isolated PHF that had been repeatedly washed with NaDodSO4. Dephosphorylation of the tissue sections with alkaline phosphatase prior to immunolabeling dramatically increased the number of tangles and plaques recognized by the antibody. The plaque core amyloid was not stained in either dephosphorylated or nondephosphorylated tissue sections. On immunoblots PHF polypeptides were labeled readily only when dephosphorylated. In contrast, a commercially available monoclonal antibody to a phosphorylated epitope of neurofilaments that labeled the tangles and the plaque neurites in tissue did not label any PHF polypeptides on immunoblots. The PHF polypeptides, labeled with the monoclonal antibody to τ , electrophoresed with those polypeptides recognized by antibodies to isolated PHF. The antibody to τ -labeled microtubules from normal human brains assembled in vitro but identically treated Alzheimer brain preparations had to be dephosphorylated to be completely recognized by this antibody. These findings suggest that τ in Alzheimer brain is an abnormally phosphorylated protein component of PHF.

  6. Deficiency of Lipoprotein Lipase in Neurons Decreases AMPA Receptor Phosphorylation and Leads to Neurobehavioral Abnormalities in Mice

    PubMed Central

    Yu, Tian; Taussig, Matthew D.; DiPatrizio, Nicholas V.; Astarita, Giuseppe; Piomelli, Daniele; Bergman, Bryan C.; Dell’Acqua, Mark L.; Eckel, Robert H.; Wang, Hong

    2015-01-01

    Alterations in lipid metabolism have been found in several neurodegenerative disorders, including Alzheimer’s disease. Lipoprotein lipase (LPL) hydrolyzes triacylglycerides in lipoproteins and regulates lipid metabolism in multiple organs and tissues, including the central nervous system (CNS). Though many brain regions express LPL, the functions of this lipase in the CNS remain largely unknown. We developed mice with neuron-specific LPL deficiency that became obese on chow by 16 wks in homozygous mutant mice (NEXLPL-/-) and 10 mo in heterozygous mice (NEXLPL+/-). In the present study, we show that 21 mo NEXLPL+/- mice display substantial cognitive function decline including poorer learning and memory, and increased anxiety with no difference in general motor activities and exploratory behavior. These neurobehavioral abnormalities are associated with a reduction in the 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl) propanoic acid (AMPA) receptor subunit GluA1 and its phosphorylation, without any alterations in amyloid β accumulation. Importantly, a marked deficit in omega-3 and omega-6 polyunsaturated fatty acids (PUFA) in the hippocampus precedes the development of the neurobehavioral phenotype of NEXLPL+/- mice. And, a diet supplemented with n-3 PUFA can improve the learning and memory of NEXLPL+/- mice at both 10 mo and 21 mo of age. We interpret these findings to indicate that LPL regulates the availability of PUFA in the CNS and, this in turn, impacts the strength of synaptic plasticity in the brain of aging mice through the modification of AMPA receptor and its phosphorylation. PMID:26263173

  7. Normal rat kidney cells secrete both phosphorylated and nonphosphorylated forms of osteopontin showing different physiological properties

    SciTech Connect

    Nemir, M.; DeVouge, M.W.; Mukherjee, B.B. )

    1989-10-25

    We have reported previously that the 69-kDa major phosphoprotein, secreted by normal rat kidney (NRK) cells, is osteopontin, a glycosylated bone matrix protein. Here we show that this 69-kDa osteopontin is secreted by NRK cells in both phosphorylated (pp69) and nonphosphorylated (np69) forms, with estimated isoelectric points of 3.8 and 4.5, respectively. Electrophoretic analysis of radioiodinated cell surface proteins immunoprecipitated with an anti-69-kDa osteopontin serum, demonstrates that the 69-kDa osteopontin is also present on the cell surface, but only its phosphorylated form (pp69) shows such cell surface association. Because osteopontin mediates cell adhesion and spreading, and contains an Arg-Gly-Asp-Ser cell-binding sequence, our observations strongly suggest that the cell surface localization of pp69 osteopontin is receptor-mediated, and the modification by phosphorylation may be crucial for its receptor binding activity. We also report that antisera directed against either fibronectin or 69-kDa osteopontin co-immunoprecipitate both np69 osteopontin and fibronectin as a heat-dissociable complex. In contrast, pp69 osteopontin does not co-precipitate with fibronectin. Furthermore, compared to NRK cells, vanadyl sulfate-treated NRK cells which acquire a reversible transformed phenotype, including anchorage-independent growth, show increased levels of pp69 on the cell surface, concomitant with significantly decreased levels of pp69 and elevated levels of np69 in the conditioned media. The data presented here establish transformation sensitivity of NRK cell-secreted osteopontin with respect to its secretion and cell surface localization, and demonstrate that phosphorylated and nonphosphorylated forms of osteopontin have different physiological properties, which may regulate the functional roles of this extracellular matrix protein.

  8. Cholangiolocellular carcinoma with rapid progression initially showing abnormally elevated serum alfa-fetoprotein.

    PubMed

    Yoh, Tomoaki; Kato, Tatsushi; Hirohata, Yoshiaki; Nakamura, Yuya; Nakayama, Hiroyuki; Okamura, Ryuji

    2016-08-01

    Cholangiolocellular carcinoma (CoCC) is a rare malignant liver tumor derived from hepatic progenitor cells, which exist in the canals of Hering. We encountered a case of CoCC with an extremely poor clinical course, initially showing abnormally elevated serum alfa-fetoprotein (AFP). A 72-year-old male presented with a liver tumor and abnormally elevated serum AFP levels (16,399 ng/ml). We preoperatively diagnosed hepatocellular carcinoma and performed extended right hepatectomy, after which the serum AFP levels remarkably decreased to 97 ng/ml. Postoperatively, the disease was pathologically diagnosed as CoCC. Furthermore, immunohistochemical pathological findings were alcian blue negative, cytokeratin (CK) 7 partially positive, CK19 positive, hepatocyte paraffin-1 negative, membranous negative for epithelial membrane antigen, and AFP negative. Fifty-five days later, intra- and extrahepatic recurrence developed, and the patient died 65 days after surgery. Although CoCCs show favorable outcomes, these characteristics of our case were not previously reported. It is necessary to accumulate more information on CoCC. PMID:27363839

  9. Eya1-deficient mice lack ears and kidneys and show abnormal apoptosis of organ primordia.

    PubMed

    Xu, P X; Adams, J; Peters, H; Brown, M C; Heaney, S; Maas, R

    1999-09-01

    Haploinsufficiency for human EYA1, a homologue of the Drosophila melanogaster gene eyes absent (eya), results in the dominantly inherited disorders branchio-oto-renal (BOR) syndrome and branchio-oto (BO) syndrome, which are characterized by craniofacial abnormalities and hearing loss with (BOR) or without (BO) kidney defects. To understand the developmental pathogenesis of organs affected in these syndromes, we inactivated the gene Eya1 in mice. Eya1 heterozygotes show renal abnormalities and a conductive hearing loss similar to BOR syndrome, whereas Eya1 homozygotes lack ears and kidneys due to defective inductive tissue interactions and apoptotic regression of the organ primordia. Inner ear development in Eya1 homozygotes arrests at the otic vesicle stage and all components of the inner ear and specific cranial sensory ganglia fail to form. In the kidney, Eya1 homozygosity results in an absence of ureteric bud outgrowth and a subsequent failure of metanephric induction. Gdnf expression, which is required to direct ureteric bud outgrowth via activation of the c-ret Rtk (refs 5, 6, 7, 8), is not detected in Eya1-/- metanephric mesenchyme. In Eya1-/- ear and kidney development, Six but not Pax expression is Eya1 dependent, similar to a genetic pathway elucidated in the Drosophila eye imaginal disc. Our results indicate that Eya1 controls critical early inductive signalling events involved in ear and kidney formation and integrate Eya1 into the genetic regulatory cascade controlling kidney formation upstream of Gdnf. In addition, our results suggest that an evolutionarily conserved Pax-Eya-Six regulatory hierarchy is used in mammalian ear and kidney development. PMID:10471511

  10. Phosphorylation of Ser-180 of rat aquaporin-4 shows marginal affect on regulation of water permeability: molecular dynamics study.

    PubMed

    Sachdeva, Ruchi; Singh, Balvinder

    2014-04-01

    Water permeation through rat aquaporin-4 (rAQP4), predominantly found in mammalian brain is regulated by phosphorylation of Ser-180. The present study has been carried out to understand the structural mechanism of regulation of water permeability across the channel. Molecular dynamics (MD) simulations have been carried out to investigate the structural changes caused due to phosphorylation of Ser-180 in the tetrameric assembly of rAQP4 along with predicted C-terminal region (255-323). The interactions involving opposite charges are observed between cytoplasmic loops and the C-terminal region during MD simulations. This results in movement of C-terminal region of rAQP4 towards the cytoplasmic mouth of water channel. Despite this movement, there was a gap between C-terminal region and cytoplasmic mouth of the channel through which water molecules were able to gain entry into the channel. The interactions between C-terminus and loop D of neighboring monomers in a tetrameric assembly appear to prevent the complete closure of cytoplasmic mouth of the water channel. Further, the rates of water permeation through phosphorylated and unphosphorylated rAQP4 have also been compared. The simulation studies showed a continuous movement of water in a single file across pore of unphosphorylated as well as phosphorylated rAQP4. PMID:23651078

  11. Individuals with autism spectrum disorder show abnormalities during initial and subsequent phases of precision gripping.

    PubMed

    Wang, Zheng; Magnon, Grant C; White, Stormi P; Greene, Rachel K; Vaillancourt, David E; Mosconi, Matthew W

    2015-04-01

    Sensorimotor impairments are common in autism spectrum disorder (ASD), but they are not well understood. Here we examined force control during initial pulses and the subsequent rise, sustained, and relaxation phases of precision gripping in 34 individuals with ASD and 25 healthy control subjects. Participants pressed on opposing load cells with their thumb and index finger while receiving visual feedback regarding their performance. They completed 2- and 8-s trials during which they pressed at 15%, 45%, or 85% of their maximum force. Initial pulses guided by feedforward control mechanisms, sustained force output controlled by visual feedback processes, and force relaxation rates all were examined. Control subjects favored an initial pulse strategy characterized by a rapid increase in and then relaxation of force when the target force was low (Type 1). When the target force level or duration of trials was increased, control subjects transitioned to a strategy in which they more gradually increased their force, paused, and then increased their force again. Individuals with ASD showed a more persistent bias toward the Type 1 strategy at higher force levels and during longer trials, and their initial force output was less accurate than that of control subjects. Patients showed increased force variability compared with control subjects when attempting to sustain a constant force level. During the relaxation phase, they showed reduced rates of force decrease. These findings suggest that both feedforward and feedback motor control mechanisms are compromised in ASD and these deficits may contribute to the dyspraxia and sensorimotor abnormalities often seen in this disorder. PMID:25552638

  12. Individuals with autism spectrum disorder show abnormalities during initial and subsequent phases of precision gripping

    PubMed Central

    Magnon, Grant C.; White, Stormi P.; Greene, Rachel K.; Vaillancourt, David E.

    2014-01-01

    Sensorimotor impairments are common in autism spectrum disorder (ASD), but they are not well understood. Here we examined force control during initial pulses and the subsequent rise, sustained, and relaxation phases of precision gripping in 34 individuals with ASD and 25 healthy control subjects. Participants pressed on opposing load cells with their thumb and index finger while receiving visual feedback regarding their performance. They completed 2- and 8-s trials during which they pressed at 15%, 45%, or 85% of their maximum force. Initial pulses guided by feedforward control mechanisms, sustained force output controlled by visual feedback processes, and force relaxation rates all were examined. Control subjects favored an initial pulse strategy characterized by a rapid increase in and then relaxation of force when the target force was low (Type 1). When the target force level or duration of trials was increased, control subjects transitioned to a strategy in which they more gradually increased their force, paused, and then increased their force again. Individuals with ASD showed a more persistent bias toward the Type 1 strategy at higher force levels and during longer trials, and their initial force output was less accurate than that of control subjects. Patients showed increased force variability compared with control subjects when attempting to sustain a constant force level. During the relaxation phase, they showed reduced rates of force decrease. These findings suggest that both feedforward and feedback motor control mechanisms are compromised in ASD and these deficits may contribute to the dyspraxia and sensorimotor abnormalities often seen in this disorder. PMID:25552638

  13. Trivalent dimethylarsenic compound induces histone H3 phosphorylation and abnormal localization of Aurora B kinase in HepG2 cells

    SciTech Connect

    Suzuki, Toshihide; Miyazaki, Koichi; Kita, Kayoko; Ochi, Takafumi

    2009-12-15

    Trivalent dimethylarsinous acid [DMA(III)] has been shown to induce mitotic abnormalities, such as centrosome abnormality, multipolar spindles, multipolar division, and aneuploidy, in several cell lines. In order to elucidate the mechanisms underlying these mitotic abnormalities, we investigated DMA(III)-mediated changes in histone H3 phosphorylation and localization of Aurora B kinase, which is a key molecule in cell mitosis. DMA(III) caused the phosphorylation of histone H3 (ser10) and was distributed predominantly in mitotic cells, especially in prometaphase cells. By contrast, most of the phospho-histone H3 was found to be localized in interphase cells after treatment with inorganic arsenite [iAs(III)], suggesting the involvement of a different pathway in phosphorylation. DMA(III) activated Aurora B kinase and slightly activated ERK MAP kinase. Phosphorylation of histone H3 by DMA(III) was effectively reduced by ZM447439 (Aurora kinase inhibitor) and slightly reduced by U0126 (MEK inhibitor). By contrast, iAs(III)-dependent histone H3 phosphorylation was markedly reduced by U0126. Aurora B kinase is generally localized in the midbody during telophase and plays an important role in cytokinesis. However, in some cells treated with DMA(III), Aurora B was not localized in the midbody of telophase cells. These findings suggested that DMA(III) induced a spindle abnormality, thereby activating the spindle assembly checkpoint (SAC) through the Aurora B kinase pathway. In addition, cytokinesis was not completed because of the abnormal localization of Aurora B kinase by DMA(III), thereby resulting in the generation of multinucleated cells. These results provide insight into the mechanism of arsenic tumorigenesis.

  14. Somatosensory cortex functional connectivity abnormalities in autism show opposite trends, depending on direction and spatial scale

    PubMed Central

    Khan, Sheraz; Michmizos, Konstantinos; Tommerdahl, Mark; Ganesan, Santosh; Kitzbichler, Manfred G.; Zetino, Manuel; Garel, Keri-Lee A.; Herbert, Martha R.; Hämäläinen, Matti S.

    2015-01-01

    Functional connectivity is abnormal in autism, but the nature of these abnormalities remains elusive. Different studies, mostly using functional magnetic resonance imaging, have found increased, decreased, or even mixed pattern functional connectivity abnormalities in autism, but no unifying framework has emerged to date. We measured functional connectivity in individuals with autism and in controls using magnetoencephalography, which allowed us to resolve both the directionality (feedforward versus feedback) and spatial scale (local or long-range) of functional connectivity. Specifically, we measured the cortical response and functional connectivity during a passive 25-Hz vibrotactile stimulation in the somatosensory cortex of 20 typically developing individuals and 15 individuals with autism, all males and right-handed, aged 8–18, and the mu-rhythm during resting state in a subset of these participants (12 per group, same age range). Two major significant group differences emerged in the response to the vibrotactile stimulus. First, the 50-Hz phase locking component of the cortical response, generated locally in the primary (S1) and secondary (S2) somatosensory cortex, was reduced in the autism group (P < 0.003, corrected). Second, feedforward functional connectivity between S1 and S2 was increased in the autism group (P < 0.004, corrected). During resting state, there was no group difference in the mu-α rhythm. In contrast, the mu-β rhythm, which has been associated with feedback connectivity, was significantly reduced in the autism group (P < 0.04, corrected). Furthermore, the strength of the mu-β was correlated to the relative strength of 50 Hz component of the response to the vibrotactile stimulus (r = 0.78, P < 0.00005), indicating a shared aetiology for these seemingly unrelated abnormalities. These magnetoencephalography-derived measures were correlated with two different behavioural sensory processing scores (P < 0.01 and P < 0.02 for the autism

  15. Novel alternative splice variants of rat phosphodiesterase 7B showing unique tissue-specific expression and phosphorylation.

    PubMed Central

    Sasaki, Takashi; Kotera, Jun; Omori, Kenji

    2002-01-01

    cDNA species coding for novel variants of cyclic-AMP-specific phosphodiesterases (PDEs), namely the PDE7B family, were isolated from rats and characterized. Rat PDE7B1 (RNPDE7B1) was composed of 446 amino acid residues. Rat PDE7B2 (RNPDE7B2) and PDE7B3 (RNPDE7B3), which possessed unique N-terminal sequences, consisted of 359 and 459 residues respectively. Northern hybridization analysis showed that rat PDE7B transcripts were particularly abundant in the striatum and testis. PCR analyses revealed that rat PDE7B2 transcripts were restricted to the testis and that low levels of PDE7B3 transcripts were expressed in the heart, lung and skeletal muscle. In situ hybridization analysis demonstrated that rat PDE7B transcripts were expressed in striatal neurons and spermatocytes. In spermatocytes, rat PDE7B transcripts were expressed in a stage-specific manner during spermatogenesis. The K(m) values of recombinant rat PDE7B1, PDE7B2 and PDE7B3 for cAMP were 0.05, 0.07 and 0.05 microM respectively. Each rat PDE7B variant was the most sensitive to 3-isobutyl-1-methylxanthine (IC(50) 1.5-2.1 microM). Two phosphorylation sites for cAMP-dependent protein kinase (PKA) were found in rat PDE7B1 and PDE7B3, whereas rat PDE7B2 possessed one site. PKA-dependent phosphorylation was observed in C-terminal phosphorylation sites of three rat PDE7B variants, in addition to unique N-terminal regions of rat PDE7B1 and PDE7B3. Unique tissue distribution and PKA-dependent phosphorylation of PDE7B variants suggested that each variant has a specific role for cellular functions via cAMP signalling in various tissues. PMID:11772393

  16. Preliminary Findings Show Maternal Hypothyroidism May Contribute to Abnormal Cortical Morphology in Offspring

    PubMed Central

    Lischinsky, Julieta E.; Skocic, Jovanka; Clairman, Hayyah; Rovet, Joanne

    2016-01-01

    In rodents, insufficient thyroid hormone (TH) gestationally has adverse effects on cerebral cortex development. Comparable studies of humans examining how TH insufficiency affects cortical morphology are limited to children with congenital hypothyroidism or offspring of hypothyroxinemic women; effects on cortex of children born to women with clinically diagnosed hypothyroidism are not known. We studied archived MRI scans from 22 children aged 10–12 years born to women treated for preexisting or de novo hypothyroidism in pregnancy (HYPO) and 24 similar age and sex controls from euthyroid women. FreeSurfer Image Analysis Suite software was used to measure cortical thickness (CT) and a vertex-based approach served to compare HYPO versus control groups and Severe versus Mild HYPO subgroups as well as to perform regression analyses examining effects of trimester-specific maternal TSH on CT. Results showed that relative to controls, HYPO had multiple regions of both cortical thinning and thickening, which differed for left and right hemispheres. In HYPO, thinning was confined to medial and mid-lateral regions of each hemisphere and thickening to superior regions (primarily frontal) of the left hemisphere and inferior regions (particularly occipital and temporal) of the right. The Severe HYPO subgroup showed more thinning than Mild in frontal and temporal regions and more thickening in bilateral posterior and frontal regions. Maternal TSH values predicted degree of thinning and thickening within multiple brain regions, with the pattern and direction of correlations differing by trimester. Notably, some correlations remained when cases born to women with severe hypothyroidism were removed from the analyses, suggesting that mild variations of maternal TH may permanently affect offspring cortex. We conclude that maternal hypothyroidism during pregnancy has long-lasting manifestations on the cortical morphology of their offspring with specific effects reflecting both

  17. A 32-kDa tyrosine-phosphorylated protein shows a protease-dependent increase in dead boar spermatozoa.

    PubMed

    Tabuchi, Tomohito; Shidara, Osamu; Harayama, Hiroshi

    2008-12-01

    Boar sperm TyrP32 is a 32-kDa tyrosine-phosphorylated protein that increases during the capacitation and acrosome reaction and during cryocapacitation. However, it is still unclear whether the increase in TyrP32 is an event that is limited to the process of sperm fertilization, including cryocapacitation. The aims of the present study were to demonstrate that TyrP32 is increased in dead spermatozoa after freeze-thawing without a cryoprotectant and to find the causal factors for this increase. Washed spermatozoa were resuspended in a salt solution and then frozen. The frozen samples were rapidly thawed in a warm water bath and then used for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE)/Western blotting to detect TyrP32, SDS-PAGE/silver staining of sperm proteins and staining of acrosomal contents with fluorescein isothiocyanate (FITC)-conjugated peanut agglutinin (PNA). In the samples before freezing, TyrP32 was barely detectable, and the distribution of the acrosomal contents was normal in most spermatozoa. One cycle of freeze-thawing induced an increase in TyrP32, a decrease in major sperm proteins and disorder in the acrosomal contents. However, the addition of a protease inhibitor (APMSF, 1 mM) suppressed the increase in TyrP32 and the decrease in the major sperm proteins, although it did not have any influence on the disorder in the acrosomal contents. Additionally, the spermatozoa did not exhibit any flagellar movement after freeze-thawing, which showed that almost all of them were dead. These results indicate that TyrP32 can show a protease-dependent increase in dead spermatozoa after freeze-thawing without a cryoprotectant even though the dead spermatozoa do not undergo cryocapacitation. PMID:18787309

  18. Cystic fibrosis transmembrane conductance regulator Cl- channels with R domain deletions and translocations show phosphorylation-dependent and -independent activity.

    PubMed

    Baldursson, O; Ostedgaard, L S; Rokhlina, T; Cotten, J F; Welsh, M J

    2001-01-19

    Phosphorylation of the R domain regulates cystic fibrosis transmembrane conductance regulator Cl- channel activity. Earlier studies suggested that the R domain controls activity via more than one mechanism; a phosphorylated R domain may stimulate activity, and an unphosphorylated R domain may prevent constitutive activity, i.e. opening with ATP alone. However, the mechanisms responsible for these two regulatory properties are not understood. In this study we asked whether the two effects are dependent on its position in the protein and whether smaller regions from the R domain mediate the effects. We found that several portions of the R domain conferred phosphorylation-stimulated activity. This was true whether the R domain sequences were present in their normal location or were translocated to the C terminus. We also found that some parts of the R domain could be deleted without inducing constitutive activity. However, when residues 760-783 were deleted, channels opened without phosphorylation. Translocation of the R domain to the C terminus did not prevent constitutive activity. These results suggest that different parts of the phosphorylated R domain can stimulate activity and that their location within the protein is not critical. In contrast, prevention of constitutive activity required a short specific sequence that could not be moved to the C terminus. These results are consistent with a recent model of an R domain composed primarily of random coil in which more than one phosphorylation site is capable of stimulating channel activity, and net activity reflects interactions between multiple sites in the R domain and the rest of the channel. PMID:11038358

  19. A diurnally regulated dehydrin from Avicennia marina that shows nucleo-cytoplasmic localization and is phosphorylated by Casein kinase II in vitro.

    PubMed

    Mehta, Preeti A; Rebala, Keerthi C; Venkataraman, Gayatri; Parida, Ajay

    2009-08-01

    Dehydrins have a key role in protecting plants from dehydration stress. We report here the isolation of two cDNAs coding for the same dehydrin, AmDHN1 and AmDHN1a from salt stressed leaves of Avicennia marina (Forsk.) Vierh. by EST library screening. AmDHN1 was found to contain a retained intron that was absent in AmDHN1a. AmDHN1 expression in the context of various environmental stresses was investigated. In leaves, AmDHN1 shows a diurnal pattern of regulation and is induced only by mannitol application. In roots, AmDHN1 is rapidly induced by salinity (NaCl) and dehydration stress (PEG and mannitol). A fragment of 795 bp corresponding to the 5' upstream region of AmDHN1 was isolated by TAIL-PCR. In silico analysis of this sequence reveals the presence of putative stress regulatory elements (ABRE, DRE, MYB and MYC binding sequences). Putative phosphorylation sites for Casein kinase II were identified in the AmDHN1a ORF. In vitro phosphorylation of Escherichia coli expressed Trx-AmDHN1a by Casein kinase II was observed that was reversed by Shrimp Alkaline Phosphatase treatment. A putative nuclear targeting domain was identified in the translated AmDHN1a ORF and stably transformed AmDHNIa-GFP was found to show nucleo-cytoplasmic localization in tobacco guard cells. As observed for maize Rab 17, the phosphorylation of AmDHN1a may contribute to its nuclear localization. PMID:19398349

  20. A case of ataxic diplegia, mental retardation, congenital nystagmus and abnormal auditory brain stem responses showing only waves I and II.

    PubMed

    Aiba, K; Yokochi, K; Ishikawa, T

    1986-01-01

    A three-year-old boy who had ataxic diplegia, mental retardation, horizontal pendular nystagmus with head nodding and abnormal auditory brain stem responses showing only waves I and II was presented. His clinical features coincided with recent reports in the Japanese literature of cases of a new syndrome that is congenital in origin and seen only in boys. PMID:3826555

  1. Children with autism spectrum disorders show abnormal conditioned response timing on delay, but not trace, eyeblink conditioning

    PubMed Central

    Oristaglio, Jeff; West, Susan Hyman; Ghaffari, Manely; Lech, Melissa S.; Verma, Beeta R.; Harvey, John A.; Welsh, John P.; Malone, Richard P.

    2013-01-01

    Children with autism spectrum disorder (ASD) and age-matched typically-developing (TD) peers were tested on two forms of eyeblink conditioning (EBC), a Pavlovian associative learning paradigm where subjects learn to execute an appropriately-timed eyeblink in response to a previously neutral conditioning stimulus (CS). One version of the task, trace EBC, interposes a stimulus-free interval between the presentation of the CS and the unconditioned stimulus (US), a puff of air to the eye which causes subjects to blink. In delay EBC, the CS overlaps in time with the delivery of the US, usually with both stimuli terminating simultaneously. ASD children performed normally during trace EBC, exhibiting no differences from typically-developing (TD) subjects with regard to learning rate or the timing of the CR. However, when subsequently tested on delay EBC, subjects with ASD displayed abnormally-timed conditioned eye blinks that began earlier and peaked sooner than those of TD subjects, consistent with previous findings. The results suggest an impaired ability of children with ASD to properly time conditioned eye blinks which appears to be specific to delay EBC. We suggest that this deficit may reflect a dysfunction of cerebellar cortex in which increases in the intensity or duration of sensory input can temporarily disrupt the accuracy of motor timing over short temporal intervals. PMID:23769889

  2. Characterization of the Skeletal Fusion with Sterility (sks) Mouse Showing Axial Skeleton Abnormalities Caused by Defects of Embryonic Skeletal Development

    PubMed Central

    Akiyama, Kouyou; Katayama, Kentaro; Tsuji, Takehito; Kunieda, Tetsuo

    2014-01-01

    The development of the axial skeleton is a complex process, consisting of segmentation and differentiation of somites and ossification of the vertebrae. The autosomal recessive skeletal fusion with sterility (sks) mutation of the mouse causes skeletal malformations due to fusion of the vertebrae and ribs, but the underlying defects of vertebral formation during embryonic development have not yet been elucidated. For the present study, we examined the skeletal phenotypes of sks/sks mice during embryonic development and the chromosomal localization of the sks locus. Multiple defects of the axial skeleton, including fusion of vertebrae and fusion and bifurcation of ribs, were observed in adult and neonatal sks/sks mice. In addition, we also found polydactyly and delayed skull ossification in the sks/sks mice. Morphological defects, including disorganized vertebral arches and fusions and bifurcations of the axial skeletal elements, were observed during embryonic development at embryonic day 12.5 (E12.5) and E14.5. However, no morphological abnormality was observed at E11.5, indicating that defects of the axial skeleton are caused by malformation of the cartilaginous vertebra and ribs at an early developmental stage after formation and segmentation of the somites. By linkage analysis, the sks locus was mapped to an 8-Mb region of chromosome 4 between D4Mit331 and D4Mit199. Since no gene has already been identified as a cause of malformation of the vertebra and ribs in this region, the gene responsible for sks is suggested to be a novel gene essential for the cartilaginous vertebra and ribs. PMID:24521859

  3. Ethanol-induced phosphorylation of cytokeratin in cultured hepatocytes

    SciTech Connect

    Kawahara, Hiromu; Cadrin, M.; French, S.W. )

    1990-01-01

    The authors studied the effect of ethanol on the phosphorylation of cytokeratins (CKs) in cultured hepatocytes since CK filaments are resulted by phosphorylation and they are abnormal in alcoholic liver disease. Hepatocytes were obtained from 14-day-old rats and cultured for 48 hrs. The hepatocytes were exposed to ethanol for 30 min. The residual insoluble cytoskeletons were analyzed by two-dimensional gel electrophoresis and autoradiography. 2D gel electrophoresis showed CK 55 and CK 49 or 8 and 18 and actin. The CKs had several isoelectric variants. The most basic spot was the dominant protein which was not phosphorylated. The more acidic spots were phosphorylated. After ethanol treatment, the phosphorylation of CK 55 and CK 49 were markedly increased over controls. They compared these results, with the effect of vasopressin, TPA and db-cAMP on the phosphorylation of CKs. Vasopressin and TPA caused the phosphorylation of CK 55 and 49 but db-cAMP did not.

  4. Transgenic rats overexpressing the human MrgX3 gene show cataracts and an abnormal skin phenotype

    SciTech Connect

    Kaisho, Yoshihiko . E-mail: Kaisho_Yoshihiko@takeda.co.jp; Watanabe, Takuya; Nakata, Mitsugu; Yano, Takashi; Yasuhara, Yoshitaka; Shimakawa, Kozo; Mori, Ikuo; Sakura, Yasufumi; Terao, Yasuko; Matsui, Hideki; Taketomi, Shigehisa

    2005-05-13

    The human MrgX3 gene, belonging to the mrgs/SNSRs (mass related genes/sensory neuron specific receptors) family, was overexpressed in transgenic rats using the actin promoter. Two animal lines showed cataracts with liquification/degeneration and swelling of the lens fiber cells. The transient epidermal desquamation was observed in line with higher gene expression. Histopathology of the transgenic rats showed acanthosis and focal parakeratosis. In the epidermis, there was an increase in cellular keratin 14, keratin 10, and loricrin, as well as PGP 9.5 in innervating nerve fibers. These phenotypes accompanied an increase in the number of proliferating cells. These results suggest that overexpression of the human MrgX3 gene causes a disturbance of the normal cell-differentiation process.

  5. Fibroblasts from patients with Diamond-Blackfan anaemia show abnormal expression of genes involved in protein synthesis, amino acid metabolism and cancer

    PubMed Central

    Avondo, Federica; Roncaglia, Paola; Crescenzio, Nicoletta; Krmac, Helena; Garelli, Emanuela; Armiraglio, Marta; Castagnoli, Carlotta; Campagnoli, Maria Francesca; Ramenghi, Ugo; Gustincich, Stefano; Santoro, Claudio; Dianzani, Irma

    2009-01-01

    Background Diamond-Blackfan anaemia (DBA) is a rare inherited red cell hypoplasia characterised by a defect in the maturation of erythroid progenitors and in some cases associated with malformations. Patients have an increased risk of solid tumors. Mutations have been found in several ribosomal protein (RP) genes, i.e RPS19, RPS24, RPS17, RPL5, RPL11, RPL35A. Studies in haematopoietic progenitors from patients show that haplo-insufficiency of an RP impairs rRNA processing and ribosome biogenesis. DBA lymphocytes show reduced protein synthesis and fibroblasts display abnormal rRNA processing and impaired proliferation. Results To evaluate the involvement of non-haematopoietic tissues in DBA, we have analysed global gene expression in fibroblasts from DBA patients compared to healthy controls. Microarray expression profiling using Affymetrix GeneChip Human Genome U133A 2.0 Arrays revealed that 421 genes are differentially expressed in DBA patient fibroblasts. These genes include a large cluster of ribosomal proteins and factors involved in protein synthesis and amino acid metabolism, as well as genes associated to cell death, cancer and tissue development. Conclusion This analysis reports for the first time an abnormal gene expression profile in a non-haematopoietic cell type in DBA. These data support the hypothesis that DBA may be due to a defect in general or specific protein synthesis. PMID:19765279

  6. Modulation of Calcium Oxalate Dihydrate Growth by Selective Crystal-face Binding of Phosphorylated Osteopontin and Polyaspartate Peptide Showing Occlusion by Sectoral (Compositional) Zoning*

    PubMed Central

    Chien, Yung-Ching; Masica, David L.; Gray, Jeffrey J.; Nguyen, Sarah; Vali, Hojatollah; McKee, Marc D.

    2009-01-01

    Calcium oxalate dihydrate (COD) mineral and the urinary protein osteopontin/uropontin (OPN) are commonly found in kidney stones. To investigate the effects of OPN on COD growth, COD crystals were grown with phosphorylated OPN or a polyaspartic acid-rich peptide of OPN (DDLDDDDD, poly-Asp86–93). Crystals grown with OPN showed increased dimensions of the {110} prismatic faces attributable to selective inhibition at this crystallographic face. At high concentrations of OPN, elongated crystals with dominant {110} faces were produced, often with intergrown, interpenetrating twin crystals. Poly-Asp86–93 dose-dependently elongated crystal morphology along the {110} faces in a manner similar to OPN. In crystal growth studies using fluorescently tagged poly-Asp86–93 followed by imaging of crystal interiors using confocal microscopy, sectoral (compositional) zoning in COD was observed resulting from selective binding and incorporation (occlusion) of peptide exclusively into {110} crystal sectors. Computational modeling of poly-Asp86–93 adsorption to COD {110} and {101} surfaces also suggests increased stabilization of the COD {110} surface and negligible change to the natively stable {101} surface. Ultrastructural, colloidal-gold immunolocalization of OPN by transmission electron microscopy in human stones confirmed an intracrystalline distribution of OPN. In summary, OPN and its poly-Asp86–93 sequence similarly affect COD mineral growth; the {110} crystallographic faces become enhanced and dominant attributable to {110} face inhibition by the protein/peptide, and peptides can incorporate into the mineral phase. We, thus, conclude that the poly-Asp86–93 domain is central to the OPN ability to interact with the {110} faces of COD, where it binds to inhibit crystal growth with subsequent intracrystalline incorporation (occlusion). PMID:19581305

  7. Immunochemical Analysis of the Electronegative LDL Subfraction Shows That Abnormal N-terminal Apolipoprotein B Conformation Is Involved in Increased Binding to Proteoglycans*

    PubMed Central

    Bancells, Cristina; Benítez, Sònia; Ordóñez-Llanos, Jordi; Öörni, Katariina; Kovanen, Petri T.; Milne, Ross W.; Sánchez-Quesada, José L.

    2011-01-01

    Electronegative LDL (LDL(−)) is a minor subfraction of modified LDL present in plasma. Among its atherogenic characteristics, low affinity to the LDL receptor and high binding to arterial proteoglycans (PGs) could be related to abnormalities in the conformation of its main protein, apolipoprotein B-100 (apoB-100). In the current study, we have performed an immunochemical analysis using monoclonal antibody (mAb) probes to analyze the conformation of apoB-100 in LDL(−). The study, performed with 28 anti-apoB-100 mAbs, showed that major differences of apoB-100 immunoreactivity between native LDL and LDL(−) concentrate in both terminal extremes. The mAbs Bsol 10, Bsol 14 (which recognize the amino-terminal region), Bsol 2, and Bsol 7 (carboxyl-terminal region) showed increased immunoreactivity in LDL(−), suggesting that both terminal extremes are more accessible in LDL(−) than in native LDL. The analysis of in vitro-modified LDLs, including LDL lipolyzed with sphingomyelinase (SMase-LDL) or phospholipase A2 (PLA2-LDL) and oxidized LDL (oxLDL), suggested that increased amino-terminal immunoreactivity was related to altered conformation due to aggregation. This was confirmed when the aggregated subfractions of LDL(−) (agLDL(−)) and oxLDL (ag-oxLDL) were isolated and analyzed. Thus, Bsol 10 and Bsol 14 immunoreactivity was high in SMase-LDL, ag-oxLDL, and agLDL(−). The altered amino-terminal apoB-100 conformation was involved in the increased PG binding affinity of agLDL(−) because Bsol 10 and Bsol 14 blocked its high PG-binding. These observations suggest that an abnormal conformation of the amino-terminal region of apoB-100 is responsible for the increased PG binding affinity of agLDL(−). PMID:21078674

  8. Translation elongation factor 1A mutants with altered actin bundling activity show reduced aminoacyl-tRNA binding and alter initiation via eIF2α phosphorylation.

    PubMed

    Perez, Winder B; Kinzy, Terri Goss

    2014-07-25

    Apart from its canonical function in translation elongation, eukaryotic translation elongation factor 1A (eEF1A) has been shown to interact with the actin cytoskeleton. Amino acid substitutions in eEF1A that reduce its ability to bind and bundle actin in vitro cause improper actin organization in vivo and reduce total translation. Initial in vivo analysis indicated the reduced translation was through initiation. The mutant strains exhibit increased levels of phosphorylated initiation factor 2α (eIF2α) dependent on the presence of the general control non-derepressible 2 (Gcn2p) protein kinase. Gcn2p causes downregulation of total protein synthesis at initiation in response to increases in deacylated tRNA levels in the cell. Increased levels of eIF2α phosphorylation are not due to a general reduction in translation elongation as eEF2 and eEF3 mutants do not exhibit this effect. Deletion of GCN2 from the eEF1A actin bundling mutant strains revealed a second defect in translation. The eEF1A actin-bundling proteins exhibit changes in their elongation activity at the level of aminoacyl-tRNA binding in vitro. These findings implicate eEF1A in a feedback mechanism for regulating translation at initiation. PMID:24936063

  9. Caveolin-3 null mice show a loss of caveolae, changes in the microdomain distribution of the dystrophin-glycoprotein complex, and t-tubule abnormalities.

    PubMed

    Galbiati, F; Engelman, J A; Volonte, D; Zhang, X L; Minetti, C; Li, M; Hou, H; Kneitz, B; Edelmann, W; Lisanti, M P

    2001-06-15

    Caveolin-3, a muscle-specific caveolin-related protein, is the principal structural protein of caveolae membrane domains in striated muscle cells. Recently, we identified a novel autosomal dominant form of limb-girdle muscular dystrophy (LGMD-1C) in humans that is due to mutations within the coding sequence of the human caveolin-3 gene (3p25). These LGMD-1C mutations lead to an approximately 95% reduction in caveolin-3 protein expression, i.e. a caveolin-3 deficiency. Here, we created a caveolin-3 null (CAV3 -/-) mouse model, using standard homologous recombination techniques, to mimic a caveolin-3 deficiency. We show that these mice lack caveolin-3 protein expression and sarcolemmal caveolae membranes. In addition, analysis of skeletal muscle tissue from these caveolin-3 null mice reveals: (i) mild myopathic changes; (ii) an exclusion of the dystrophin-glycoprotein complex from lipid raft domains; and (iii) abnormalities in the organization of the T-tubule system, with dilated and longitudinally oriented T-tubules. These results have clear mechanistic implications for understanding the pathogenesis of LGMD-1C at a molecular level. PMID:11259414

  10. Rothmund-Thomson syndrome: two case reports show heterogeneous cutaneous abnormalities, an association with genetically programmed ageing changes, and increased chromosomal radiosensitivity.

    PubMed Central

    Kerr, B; Ashcroft, G S; Scott, D; Horan, M A; Ferguson, M W; Donnai, D

    1996-01-01

    Rothmund-Thomson syndrome is a rare, autosomal recessive disorder associated with characteristic cutaneous changes, sparse hair, juvenile cataracts, short stature, skeletal defects, dystrophic teeth and nails, and hypogonadism. Mental retardation is unusual. An increased incidence of certain malignancies has been reported. Clonal or mosaic chromosome abnormalities and abnormalities in DNA repair mechanisms have been reported in some cases. We report two cases of Rothmund-Thomson syndrome, both with intellectual handicap, associated in one with a previously undescribed histological appearance of involved skin, suggesting that the spectrum of abnormalities is even more heterogeneous than previously presumed. Both cases exhibited chromosomal radiosensitivity of lymphocytes which may be an indication of a DNA repair defect. This is the first report of an association between Rothmund-Thomson syndrome and unique, intrinsic, age related skin changes. Images PMID:8950673

  11. Mutant forms of tumour necrosis factor receptor I that occur in TNF-receptor-associated periodic syndrome retain signalling functions but show abnormal behaviour

    PubMed Central

    Todd, Ian; Radford, Paul M; Draper-Morgan, Kelly-Ann; McIntosh, Richard; Bainbridge, Susan; Dickinson, Peter; Jamhawi, Lama; Sansaridis, Marios; Huggins, Mary L; Tighe, Patrick J; Powell, Richard J

    2004-01-01

    Tumour necrosis factor (TNF)-receptor-associated periodic syndrome (TRAPS) is a hereditary autoinflammatory disorder involving autosomal-dominant missense mutations in TNF receptor superfamily 1A (TNFRSF1A) ectodomains. To elucidate the molecular effects of TRAPS-related mutations, we transfected HEK-293 cells to produce lines stably expressing high levels of either wild-type (WT) or single mutant recombinant forms of TNFRSF1A. Mutants with single amino acid substitutions in the first cysteine-rich domain (CRD1) were produced both as full-length receptor proteins and as truncated forms lacking the cytoplasmic signalling domain (Δsig). High-level expression of either WT or mutant full-length TNFRSF1A spontaneously induced apoptosis and interleukin-8 production, indicating that the mutations in CRD1 did not abrogate signalling. Consistent with this, WT and mutant full-length TNFRSF1A formed cytoplasmic aggregates that co-localized with ubiquitin and chaperones, and with the signal transducer TRADD, but not with the inhibitor, silencer of death domain (SODD). Furthermore, as expected, WT and mutant Δsig forms of TNFRSF1A did not induce apoptosis or interleukin-8 production. However, whereas the WT full-length TNFRSF1A was expressed both in the cytoplasm and on the cell surface, the mutant receptors showed strong cytoplasmic expression but reduced cell-surface expression. The WT and mutant Δsig forms of TNFRSF1A were all expressed at the cell surface, but a proportion of the mutant receptors were also retained in the cytoplasm and co-localized with BiP. Furthermore, the mutant forms of surface-expressed Δsig TNFRSF1A were defective in binding TNF-α. We conclude that TRAPS-related CRD1 mutants of TNFRSF1A possess signalling properties associated with the cytoplasmic death domain, but other behavioural features of the mutant receptors are abnormal, including intracellular trafficking and TNF binding. PMID:15312137

  12. [An acute severe heat stroke patient showing abnormal diffuse high intensity of the cerebellar cortex in diffusion weighted image: a case report].

    PubMed

    Fujioka, Yusuke; Yasui, Keizo; Hasegawa, Yasuhiro; Takahashi, Akira; Sobue, Gen

    2009-10-01

    A 47-year-old man was admitted to the hospital because of general convulsion, loss of consciousness and hyperthermia. A diagnosis of acute heat stroke was made clinically and neuroradiologically. As the consciousness level ameliorated, he developed severe abulia and mutism, then cerebellar ataxic syndrome (viz. truncal ataxia, hypermetria, ataxic speech and nystagmus). An MRI (diffusion weighted image; DWI) disclosed abnormal diffuse high signal intensity of the cerebellar cortex with reduced apparent diffusion coefficient (ADC). Two months later after the onset, truncal ataxia and dysarthria significantly improved, while dysmetria of the extremities rather worsened. At that time, the abnormal signal intensity of the cerebellar cortex disappeared, and the cerebellum became atrophic. The cerebellar blood flow was significantly decreased on brain SPECT (99mTc-ECD). The abnormal DWI signal intensity of the cerebellar cortex in the present patient may represent the cytotoxic edema of Purkinje cells resulting from heat stroke-related hyperthermia It is essential to repeat MRI examination for cerebellar pathology and to obtain better insight into sequelae in patients with acute heat stroke. Protirelin tartrate seemed to be valid for improvement of abulia in the present patient. Further study is indicated. PMID:19999144

  13. Mitotic-like tau phosphorylation by p25-Cdk5 kinase complex.

    PubMed

    Hamdane, Malika; Sambo, Anne-Véronique; Delobel, Patrice; Bégard, Séverine; Violleau, Anne; Delacourte, André; Bertrand, Philippe; Benavides, Jesus; Buée, Luc

    2003-09-01

    Among tau phosphorylation sites, some phosphoepitopes referred to as abnormal ones are exclusively found on tau aggregated into filaments in Alzheimer's disease. Recent data suggested that molecular mechanisms similar to those encountered during mitosis may play a role in abnormal tau phosphorylation. In particular, TG-3 phosphoepitope is associated with early stages of neurofibrillary tangles (NFTs). In this study, we reported a suitable cell model consisting of SH-SY5Y cells stably transfected with an inducible p25 expression vector. It allows investigation of tau phosphorylation by p25-Cdk5 kinase complex in a neuronal context and avoiding p25-induced cytotoxicity. Immunoblotting analyses showed that p25-Cdk5 strongly phosphorylates tau protein not only at the AT8 epitope but also at the AT180 epitope and at the Alzheimer's mitotic epitope TG-3. Further biochemical analyses showed that abnormal phosphorylated tau accumulated in cytosol as a microtubule-free form, suggesting its impact on tau biological activity. Since tau abnormal phosphorylation occurred in dividing cells, TG-3 immunoreactivity was also investigated in differentiated neuronal ones, and both TG-3-immunoreactive tau and nucleolin, another early marker for NFT, were also generated. These data suggest that p25-Cdk5 is responsible for the mitotic-like phosphoepitopes present in NFT and argue for a critical role of Cdk5 in neurodegenerative mechanisms. PMID:12826674

  14. Histone phosphorylation

    PubMed Central

    Rossetto, Dorine; Avvakumov, Nikita; Côté, Jacques

    2012-01-01

    Histone posttranslational modifications are key components of diverse processes that modulate chromatin structure. These marks function as signals during various chromatin-based events, and act as platforms for recruitment, assembly or retention of chromatin-associated factors. The best-known function of histone phosphorylation takes place during cellular response to DNA damage, when phosphorylated histone H2A(X) demarcates large chromatin domains around the site of DNA breakage. However, multiple studies have also shown that histone phosphorylation plays crucial roles in chromatin remodeling linked to other nuclear processes. In this review, we summarize the current knowledge of histone phosphorylation and describe the many kinases and phosphatases that regulate it. We discuss the key roles played by this histone mark in DNA repair, transcription and chromatin compaction during cell division and apoptosis. Additionally, we describe the intricate crosstalk that occurs between phosphorylation and other histone modifications and allows for sophisticated control over the chromatin remodeling processes. PMID:22948226

  15. Meiotic abnormalities

    SciTech Connect

    1993-12-31

    Chapter 19, describes meiotic abnormalities. These include nondisjunction of autosomes and sex chromosomes, genetic and environmental causes of nondisjunction, misdivision of the centromere, chromosomally abnormal human sperm, male infertility, parental age, and origin of diploid gametes. 57 refs., 2 figs., 1 tab.

  16. Oxidative phosphorylation and lacunar stroke

    PubMed Central

    Anderson, Christopher D.; Hurford, Robert; Bevan, Steve; Markus, Hugh S.

    2016-01-01

    Objective: We investigated whether oxidative phosphorylation (OXPHOS) abnormalities were associated with lacunar stroke, hypothesizing that these would be more strongly associated in patients with multiple lacunar infarcts and leukoaraiosis (LA). Methods: In 1,012 MRI-confirmed lacunar stroke cases and 964 age-matched controls recruited from general practice surgeries, we investigated associations between common genetic variants within the OXPHOS pathway and lacunar stroke using a permutation-based enrichment approach. Cases were phenotyped using MRI into those with multiple infarcts or LA (MLI/LA) and those with isolated lacunar infarcts (ILI) based on the number of subcortical infarcts and degree of LA, using the Fazekas grading. Using gene-level association statistics, we tested for enrichment of genes in the OXPHOS pathway with all lacunar stroke and the 2 subtypes. Results: There was a specific association with strong evidence of enrichment in the top 1% of genes in the MLI/LA (subtype p = 0.0017) but not in the ILI subtype (p = 1). Genes in the top percentile for the all lacunar stroke analysis were not significantly enriched (p = 0.07). Conclusions: Our results implicate the OXPHOS pathway in the pathogenesis of lacunar stroke, and show the association is specific to patients with the MLI/LA subtype. They show that MRI-based subtyping of lacunar stroke can provide insights into disease pathophysiology, and imply that different radiologic subtypes of lacunar stroke subtypes have distinct underlying pathophysiologic processes. PMID:26674331

  17. AMP-activated protein kinase modulates tau phosphorylation and tau pathology in vivo

    PubMed Central

    Domise, Manon; Didier, Sébastien; Marinangeli, Claudia; Zhao, Haitian; Chandakkar, Pallavi; Buée, Luc; Viollet, Benoit; Davies, Peter; Marambaud, Philippe; Vingtdeux, Valérie

    2016-01-01

    Neurofibrillary tangles (NFTs) are the pathological hallmark of neurodegenerative diseases commonly known as tauopathies. NFTs result from the intracellular aggregation of abnormally and hyperphosphorylated tau proteins. Tau functions, which include the regulation of microtubules dynamics, are dependent on its phosphorylation status. As a consequence, any changes in tau phosphorylation can have major impacts on synaptic plasticity and memory. Recently, it has been demonstrated that AMP-activated protein kinase (AMPK) was deregulated in the brain of Alzheimer’s disease (AD) patients where it co-localized with phosphorylated tau in pre-tangle and tangle-bearing neurons. Besides, it was found that AMPK was a tau kinase in vitro. Here, we find that endogenous AMPK activation in mouse primary neurons induced an increase of tau phosphorylation at multiple sites, whereas AMPK inhibition led to a rapid decrease of tau phosphorylation. We further show that AMPK mice deficient for one of the catalytic alpha subunits displayed reduced endogenous tau phosphorylation. Finally, we found that AMPK deficiency reduced tau pathology in the PS19 mouse model of tauopathy. These results show that AMPK regulates tau phosphorylation in mouse primary neurons as well as in vivo, and thus suggest that AMPK could be a key player in the development of AD pathology. PMID:27230293

  18. AMP-activated protein kinase modulates tau phosphorylation and tau pathology in vivo.

    PubMed

    Domise, Manon; Didier, Sébastien; Marinangeli, Claudia; Zhao, Haitian; Chandakkar, Pallavi; Buée, Luc; Viollet, Benoit; Davies, Peter; Marambaud, Philippe; Vingtdeux, Valérie

    2016-01-01

    Neurofibrillary tangles (NFTs) are the pathological hallmark of neurodegenerative diseases commonly known as tauopathies. NFTs result from the intracellular aggregation of abnormally and hyperphosphorylated tau proteins. Tau functions, which include the regulation of microtubules dynamics, are dependent on its phosphorylation status. As a consequence, any changes in tau phosphorylation can have major impacts on synaptic plasticity and memory. Recently, it has been demonstrated that AMP-activated protein kinase (AMPK) was deregulated in the brain of Alzheimer's disease (AD) patients where it co-localized with phosphorylated tau in pre-tangle and tangle-bearing neurons. Besides, it was found that AMPK was a tau kinase in vitro. Here, we find that endogenous AMPK activation in mouse primary neurons induced an increase of tau phosphorylation at multiple sites, whereas AMPK inhibition led to a rapid decrease of tau phosphorylation. We further show that AMPK mice deficient for one of the catalytic alpha subunits displayed reduced endogenous tau phosphorylation. Finally, we found that AMPK deficiency reduced tau pathology in the PS19 mouse model of tauopathy. These results show that AMPK regulates tau phosphorylation in mouse primary neurons as well as in vivo, and thus suggest that AMPK could be a key player in the development of AD pathology. PMID:27230293

  19. Congenital Abnormalities

    MedlinePlus

    ... serious health problems (e.g. Down syndrome ). Single-Gene Abnormalities Sometimes the chromosomes are normal in number, ... blood flow to the fetus impair fetal growth. Alcohol consumption and certain drugs during pregnancy significantly increase ...

  20. Craniofacial Abnormalities

    MedlinePlus

    ... of the skull and face. Craniofacial abnormalities are birth defects of the face or head. Some, like cleft ... palate, are among the most common of all birth defects. Others are very rare. Most of them affect ...

  1. Walking abnormalities

    MedlinePlus

    ... include: Arthritis of the leg or foot joints Conversion disorder (a psychological disorder) Foot problems (such as a ... injuries. For an abnormal gait that occurs with conversion disorder, counseling and support from family members are strongly ...

  2. Chromosome Abnormalities

    MedlinePlus

    ... decade, newer techniques have been developed that allow scientists and doctors to screen for chromosomal abnormalities without using a microscope. These newer methods compare the patient's DNA to a normal DNA ...

  3. Nail abnormalities

    MedlinePlus

    Nail abnormalities are problems with the color, shape, texture, or thickness of the fingernails or toenails. ... Fungus or yeast cause changes in the color, texture, and shape of the nails. Bacterial infection may ...

  4. Abnormal pressures as hydrodynamic phenomena

    USGS Publications Warehouse

    Neuzil, C.E.

    1995-01-01

    So-called abnormal pressures, subsurface fluid pressures significantly higher or lower than hydrostatic, have excited speculation about their origin since subsurface exploration first encountered them. Two distinct conceptual models for abnormal pressures have gained currency among earth scientists. The static model sees abnormal pressures generally as relict features preserved by a virtual absence of fluid flow over geologic time. The hydrodynamic model instead envisions abnormal pressures as phenomena in which flow usually plays an important role. This paper develops the theoretical framework for abnormal pressures as hydrodynamic phenomena, shows that it explains the manifold occurrences of abnormal pressures, and examines the implications of this approach. -from Author

  5. Mildly abnormal general movement quality in infants is associated with higher Mead acid and lower arachidonic acid and shows a U-shaped relation with the DHA/AA ratio.

    PubMed

    van Goor, S A; Schaafsma, A; Erwich, J J H M; Dijck-Brouwer, D A J; Muskiet, F A J

    2010-01-01

    We showed that docosahexaenoic acid (DHA) supplementation during pregnancy and lactation was associated with more mildly abnormal (MA) general movements (GMs) in the infants. Since this finding was unexpected and inter-individual DHA intakes are highly variable, we explored the relationship between GM quality and erythrocyte DHA, arachidonic acid (AA), DHA/AA and Mead acid in 57 infants of this trial. MA GMs were inversely related to AA, associated with Mead acid, and associated with DHA/AA in a U-shaped manner. These relationships may indicate dependence of newborn AA status on synthesis from linoleic acid. This becomes restricted during the intrauterine period by abundant de novo synthesis of oleic and Mead acids from glucose, consistent with reduced insulin sensitivity during the third trimester. The descending part of the U-shaped relation between MA GMs and DHA/AA probably indicates DHA shortage next to AA shortage. The ascending part may reflect a different developmental trajectory that is not necessarily unfavorable. PMID:20022733

  6. Mycobacterium tuberculosis supports protein tyrosine phosphorylation

    PubMed Central

    Kusebauch, Ulrike; Ortega, Corrie; Ollodart, Anja; Rogers, Richard S.; Sherman, David R.; Moritz, Robert L.; Grundner, Christoph

    2014-01-01

    Reversible protein phosphorylation determines growth and adaptive decisions in Mycobacterium tuberculosis (Mtb). At least 11 two-component systems and 11 Ser/Thr protein kinases (STPKs) mediate phosphorylation on Asp, His, Ser, and Thr. In contrast, protein phosphorylation on Tyr has not been described previously in Mtb. Here, using a combination of phospho-enrichment and highly sensitive mass spectrometry, we show extensive protein Tyr phosphorylation of diverse Mtb proteins, including STPKs. Several STPKs function as dual-specificity kinases that phosphorylate Tyr in cis and in trans, suggesting that dual-specificity kinases have a major role in bacterial phospho-signaling. Mutation of a phosphotyrosine site of the essential STPK PknB reduces its activity in vitro and in live Mtb, indicating that Tyr phosphorylation has a functional role in bacterial growth. These data identify a previously unrecognized phosphorylation system in a human pathogen that claims ∼1.4 million lives every year. PMID:24927537

  7. Abnormalities in Hippocampal Functioning with Persistent Pain

    PubMed Central

    Mutso, Amelia A.; Radzicki, Daniel; Baliki, Marwan N.; Huang, Lejian; Banisadr, Ghazal; Centeno, Maria Virginia; Radulovic, Jelena; Martina, Marco; Miller, Richard J.; Apkarian, A. Vania

    2012-01-01

    Chronic pain patients exhibit increased anxiety, depression, and deficits in learning and memory. Yet how persistent pain affects the key brain area regulating these behaviors, the hippocampus, has remained minimally explored. In this study we investigated the impact of spared nerve injury (SNI) neuropathic pain in mice on hippocampal-dependent behavior and underlying cellular and molecular changes. In parallel, we measured the hippocampal volume of three groups of chronic pain patients. We found that SNI animals were unable to extinguish to contextual fear and showed increased anxiety-like behavior. Additionally, SNI mice in comparison to sham animals exhibited hippocampal 1) reduced extracellular signal-regulated kinase (ERK) expression and phosphorylation, 2) decreased neurogenesis and 3) altered short-term synaptic plasticity. In order to relate the observed hippocampal abnormalities with human chronic pain, we measured the volume of human hippocampus in chronic back pain (CBP), complex regional pain syndrome (CRPS), and osteoarthritis patients (OA). Compared to controls, CBP and CRPS, but not OA, had significantly less bilateral hippocampal volume. These results indicate that hippocampus-mediated behavior, synaptic plasticity and neurogenesis are abnormal in neuropathic rodents. The changes may be related to the reduction in hippocampal volume we see in chronic pain patients, and these abnormalities may underlie learning and emotional deficits commonly observed in such patients. PMID:22539837

  8. Properties of phosphorylated thymidylate synthase.

    PubMed

    Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr; Palmowski, Paweł; Rogowska-Wrzesinska, Adelina; Cieśla, Joanna; Zieliński, Zbigniew; Nizioł, Joanna; Jarmuła, Adam; Maj, Piotr; Gołos, Barbara; Wińska, Patrycja; Ostafil, Sylwia; Wałajtys-Rode, Elżbieta; Shugar, David; Rode, Wojciech

    2015-12-01

    Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichinella spiralis and Caenorhabditis elegans TSs, expressed in Escherichia coli, the phosphorylated, compared to non-phosphorylated recombinant enzyme forms, showed a decrease in Vmax(app), bound their cognate mRNA (only rat enzyme studied), and repressed translation of their own and several heterologous mRNAs (human, rat and mouse enzymes studied). However, attempts to determine the modification site(s), whether endogenously expressed in mammalian cells, or recombinant proteins, did not lead to unequivocal results. Comparative ESI-MS/analysis of IEF fractions of TS preparations from parental and FdUrd-resistant mouse leukemia L1210 cells, differing in sensitivity to inactivation by FdUMP, demonstrated phosphorylation of Ser(10) and Ser(16) in the resistant enzyme only, although PGS staining pointed to the modification of both L1210 TS proteins. The TS proteins phosphorylated in bacterial cells were shown by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent. PMID:26315778

  9. Phosphorylated nano-diamond/ Polyimide Nanocomposites

    NASA Astrophysics Data System (ADS)

    Beyler-Çiǧil, Asli; Çakmakçi, Emrah; Vezir Kahraman, Memet

    2014-08-01

    In this study, a novel route to synthesize polyimide (PI)/phosphorylated nanodiamond films with improved thermal and mechanical properties was developed. Surface phosphorylation of nano-diamond was performed in dichloromethane. Phosphorylation dramatically enhanced the thermal stability of nano-diamond. Poly(amic acid) (PAA), which is the precursor of PI, was successfully synthesized with 3,3',4,4'-Benzophenonetetracarboxylic dianhydride (BTDA) and 4,4'-oxydianiline (4,4'-ODA) in the solution of N,N- dimethylformamide (DMF). Pure BTDA-ODA polyimide films and phosphorylated nanodiamond containing BTDA-ODA PI films were prepared. The PAA displayed good compatibility with phosphorylated nano-diamond. The morphology of the polyimide (PI)/phosphorylated nano-diamond was characterized by scanning electron microscopy (SEM). Chemical structure of polyimide and polyimide (PI)/phosphorylated nano-diamond was characterized by FTIR. SEM and FTIR results showed that the phosphorylated nano-diamond was successfully prepared. Thermal properties of the polyimide (PI)/phosphorylated nanodiamond was characterized by thermogravimetric analysis (TGA). TGA results showed that the thermal stability of (PI)/phosphorylated nano-diamond film was increased.

  10. Phosphorylated TDP-43 in frontotemporal lobar degeneration and ALS

    PubMed Central

    Hasegawa, Masato; Arai, Tetsuaki; Nonaka, Takashi; Kametani, Fuyuki; Yoshida, Mari; Hashizume, Yoshio; Beach, Thomas G.; Buratti, Emanuele; Baralle, Francisco; Morita, Mitsuya; Nakano, Imaharu; Oda, Tatsuro; Tsuchiya, Kuniaki; Akiyama, Haruhiko

    2009-01-01

    Objective TDP-43 is deposited as cytoplasmic and intranuclear inclusions in brains of subjects with frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). Previous studies reported that abnormal phosphorylation takes place in deposited TDP-43. The aim of this study was to identify the phosphorylation sites and responsible kinases, and to clarify the pathological significance of phosphorylation of TDP-43. Methods We generated multiple antibodies specific to phosphorylated TDP-43 by immunizing phosphopeptides of TDP-43, and analyzed FTLD-U and ALS brains by immunohistochemistry, immunoelectron microscopy and immunoblots. Additionally, we performed investigations aimed at identifying the responsible kinases and we assessed the effects of phosphorylation on TDP-43 oligomerization and fibrillization. Results We identified multiple phosphorylation sites in carboxyl-terminal regions of deposited TDP-43. Phosphorylation-specific antibodies stained more inclusions than antibodies to ubiquitin and, unlike existing commercially-available anti-TDP-43 antibodies, did not stain normal nuclei. Ultrastructurally, these antibodies labeled abnormal fibers of 15 nm diameter, and on immunoblots recognized hyperphosphorylated TDP-43 at 45 kDa, with additional 22–28 kDa fragments in sarkosyl-insoluble fractions from FTLD-U and ALS brains. The phosphorylated epitopes were generated by casein kinase 1 and 2, and phosphorylation led to increased oligomerization and fibrillization of TDP-43. Interpretation These results suggest that phosphorylated TDP-43 is a major component of the inclusions, and that abnormal phosphorylation of TDP-43 is a critical step in the pathogenesis of FTLD-U and ALS. Phosphorylation-specific antibodies will be powerful tools for the investigation of these disorders. PMID:18546284

  11. Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly*

    PubMed Central

    Shim, Su Yeon; de Castro, Ignacio Perez; Neumayer, Gernot; Wang, Jian; Park, Sang Ki; Sanada, Kamon; Nguyen, Minh Dang

    2015-01-01

    The human ortholog of the targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a cytoskeletal protein that plays a major role in spindle assembly and is required for mitosis. During spindle morphogenesis, TPX2 cooperates with Aurora A kinase and Eg5 kinesin to regulate microtubule organization. TPX2 displays over 40 putative phosphorylation sites identified from various high-throughput proteomic screenings. In this study, we characterize the phosphorylation of threonine 72 (Thr72) in human TPX2, a residue highly conserved across species. We find that Cdk1/2 phosphorylate TPX2 in vitro and in vivo. Using homemade antibodies specific for TPX2 phosphorylated at Thr72, we show that this phosphorylation is cell cycle-dependent and peaks at M phase. Endogenous TPX2 phosphorylated at Thr72 does not associate with the mitotic spindle. Furthermore, ectopic GFP-TPX2 T72A preferentially concentrates on the spindle, whereas GFP-TPX2 WT distributes to both spindle and cytosol. The T72A mutant also increases the proportion of cells with multipolar spindles phenotype. This effect is associated with increased Aurora A activity and abnormally elongated spindles, indicative of higher Eg5 activity. In summary, we propose that phosphorylation of Thr72 regulates TPX2 localization and impacts spindle assembly via Aurora A and Eg5. PMID:25688093

  12. Bioactives from Artemisia dracunculus L. Enhance Insulin Sensitivity via Modulation of Skeletal Muscle Protein Phosphorylation

    PubMed Central

    Kheterpal, Indu; Scherp, Peter; Kelley, Lauren; Wang, Zhong; Johnson, William; Ribnicky, David; Cefalu, William T.

    2014-01-01

    A botanical extract from Artemisia dracunculus L., termed PMI 5011, has been shown previously to improve insulin sensitivity by increasing cellular insulin signaling in in vitro and in vivo studies. These studies suggest that PMI 5011 effects changes in phosphorylation levels of proteins involved in insulin signaling. To explore effects of this promising botanical extract on the human skeletal muscle phosphoproteome, changes in site-specific protein phosphorylation levels in primary skeletal muscle cultures from obese, insulin resistant individuals were evaluated with and without insulin stimulation. Insulin resistance is a condition in which a normal or elevated insulin level results in an abnormal biologic response, e.g., glucose uptake. Using isobaric tagging for relative and absolute quantification (iTRAQ™) followed by phosphopeptide enrichment and liquid chromatography – tandem mass spectrometry, 125 unique phosphopeptides and 159 unique phosphorylation sites from 80 unique proteins were identified and quantified. Insulin stimulation of primary cultured muscle cells from insulin resistant individuals resulted in minimal increase in phosphorylation, demonstrating impaired insulin action in this condition. Treatment with PMI 5011 resulted in significant up regulation of 35 phosphopeptides that were mapped to proteins participating in the regulation of transcription, translation, actin cytoskeleton signaling, caveolae translocation and GLUT4 transport. These data further showed that PMI 5011 increased phosphorylation levels of specific amino acids in proteins in the insulin resistant state that are normally phosphorylated by insulin (thus, increasing cellular insulin signaling) and PMI 5011 also increased the abundance of phosphorylation sites of proteins regulating anti-apoptotic effects. Thus, the phosphoproteomics analysis demonstrated conclusively that PMI 5011 effects changes in phosphorylation levels of proteins and identified novel pathways by which

  13. Uncouplers of oxidative phosphorylation.

    PubMed

    Terada, H

    1990-07-01

    Uncouplers of oxidative phosphorylation in mitochondria inhibit the coupling between the electron transport and phosphorylation reactions and thus inhibit ATP synthesis without affecting the respiratory chain and ATP synthase (H(+)-ATPase). Miscellaneous compounds are known to be uncouplers, but weakly acidic uncouplers are representative because they show very potent activities. The most potent uncouplers discovered so far are the hindered phenol SF 6847, and hydrophobic salicylanilide S-13, which are active in vitro at concentrations in the 10 nM range. For induction of uncoupling, an acid dissociable group, bulky hydrophobic moiety and strong electron-withdrawing group are required. Weakly acidic uncouplers are considered to produce uncoupling by their protonophoric action in the H(+)-impermeable mitochondrial membrane. For exerting these effects, the stability of the respective uncoupler anions in the hydrophobic membrane is very important. High stability is achieved by delocalization of the polar ionic charge through uncoupler (chemical)-specific mechanisms. Such an action of weakly acidic uncouplers is characteristic of the highly efficient membrane targeting action of a nonsite-specific type of bioactive compound. PMID:2176586

  14. Uncouplers of oxidative phosphorylation.

    PubMed Central

    Terada, H

    1990-01-01

    Uncouplers of oxidative phosphorylation in mitochondria inhibit the coupling between the electron transport and phosphorylation reactions and thus inhibit ATP synthesis without affecting the respiratory chain and ATP synthase (H(+)-ATPase). Miscellaneous compounds are known to be uncouplers, but weakly acidic uncouplers are representative because they show very potent activities. The most potent uncouplers discovered so far are the hindered phenol SF 6847, and hydrophobic salicylanilide S-13, which are active in vitro at concentrations in the 10 nM range. For induction of uncoupling, an acid dissociable group, bulky hydrophobic moiety and strong electron-withdrawing group are required. Weakly acidic uncouplers are considered to produce uncoupling by their protonophoric action in the H(+)-impermeable mitochondrial membrane. For exerting these effects, the stability of the respective uncoupler anions in the hydrophobic membrane is very important. High stability is achieved by delocalization of the polar ionic charge through uncoupler (chemical)-specific mechanisms. Such an action of weakly acidic uncouplers is characteristic of the highly efficient membrane targeting action of a nonsite-specific type of bioactive compound. PMID:2176586

  15. Clinical applications of basic research that shows reducing skin tension could prevent and treat abnormal scarring: the importance of fascial/subcutaneous tensile reduction sutures and flap surgery for keloid and hypertrophic scar reconstruction.

    PubMed

    Ogawa, Rei; Akaishi, Satoshi; Huang, Chenyu; Dohi, Teruyuki; Aoki, Masayo; Omori, Yasutaka; Koike, Sachiko; Kobe, Kyoko; Akimoto, Masataka; Hyakusoku, Hiko

    2011-01-01

    We use evidence-based algorithms to treat abnormal scarring, including keloids and hypertrophic scars (HSs). This involves a multimodal approach that employs traditional methods such as surgical removal, postoperative radiotherapy, corticosteroid injection, laser, and silicone gel sheets. As a result, the rate of abnormal scarring recurrence has decreased dramatically over the past 10 years. However, several problems remain to be solved. First, despite the optimization of a radiotherapy protocol, over 10% of cases who are treated with surgery and postoperative radiotherapy still recur in our facility. Second, the treatment options for cases with huge keloids are very limited. To address these problems, we performed basic research on the mechanisms that drive the formation of keloids and HSs. Extrapolation of these research observations to the clinic has led to the development of two treatment strategies that have reduced the rate of abnormal scar recurrence further and provided a means to remove large scars. Our finite element analysis of the mechanical force distribution around keloids revealed high skin tension at the keloid edges and lower tension in the keloid center. Moreover, when a sophisticated servo-controlled device was used to stretch wounded murine dorsal skin, it was observed that the stretched samples exhibited upregulated epidermal proliferation and angiogenesis, which are also observed in keloids and HSs. Real-time RT-PCR also revealed that growth factors and neuropeptides are more strongly expressed in cyclically stretched skin than in statically stretched skin. These findings support the well-established notion that mechanical forces on the skin strongly influence the cellular behavior that leads to scarring. These observations led us to focus on the importance of reducing skin tension when keloids/HSs are surgically removed to prevent their recurrence. Clinical trials revealed that subcutaneous/fascial tensile reduction sutures, which apply

  16. Histone deacetylase 2 is phosphorylated, ubiquitinated, and degraded by cigarette smoke.

    PubMed

    Adenuga, David; Yao, Hongwei; March, Thomas H; Seagrave, Jeanclare; Rahman, Irfan

    2009-04-01

    Cigarette smoke (CS)-induced lung inflammation involves the reduction of histone deacetylase 2 (HDAC2) abundance, which is associated with steroid resistance in patients with chronic obstructive pulmonary disease and in individuals with severe asthma who smoke cigarettes. However, the molecular mechanism of CS-mediated reduction of HDAC2 is not clearly known. We hypothesized that HDAC2 is phosphorylated and subsequently degraded by the proteasome in vitro in macrophages (MonoMac6), human bronchial and primary small airway epithelial cells, and in vivo in mouse lungs in response to chronic CS exposure. Cigarette smoke extract (CSE) exposure in MonoMac6 and in bronchial and airway epithelial cells led to phosphorylation of HDAC2 on serine/threonine residues by a protein kinase CK2-mediated mechanism, decreased HDAC2 activity, and increased ubiquitin-proteasome-dependent HDAC2 degradation. CK2 and proteasome inhibitors reversed CSE-mediated HDAC2 degradation, whereas serine/threonine phosphatase inhibitor, okadaic acid, caused phosphorylation and subsequent ubiquitination of HDAC2. CS-induced HDAC2 phosphorylation was detected in mouse lungs from 2 weeks to 4 months of CS exposure, and mice showed significantly lower lung HDAC2 levels. Thus, CS-mediated down-regulation of HDAC2 in human macrophages and lung epithelial cells in vitro and in mouse lung in vivo involves the induction of serine/threonine phosphorylation and proteasomal degradation, which may have implications for steroid resistance and abnormal inflammation caused by cigarette smoke. PMID:18927347

  17. Neuroinflammation is not a Prerequisite for Diabetes-induced Tau Phosphorylation

    PubMed Central

    van der Harg, Judith M.; Eggels, Leslie; Ruigrok, Silvie R.; Hoozemans, Jeroen J. M.; la Fleur, Susanne E.; Scheper, Wiep

    2015-01-01

    Abnormal phosphorylation and aggregation of tau is a key hallmark of Alzheimer's disease (AD). AD is a multifactorial neurodegenerative disorder for which Diabetes Mellitus (DM) is a risk factor. In animal models for DM, the phosphorylation and aggregation of tau is induced or exacerbated, however the underlying mechanism is unknown. In addition to the metabolic dysfunction, DM is characterized by chronic low-grade inflammation. This was reported to be associated with a neuroinflammatory response in the hypothalamus of DM animal models. Neuroinflammation is also implicated in the development and progression of AD. It is unknown whether DM also induces neuroinflammation in brain areas affected in AD, the cortex and hippocampus. Here we investigated whether neuroinflammation could be the mechanistic trigger to induce tau phosphorylation in the brain of DM animals. Two distinct diabetic animal models were used; rats on free-choice high-fat high-sugar (fcHFHS) diet that are insulin resistant and streptozotocin-treated rats that are insulin deficient. The streptozotocin-treated animals demonstrated increased tau phosphorylation in the brain as expected, whereas the fcHFHS diet fed animals did not. Remarkably, neither of the diabetic animal models showed reactive microglia or increased GFAP and COX-2 levels in the cortex or hippocampus. From this, we conclude: 1. DM does not induce neuroinflammation in brain regions affected in AD, and 2. Neuroinflammation is not a prerequisite for tau phosphorylation. Neuroinflammation is therefore not the mechanism that explains the close connection between DM and AD. PMID:26617484

  18. Phosphorylation and RLK signaling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant genomes encode hundreds of receptor-like kinases (RLKs) with an organization of functional domains similar to that of animal receptor kinases. Ligand-dependent phosphorylation has now been demonstrated for several plant RLKs and identification of specific phosphorylation sites followed by thei...

  19. Autophagy proteins regulate ERK phosphorylation

    PubMed Central

    Martinez-Lopez, Nuria; Athonvarangkul, Diana; Mishall, Priti; Sahu, Srabani; Singh, Rajat

    2013-01-01

    Autophagy is a conserved pathway that maintains cellular quality control. Extracellular signal-regulated kinase (ERK) controls various aspects of cell physiology including proliferation. Multiple signalling cascades, including ERK, have been shown to regulate autophagy, however whether autophagy proteins (ATG) regulate cell signalling is unknown. Here we show that growth factor exposure increases the interaction of ERK cascade components with ATG proteins in the cytosol and nucleus. ERK and its upstream kinase MEK localize to the extra-luminal face of autophagosomes. ERK2 interacts with ATG proteins via its substrate-binding domains. Deleting Atg7 or Atg5 or blocking LC3 lipidation or ATG5–ATG12 conjugation decreases ERK phosphorylation. Conversely, increasing LC3-II availability by silencing the cysteine protease ATG4B or acute trehalose exposure increases ERK phosphorylation. Decreased ERK phosphorylation in Atg5−/− cells does not occur from overactive phosphatases. Our findings thus reveal an unconventional function of ATG proteins as cellular scaffolds in the regulation of ERK phosphorylation. PMID:24240988

  20. miR-106b inhibits tau phosphorylation at Tyr18 by targeting Fyn in a model of Alzheimer's disease.

    PubMed

    Liu, Wei; Zhao, Jingya; Lu, Guangxiu

    2016-09-16

    Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by β-amyloid deposits and neurofibrillary tangles consisting of hyperphosphorylated tau protein. Increasing evidence has revealed that microRNAs (miRNAs) are implicated in the pathogenesis of AD. However, the effect of miRNAs on abnormal tau phosphorylation remains largely unclear so far. In this study, we investigated the role of miR-106b in tau phosphorylation and identified a new molecular mechanism of the hyperphosphorylation of tau. The results of qRT-PCR showed that the expression level of miR-106b was decreased, but Fyn was increased in the temporal cortex of AD patients. Overexpression of miR-106b inhibited Aβ1-42-induced tau phosphorylation at Tyr18 in SH-SY5Y cells stably expressing tau (SH-SY5Y/tau), whereas no changes were observed in tau phosphorylation at Ser396/404. Dual-luciferase reporter gene assay validated that Fyn was a direct target gene of miR-106b. In addition, western blot analysis revealed that Fyn protein expression was suppressed when SH-SY5Y cells were transfected with miR-106b mimics. Endogenous Fyn expression was knockdown by transfection with a small interfering RNA specific for Fyn (si-Fyn). The phosphorylation level of tau at Tyr 18 was decreased in the si-Fyn group compared with the negative control group, but the inhibitory effect of si-Fyn on tau phosphorylation was attenuated when miR-106b expression was inhibited. Taken together, these data suggest that miR-106b inhibits Aβ1-42-induced tau phosphorylation at Tyr18 by targeting Fyn. Our findings extend the knowledge about the regulation of tau phosphorylation and the regulatory mechanism of Fyn gene expression. PMID:27520374

  1. PKA regulates calcineurin function through the phosphorylation of RCAN1: Identification of a novel phosphorylation site

    SciTech Connect

    Kim, Seon Sook; Lee, Eun Hye; Lee, Kooyeon; Jo, Su-Hyun; Seo, Su Ryeon

    2015-04-17

    Calcineurin is a calcium/calmodulin-dependent phosphatase that has been implicated in T cell activation through the induction of nuclear factors of activated T cells (NFAT). We have previously suggested that endogenous regulator of calcineurin (RCAN1, also known as DSCR1) is targeted by protein kinase A (PKA) for the control of calcineurin activity. In the present study, we characterized the PKA-mediated phosphorylation site in RCAN1 by mass spectrometric analysis and revealed that PKA directly phosphorylated RCAN1 at the Ser 93. PKA-induced phosphorylation and the increase in the half-life of the RCAN1 protein were prevented by the substitution of Ser 93 with Ala (S93A). Furthermore, the PKA-mediated phosphorylation of RCAN1 at Ser 93 potentiated the inhibition of calcineurin-dependent pro-inflammatory cytokine gene expression by RCAN1. Our results suggest the presence of a novel phosphorylation site in RCAN1 and that its phosphorylation influences calcineurin-dependent inflammatory target gene expression. - Highlights: • We identify novel phosphorylation sites in RCAN1 by LC-MS/MS analysis. • PKA-dependent phosphorylation of RCAN1 at Ser 93 inhibits calcineurin-mediated intracellular signaling. • We show the immunosuppressive function of RCAN1 phosphorylation at Ser 93 in suppressing cytokine expression.

  2. Mining Conditional Phosphorylation Motifs.

    PubMed

    Liu, Xiaoqing; Wu, Jun; Gong, Haipeng; Deng, Shengchun; He, Zengyou

    2014-01-01

    Phosphorylation motifs represent position-specific amino acid patterns around the phosphorylation sites in the set of phosphopeptides. Several algorithms have been proposed to uncover phosphorylation motifs, whereas the problem of efficiently discovering a set of significant motifs with sufficiently high coverage and non-redundancy still remains unsolved. Here we present a novel notion called conditional phosphorylation motifs. Through this new concept, the motifs whose over-expressiveness mainly benefits from its constituting parts can be filtered out effectively. To discover conditional phosphorylation motifs, we propose an algorithm called C-Motif for a non-redundant identification of significant phosphorylation motifs. C-Motif is implemented under the Apriori framework, and it tests the statistical significance together with the frequency of candidate motifs in a single stage. Experiments demonstrate that C-Motif outperforms some current algorithms such as MMFPh and Motif-All in terms of coverage and non-redundancy of the results and efficiency of the execution. The source code of C-Motif is available at: https://sourceforge. net/projects/cmotif/. PMID:26356863

  3. Cellular regulation by protein phosphorylation.

    PubMed

    Fischer, Edmond H

    2013-01-11

    A historical account of the discovery of reversible protein phosphorylation is presented. This process was uncovered in the mid 1950s in a study undertaken with Edwin G. Krebs to elucidate the complex hormonal regulation of skeletal muscle glycogen phosphorylase. Contrary to the known activation of this enzyme by AMP which serves as an allosteric effector, its hormonal regulation results from a phosphorylation of the protein by phosphorylase kinase following the activation of the latter by Ca(2+) and ATP. The study led to the establishment of the first hormonal cascade of successive enzymatic reactions, kinases acting on kinases, initiated by cAMP discovered by Earl Sutherland. It also showed how two different physiological processes, carbohydrate metabolism and muscle contraction, could be regulated in concert. PMID:23058924

  4. Mitotic Phosphorylation of Histone H3: Spatio-Temporal Regulation by Mammalian Aurora Kinases

    PubMed Central

    Crosio, Claudia; Fimia, Gian Maria; Loury, Romain; Kimura, Masashi; Okano, Yukio; Zhou, Hongyi; Sen, Subrata; Allis, C. David; Sassone-Corsi, Paolo

    2002-01-01

    Phosphorylation at a highly conserved serine residue (Ser-10) in the histone H3 tail is considered to be a crucial event for the onset of mitosis. This modification appears early in the G2 phase within pericentromeric heterochromatin and spreads in an ordered fashion coincident with mitotic chromosome condensation. Mutation of Ser-10 is essential in Tetrahymena, since it results in abnormal chromosome segregation and extensive chromosome loss during mitosis and meiosis, establishing a strong link between signaling and chromosome dynamics. Although mitotic H3 phosphorylation has been long recognized, the transduction routes and the identity of the protein kinases involved have been elusive. Here we show that the expression of Aurora-A and Aurora-B, two kinases of the Aurora/AIK family, is tightly coordinated with H3 phosphorylation during the G2/M transition. During the G2 phase, the Aurora-A kinase is coexpressed while the Aurora-B kinase colocalizes with phosphorylated histone H3. At prophase and metaphase, Aurora-A is highly localized in the centrosomic region and in the spindle poles while Aurora-B is present in the centromeric region concurrent with H3 phosphorylation, to then translocate by cytokinesis to the midbody region. Both Aurora-A and Aurora-B proteins physically interact with the H3 tail and efficiently phosphorylate Ser10 both in vitro and in vivo, even if Aurora-A appears to be a better H3 kinase than Aurora-B. Since Aurora-A and Aurora-B are known to be overexpressed in a variety of human cancers, our findings provide an attractive link between cell transformation, chromatin modifications and a specific kinase system. PMID:11784863

  5. Spirometric abnormalities among welders

    SciTech Connect

    Rastogi, S.K.; Gupta, B.N.; Husain, T.; Mathur, N.; Srivastava, S. )

    1991-10-01

    A group of manual welders age group 13-60 years having a mean exposure period of 12.4 {plus minus} 1.12 years were subjected to spirometry to evaluate the prevalence of spirometric abnormalities. The welders showed a significantly higher prevalence of respiratory impairment than that observed among the unexposed controls as a result of exposure to welding gases which comprised fine particles of lead, zinc, chromium, and manganese. This occurred despite the lower concentration of the pollutants at the work place. In the expose group, the smoking welders showed a prevalence of respiratory impairment significantly higher than that observed in the nonsmoking welders. The results of the pulmonary function tests showed a predominantly restrictive type of pulmonary impairment followed by a mixed ventilatory defect among the welders. The effect of age on pulmonary impairment was not discernible. Welders exposed for over 10 years showed a prevalence of respiratory abnormalities significantly higher than those exposed for less than 10 years. Smoking also had a contributory role.

  6. Phosphorylation of intact erythrocytes in human muscular dystrophy

    SciTech Connect

    Johnson, R.M.; Nigro, M.

    1986-04-01

    The uptake of exogenous /sup 32/Pi into the membrane proteins of intact erythrocytes was measured in 8 patients with Duchenne muscular dystrophy. No abnormalities were noted after autoradiographic analysis. This contrasts with earlier results obtained when isolated membranes were phosphorylated with gamma-(/sup 32/P)ATP, and suggests a possible reinterpretation of those experiments.

  7. Transforming growth factor-{beta}-inducible phosphorylation of Smad3.

    PubMed

    Wang, Guannan; Matsuura, Isao; He, Dongming; Liu, Fang

    2009-04-10

    Smad proteins transduce the transforming growth factor-beta (TGF-beta) signal at the cell surface into gene regulation in the nucleus. Upon TGF-beta treatment, the highly homologous Smad2 and Smad3 are phosphorylated by the TGF-beta receptor at the SSXS motif in the C-terminal tail. Here we show that in addition to the C-tail, three (S/T)-P sites in the Smad3 linker region, Ser(208), Ser(204), and Thr(179) are phosphorylated in response to TGF-beta. The linker phosphorylation peaks at 1 h after TGF-beta treatment, behind the peak of the C-tail phosphorylation. We provide evidence suggesting that the C-tail phosphorylation by the TGF-beta receptor is necessary for the TGF-beta-induced linker phosphorylation. Although the TGF-beta receptor is necessary for the linker phosphorylation, the receptor itself does not phosphorylate these sites. We further show that ERK is not responsible for TGF-beta-dependent phosphorylation of these three sites. We show that GSK3 accounts for TGF-beta-inducible Ser(204) phosphorylation. Flavopiridol, a pan-CDK inhibitor, abolishes TGF-beta-induced phosphorylation of Thr(179) and Ser(208), suggesting that the CDK family is responsible for phosphorylation of Thr(179) and Ser(208) in response to TGF-beta. Mutation of the linker phosphorylation sites to nonphosphorylatable residues increases the ability of Smad3 to activate a TGF-beta/Smad-target gene as well as the growth-inhibitory function of Smad3. Thus, these observations suggest that TGF-beta-induced phosphorylation of Smad3 linker sites inhibits its antiproliferative activity. PMID:19218245

  8. Phosphorylation of human skeletal muscle myosin

    SciTech Connect

    Houston, M.E.; Lingley, M.D.; Stuart, D.S.; Hoffman-Goetz, L.

    1986-03-01

    Phosphorylation of the P-light chains (phosphorylatable light chains) in human skeletal muscle myosin was studied in vitro and in vivo under resting an d contracted conditions. biopsy samples from rested vastus lateralis muscle of male and female subjects were incubated in oxygenated physiological solution at 30/sup 0/C. Samples frozen following a quiescent period showed the presence of only unphosphorylated P-light chains designated LC2f (light chain two of fast myosin) CL2s and LC2s'(light chains two of slow myosin). Treatment with caffeine (10 mM) or direct electrical stimulation resulted in the appearance of three additional bands which were identified as the phosphorylated forms of the P-light chains i.e. LC2f-P, LC2s-P and LC2s'-P. The presence of phosphate was confirmed by prior incubation with (/sup 30/P) orthophosphate. Muscle samples rapidly frozen from resting vastus lateralis muscle revealed the presence of unphosphorylated and phosphorylated P-light chains in approximately equal ratios. Muscle samples rapidly frozen following a maximal 10 second isometric contraction showed virtually only phosphorylated fast and slow P-light chains. These results reveal that the P-light chains in human fast and slow myosin may be rapidly phosphorylated, but the basal level of phosphorylation in rested human muscle considerably exceeds that observed in animal muscles studied in vitro or in situ.

  9. Abnormal Head Position

    MedlinePlus

    ... cause. Can a longstanding head turn lead to any permanent problems? Yes, a significant abnormal head posture could cause permanent ... occipitocervical synostosis and unilateral hearing loss. Are there any ... postures? Yes. Abnormal head postures can usually be improved depending ...

  10. Urine - abnormal color

    MedlinePlus

    ... straw-yellow. Abnormally colored urine may be cloudy, dark, or blood-colored. Causes Abnormal urine color may ... red blood cells, or mucus in the urine. Dark brown but clear urine is a sign of ...

  11. A strategy to quantitate global phosphorylation of bone matrix proteins.

    PubMed

    Sroga, Grażyna E; Vashishth, Deepak

    2016-04-15

    Current studies of protein phosphorylation focus primarily on the importance of specific phosphoproteins and their landscapes of phosphorylation in the regulation of different cellular functions. However, global changes in phosphorylation of extracellular matrix phosphoproteins measured "in bulk" are equally important. For example, correct global phosphorylation of different bone matrix proteins is critical to healthy tissue biomineralization. To study changes of bone matrix global phosphorylation, we developed a strategy that combines a procedure for in vitro phosphorylation/dephosphorylation of fully mineralized bone in addition to quantitation of the global phosphorylation levels of bone matrix proteins. For the first time, we show that it is possible to enzymatically phosphorylate/dephosphorylate fully mineralized bone originating from either cadaveric human donors or laboratory animals (mice). Using our strategy, we detected the difference in the global phosphorylation levels of matrix proteins isolated from wild-type and osteopontin knockout mice. We also observed that the global phosphorylation levels of matrix proteins isolated from human cortical bone were lower than those isolated from trabecular bone. The developed strategy has the potential to open new avenues for studies on the global phosphorylation of bone matrix proteins and their role in biomineralization as well for other tissues/cells and protein-based materials. PMID:26851341

  12. Struvite and prebiotic phosphorylation.

    NASA Technical Reports Server (NTRS)

    Handschuh, G. J.; Orgel, L. E.

    1973-01-01

    Struvite rather than apatite or amorphous calcium phosphate is precipitated when phosphate is added to seawater containing more than 0.01M NH4+ ions. Struvite may have precipitated from evaporating seawater on the primitive earth, and may have been important for prebiotic phosphorylation.

  13. Phosphorylation state-dependent interaction between AKAP7δ/γ and phospholamban increases phospholamban phosphorylation.

    PubMed

    Rigatti, Marc; Le, Andrew V; Gerber, Claire; Moraru, Ion I; Dodge-Kafka, Kimberly L

    2015-09-01

    Changes in heart rate and contractility in response to sympathetic stimulation occur via activation of cAMP dependent protein kinase A (PKA), leading to phosphorylation of numerous substrates that alter Ca(2+) cycling. Phosphorylation of these substrates is coordinated by A-kinase anchoring proteins (AKAPs), which recruit PKA to specific substrates [1]. Phosphorylation of the PKA substrate phospholamban (PLB) is a critical determinant of Ca(2+) re-entry into the sarcoplasmic reticulum and is coordinated by AKAP7δ/γ [2,3]. Here, we further these findings by showing that phosphorylation of PLB requires interaction with AKAP7δ/γ and that this interaction occurs only when PLB is unphosphorylated. Additionally, we find that two mutants of PLB (R9C and Δ14), which are associated with dilated cardiomyopathy in humans, prevent association with AKAP7δ/γ and display reduced phosphorylation in vitro. This finding implicates the AKAP7δ/γ-PLB interaction in the pathology of the disease phenotype. Further exploration of the AKAP7δ/γ-PLB association demonstrated a phosphorylation state-dependence of the interaction. Computational modeling revealed that this mode of interaction allows for small amounts of AKAP and PKA (100-200nM) to regulate the phosphorylation of large quantities of PLB (50μM). Our results confirm that AKAP7γ/δ binding to PLB is important for phosphorylation of PLB, and describe a novel phosphorylation state-dependent binding mechanism that explains how phosphorylation of highly abundant PKA substrates can be regulated by AKAPs present at ~100-200 fold lower concentrations. PMID:26027516

  14. "The Show"

    ERIC Educational Resources Information Center

    Gehring, John

    2004-01-01

    For the past 16 years, the blue-collar city of Huntington, West Virginia, has rolled out the red carpet to welcome young wrestlers and their families as old friends. They have come to town chasing the same dream for a spot in what many of them call "The Show". For three days, under the lights of an arena packed with 5,000 fans, the state's best…

  15. Evidence that phosphorylation by the mitotic kinase Cdk1 promotes ICER monoubiquitination and nuclear delocalization

    SciTech Connect

    Memin, Elisabeth; Genzale, Megan; Crow, Marni; Molina, Carlos A.

    2011-10-15

    In contrast to normal prostatic cells, the transcriptional repressor Inducible cAMP Early Repressor (ICER) is undetected in the nuclei of prostate cancer cells. The molecular mechanisms for ICER abnormal expression in prostate cancer cells remained largely unknown. In this report data is presented demonstrating that ICER is phosphorylated by the mitotic kinase cdk1. Phosphorylation of ICER on a discrete residue targeted ICER to be monoubiquitinated. Different from unphosphorylated, phosphorylated and polyubiquitinated ICER, monoubiquitinated ICER was found to be cytosolic. Taken together, these results hinted on a mechanism for the observed abnormal subcellular localization of ICER in human prostate tumors.

  16. Evidence that phosphorylation by the mitotic kinase Cdk1 promotes ICER monoubiquitination and nuclear delocalization.

    PubMed

    Mémin, Elisabeth; Genzale, Megan; Crow, Marni; Molina, Carlos A

    2011-10-15

    In contrast to normal prostatic cells, the transcriptional repressor Inducible cAMP Early Repressor (ICER) is undetected in the nuclei of prostate cancer cells. The molecular mechanisms for ICER abnormal expression in prostate cancer cells remained largely unknown. In this report data is presented demonstrating that ICER is phosphorylated by the mitotic kinase cdk1. Phosphorylation of ICER on a discrete residue targeted ICER to be monoubiquitinated. Different from unphosphorylated, phosphorylated and polyubiquitinated ICER, monoubiquitinated ICER was found to be cytosolic. Taken together, these results hinted on a mechanism for the observed abnormal subcellular localization of ICER in human prostate tumors. PMID:21767532

  17. Immunodetection of phosphorylation sites gives new insights into the mechanisms underlying phospholamban phosphorylation in the intact heart.

    PubMed

    Mundiña-Weilenmann, C; Vittone, L; Ortale, M; de Cingolani, G C; Mattiazzi, A

    1996-12-27

    Phosphorylation site-specific antibodies, quantification of 32P incorporation into phospholamban, and simultaneous measurements of mechanical activity were used in Langendorff-perfused rat hearts to provide further insights into the underlying mechanisms of phospholamban phosphorylation. Immunological detection of phospholamban phosphorylation sites showed that the isoproterenol concentration-dependent increase in phospholamban phosphorylation was due to increases in phosphorylation of both Ser16 and Thr17 residues. When isoproterenol concentration was increased at extremely low Ca2+ supply to the myocardium, phosphorylation of Thr17 was virtually absent. Under these conditions, 32P incorporation into phospholamban, due to Ser16, decreased by 50%. Changes in Ca2+ supply to the myocardium either at constant beta-adrenergic stimulation or in the presence of okadaic acid, a phosphatase inhibitor, exclusively modified Thr17 phosphorylation. Changes in phospholamban phosphorylation due to either Ser16 and/or Thr17 were paralleled by changes in myocardial relaxation. The results indicate that cAMP- (Ser16) and Ca2+-calmodulin (Thr17)-dependent pathways of phospholamban phosphorylation can occur independently of each other. However, in the absence of beta-adrenergic stimulation, phosphorylation of Thr17 could only be detected after simultaneous activation of Ca2+-calmodulin-dependent protein kinase and inactivation of phosphatase. It is suggested that under physiological conditions, this requisite is only filled by cAMP-dependent mechanisms. PMID:8969222

  18. Tooth - abnormal shape

    MedlinePlus

    Hutchinson incisors; Abnormal tooth shape; Peg teeth; Mulberry teeth; Conical teeth ... The appearance of normal teeth varies, especially the molars. ... conditions. Specific diseases can affect tooth shape, tooth ...

  19. Tooth - abnormal shape

    MedlinePlus

    Hutchinson incisors; Abnormal tooth shape; Peg teeth; Mulberry teeth; Conical teeth ... from many different conditions. Specific diseases can affect tooth shape, tooth color, time of appearance, or absence ...

  20. Phosphorylation modifies the molecular stability of β-amyloid deposits

    NASA Astrophysics Data System (ADS)

    Rezaei-Ghaleh, Nasrollah; Amininasab, Mehriar; Kumar, Sathish; Walter, Jochen; Zweckstetter, Markus

    2016-04-01

    Protein aggregation plays a crucial role in neurodegenerative diseases. A key feature of protein aggregates is their ubiquitous modification by phosphorylation. Little is known, however, about the molecular consequences of phosphorylation of protein aggregates. Here we show that phosphorylation of β-amyloid at serine 8 increases the stability of its pathogenic aggregates against high-pressure and SDS-induced dissociation. We further demonstrate that phosphorylation results in an elevated number of hydrogen bonds at the N terminus of β-amyloid, the region that is critically regulated by a variety of post-translational modifications. Because of the increased lifetime of phosphorylated β-amyloid aggregates, phosphorylation can promote the spreading of β-amyloid in Alzheimer pathogenesis. Our study suggests that regulation of the molecular stability of protein aggregates by post-translational modifications is a crucial factor for disease progression in the brain.

  1. Phosphorylation modifies the molecular stability of β-amyloid deposits

    PubMed Central

    Rezaei-Ghaleh, Nasrollah; Amininasab, Mehriar; Kumar, Sathish; Walter, Jochen; Zweckstetter, Markus

    2016-01-01

    Protein aggregation plays a crucial role in neurodegenerative diseases. A key feature of protein aggregates is their ubiquitous modification by phosphorylation. Little is known, however, about the molecular consequences of phosphorylation of protein aggregates. Here we show that phosphorylation of β-amyloid at serine 8 increases the stability of its pathogenic aggregates against high-pressure and SDS-induced dissociation. We further demonstrate that phosphorylation results in an elevated number of hydrogen bonds at the N terminus of β-amyloid, the region that is critically regulated by a variety of post-translational modifications. Because of the increased lifetime of phosphorylated β-amyloid aggregates, phosphorylation can promote the spreading of β-amyloid in Alzheimer pathogenesis. Our study suggests that regulation of the molecular stability of protein aggregates by post-translational modifications is a crucial factor for disease progression in the brain. PMID:27072999

  2. Matefin/SUN-1 Phosphorylation on Serine 43 Is Mediated by CDK-1 and Required for Its Localization to Centrosomes and Normal Mitosis in C. elegans Embryos

    PubMed Central

    Zuela, Noam; Gruenbaum, Yosef

    2016-01-01

    Matefin/SUN-1 is an evolutionary conserved C. elegans inner nuclear membrane SUN-domain protein. By creating a bridge with the KASH-domain protein ZYG-12, it connects the nucleus to cytoplasmic filaments and organelles. Matefin/SUN-1 is expressed in the germline where it undergoes specific phosphorylation at its N-terminal domain, which is required for germline development and homologous chromosome pairing. The maternally deposited matefin/SUN-1 is then essential for embryonic development. Here, we show that in embryos, serine 43 of matefin/SUN-1 (S43) is phosphorylated in a CDK-1 dependent manner and is localized throughout the cell cycle mostly to centrosomes. By generating animals expressing phosphodead S43A and phosphomimetic S43E mutations, we show that phosphorylation of S43 is required to maintain centrosome integrity and function, as well as for the localization of ZYG-12 and lamin. Expression of S43E in early embryos also leads to an increase in chromatin structural changes, decreased progeny and to almost complete embryonic lethality. Down regulation of emerin further increases the occurrence of chromatin organization abnormalities, indicating possible collaborative roles for these proteins that is regulated by S43 phosphorylation. Taken together, these results support a role for phosphorylation of serine 43 in matefin/SUN-1 in mitosis. PMID:26927181

  3. HSP90 inhibition blocks ERBB3 and RET phosphorylation in myxoid/round cell liposarcoma and causes massive cell death in vitro and in vivo

    PubMed Central

    Safavi, Setareh; Järnum, Sofia; Vannas, Christoffer; Udhane, Sameer; Jonasson, Emma; Tomic, Tajana Tesan; Grundevik, Pernilla; Fagman, Henrik; Hansson, Magnus; Kalender, Zeynep; Jauhiainen, Alexandra; Dolatabadi, Soheila; Stratford, Eva Wessel; Myklebost, Ola; Eriksson, Mikael; Stenman, Göran; Stock, Regine Schneider; Ståhlberg, Anders; Åman, Pierre

    2016-01-01

    Myxoid sarcoma (MLS) is one of the most common types of malignant soft tissue tumors. MLS is characterized by the FUS-DDIT3 or EWSR1-DDIT3 fusion oncogenes that encode abnormal transcription factors. The receptor tyrosine kinase (RTK) encoding RET was previously identified as a putative downstream target gene to FUS-DDIT3 and here we show that cultured MLS cells expressed phosphorylated RET together with its ligand Persephin. Treatment with RET specific kinase inhibitor Vandetanib failed to reduce RET phosphorylation and inhibit cell growth, suggesting that other RTKs may phosphorylate RET. A screening pointed out EGFR and ERBB3 as the strongest expressed phosphorylated RTKs in MLS cells. We show that ERBB3 formed nuclear and cytoplasmic complexes with RET and both RTKs were previously reported to form complexes with EGFR. The formation of RTK hetero complexes could explain the observed Vandetanib resistence in MLS. EGFR and ERBB3 are clients of HSP90 that help complex formation and RTK activation. Treatment of cultured MLS cells with HSP90 inhibitor 17-DMAG, caused loss of RET and ERBB3 phosphorylation and lead to rapid cell death. Treatment of MLS xenograft carrying Nude mice resulted in massive necrosis, rupture of capillaries and hemorrhages in tumor tissues. We conclude that complex formation between RET and other RTKs may cause RTK inhibitor resistance. HSP90 inhibitors can overcome this resistance and are thus promising drugs for treatment of MLS/RCLS. PMID:26595521

  4. Synaptic plasticity and phosphorylation

    PubMed Central

    Lee, Hey-Kyoung

    2009-01-01

    A number of neuronal functions, including synaptic plasticity, depend on proper regulation of synaptic proteins, many of which can be rapidly regulated by phosphorylation. Neuronal activity controls the function of these synaptic proteins by exquisitely regulating the balance of various protein kinase and protein phosphatase activity. Recent understanding of synaptic plasticity mechanisms underscores important roles that these synaptic phosphoproteins play in regulating both pre- and post-synaptic functions. This review will focus on key postsynaptic phosphoproteins that have been implicated to play a role in synaptic plasticity. PMID:16904750

  5. Role of Runx2 Phosphorylation in Prostate Cancer and Association with Metastatic Disease

    PubMed Central

    Ge, Chunxi; Zhao, Guisheng; Li, Yan; Li, Hui; Zhao, Xiang; Pannone, Giuseppe; Bufo, Pantaleo; Santoro, Angela; Sanguedolce, Francesca; Tortorella, Simona; Mattoni, Marilena; Papagerakis, Silvana; Keller, Evan T.; Franceschi, Renny T.

    2015-01-01

    The osteogenic transcription factor, Runx2, is abnormally expressed in prostate cancer (PCa) and associated with metastatic disease. During bone development, Runx2 is activated by signals known to be hyperactive in PCa including the RAS/MAP kinase pathway, which phosphorylates Runx2 on multiple serine residues including S301 and S319 (equivalent to S294 and S312 in human Runx2). This study examines the role of these phosphorylation sites in PCa. Runx2 was preferentially expressed in more invasive prostate cancer cell lines (PC3 > C4-2B > LNCaP). Furthermore, analysis using a P-S319-Runx2-specific antibody revealed that the ratio of P-S319-Runx2/total Runx2 as well as P-ERK/total ERK was highest in PC3 followed by C4-2B and LNCaP cells. These results were confirmed by immunofluorescence confocal microscopy, which showed a higher percentage of PC3 cells staining positive for P-S319-Runx2 relative to C4-2B and LNCaP cells. Phosphorylated Runx2 had an exclusively nuclear localization. When expressed in prostate cell lines, wild type Runx2 increased metastasis-associated gene expression, in vitro migratory and invasive activity as well as in vivo growth of tumor cell xenografts. In contrast, S301A/S319A phosphorylation site mutations greatly attenuated these Runx2 responses. Analysis of tissue microarrays from 129 patients revealed strong nuclear staining with the P-S319-Runx2 antibody in primary prostate cancers and metastases. P-S319-Runx2 staining was positively correlated with Gleason score and occurrence of lymph node metastases while little or no Runx2 phosphorylation was seen in normal prostate, benign prostate hyperplasia or prostatitis indicating that Runx2 S319 phosphorylation is closely associated with prostate cancer induction and progression towards an aggressive phenotype. These studies establish the importance of Runx2 phosphorylation in prostate tumor growth and highlight its value as a potential diagnostic marker and therapeutic target. PMID:25867060

  6. Role of Runx2 phosphorylation in prostate cancer and association with metastatic disease.

    PubMed

    Ge, C; Zhao, G; Li, Y; Li, H; Zhao, X; Pannone, G; Bufo, P; Santoro, A; Sanguedolce, F; Tortorella, S; Mattoni, M; Papagerakis, S; Keller, E T; Franceschi, R T

    2016-01-21

    The osteogenic transcription factor, Runx2, is abnormally expressed in prostate cancer (PCa) and associated with metastatic disease. During bone development, Runx2 is activated by signals known to be hyperactive in PCa including the RAS/MAP kinase pathway, which phosphorylates Runx2 on multiple serine residues including S301 and S319 (equivalent to S294 and S312 in human Runx2). This study examines the role of these phosphorylation sites in PCa. Runx2 was preferentially expressed in more invasive PCa cell lines (PC3>C4-2B>LNCaP). Furthermore, analysis using a P-S319-Runx2-specific antibody revealed that the ratio of P-S319-Runx2/total Runx2 as well as P-ERK/total ERK was highest in PC3 followed by C4-2B and LNCaP cells. These results were confirmed by immunofluorescence confocal microscopy, which showed a higher percentage of PC3 cells staining positive for P-S319-Runx2 relative to C4-2B and LNCaP cells. Phosphorylated Runx2 had an exclusively nuclear localization. When expressed in prostate cell lines, wild-type Runx2 increased metastasis-associated gene expression, in vitro migratory and invasive activity as well as in vivo growth of tumor cell xenografts. In contrast, S301A/S319A phosphorylation site mutations greatly attenuated these Runx2 responses. Analysis of tissue microarrays from 129 patients revealed strong nuclear staining with the P-S319-Runx2 antibody in primary PCas and metastases. P-S319-Runx2 staining was positively correlated with Gleason score and occurrence of lymph node metastases while little or no Runx2 phosphorylation was seen in normal prostate, benign prostate hyperplasia or prostatitis indicating that Runx2 S319 phosphorylation is closely associated with PCa induction and progression towards an aggressive phenotype. These studies establish the importance of Runx2 phosphorylation in prostate tumor growth and highlight its value as a potential diagnostic marker and therapeutic target. PMID:25867060

  7. Protein phosphorylation is involved in bacterial chemotaxis.

    PubMed Central

    Hess, J F; Oosawa, K; Matsumura, P; Simon, M I

    1987-01-01

    The nature of the biochemical signal that is involved in the excitation response in bacterial chemotaxis is not known. However, ATP is required for chemotaxis. We have purified all of the proteins involved in signal transduction and show that the product of the cheA gene is rapidly autophosphorylated, while some mutant CheA proteins cannot be phosphorylated. The presence of stoichiometric levels of two other purified components in the chemotaxis system, the CheY and CheZ proteins, induces dephosphorylation. We suggest that the phosphorylation of CheA by ATP plays a central role in signal transduction in chemotaxis. Images PMID:3313398

  8. Structurally abnormal human autosomes

    SciTech Connect

    1993-12-31

    Chapter 25, discusses structurally abnormal human autosomes. This discussion includes: structurally abnormal chromosomes, chromosomal polymorphisms, pericentric inversions, paracentric inversions, deletions or partial monosomies, cri du chat (cat cry) syndrome, ring chromosomes, insertions, duplication or pure partial trisomy and mosaicism. 71 refs., 8 figs.

  9. Activation loop phosphorylation regulates B-Raf in vivo and transformation by B-Raf mutants.

    PubMed

    Köhler, Martin; Röring, Michael; Schorch, Björn; Heilmann, Katharina; Stickel, Natalie; Fiala, Gina J; Schmitt, Lisa C; Braun, Sandra; Ehrenfeld, Sophia; Uhl, Franziska M; Kaltenbacher, Thorsten; Weinberg, Florian; Herzog, Sebastian; Zeiser, Robert; Schamel, Wolfgang W; Jumaa, Hassan; Brummer, Tilman

    2016-01-18

    Despite being mutated in cancer and RASopathies, the role of the activation segment (AS) has not been addressed for B-Raf signaling in vivo. Here, we generated a conditional knock-in mouse allowing the expression of the B-Raf(AVKA) mutant in which the AS phosphoacceptor sites T599 and S602 are replaced by alanine residues. Surprisingly, despite producing a kinase-impaired protein, the Braf(AVKA) allele does not phenocopy the lethality of Braf-knockout or paradoxically acting knock-in alleles. However, Braf(AVKA) mice display abnormalities in the hematopoietic system, a distinct facial morphology, reduced ERK pathway activity in the brain, and an abnormal gait. This phenotype suggests that maximum B-Raf activity is required for the proper development, function, and maintenance of certain cell populations. By establishing conditional murine embryonic fibroblast cultures, we further show that MEK/ERK phosphorylation and the immediate early gene response toward growth factors are impaired in the presence of B-Raf(AVKA). Importantly, alanine substitution of T599/S602 impairs the transformation potential of oncogenic non-V600E B-Raf mutants and a fusion protein, suggesting that blocking their phosphorylation could represent an alternative strategy to ATP-competitive inhibitors. PMID:26657898

  10. Determination of GPCR Phosphorylation Status: Establishing a Phosphorylation Barcode.

    PubMed

    Prihandoko, Rudi; Bradley, Sophie J; Tobin, Andrew B; Butcher, Adrian J

    2015-01-01

    G protein-coupled receptors (GPCRs) are rapidly phosphorylated following agonist occupation in a process that mediates receptor uncoupling from its cognate G protein, a process referred to as desensitization. In addition, this process provides a mechanism by which receptors can engage with arrestin adaptor molecules and couple to downstream signaling pathways. The importance of this regulatory process has been highlighted recently by the understanding that ligands can direct receptor signaling along one pathway in preference to another, the phenomenon of signaling bias that is partly mediated by the phosphorylation status or phosphorylation barcode of the receptor. Methods to determine the phosphorylation status of a GPCR in vitro and in vivo are necessary to understand not only the physiological mechanisms involved in GPCR signaling, but also to fully examine the signaling properties of GPCR ligands. This unit describes detailed methods for determining the overall phosphorylation pattern on a receptor (the phosphorylation barcode), as well as mass spectrometry approaches that can define the precise sites that become phosphorylated. These techniques, coupled with the generation and characterization of receptor phosphorylation-specific antibodies, provide a full palate of techniques necessary to determine the phosphorylation status of any given GPCR subtype. PMID:26344213

  11. Breathing abnormalities in sleep in achondroplasia.

    PubMed Central

    Waters, K A; Everett, F; Sillence, D; Fagan, E; Sullivan, C E

    1993-01-01

    Overnight sleep studies were performed in 20 subjects with achondroplasia to document further the respiratory abnormalities present in this group. Somatosensory evoked potentials (SEPs) were recorded in 19 of the subjects to screen for the presence of brainstem abnormalities, which are one of the potential aetiological mechanisms. Fifteen children aged 1 to 14 years, and five young adults, aged 20 to 31 years were included. All had upper airway obstruction and 15 (75%) had a pathological apnoea index (greater than five per hour). Other sleep associated respiratory abnormalities, including partial obstruction, central apnoea, and abnormal electromyographic activity of accessory muscles of respiration, also showed a high prevalence. SEPs were abnormal in eight (42%), but there was no correlation between abnormal SEPs and apnoea during sleep, either qualitatively or quantitatively. A high prevalence of both sleep related respiratory abnormalities and abnormal SEPs in young subjects with achondroplasia was demonstrated. However, the sleep related respiratory abnormalities do not always result in significant blood gas disturbances or correlate with abnormal SEPs in this group. PMID:8215519

  12. The self-assembly ability of First microtubule-binding repeat from tau and its modulation by phosphorylation

    SciTech Connect

    Zhou Lianxiu; Zeng Zhiyang; Du Jintang; Zhao Yufen; Li Yanmei . E-mail: liym@mail.tsinghua.edu.cn

    2006-09-22

    Aggregation of abnormally phosphorylated tau in the form of tangs of paired helical filaments (PHFs) is one of the hallmarks of Alzheimer's disease (AD) and other tauopathies. It is of fundamental importance to study the mechanism of PHF formation and its modulation by phosphorylation. In this work, we have focused on First microtubule-binding repeat of tau encompassing an abnormal phosphorylation site Ser{sup 262}. The assembly propensities of this repeat and its corresponding phosphorylated form were investigated by turbidity and electron microscopy. Additionally, conformation of the two peptides is also analyzed through circular dichroism (CD) and NMR spectroscopy. Our results reveal that both of them are capable of self-assembly and phosphorylation at Ser{sup 262} could speed up the process of assembly. A possible mechanism of PHF formation is proposed and enhancing effect of phosphorylation on assembly provides an explanation to its toxicity in Alzheimer's disease.

  13. Constitutive phosphorylation of cardiac myosin regulatory light chain in vivo.

    PubMed

    Chang, Audrey N; Battiprolu, Pavan K; Cowley, Patrick M; Chen, Guohua; Gerard, Robert D; Pinto, Jose R; Hill, Joseph A; Baker, Anthony J; Kamm, Kristine E; Stull, James T

    2015-04-24

    In beating hearts, phosphorylation of myosin regulatory light chain (RLC) at a single site to 0.45 mol of phosphate/mol by cardiac myosin light chain kinase (cMLCK) increases Ca(2+) sensitivity of myofilament contraction necessary for normal cardiac performance. Reduction of RLC phosphorylation in conditional cMLCK knock-out mice caused cardiac dilation and loss of cardiac performance by 1 week, as shown by increased left ventricular internal diameter at end-diastole and decreased fractional shortening. Decreased RLC phosphorylation by conventional or conditional cMLCK gene ablation did not affect troponin-I or myosin-binding protein-C phosphorylation in vivo. The extent of RLC phosphorylation was not changed by prolonged infusion of dobutamine or treatment with a β-adrenergic antagonist, suggesting that RLC is constitutively phosphorylated to maintain cardiac performance. Biochemical studies with myofilaments showed that RLC phosphorylation up to 90% was a random process. RLC is slowly dephosphorylated in both noncontracting hearts and isolated cardiac myocytes from adult mice. Electrically paced ventricular trabeculae restored RLC phosphorylation, which was increased to 0.91 mol of phosphate/mol of RLC with inhibition of myosin light chain phosphatase (MLCP). The two RLCs in each myosin appear to be readily available for phosphorylation by a soluble cMLCK, but MLCP activity limits the amount of constitutive RLC phosphorylation. MLCP with its regulatory subunit MYPT2 bound tightly to myofilaments was constitutively phosphorylated in beating hearts at a site that inhibits MLCP activity. Thus, the constitutive RLC phosphorylation is limited physiologically by low cMLCK activity in balance with low MLCP activity. PMID:25733667

  14. Evolutionary constraints of phosphorylation in eukaryotes, prokaryotes, and mitochondria.

    PubMed

    Gnad, Florian; Forner, Francesca; Zielinska, Dorota F; Birney, Ewan; Gunawardena, Jeremy; Mann, Matthias

    2010-12-01

    High accuracy mass spectrometry has proven to be a powerful technology for the large scale identification of serine/threonine/tyrosine phosphorylation in the living cell. However, despite many described phosphoproteomes, there has been no comparative study of the extent of phosphorylation and its evolutionary conservation in all domains of life. Here we analyze the results of phosphoproteomics studies performed with the same technology in a diverse set of organisms. For the most ancient organisms, the prokaryotes, only a few hundred proteins have been found to be phosphorylated. Applying the same technology to eukaryotic species resulted in the detection of thousands of phosphorylation events. Evolutionary analysis shows that prokaryotic phosphoproteins are preferentially conserved in all living organisms, whereas-site specific phosphorylation is not. Eukaryotic phosphosites are generally more conserved than their non-phosphorylated counterparts (with similar structural constraints) throughout the eukaryotic domain. Yeast and Caenorhabditis elegans are two exceptions, indicating that the majority of phosphorylation events evolved after the divergence of higher eukaryotes from yeast and reflecting the unusually large number of nematode-specific kinases. Mitochondria present an interesting intermediate link between the prokaryotic and eukaryotic domains. Applying the same technology to this organelle yielded 174 phosphorylation sites mapped to 74 proteins. Thus, the mitochondrial phosphoproteome is similarly sparse as the prokaryotic phosphoproteomes. As expected from the endosymbiotic theory, phosphorylated as well as non-phosphorylated mitochondrial proteins are significantly conserved in prokaryotes. However, mitochondrial phosphorylation sites are not conserved throughout prokaryotes, consistent with the notion that serine/threonine phosphorylation in prokaryotes occurred relatively recently in evolution. Thus, the phosphoproteome reflects major events in the

  15. Mapping of phosphorylation sites in polyomavirus large T antigen

    SciTech Connect

    Hassauer, M.; Scheidtmann, K.H.; Walter, G.

    1986-06-01

    The phosphorylation sites of polyomavirus large T antigen from infected or transformed cells were investigated. Tryptic digestion of large T antigen from infected, /sup 32/P/sub i/-labeled cells revealed seven major phosphopeptides. Five of these were phosphorylated only at serine residues, and two were phosphorylated at serine and threonine residues. The overall ratio of phosphoserine to phosphothreonine was 6:1. The transformed cell line B4 expressed two polyomavirus-specific phosphoproteins: large T antigen, which was only weakly phosphorylated, and a truncated form of large T antigen of 34,000 molecular weight which was heavily phosphorylated. Both showed phosphorylation patterns similar to that of large T antigen from infected cells. Peptide analyses of large T antigens encoded by the deletion mutants dl8 and dl23 or of specific fragments of wild-type large T antigen indicated that the phosphorylation sites are located in an amino-terminal region upstream of residue 194. The amino acid composition of the phosphopeptides as revealed by differential labeling with various amino acids indicated that several phosphopeptides contain overlapping sequences and that all phosphorylation sites are located in four tryptic peptides derived from a region between Met71 and Arg191. Two of the potential phosphorylation sites were identified as Ser81 and Thr187. The possible role of this modification of large T antigen is discussed.

  16. Sequential Phosphorylation of Smoothened Transduces Graded Hedgehog Signaling

    PubMed Central

    Su, Ying; Ospina, Jason K.; Zhang, Junzheng; Michelson, Andrew P.; Schoen, Adam M.; Zhu, Alan Jian

    2012-01-01

    The correct interpretation of a gradient of the morphogen Hedgehog (Hh) during development requires phosphorylation of the Hh signaling activator Smoothened (Smo); however, the molecular mechanism by which Smo transduces graded Hh signaling is not well understood. We show that regulation of the phosphorylation status of Smo by distinct phosphatases at specific phosphorylated residues creates differential thresholds of Hh signaling. Phosphorylation of Smo was initiated by adenosine 3′,5′-monophosphate (cAMP)–dependent protein kinase (PKA) and further enhanced by casein kinase I (CKI). We found that protein phosphatase 1 (PP1) directly dephosphorylated PKA-phosphorylated Smo to reduce signaling mediated by intermediate concentrations of Hh, whereas PP2A specifically dephosphorylated PKA-primed, CKI-phosphorylated Smo to restrict signaling by high concentrations of Hh. We also established a functional link between sequentially phosphorylated Smo species and graded Hh activity. Thus, we propose a sequential phosphorylation model in which precise interpretation of morphogen concentration can be achieved upon versatile phosphatase-mediated regulation of the phosphorylation status of an essential activator in developmental signaling. PMID:21730325

  17. Protein phosphorylation in stomatal movement

    PubMed Central

    Zhang, Tong; Chen, Sixue; Harmon, Alice C

    2014-01-01

    As research progresses on how guard cells perceive and transduce environmental cues to regulate stomatal movement, plant biologists are discovering key roles of protein phosphorylation. Early research efforts focused on characterization of ion channels and transporters in guard cell hormonal signaling. Subsequent genetic studies identified mutants of kinases and phosphatases that are defective in regulating guard cell ion channel activities, and recently proteins regulated by phosphorylation have been identified. Here we review the essential role of protein phosphorylation in ABA-induced stomatal closure and in blue light-induced stomatal opening. We also highlight evidence for the cross-talk between different pathways, which is mediated by protein phosphorylation. PMID:25482764

  18. Phosphorylation site prediction in plants.

    PubMed

    Yao, Qiuming; Schulze, Waltraud X; Xu, Dong

    2015-01-01

    Protein phosphorylation events on serine, threonine, and tyrosine residues are the most pervasive protein covalent bond modifications in plant signaling. Both low and high throughput studies reveal the importance of phosphorylation in plant molecular biology. Although becoming more and more common, the proteome-wide screening on phosphorylation by experiments remains time consuming and costly. Therefore, in silico prediction methods are proposed as a complementary analysis tool to enhance the phosphorylation site identification, develop biological hypothesis, or help experimental design. These methods build statistical models based on the experimental data, and they do not have some of the technical-specific bias, which may have advantage in proteome-wide analysis. More importantly computational methods are very fast and cheap to run, which makes large-scale phosphorylation identifications very practical for any types of biological study. Thus, the phosphorylation prediction tools become more and more popular. In this chapter, we will focus on plant specific phosphorylation site prediction tools, with essential illustration of technical details and application guidelines. We will use Musite, PhosPhAt and PlantPhos as the representative tools. We will present the results on the prediction of the Arabidopsis protein phosphorylation events to give users a general idea of the performance range of the three tools, together with their strengths and limitations. We believe these prediction tools will contribute more and more to the plant phosphorylation research community. PMID:25930706

  19. Protein phosphorylation in stomatal movement.

    PubMed

    Zhang, Tong; Chen, Sixue; Harmon, Alice C

    2014-01-01

    As research progresses on how guard cells perceive and transduce environmental cues to regulate stomatal movement, plant biologists are discovering key roles of protein phosphorylation. Early research efforts focused on characterization of ion channels and transporters in guard cell hormonal signaling. Subsequent genetic studies identified mutants of kinases and phosphatases that are defective in regulating guard cell ion channel activities, and recently proteins regulated by phosphorylation have been identified. Here we review the essential role of protein phosphorylation in ABA-induced stomatal closure and in blue light-induced stomatal opening. We also highlight evidence for the cross-talk between different pathways, which is mediated by protein phosphorylation. PMID:25482764

  20. What Do Blood Tests Show?

    MedlinePlus

    ... shows the ranges for blood glucose levels after 8 to 12 hours of fasting (not eating). It shows the normal range and the abnormal ranges that are a sign of prediabetes or diabetes. Plasma Glucose Results (mg/dL)* Diagnosis 70 to 99 ...

  1. Pancreatic and duodenal homeobox 1 (PDX1) phosphorylation at serine-269 is HIPK2-dependent and affects PDX1 subnuclear localization.

    PubMed

    An, Rong; da Silva Xavier, Gabriela; Semplici, Francesca; Vakhshouri, Saharnaz; Hao, Huai-Xiang; Rutter, Jared; Pagano, Mario A; Meggio, Flavio; Pinna, Lorenzo A; Rutter, Guy A

    2010-08-20

    Pancreatic and duodenal homeobox 1 (PDX1) regulates pancreatic development and mature beta-cell function. We demonstrate by mass spectrometry that serine residue at position 269 in the C-terminal domain of PDX1 is phosphorylated in beta-cells. Besides we show that the degree of phosphorylation, assessed with a phospho-Ser-269-specific antibody, is decreased by elevated glucose concentrations in both MIN6 beta-cells and primary mouse pancreatic islets. Homeodomain interacting protein kinase 2 (HIPK2) phosphorylates PDX1 in vitro; phosphate incorporation substantially decreases in PDX1 S269A mutant. Silencing of HIPK2 led to a 51+/-0.2% decrease in Ser-269 phosphorylation in MIN6 beta-cells. Mutation of Ser-269 to phosphomimetic residue glutamic acid (S269E) or de-phosphomimetic residue alanine (S269A) exerted no effect on PDX1 half-life. Instead, PDX1 S269E mutant displayed abnormal changes in subnuclear localization in response to high glucose. Our results suggest that HIPK2-mediated phosphorylation of PDX1 at Ser-269 might be a regulatory mechanism connecting signals generated by changes in extracellular glucose concentration to downstream effectors via changes in subnuclear localization of PDX1, thereby influencing islet cell differentiation and function. PMID:20637728

  2. Phosphorylated. beta. -dicarbonyl compounds

    SciTech Connect

    Liorber, B.G.; Tarzivolova, T.A.; Pavlov, V.A.; Zykova, T.V.; Kisilev, V.V.; Tumasheva, N.A.; Slizkii, A.Yu.; Shagvaleev, F.S.

    1987-08-20

    The reaction of trialkyl phosphites with alkyl malonyl chlorides leads to alkyl 3-dialkoxyphosphoryl-3-oxopropionates, which exist in the stable E-enol form. Depending on the basicities of the bases, the reactions of alkyl 3-dialkoxyphosphoryl-3-oxopropionates with nitrogen bases proceed with retention of the C-P bond and the formation of phosphorylated azomethine derivatives or with cleavage of the C-P bond and the liberation of nitrogen-containing derivatives of malonic acid. The /sup 1/H, /sup 13/C, and /sup 13/P NMR spectra were recorded with a Bruker WP-80 NMR spectrometer. The chemical shifts of the protons and carbon atoms are presented relative to tetramethylsilane (TMS). The chemical shifts of the /sup 31/P nuclei were determined relative to H/sub 3/PO/sub 4/.

  3. Involvement of I2PP2A in the abnormal hyperphosphorylation of tau and its reversal by Memantine.

    PubMed

    Chohan, Muhammad Omar; Khatoon, Sabiha; Iqbal, Inge-Grundke; Iqbal, Khalid

    2006-07-10

    The activity of protein phosphatase (PP)-2A, which regulates tau phosphorylation, is compromised in Alzheimer disease brain. Here we show that the transient transfection of PC12 cells with inhibitor-2 (I2PP2A) of PP2A causes abnormal hyperphosphorylation of tau at Ser396/Ser404 and Ser262/Ser356. This hyperphosphorylation of tau is observed only when a sub-cellular shift of I2PP2A takes place from the nucleus to the cytoplasm and is accompanied by cleavage of I2PP2A into a 20 kDa fragment. Memantine, an un-competitive inhibitor of N-methyl-D-aspartate receptors, inhibits this abnormal phosphorylation of tau and cell death and prevents the I2PP2A-induced inhibition of PP2A activity in vitro. These findings demonstrate novel mechanisms by which I2PP2A regulates the intracellular activity of PP2A and phosphorylation of tau, and by which Memantine modulates PP2A signaling and inhibits neurofibrillary degeneration. PMID:16806196

  4. PSEA: Kinase-specific prediction and analysis of human phosphorylation substrates

    NASA Astrophysics Data System (ADS)

    Suo, Sheng-Bao; Qiu, Jian-Ding; Shi, Shao-Ping; Chen, Xiang; Liang, Ru-Ping

    2014-03-01

    Protein phosphorylation catalysed by kinases plays crucial regulatory roles in intracellular signal transduction. With the increasing number of kinase-specific phosphorylation sites and disease-related phosphorylation substrates that have been identified, the desire to explore the regulatory relationship between protein kinases and disease-related phosphorylation substrates is motivated. In this work, we analysed the kinases' characteristic of all disease-related phosphorylation substrates by using our developed Phosphorylation Set Enrichment Analysis (PSEA) method. We evaluated the efficiency of our method with independent test and concluded that our approach is reliable for identifying kinases responsible for phosphorylated substrates. In addition, we found that Mitogen-activated protein kinase (MAPK) and Glycogen synthase kinase (GSK) families are more associated with abnormal phosphorylation. It can be anticipated that our method might be helpful to identify the mechanism of phosphorylation and the relationship between kinase and phosphorylation related diseases. A user-friendly web interface is now freely available at http://bioinfo.ncu.edu.cn/PKPred_Home.aspx.

  5. PSEA: Kinase-specific prediction and analysis of human phosphorylation substrates.

    PubMed

    Suo, Sheng-Bao; Qiu, Jian-Ding; Shi, Shao-Ping; Chen, Xiang; Liang, Ru-Ping

    2014-01-01

    Protein phosphorylation catalysed by kinases plays crucial regulatory roles in intracellular signal transduction. With the increasing number of kinase-specific phosphorylation sites and disease-related phosphorylation substrates that have been identified, the desire to explore the regulatory relationship between protein kinases and disease-related phosphorylation substrates is motivated. In this work, we analysed the kinases' characteristic of all disease-related phosphorylation substrates by using our developed Phosphorylation Set Enrichment Analysis (PSEA) method. We evaluated the efficiency of our method with independent test and concluded that our approach is reliable for identifying kinases responsible for phosphorylated substrates. In addition, we found that Mitogen-activated protein kinase (MAPK) and Glycogen synthase kinase (GSK) families are more associated with abnormal phosphorylation. It can be anticipated that our method might be helpful to identify the mechanism of phosphorylation and the relationship between kinase and phosphorylation related diseases. A user-friendly web interface is now freely available at http://bioinfo.ncu.edu.cn/PKPred_Home.aspx. PMID:24681538

  6. PSEA: Kinase-specific prediction and analysis of human phosphorylation substrates

    PubMed Central

    Suo, Sheng-Bao; Qiu, Jian-Ding; Shi, Shao-Ping; Chen, Xiang; Liang, Ru-Ping

    2014-01-01

    Protein phosphorylation catalysed by kinases plays crucial regulatory roles in intracellular signal transduction. With the increasing number of kinase-specific phosphorylation sites and disease-related phosphorylation substrates that have been identified, the desire to explore the regulatory relationship between protein kinases and disease-related phosphorylation substrates is motivated. In this work, we analysed the kinases' characteristic of all disease-related phosphorylation substrates by using our developed Phosphorylation Set Enrichment Analysis (PSEA) method. We evaluated the efficiency of our method with independent test and concluded that our approach is reliable for identifying kinases responsible for phosphorylated substrates. In addition, we found that Mitogen-activated protein kinase (MAPK) and Glycogen synthase kinase (GSK) families are more associated with abnormal phosphorylation. It can be anticipated that our method might be helpful to identify the mechanism of phosphorylation and the relationship between kinase and phosphorylation related diseases. A user-friendly web interface is now freely available at http://bioinfo.ncu.edu.cn/PKPred_Home.aspx. PMID:24681538

  7. "Jeopardy" in Abnormal Psychology.

    ERIC Educational Resources Information Center

    Keutzer, Carolin S.

    1993-01-01

    Describes the use of the board game, Jeopardy, in a college level abnormal psychology course. Finds increased student interaction and improved application of information. Reports generally favorable student evaluation of the technique. (CFR)

  8. Abnormal Uterine Bleeding

    MedlinePlus

    ... Abnormal uterine bleeding is any bleeding from the uterus (through your vagina) other than your normal monthly ... or fibroids (small and large growths) in the uterus can also cause bleeding. Rarely, a thyroid problem, ...

  9. Abnormal Uterine Bleeding FAQ

    MedlinePlus

    ... as cancer of the uterus, cervix, or vagina • Polycystic ovary syndrome How is abnormal bleeding diagnosed? Your health care ... before the fetus can survive outside the uterus. Polycystic Ovary Syndrome: A condition characterized by two of the following ...

  10. FSCB phosphorylation regulates mouse spermatozoa capacitation through suppressing SUMOylation of ROPN1/ROPN1L

    PubMed Central

    Zhang, Xinqi; Chen, Mingrui; Yu, Renyi; Liu, Benli; Tian, Zhiqiang; Liu, Shunli

    2016-01-01

    Fibrous sheath CABYR binding protein (FSCB) is regulated by protein kinase A (PKA)-mediated tyrosine phosphorylation in the spermatozoa capacitation. Recently, we showed that FSCB phosphorylation activated spermatozoa motility. Nevertheless, the underlying mechanisms have not been completely elucidated. Here, we showed that FSCB phosphorylation inhibited SUMOylation of two crucial proteins ROPN1/ROPN1L that are associated with PKA/A kinase activity and spermatozoa motility. Suppression of SUMOylation of ROPN1/ROPN1L mimicked the effects of FSCB phosphorylation on spermatozoa motility. Immunoprecipitation assay showed that phosphorylated FSCB had a significantly higher affinity to ROPN1/ROPN1L than non-phosphorylated FSCB. Together, our data suggest that FSCB phosphorylation may regulate mouse spermatozoa capacitation through suppressing SUMOylation of ROPN1/ROPN1L, which sheds new light on creating a therapeutic strategy targeting FSCB phosphorylation in the study of infertility.

  11. Stat5a serine phosphorylation. Serine 779 is constitutively phosphorylated in the mammary gland, and serine 725 phosphorylation influences prolactin-stimulated in vitro DNA binding activity.

    PubMed

    Beuvink, I; Hess, D; Flotow, H; Hofsteenge, J; Groner, B; Hynes, N E

    2000-04-01

    The activity of transcription factors of the Stat family is controlled by phosphorylation of a conserved, carboxyl-terminal tyrosine residue. Tyrosine phosphorylation is essential for Stat dimerization, nuclear translocation, DNA binding, and transcriptional activation. Phosphorylation of Stats on specific serine residues has also been described. We have previously shown that in HC11 mammary epithelial cells Stat5a is phosphorylated on Tyr(694) in a prolactin-sensitive manner, whereas serine phosphorylation is constitutive (Wartmann, M., Cella, N., Hofer, P., Groner, B., Xiuwen, L., Hennighausen, L., and Hynes, N. E. (1996) J. Biol. Chem. 271, 31863-31868). By using mass spectrometry and site-directed mutagenesis, we have now identified Ser(779), located in a unique Stat5a SP motif, as the site of serine phosphorylation. By using phospho-Ser(779)-specific antiserum, we have determined that Ser(779) is constitutively phosphorylated in mammary glands taken from different developmental stages. Stat5a isolated from spleen, heart, brain, and lung was also found to be phosphorylated on Ser(779). Ser(725) in Stat5a has also been identified as a phosphorylation site (Yamashita, H., Xu, J., Erwin, R. A., Farrar, W. L., Kirken, R. A., and Rui, H. (1998) J. Biol. Chem. 273, 30218-30224). Here we show that mutagenesis of Ser(725), Ser(779), or a combination of Ser(725/779) to an Ala had no effect on prolactin-induced transcriptional activation of a beta-casein reporter construct. However, following prolactin induction the Ser(725) mutant displayed sustained DNA binding activity compared with that of wild type Stat5a. The results suggest that Ser(725) phosphorylation has an impact on signal duration. PMID:10744710

  12. Phosphorylation Regulates Functions of ZEB1 Transcription Factor.

    PubMed

    Llorens, M Candelaria; Lorenzatti, Guadalupe; Cavallo, Natalia L; Vaglienti, Maria V; Perrone, Ana P; Carenbauer, Anne L; Darling, Douglas S; Cabanillas, Ana M

    2016-10-01

    ZEB1 transcription factor is important in both development and disease, including many TGFβ-induced responses, and the epithelial-to-mesenchymal transition (EMT) by which many tumors undergo metastasis. ZEB1 is differentially phosphorylated in different cell types; however the role of phosphorylation in ZEB1 activity is unknown. Luciferase reporter studies and electrophoresis mobility shift assays (EMSA) show that a decrease in phosphorylation of ZEB1 increases both DNA-binding and transcriptional repression of ZEB1 target genes. Functional analysis of ZEB1 phosphorylation site mutants near the second zinc finger domain (termed ZD2) show that increased phosphorylation (due to either PMA plus ionomycin, or IGF-1) can inhibit transcriptional repression by either a ZEB1-ZD2 domain clone, or full-length ZEB1. This approach identifies phosphosites that have a substantial effect regulating the transcriptional and DNA-binding activity of ZEB1. Immunoprecipitation with anti-ZEB1 antibodies followed by western analysis with a phospho-Threonine-Proline-specific antibody indicates that the ERK consensus site at Thr-867 is phosphorylated in ZEB1. In addition to disrupting in vitro DNA-binding measured by EMSA, IGF-1-induced MEK/ERK phosphorylation is sufficient to disrupt nuclear localization of GFP-ZEB1 fusion clones. These data suggest that phosphorylation of ZEB1 integrates TGFβ signaling with other signaling pathways such as IGF-1. J. Cell. Physiol. 231: 2205-2217, 2016. © 2016 Wiley Periodicals, Inc. PMID:26868487

  13. Chromosomal Abnormalities and Schizophrenia

    PubMed Central

    BASSETT, ANNE S.; CHOW, EVA W.C.; WEKSBERG, ROSANNA

    2011-01-01

    Schizophrenia is a common and serious psychiatric illness with strong evidence for genetic causation, but no specific loci yet identified. Chromosomal abnormalities associated with schizophrenia may help to understand the genetic complexity of the illness. This paper reviews the evidence for associations between chromosomal abnormalities and schizophrenia and related disorders. The results indicate that 22q11.2 microdeletions detected by fluorescence in-situ hybridization (FISH) are significantly associated with schizophrenia. Sex chromosome abnormalities seem to be increased in schizophrenia but insufficient data are available to indicate whether schizophrenia or related disorders are increased in patients with sex chromosome aneuploidies. Other reports of chromosomal abnormalities associated with schizophrenia have the potential to be important adjuncts to linkage studies in gene localization. Advances in molecular cytogenetic techniques (i.e., FISH) have produced significant increases in rates of identified abnormalities in schizophrenia, particularly in patients with very early age at onset, learning difficulties or mental retardation, or dysmorphic features. The results emphasize the importance of considering behavioral phenotypes, including adult onset psychiatric illnesses, in genetic syndromes and the need for clinicians to actively consider identifying chromosomal abnormalities and genetic syndromes in selected psychiatric patients. PMID:10813803

  14. Protein Synthesis Initiation Factors: Phosphorylation and Regulation

    SciTech Connect

    Karen S. Browning

    2009-06-15

    The initiation of the synthesis of proteins is a fundamental process shared by all living organisms. Each organism has both shared and unique mechanisms for regulation of this vital process. Higher plants provide for a major amount of fixation of carbon from the environment and turn this carbon into food and fuel sources for our use. However, we have very little understanding of how plants regulate the synthesis of the proteins necessary for these metabolic processes. The research carried out during the grant period sought to address some of these unknowns in the regulation of protein synthesis initiation. Our first goal was to determine if phosphorylation plays a significant role in plant initiation of protein synthesis. The role of phosphorylation, although well documented in mammalian protein synthesis regulation, is not well studied in plants. We showed that several of the factors necessary for the initiation of protein synthesis were targets of plant casein kinase and showed differential phosphorylation by the plant specific isoforms of this kinase. In addition, we identified and confirmed the phosphorylation sites in five of the plant initiation factors. Further, we showed that phosphorylation of one of these factors, eIF5, affected the ability of the factor to participate in the initiation process. Our second goal was to develop a method to make initiation factor 3 (eIF3) using recombinant methods. To date, we successfully cloned and expressed 13/13 subunits of wheat eIF3 in E. coli using de novo gene construction methods. The final step in this process is to place the subunits into three different plasmid operons for co-expression. Successful completion of expression of eIF3 will be an invaluable tool to the plant translation community.

  15. Involvement of histamine receptors in SAPK/JNK phosphorylation.

    PubMed

    Dandekar, Radhika D; Khan, Manzoor M

    2012-06-01

    Histamine is a mediator of inflammation in allergic disease and asthma. Stress activated protein kinases/c-jun N-terminal kinases (SAPK/JNK) are involved in asthma. This study examined the role of histamine receptors on the phosphorylation of SAPK/JNK in splenocytes. C57BL/6 mice splenocytes were treated with histamine (10⁻⁴ M to 10⁻¹¹ M), and its selective receptor agonists, phorbol 12 myristate 13-acetate (PMA) was used as a positive control, and phosphorylation of SAPK/JNK was determined. Histamine (10⁻⁴ M-10⁻⁸ M) inhibited phosphorylation of SAPK/JNK. H1R agonist betahistine (10⁻⁵ M) decreased SAPK/JNK phosphorylation and H2R agonist amthamine (10⁻⁵ M) did not show any significant effect. However, H3R agonist methimepip (10⁻⁶ M) and H4R agonist 4-methyl histamine (10⁻⁶ M), increased SAPK/JNK phosphorylation. We used TNFα knockout mice to determine if histamine regulated SAPK/JNK phosphorylation via TNFα. While the effects of histamine and H1 agonists were similar to that of wild type mice in inhibiting the phosphorylation of SAPK/JNK, the effects of H3 and H4 agonists differed in TNFα knockout mice splenocytes. Activation of H3 receptors decreased SAPK/JNK phosphorylation in TNFα knockout mice, as opposed to an increase in wild type mice, whereas H4 agonist did not show any significant effect on the phosphorylation of SAPK/JNK. This data showed that histamine acting through H4 receptors caused the phosphorylation of SAPK/JNK via TNFα. The role of H4 receptors in pro-inflammatory response is intriguing. PMID:22487127

  16. Inhibition of Bcr serine kinase by tyrosine phosphorylation.

    PubMed Central

    Liu, J; Wu, Y; Ma, G Z; Lu, D; Haataja, L; Heisterkamp, N; Groffen, J; Arlinghaus, R B

    1996-01-01

    The first exon of the BCR gene encodes a new serine/threonine protein kinase. Abnormal fusion of the BCR and ABL genes, resulting from the formation of the Philadelphia chromosome (Ph), is the hallmark of Ph-positive leukemia. We have previously demonstrated that the Bcr protein is tyrosine phosphorylated within first-exon sequences by the Bcr-Abl oncoprotein. Here we report that in addition to tyrose 177 (Y-177), Y-360 and Y283 are phosphorylated in Bcr-Abl proteins in vitro. Moreover, Bcr tyrosine 360 is phosphorylated in vivo within both Bcr-Abl and Bcr. Bcr mutant Y177F had a greatly reduced ability to transphosphorylate casein and histone H1, whereas Bcr mutants Y177F and Y283F had wild-type activities. In contrast, the Y360F mutation had little effect on Bcr's autophosphorylation activity. Tyrosine-phosphorylated Bcr, phosphorylated in vitro by Bcr-Abl, was greatly inhibited in its serine/threonine kinase activity, impairing both auto- and transkinase activities of Bcr. Similarly, the isolation of Bcr from cells expressing Bcr-Abl under conditions that preserve phosphotyrosine residues also reduced Bcr's kinase activity. These results indicate that tyrosine 360 of Bcr is critical for the transphosphorylation activity of Bcr and that in Ph-positive leukemia, Bcr serine/threonine kinase activity is seriously impaired. PMID:8622703

  17. Synaptic Activation of Ribosomal Protein S6 Phosphorylation Occurs Locally in Activated Dendritic Domains

    ERIC Educational Resources Information Center

    Pirbhoy, Patricia Salgado; Farris, Shannon; Steward, Oswald

    2016-01-01

    Previous studies have shown that induction of long-term potentiation (LTP) induces phosphorylation of ribosomal protein S6 (rpS6) in postsynaptic neurons, but the functional significance of rpS6 phosphorylation is poorly understood. Here, we show that synaptic stimulation that induces perforant path LTP triggers phosphorylation of rpS6 (p-rpS6)…

  18. Isolation of regulatory-competent, phosphorylated cytochrome C oxidase.

    PubMed

    Lee, Icksoo; Salomon, Arthur R; Yu, Kebing; Samavati, Lobelia; Pecina, Petr; Pecinova, Alena; Hüttemann, Maik

    2009-01-01

    The role of posttranslational modifications, specifically reversible phosphorylation as a regulatory mechanism operating in the mitochondria, is a novel research direction. The mitochondrial oxidative phosphorylation system is a particularly interesting unit because it is responsible for the production of the vast majority of cellular energy in addition to free radicals, two factors that are aberrant in numerous human diseases and that may be influenced by reversible phosphorylation of the oxidative phosphorylation complexes. We here describe a detailed protocol for the isolation of mammalian liver and heart mitochondria and subsequently cytochrome c oxidase (CcO) under conditions maintaining the physiological phosphorylation state. The protocol employs the use of activated vanadate, an unspecific tyrosine phosphatase inhibitor, fluoride, an unspecific serine/threonine phosphatase inhibitor, and EGTA, a calcium chelator to prevent the activation of calcium-dependent protein phosphatases. CcO purified without manipulation of signaling pathways shows strong tyrosine phosphorylation on subunits II and IV, whereas tyrosine phosphorylation of subunit I can be induced by the cAMP- and TNFalpha-dependent pathways in liver. Using our protocol on cow liver tissue we further show the identification of a new phosphorylation site on CcO subunit IV tyrosine 11 of the mature protein (corresponding to tyrosine 33 of the precursor peptide) via immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ESI-MS). This phosphorylation site is located close to the ATP and ADP binding site, which adjusts CcO activity to cellular energy demand, and we propose that phosphorylation of tyrosine 11 enables allosteric regulation. PMID:19426869

  19. Phosphorylation of FEZ1 by Microtubule Affinity Regulating Kinases regulates its function in presynaptic protein trafficking

    PubMed Central

    Butkevich, Eugenia; Härtig, Wolfgang; Nikolov, Miroslav; Erck, Christian; Grosche, Jens; Urlaub, Henning; Schmidt, Christoph F.; Klopfenstein, Dieter R.; Chua, John Jia En

    2016-01-01

    Adapters bind motor proteins to cargoes and therefore play essential roles in Kinesin-1 mediated intracellular transport. The regulatory mechanisms governing adapter functions and the spectrum of cargoes recognized by individual adapters remain poorly defined. Here, we show that cargoes transported by the Kinesin-1 adapter FEZ1 are enriched for presynaptic components and identify that specific phosphorylation of FEZ1 at its serine 58 regulatory site is mediated by microtubule affinity-regulating kinases (MARK/PAR-1). Loss of MARK/PAR-1 impairs axonal transport, with adapter and cargo abnormally co-aggregating in neuronal cell bodies and axons. Presynaptic specializations are markedly reduced and distorted in FEZ1 and MARK/PAR-1 mutants. Strikingly, abnormal co-aggregates of unphosphorylated FEZ1, Kinesin-1 and its putative cargoes are present in brains of transgenic mice modelling aspects of Alzheimer’s disease, a neurodegenerative disorder exhibiting impaired axonal transport and altered MARK activity. Our findings suggest that perturbed FEZ1-mediated synaptic delivery of proteins arising from abnormal signalling potentially contributes to the process of neurodegeneration. PMID:27247180

  20. Phosphorylation of FEZ1 by Microtubule Affinity Regulating Kinases regulates its function in presynaptic protein trafficking.

    PubMed

    Butkevich, Eugenia; Härtig, Wolfgang; Nikolov, Miroslav; Erck, Christian; Grosche, Jens; Urlaub, Henning; Schmidt, Christoph F; Klopfenstein, Dieter R; Chua, John Jia En

    2016-01-01

    Adapters bind motor proteins to cargoes and therefore play essential roles in Kinesin-1 mediated intracellular transport. The regulatory mechanisms governing adapter functions and the spectrum of cargoes recognized by individual adapters remain poorly defined. Here, we show that cargoes transported by the Kinesin-1 adapter FEZ1 are enriched for presynaptic components and identify that specific phosphorylation of FEZ1 at its serine 58 regulatory site is mediated by microtubule affinity-regulating kinases (MARK/PAR-1). Loss of MARK/PAR-1 impairs axonal transport, with adapter and cargo abnormally co-aggregating in neuronal cell bodies and axons. Presynaptic specializations are markedly reduced and distorted in FEZ1 and MARK/PAR-1 mutants. Strikingly, abnormal co-aggregates of unphosphorylated FEZ1, Kinesin-1 and its putative cargoes are present in brains of transgenic mice modelling aspects of Alzheimer's disease, a neurodegenerative disorder exhibiting impaired axonal transport and altered MARK activity. Our findings suggest that perturbed FEZ1-mediated synaptic delivery of proteins arising from abnormal signalling potentially contributes to the process of neurodegeneration. PMID:27247180

  1. Phosphorylation of Ser78 of Hsp27 correlated with HER-2/neu status and lymph node positivity in breast cancer

    PubMed Central

    Zhang, Daohai; Wong, Lee Lee; Koay, Evelyn SC

    2007-01-01

    Background Abnormal amplification/expression of HER-2/neu oncogene has been causally linked with tumorigenesis and metastasis in breast cancer and associated with shortened overall survival of patients. Recently, heat shock protein 27 (Hsp27) was reported to be highly expressed in HER-2/neu positive tumors and cell lines. However, putative functional links between phosphorylation of Hsp27 with HER-2/neu status and other clinicopathological features remain to be elucidated. Results Comparative phosphoproteomic studies of HER-2/neu positive and -negative breast tumors revealed that Hsp27, one of the identified phosphoproteins, was highly phosphorylated in HER-2/neu positive tumors. The extent of Hsp27 phosphorylation at its Ser15, Ser78 and Ser82 residues were further evaluated with site-specific antibodies in tumor samples by tissue lysate array- and tissue microarray-based analyses, and in the BT474 breast cancer cell line treated with heregulin α1 (HRG α1) or the p38 MAPK inhibitor, SB203580. The tissue lysate array study indicated that only the level of pSer78 in HER-2/neu positive tumors was more than 2-fold that in HER-2/neu negative tumors. Treatment of BT474 cells with HRG α1 and SB203580 indicated that Ser78 phosphorylation was mainly regulated by the HER-2/neu-p38 MAPK pathway. Immunohistochemical staining of sections from a tissue microarray with 97 breast tumors showed that positive staining of pSer78 significantly correlated with HER-2/neu (p = 0.004) and lymph node positivity (p = 0.026). Conclusion This investigation demonstrated the significant correlation of enhanced phosphorylation of the Ser78 residue of Hsp27 with HER-2/neu and lymph node positivity in breast cancer. PMID:17697330

  2. Curcumin attenuates high glucose-induced podocyte apoptosis by regulating functional connections between caveolin-1 phosphorylation and ROS

    PubMed Central

    Sun, Li-na; Liu, Xiang-chun; Chen, Xiang-jun; Guan, Guang-ju; Liu, Gang

    2016-01-01

    Aim: Caveolin-1 (cav-1) is a major multifunctional scaffolding protein of caveolae. Cav-1 is primarily expressed in mesangial cells, renal proximal tubule cells and podocytes in kidneys. Recent evidence shows that the functional connections between cav-1 and ROS play a key role in many diseases. In this study we investigated whether regulating the functional connections between cav-1 and ROS in kidneys contributed to the beneficial effects of curcumin in treating diabetic nephropathy in vitro and in vivo. Methods: Cultured mouse podocytes (mpc5) were incubated in a high glucose (HG, 30 mmol/L) medium for 24, 48 or 72 h. Male rats were injected with STZ (60 mg/kg, ip) to induce diabetes. ROS generation, SOD activity, MDA content and caspase-3 activity in the cultured cells and kidney cortex homogenate were determined. Apoptotic proteins and cav-1 phosphorylation were analyzed using Western blot analyses. Results: Incubation in HG-containing medium time-dependently increased ROS production, oxidative stress, apoptosis, and cav-1 phosphorylation in podocytes. Pretreatment with curcumin (1, 5, and 10 μmol/L) dose-dependently attenuated these abnormalities in HG-treated podocytes. Furthermore, in HG-containing medium, the podocytes transfected with a recombinant plasmid GFP-cav-1 Y14F (mutation at a cav-1 phosphorylation site) exhibited significantly decreased ROS production and apoptosis compared with the cells transfected with empty vector. In diabetic rats, administration of curcumin (100 or 200 mg/kg body weight per day, ig, for 8 weeks) not only significantly improved the renal function, but also suppressed ROS levels, oxidative stress, apoptosis and cav-1 phosphorylation in the kidneys. Conclusion: Curcumin attenuates high glucose-induced podocyte apoptosis in vitro and diabetic nephropathy in vivo partly through regulating the functional connections between cav-1 phosphorylation and ROS. PMID:26838071

  3. Phosphorylated testis-specific serine/threonine kinase 4 may phosphorylate Crem at Ser-117.

    PubMed

    Fu, Guolong; Wei, Youheng; Wang, Xiaoli; Yu, Long

    2016-06-01

    We aimed to investigate the internal existence status of testis-specific serine/threonine kinase 4 (Tssk4) and the interaction of Tssk4 and Cre-responsive element modulator (Crem). The internal existence status of Tssk4 in testis of mice was detected using western blotting and dephosphorylation method. The interaction of Tssk4 and Crem was analyzed by western blotting, immunohistochemistry, immunofluorescence, in vitro co-immunoprecipitation assays, and in vitro kinase assay. The results revealed that Tssk4 existed in testis both in phosphorylation and unphosphorylation status by a temporal manner with the development of testis. Immunofluorescence results showed that Tssk4 had identical distribution pattern with Crem in testis, which was utterly different to the localization of Cre-responsive element binding (Creb). In conclusion, our study demonstrated that phosphorylated Tssk4 might participate in testis genes expressions by phosphorylating Crem at Ser-117. PMID:26940607

  4. Phosphorylated TDP-43 becomes resistant to cleavage by calpain: A regulatory role for phosphorylation in TDP-43 pathology of ALS/FTLD.

    PubMed

    Yamashita, Takenari; Teramoto, Sayaka; Kwak, Shin

    2016-06-01

    TAR DNA-binding protein-43 (TDP-43) pathology, which includes the presence of abnormal TDP-43-containing inclusions with a loss of nuclear TDP-43 in affected neurons, is a pathological hallmark of amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobar degeneration (FTLD). TDP-43 in the pathological brains and spinal cords of ALS/FTLD patients is abnormally fragmented and phosphorylated. It is believed that the generation of aggregation-prone TDP-43 fragments initiates TDP-43 pathology, and we previously reported that calpain has an important role in the generation of such aggregation-prone TDP-43 fragments. However, the role of phosphorylation in TDP-43 pathology has not been largely elucidated, despite previous observations that several kinases and their kinases are involved in TDP-43 phosphorylation. Here, we investigated the role of TDP-43 phosphorylation in the calpain-dependent cleavage of TDP-43 and found that phosphorylated, full-length TDP-43 and calpain-dependent TDP-43 fragments were more resistant to cleavage by calpain than endogenous full-length TDP-43 was. These results suggest that both phosphorylated and calpain-cleaved TDP-43 fragments persist intracellularly for a length of time that is sufficient for self-aggregation, thereby serving as seeds for inclusions. PMID:26723245

  5. Oxidative and Photosynthetic Phosphorylation Mechanisms

    ERIC Educational Resources Information Center

    Wang, Jui H.

    1970-01-01

    Proposes a molecular mechanism for the coupling of phosphorylation to electron transport in both mitochondria and chloroplasts. Justifies the proposed reaction schemes in terms of thermodynamics and biochemical data. Suggests how areobic respiration could have evolved. (EB)

  6. Phosphorylation in protein-protein binding: effect on stability and function

    PubMed Central

    Nishi, Hafumi; Hashimoto, Kosuke; Panchenko, Anna R.

    2011-01-01

    Summary Post-translational modifications offer a dynamic way to regulate protein activity, subcellular localization and stability. Here we estimate the effect of phosphorylation on protein binding and function for different types of complexes from human proteome. We find that phosphorylation sites have a tendency to be located on binding interfaces in heterooligomeric and weak transient homooligomeric complexes. The analysis of molecular mechanisms of phosphorylation shows that phosphorylation may modulate the strength of interactions directly on interfaces and binding hotspots have a tendency to be phosphorylated in heterooligomers. Although majority of phosphosites do not show significant estimated stability differences upon attaching the phosphate groups, for about one third of all complexes it causes relatively large changes in binding energy. We discuss the cases where phosphorylation mediates the complex formation and regulates the function. We show that phosphorylation sites are not only more likely to be evolutionary conserved than surface residues but even more so than other interfacial residues. PMID:22153503

  7. Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation

    PubMed Central

    Kazlauskaite, Agne; Martínez-Torres, R Julio; Wilkie, Scott; Kumar, Atul; Peltier, Julien; Gonzalez, Alba; Johnson, Clare; Zhang, Jinwei; Hope, Anthony G; Peggie, Mark; Trost, Matthias; van Aalten, Daan MF; Alessi, Dario R; Prescott, Alan R; Knebel, Axel; Walden, Helen; Muqit, Miratul MK

    2015-01-01

    Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser65)—which lies within its ubiquitin-like domain (Ubl)—and indirectly through phosphorylation of ubiquitin at Ser65. How Ser65-phosphorylated ubiquitin (ubiquitinPhospho-Ser65) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitinPhospho-Ser65 binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser65 by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site-directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitinPhospho-Ser65, thereby promoting Parkin Ser65 phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser65 phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitinPhospho-Ser65 to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser65. Finally, purified Parkin maximally phosphorylated at Ser65 in vitro cannot be further activated by the addition of ubiquitinPhospho-Ser65. Our results thus suggest that a major role of ubiquitinPhospho-Ser65 is to promote PINK1-mediated phosphorylation of Parkin at Ser65, leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser65-binding pocket on the surface of Parkin that is critical for the ubiquitinPhospho-Ser65 interaction. This study provides new mechanistic insights into Parkin activation by ubiquitinPhospho-Ser65, which could aid in the development of Parkin activators that mimic the effect of

  8. Tau phosphorylation at Alzheimer's disease-related Ser356 contributes to tau stabilization when PAR-1/MARK activity is elevated.

    PubMed

    Ando, Kanae; Oka, Mikiko; Ohtake, Yosuke; Hayashishita, Motoki; Shimizu, Sawako; Hisanaga, Shin-Ichi; Iijima, Koichi M

    2016-09-16

    Abnormal phosphorylation of the microtubule-associated protein tau is observed in many neurodegenerative diseases, including Alzheimer's disease (AD). AD-related phosphorylation of two tau residues, Ser262 and Ser356, by PAR-1/MARK stabilizes tau in the initial phase of mismetabolism, leading to subsequent phosphorylation events, accumulation, and toxicity. However, the relative contribution of phosphorylation at each of these sites to tau stabilization has not yet been elucidated. In a Drosophila model of human tau toxicity, we found that tau was phosphorylated at Ser262, but not at Ser356, and that blocking Ser262 phosphorylation decreased total tau levels. By contrast, when PAR-1 was co-overexpressed with tau, tau was hyperphosphorylated at both Ser262 and Ser356. Under these conditions, the protein levels of tau were significantly elevated, and prevention of tau phosphorylation at both residues was necessary to completely suppress this elevation. These results suggest that tau phosphorylation at Ser262 plays the predominant role in tau stabilization when PAR-1/MARK activity is normal, whereas Ser356 phosphorylation begins to contribute to this process when PAR-1/MARK activity is abnormally elevated, as in diseased brains. PMID:27520376

  9. Acute exercise modifies titin phosphorylation and increases cardiac myofilament stiffness.

    PubMed

    Müller, Anna E; Kreiner, Matthias; Kötter, Sebastian; Lassak, Philipp; Bloch, Wilhelm; Suhr, Frank; Krüger, Martina

    2014-01-01

    Titin-based myofilament stiffness is largely modulated by phosphorylation of its elastic I-band regions N2-Bus (decreases passive stiffness, PT) and PEVK (increases PT). Here, we tested the hypothesis that acute exercise changes titin phosphorylation and modifies myofilament stiffness. Adult rats were exercised on a treadmill for 15 min, untrained animals served as controls. Titin phosphorylation was determined by Western blot analysis using phosphospecific antibodies to Ser4099 and Ser4010 in the N2-Bus region (PKG and PKA-dependent. respectively), and to Ser11878 and Ser 12022 in the PEVK region (PKCα and CaMKIIδ-dependent, respectively). Passive tension was determined by step-wise stretching of isolated skinned cardiomyocytes to sarcomere length (SL) ranging from 1.9 to 2.4 μm and showed a significantly increased PT from exercised samples, compared to controls. In cardiac samples titin N2-Bus phosphorylation was significantly decreased by 40% at Ser4099, however, no significant changes were observed at Ser4010. PEVK phosphorylation at Ser11878 was significantly increased, which is probably mediated by the observed exercise-induced increase in PKCα activity. Interestingly, relative phosphorylation of Ser12022 was substantially decreased in the exercised samples. Surprisingly, in skeletal samples from acutely exercised animals we detected a significant decrease in PEVK phosphorylation at Ser11878 and an increase in Ser12022 phosphorylation; however, PKCα activity remained unchanged. In summary, our data show that a single exercise bout of 15 min affects titin domain phosphorylation and titin-based myocyte stiffness with obviously divergent effects in cardiac and skeletal muscle tissues. The observed changes in titin stiffness could play an important role in adapting the passive and active properties of the myocardium and the skeletal muscle to increased physical activity. PMID:25477822

  10. Acute exercise modifies titin phosphorylation and increases cardiac myofilament stiffness

    PubMed Central

    Müller, Anna E.; Kreiner, Matthias; Kötter, Sebastian; Lassak, Philipp; Bloch, Wilhelm; Suhr, Frank; Krüger, Martina

    2014-01-01

    Titin-based myofilament stiffness is largely modulated by phosphorylation of its elastic I-band regions N2-Bus (decreases passive stiffness, PT) and PEVK (increases PT). Here, we tested the hypothesis that acute exercise changes titin phosphorylation and modifies myofilament stiffness. Adult rats were exercised on a treadmill for 15 min, untrained animals served as controls. Titin phosphorylation was determined by Western blot analysis using phosphospecific antibodies to Ser4099 and Ser4010 in the N2-Bus region (PKG and PKA-dependent. respectively), and to Ser11878 and Ser 12022 in the PEVK region (PKCα and CaMKIIδ-dependent, respectively). Passive tension was determined by step-wise stretching of isolated skinned cardiomyocytes to sarcomere length (SL) ranging from 1.9 to 2.4 μm and showed a significantly increased PT from exercised samples, compared to controls. In cardiac samples titin N2-Bus phosphorylation was significantly decreased by 40% at Ser4099, however, no significant changes were observed at Ser4010. PEVK phosphorylation at Ser11878 was significantly increased, which is probably mediated by the observed exercise-induced increase in PKCα activity. Interestingly, relative phosphorylation of Ser12022 was substantially decreased in the exercised samples. Surprisingly, in skeletal samples from acutely exercised animals we detected a significant decrease in PEVK phosphorylation at Ser11878 and an increase in Ser12022 phosphorylation; however, PKCα activity remained unchanged. In summary, our data show that a single exercise bout of 15 min affects titin domain phosphorylation and titin-based myocyte stiffness with obviously divergent effects in cardiac and skeletal muscle tissues. The observed changes in titin stiffness could play an important role in adapting the passive and active properties of the myocardium and the skeletal muscle to increased physical activity. PMID:25477822

  11. Predicting and analyzing protein phosphorylation sites in plants using musite.

    PubMed

    Yao, Qiuming; Gao, Jianjiong; Bollinger, Curtis; Thelen, Jay J; Xu, Dong

    2012-01-01

    Although protein phosphorylation sites can be reliably identified with high-resolution mass spectrometry, the experimental approach is time-consuming and resource-dependent. Furthermore, it is unlikely that an experimental approach could catalog an entire phosphoproteome. Computational prediction of phosphorylation sites provides an efficient and flexible way to reveal potential phosphorylation sites and provide hypotheses in experimental design. Musite is a tool that we previously developed to predict phosphorylation sites based solely on protein sequence. However, it was not comprehensively applied to plants. In this study, the phosphorylation data from Arabidopsis thaliana, B. napus, G. max, M. truncatula, O. sativa, and Z. mays were collected for cross-species testing and the overall plant-specific prediction as well. The results show that the model for A. thaliana can be extended to other organisms, and the overall plant model from Musite outperforms the current plant-specific prediction tools, Plantphos, and PhosphAt, in prediction accuracy. Furthermore, a comparative study of predicted phosphorylation sites across orthologs among different plants was conducted to reveal potential evolutionary features. A bipolar distribution of isolated, non-conserved phosphorylation sites, and highly conserved ones in terms of the amino acid type was observed. It also shows that predicted phosphorylation sites conserved within orthologs do not necessarily share more sequence similarity in the flanking regions than the background, but they often inherit protein disorder, a property that does not necessitate high sequence conservation. Our analysis also suggests that the phosphorylation frequencies among serine, threonine, and tyrosine correlate with their relative proportion in disordered regions. Musite can be used as a web server (http://musite.net) or downloaded as an open-source standalone tool (http://musite.sourceforge.net/). PMID:22934099

  12. Abiotic regioselective phosphorylation of adenosine with borate in formamide.

    PubMed

    Furukawa, Yoshihiro; Kim, Hyo-Joong; Hutter, Daniel; Benner, Steven A

    2015-04-01

    Nearly 40 years ago, Schoffstall and his coworkers used formamide as a solvent to permit the phosphorylation of nucleosides by inorganic phosphate to give nucleoside phosphates, which (due to their thermodynamic instability with respect to hydrolysis) cannot be easily created in water by an analogous phosphorylation (the "water problem" in prebiotic chemistry). More recently, we showed that borate could stabilize certain carbohydrates against degradation (the "asphalt problem"). Here, we combine the two concepts to show that borate can work in formamide to guide the reactivity of nucleosides under conditions where they are phosphorylated. Specifically, reaction of adenosine in formamide with inorganic phosphate and pyrophosphate in the presence of borate gives adenosine-5'-phosphate as the only detectable phosphorylated product, with formylation (as opposed to hydrolysis) being the competing reaction. PMID:25826074

  13. Protein phosphorylation in chloroplasts - a survey of phosphorylation targets.

    PubMed

    Baginsky, Sacha

    2016-06-01

    The development of new software tools, improved mass spectrometry equipment, a suite of optimized scan types, and better-quality phosphopeptide affinity capture have paved the way for an explosion of mass spectrometry data on phosphopeptides. Because phosphoproteomics achieves good sensitivity, most studies use complete cell extracts for phosphopeptide enrichment and identification without prior enrichment of proteins or subcellular compartments. As a consequence, the phosphoproteome of cell organelles often comes as a by-product from large-scale studies and is commonly assembled from these in meta-analyses. This review aims at providing some guidance on the limitations of meta-analyses that combine data from analyses with different scopes, reports on the current status of knowledge on chloroplast phosphorylation targets, provides initial insights into phosphorylation site conservation in different plant species, and highlights emerging information on the integration of gene expression with metabolism and photosynthesis by means of protein phosphorylation. PMID:26969742

  14. Morphological abnormalities in elasmobranchs.

    PubMed

    Moore, A B M

    2015-08-01

    A total of 10 abnormal free-swimming (i.e., post-birth) elasmobranchs are reported from The (Persian-Arabian) Gulf, encompassing five species and including deformed heads, snouts, caudal fins and claspers. The complete absence of pelvic fins in a milk shark Rhizoprionodon acutus may be the first record in any elasmobranch. Possible causes, including the extreme environmental conditions and the high level of anthropogenic pollution particular to The Gulf, are briefly discussed. PMID:25903257

  15. Mutations of cellulose synthase (CESA1) phosphorylation sites modulate anisotropic cell expansion and bidirectional mobility of cellulose synthase

    PubMed Central

    Chen, Shaolin; Ehrhardt, David W.; Somerville, Chris R.

    2010-01-01

    The CESA1 component of cellulose synthase is phosphorylated at sites clustered in two hypervariable regions of the protein. Mutations of the phosphorylated residues to Ala (A) or Glu (E) alter anisotropic cell expansion and cellulose synthesis in rapidly expanding roots and hypocotyls. Expression of T166E, S686E, or S688E mutants of CESA1 fully rescued the temperature sensitive cesA1-1 allele (rsw1) at a restrictive temperature whereas mutations to A at these positions caused defects in anisotropic cell expansion. However, mutations to E at residues surrounding T166 (i.e., S162, T165, and S167) caused opposite effects. Live-cell imaging of fluorescently labeled CESA showed close correlations between tissue or cell morphology and patterns of bidirectional motility of CESA complexes in the plasma membrane. In the WT, CESA complexes moved at similar velocities in both directions along microtubule tracks. By contrast, the rate of movement of CESA particles was directionally asymmetric in mutant lines that exhibited abnormal tissue or cell expansion, and the asymmetry was removed upon depolymerizing microtubules with oryzalin. This suggests that phosphorylation of CESA differentially affects a polar interaction with microtubules that may regulate the length or quantity of a subset of cellulose microfibrils and that this, in turn, alters microfibril structure in the primary cell wall resulting in or contributing to the observed defect in anisotropic cell expansion. PMID:20855602

  16. Chromosome abnormalities in glioma

    SciTech Connect

    Li, Y.S.; Ramsay, D.A.; Fan, Y.S.

    1994-09-01

    Cytogenetic studies were performed in 25 patients with gliomas. An interesting finding was a seemingly identical abnormality, an extra band on the tip of the short arm of chromosome 1, add(1)(p36), in two cases. The abnormality was present in all cells from a patient with a glioblastoma and in 27% of the tumor cells from a patient with a recurrent irradiated anaplastic astrocytoma; in the latter case, 7 unrelated abnormal clones were identified except 4 of those clones shared a common change, -Y. Three similar cases have been described previously. In a patient with pleomorphic astrocytoma, the band 1q42 in both homologues of chromosome 1 was involved in two different rearrangements. A review of the literature revealed that deletion of the long arm of chromosome 1 including 1q42 often occurs in glioma. This may indicate a possible tumor suppressor gene in this region. Cytogenetic follow-up studies were carried out in two patients and emergence of unrelated clones were noted in both. A total of 124 clonal breakpoints were identified in the 25 patients. The breakpoints which occurred three times or more were: 1p36, 1p22, 1q21, 1q25, 3q21, 7q32, 8q22, 9q22, 16q22, and 22q13.

  17. [Congenital foot abnormalities].

    PubMed

    Delpont, M; Lafosse, T; Bachy, M; Mary, P; Alves, A; Vialle, R

    2015-03-01

    The foot may be the site of birth defects. These abnormalities are sometimes suspected prenatally. Final diagnosis depends on clinical examination at birth. These deformations can be simple malpositions: metatarsus adductus, talipes calcaneovalgus and pes supinatus. The prognosis is excellent spontaneously or with a simple orthopedic treatment. Surgery remains outstanding. The use of a pediatric orthopedist will be considered if malposition does not relax after several weeks. Malformations (clubfoot, vertical talus and skew foot) require specialized care early. Clubfoot is characterized by an equine and varus hindfoot, an adducted and supine forefoot, not reducible. Vertical talus combines equine hindfoot and dorsiflexion of the forefoot, which is performed in the midfoot instead of the ankle. Skew foot is suspected when a metatarsus adductus is resistant to conservative treatment. Early treatment is primarily orthopedic at birth. Surgical treatment begins to be considered after walking age. Keep in mind that an abnormality of the foot may be associated with other conditions: malposition with congenital hip, malformations with syndromes, neurological and genetic abnormalities. PMID:25524290

  18. Effects of phosphorylation on the intrinsic propensity of backbone conformations of serine/threonine.

    PubMed

    He, Erbin; Yan, Guanghui; Zhang, Jian; Wang, Jun; Li, Wenfei

    2016-03-01

    Each amino acid has its intrinsic propensity for certain local backbone conformations, which can be further modulated by the physicochemical environment and post-translational modifications. In this work, we study the effects of phosphorylation on the intrinsic propensity for different local backbone conformations of serine/threonine by molecular dynamics simulations. We showed that phosphorylation has very different effects on the intrinsic propensity for certain local backbone conformations for the serine and threonine. The phosphorylation of serine increases the propensity of forming polyproline II, whereas that of threonine has the opposite effect. Detailed analysis showed that such different responses to phosphorylation mainly arise from their different perturbations to the backbone hydration and the geometrical constraints by forming side-chain-backbone hydrogen bonds due to phosphorylation. Such an effect of phosphorylation on backbone conformations can be crucial for understanding the molecular mechanism of phosphorylation-regulated protein structures/dynamics and functions. PMID:26759163

  19. A mathematical model of phosphorylation AKT in Acute Myeloid Leukemia

    NASA Astrophysics Data System (ADS)

    Adi, Y. A.; Kusumo, F. A.; Aryati, L.; Hardianti, M. S.

    2016-04-01

    In this paper we consider a mathematical model of PI3K/AKT signaling pathways in phosphorylation AKT. PI3K/AKT pathway is an important mediator of cytokine signaling implicated in regulation of hematopoiesis. Constitutive activation of PI3K/AKT signaling pathway has been observed in Acute Meyloid Leukemia (AML) it caused by the mutation of Fms-like Tyrosine Kinase 3 in internal tandem duplication (FLT3-ITD), the most common molecular abnormality associated with AML. Depending upon its phosphorylation status, protein interaction, substrate availability, and localization, AKT can phosphorylate or inhibite numerous substrates in its downstream pathways that promote protein synthesis, survival, proliferation, and metabolism. Firstly, we present a mass action ordinary differential equation model describing AKT double phosphorylation (AKTpp) in a system with 11 equations. Finally, under the asumtion enzyme catalyst constant and steady state equilibrium, we reduce the system in 4 equation included Michaelis Menten constant. Simulation result suggested that a high concentration of PI3K and/or a low concentration of phospatase increased AKTpp activation. This result also indicates that PI3K is a potential target theraphy in AML.

  20. Interphase phosphorylation of lamin A.

    PubMed

    Kochin, Vitaly; Shimi, Takeshi; Torvaldson, Elin; Adam, Stephen A; Goldman, Anne; Pack, Chan-Gi; Melo-Cardenas, Johanna; Imanishi, Susumu Y; Goldman, Robert D; Eriksson, John E

    2014-06-15

    Nuclear lamins form the major structural elements that comprise the nuclear lamina. Loss of nuclear structural integrity has been implicated as a key factor in the lamin A/C gene mutations that cause laminopathies, whereas the normal regulation of lamin A assembly and organization in interphase cells is still undefined. We assumed phosphorylation to be a major determinant, identifying 20 prime interphase phosphorylation sites, of which eight were high-turnover sites. We examined the roles of these latter sites by site-directed mutagenesis, followed by detailed microscopic analysis - including fluorescence recovery after photobleaching, fluorescence correlation spectroscopy and nuclear extraction techniques. The results reveal three phosphorylation regions, each with dominant sites, together controlling lamin A structure and dynamics. Interestingly, two of these interphase sites are hyper-phosphorylated in mitotic cells and one of these sites is within the sequence that is missing in progerin of the Hutchinson-Gilford progeria syndrome. We present a model where different phosphorylation combinations yield markedly different effects on the assembly, subunit turnover and the mobility of lamin A between, and within, the lamina, the nucleoplasm and the cytoplasm of interphase cells. PMID:24741066

  1. Protein Phosphorylation during Coconut Zygotic Embryo Development1

    PubMed Central

    Islas-Flores, Ignacio; Oropeza, Carlos; Hernández-Sotomayor, S.M. Teresa

    1998-01-01

    Evidence was obtained on the occurrence of protein threonine, serine, and tyrosine (Tyr) kinases in developing coconut (Cocos nucifera L.) zygotic embryos, based on in vitro phosphorylation of proteins in the presence of [γ-32P]ATP, alkaline treatment, and thin-layer chromatography analysis, which showed the presence of [32P]phosphoserine, [32P]phosphothreonine, and [32P]phosphotyrosine in [32P]-labeled protein hydrolyzates. Tyr kinase activity was further confirmed in extracts of embryos at different stages of development using antiphosphotyrosine monoclonal antibodies and the synthetic peptide derived from the amino acid sequence surrounding the phosphorylation site in pp60src (RR-SRC), which is specific for Tyr kinases. Anti-phosphotyrosine western blotting revealed a changing profile of Tyr-phosphorylated proteins during embryo development. Tyr kinase activity, as assayed using RR-SRC, also changed during embryo development, showing two peaks of activity, one during early and another during late embryo development. In addition, the use of genistein, a Tyr kinase inhibitor, diminished the ability of extracts to phosphorylate RR-SRC. Results presented here show the occurrence of threonine, serine, and Tyr kinases in developing coconut zygotic embryos, and suggest that protein phosphorylation, and the possible inference of Tyr phosphorylation in particular, may play a role in the coordination of the development of embryos in this species. PMID:9733545

  2. Feeling Abnormal: Simulation of Deviancy in Abnormal and Exceptionality Courses.

    ERIC Educational Resources Information Center

    Fernald, Charles D.

    1980-01-01

    Describes activity in which student in abnormal psychology and psychology of exceptional children classes personally experience being judged abnormal. The experience allows the students to remember relevant research, become sensitized to the feelings of individuals classified as deviant, and use caution in classifying individuals as abnormal.…

  3. Phosphoproteomics Identified an NS5A Phosphorylation Site Involved in Hepatitis C Virus Replication.

    PubMed

    Chong, Weng Man; Hsu, Shih-Chin; Kao, Wei-Ting; Lo, Chieh-Wen; Lee, Kuan-Ying; Shao, Jheng-Syuan; Chen, Yi-Hung; Chang, Justin; Chen, Steve S-L; Yu, Ming-Jiun

    2016-02-19

    The non-structural protein 5A (NS5A) is a hepatitis C virus (HCV) protein indispensable for the viral life cycle. Many prior papers have pinpointed several serine residues in the low complexity sequence I region of NS5A responsible for NS5A phosphorylation; however, the functions of specific phosphorylation sites remained obscure. Using phosphoproteomics, we identified three phosphorylation sites (serines 222, 235, and 238) in the NS5A low complexity sequence I region. Reporter virus and replicon assays using phosphorylation-ablated alanine mutants of these sites showed that Ser-235 dominated over Ser-222 and Ser-238 in HCV replication. Immunoblotting using an Ser-235 phosphorylation-specific antibody showed a time-dependent increase in Ser-235 phosphorylation that correlated with the viral replication activity. Ser-235 phosphorylated NS5A co-localized with double-stranded RNA, consistent with its role in HCV replication. Mechanistically, Ser-235 phosphorylation probably promotes the replication complex formation via increasing NS5A interaction with the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein. Casein kinase Iα (CKIα) directly phosphorylated Ser-235 in vitro. Inhibition of CKIα reduced Ser-235 phosphorylation and the HCV RNA levels in the infected cells. We concluded that NS5A Ser-235 phosphorylated by CKIα probably promotes HCV replication via increasing NS5A interaction with the 33-kDa vesicle-associated membrane protein-associated protein. PMID:26702051

  4. Linker histone partial phosphorylation: effects on secondary structure and chromatin condensation.

    PubMed

    Lopez, Rita; Sarg, Bettina; Lindner, Herbert; Bartolomé, Salvador; Ponte, Inma; Suau, Pedro; Roque, Alicia

    2015-05-19

    Linker histones are involved in chromatin higher-order structure and gene regulation. We have successfully achieved partial phosphorylation of linker histones in chicken erythrocyte soluble chromatin with CDK2, as indicated by HPCE, MALDI-TOF and Tandem MS. We have studied the effects of linker histone partial phosphorylation on secondary structure and chromatin condensation. Infrared spectroscopy analysis showed a gradual increase of β-structure in the phosphorylated samples, concomitant to a decrease in α-helix/turns, with increasing linker histone phosphorylation. This conformational change could act as the first step in the phosphorylation-induced effects on chromatin condensation. A decrease of the sedimentation rate through sucrose gradients of the phosphorylated samples was observed, indicating a global relaxation of the 30-nm fiber following linker histone phosphorylation. Analysis of specific genes, combining nuclease digestion and qPCR, showed that phosphorylated samples were more accessible than unphosphorylated samples, suggesting local chromatin relaxation. Chromatin aggregation was induced by MgCl2 and analyzed by dynamic light scattering (DLS). Phosphorylated chromatin had lower percentages in volume of aggregated molecules and the aggregates had smaller hydrodynamic diameter than unphosphorylated chromatin, indicating that linker histone phosphorylation impaired chromatin aggregation. These findings provide new insights into the effects of linker histone phosphorylation in chromatin condensation. PMID:25870416

  5. SUMOylation at K340 inhibits tau degradation through deregulating its phosphorylation and ubiquitination.

    PubMed

    Luo, Hong-Bin; Xia, Yi-Yuan; Shu, Xi-Ji; Liu, Zan-Chao; Feng, Ye; Liu, Xing-Hua; Yu, Guang; Yin, Gang; Xiong, Yan-Si; Zeng, Kuan; Jiang, Jun; Ye, Keqiang; Wang, Xiao-Chuan; Wang, Jian-Zhi

    2014-11-18

    Intracellular accumulation of the abnormally modified tau is hallmark pathology of Alzheimer's disease (AD), but the mechanism leading to tau aggregation is not fully characterized. Here, we studied the effects of tau SUMOylation on its phosphorylation, ubiquitination, and degradation. We show that tau SUMOylation induces tau hyperphosphorylation at multiple AD-associated sites, whereas site-specific mutagenesis of tau at K340R (the SUMOylation site) or simultaneous inhibition of tau SUMOylation by ginkgolic acid abolishes the effect of small ubiquitin-like modifier protein 1 (SUMO-1). Conversely, tau hyperphosphorylation promotes its SUMOylation; the latter in turn inhibits tau degradation with reduction of solubility and ubiquitination of tau proteins. Furthermore, the enhanced SUMO-immunoreactivity, costained with the hyperphosphorylated tau, is detected in cerebral cortex of the AD brains, and β-amyloid exposure of rat primary hippocampal neurons induces a dose-dependent SUMOylation of the hyperphosphorylated tau. Our findings suggest that tau SUMOylation reciprocally stimulates its phosphorylation and inhibits the ubiquitination-mediated tau degradation, which provides a new insight into the AD-like tau accumulation. PMID:25378699

  6. A secretory kinase complex regulates extracellular protein phosphorylation.

    PubMed

    Cui, Jixin; Xiao, Junyu; Tagliabracci, Vincent S; Wen, Jianzhong; Rahdar, Meghdad; Dixon, Jack E

    2015-01-01

    Although numerous extracellular phosphoproteins have been identified, the protein kinases within the secretory pathway have only recently been discovered, and their regulation is virtually unexplored. Fam20C is the physiological Golgi casein kinase, which phosphorylates many secreted proteins and is critical for proper biomineralization. Fam20A, a Fam20C paralog, is essential for enamel formation, but the biochemical function of Fam20A is unknown. Here we show that Fam20A potentiates Fam20C kinase activity and promotes the phosphorylation of enamel matrix proteins in vitro and in cells. Mechanistically, Fam20A is a pseudokinase that forms a functional complex with Fam20C, and this complex enhances extracellular protein phosphorylation within the secretory pathway. Our findings shed light on the molecular mechanism by which Fam20C and Fam20A collaborate to control enamel formation, and provide the first insight into the regulation of secretory pathway phosphorylation. PMID:25789606

  7. Microfluidic IEF technique for sequential phosphorylation analysis of protein kinases

    NASA Astrophysics Data System (ADS)

    Choi, Nakchul; Song, Simon; Choi, Hoseok; Lim, Bu-Taek; Kim, Young-Pil

    2015-11-01

    Sequential phosphorylation of protein kinases play the important role in signal transduction, protein regulation, and metabolism in living cells. The analysis of these phosphorylation cascades will provide new insights into their physiological functions in many biological functions. Unfortunately, the existing methods are limited to analyze the cascade activity. Therefore, we suggest a microfluidic isoelectric focusing technique (μIEF) for the analysis of the cascade activity. Using the technique, we show that the sequential phosphorylation of a peptide by two different kinases can be successfully detected on a microfluidic chip. In addition, the inhibition assay for kinase activity and the analysis on a real sample have also been conducted. The results indicate that μIEF is an excellent means for studies on phosphorylation cascade activity.

  8. Phosphorylation of Mad controls competition between wingless and BMP signaling.

    PubMed

    Eivers, Edward; Demagny, Hadrien; Choi, Renee H; De Robertis, Edward M

    2011-01-01

    Bone morphogenetic proteins (BMPs) and Wnts are growth factors that provide essential patterning signals for cell proliferation and differentiation. Here, we describe a molecular mechanism by which the phosphorylation state of the Drosophila transcription factor Mad determines its ability to transduce either BMP or Wingless (Wg) signals. Previously, Mad was thought to function in gene transcription only when phosphorylated by BMP receptors. We found that the unphosphorylated form of Mad was required for canonical Wg signaling by interacting with the Pangolin-Armadillo transcriptional complex. Phosphorylation of the carboxyl terminus of Mad by BMP receptor directed Mad toward BMP signaling, thereby preventing Mad from functioning in the Wg pathway. The results show that Mad has distinct signal transduction roles in the BMP and Wnt pathways depending on its phosphorylation state. PMID:21990430

  9. Abnormal human sex chromosome constitutions

    SciTech Connect

    1993-12-31

    Chapter 22, discusses abnormal human sex chromosome constitution. Aneuploidy of X chromosomes with a female phenotype, sex chromosome aneuploidy with a male phenotype, and various abnormalities in X chromosome behavior are described. 31 refs., 2 figs.

  10. Exercises to Improve Gait Abnormalities

    MedlinePlus

    ... Home About iChip Articles Directories Videos Resources Contact Exercises to Improve Gait Abnormalities Home » Article Categories » Exercise and Fitness Font Size: A A A A Exercises to Improve Gait Abnormalities Next Page The manner ...

  11. Physiological and pathological phosphorylation of tau by Cdk5.

    PubMed

    Kimura, Taeko; Ishiguro, Koichi; Hisanaga, Shin-Ichi

    2014-01-01

    Hyperphosphorylation of microtubule-associated protein tau is one of the major pathological events in Alzheimer's disease (AD) and other related neurodegenerative diseases, including frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). Mutations in the tau gene MAPT are a cause of FTDP-17, and the mutated tau proteins are hyperphosphorylated in patient brains. Thus, it is important to determine the molecular mechanism of hyperphosphorylation of tau to understand the pathology of these diseases collectively called tauopathy. Tau is phosphorylated at many sites via several protein kinases, and a characteristic is phosphorylation at Ser/Thr residues in Ser/Thr-Pro sequences, which are targeted by proline-directed protein kinases such as ERK, GSK3β, and Cdk5. Among these kinases, Cdk5 is particularly interesting because it could be abnormally activated in AD. Cdk5 is a member of the cyclin-dependent kinases (Cdks), but in contrast to the major Cdks, which promote cell cycle progression in proliferating cells, Cdk5 is activated in post-mitotic neurons via the neuron-specific activator p35. Cdk5-p35 plays a critical role in brain development and physiological synaptic activity. In contrast, in disease brains, Cdk5 is thought to be hyperactivated by p25, which is the N-terminal truncated form of p35 and is generated by cleavage with calpain. Several reports have indicated that tau is hyperphosphorylated by Cdk5-p25. However, normal and abnormal phosphorylation of tau by Cdk5 is still not completely understood. In this article, we summarize the physiological and pathological phosphorylation of tau via Cdk5. PMID:25076872

  12. Physiological and pathological phosphorylation of tau by Cdk5

    PubMed Central

    Kimura, Taeko; Ishiguro, Koichi; Hisanaga, Shin-ichi

    2014-01-01

    Hyperphosphorylation of microtubule-associated protein tau is one of the major pathological events in Alzheimer’s disease (AD) and other related neurodegenerative diseases, including frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). Mutations in the tau gene MAPT are a cause of FTDP-17, and the mutated tau proteins are hyperphosphorylated in patient brains. Thus, it is important to determine the molecular mechanism of hyperphosphorylation of tau to understand the pathology of these diseases collectively called tauopathy. Tau is phosphorylated at many sites via several protein kinases, and a characteristic is phosphorylation at Ser/Thr residues in Ser/Thr-Pro sequences, which are targeted by proline-directed protein kinases such as ERK, GSK3β, and Cdk5. Among these kinases, Cdk5 is particularly interesting because it could be abnormally activated in AD. Cdk5 is a member of the cyclin-dependent kinases (Cdks), but in contrast to the major Cdks, which promote cell cycle progression in proliferating cells, Cdk5 is activated in post-mitotic neurons via the neuron-specific activator p35. Cdk5-p35 plays a critical role in brain development and physiological synaptic activity. In contrast, in disease brains, Cdk5 is thought to be hyperactivated by p25, which is the N-terminal truncated form of p35 and is generated by cleavage with calpain. Several reports have indicated that tau is hyperphosphorylated by Cdk5-p25. However, normal and abnormal phosphorylation of tau by Cdk5 is still not completely understood. In this article, we summarize the physiological and pathological phosphorylation of tau via Cdk5. PMID:25076872

  13. Diacylglycerol kinase-zeta localization in skeletal muscle is regulated by phosphorylation and interaction with syntrophins.

    PubMed

    Abramovici, Hanan; Hogan, Angela B; Obagi, Christopher; Topham, Matthew K; Gee, Stephen H

    2003-11-01

    Syntrophins are scaffolding proteins that link signaling molecules to dystrophin and the cytoskeleton. We previously reported that syntrophins interact with diacylglycerol kinase-zeta (DGK-zeta), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show syntrophins and DGK-zeta form a complex in skeletal muscle whose translocation from the cytosol to the plasma membrane is regulated by protein kinase C-dependent phosphorylation of the DGK-zeta MARCKS domain. DGK-zeta mutants that do not bind syntrophins were mislocalized, and an activated mutant of this sort induced atypical changes in the actin cytoskeleton, indicating syntrophins are important for localizing DGK-zeta and regulating its activity. Consistent with a role in actin organization, DGK-zeta and syntrophins were colocalized with filamentous (F)-actin and Rac in lamellipodia and ruffles. Moreover, extracellular signal-related kinase-dependent phosphorylation of DGK-zeta regulated its association with the cytoskeleton. In adult muscle, DGK-zeta was colocalized with syntrophins on the sarcolemma and was concentrated at neuromuscular junctions (NMJs), whereas in type IIB fibers it was found exclusively at NMJs. DGK-zeta was reduced at the sarcolemma of dystrophin-deficient mdx mouse myofibers but was specifically retained at NMJs, indicating that dystrophin is important for the sarcolemmal but not synaptic localization of DGK-zeta. Together, our findings suggest syntrophins localize DGK-zeta signaling complexes at specialized domains of muscle cells, which may be critical for the proper control of lipid-signaling pathways regulating actin organization. In dystrophic muscle, mislocalized DGK-zeta may cause abnormal cytoskeletal changes that contribute to disease pathogenesis. PMID:14551255

  14. NuMA Phosphorylation by Aurora-A Orchestrates Spindle Orientation.

    PubMed

    Gallini, Sara; Carminati, Manuel; De Mattia, Fabiola; Pirovano, Laura; Martini, Emanuele; Oldani, Amanda; Asteriti, Italia Anna; Guarguaglini, Giulia; Mapelli, Marina

    2016-02-22

    Spindle positioning is essential for tissue morphogenesis and homeostasis. The signaling network synchronizing spindle placement with mitotic progression relies on timely recruitment at the cell cortex of NuMA:LGN:Gαi complexes, in which NuMA acts as a receptor for the microtubule motor Dynein. To study the implication of Aurora-A in spindle orientation, we developed protocols for the partial inhibition of its activity. Under these conditions, in metaphase NuMA and Dynein accumulate abnormally at the spindle poles and do not reach the cortex, while the cortical distribution of LGN remains unperturbed. FRAP experiments revealed that Aurora-A governs the dynamic exchange between the cytoplasmic and the spindle pole-localized pools of NuMA. We show that Aurora-A phosphorylates directly the C terminus of NuMA on three Ser residues, of which Ser1969 determines the dynamic behavior and the spindle orientation functions of NuMA. Most interestingly, we identify a new microtubule-binding domain of NuMA, which does not overlap with the LGN-binding motif. Our study demonstrates that in metaphase the direct phosphorylation of NuMA by Aurora-A controls its cortical enrichment, and that this is the major event underlying the spindle orientation functions of Aurora-A in transformed and non-transformed cells in culture. Phosphorylation of NuMA by Aurora-A does not affect its affinity for microtubules or for LGN but rather determines the mobility of the protein at the spindle poles. The finding that NuMA can associate concomitantly with LGN and microtubules suggests that its microtubule-binding activity contributes to anchor Dynein-loaded microtubule +TIPs at cortical sites with LGN. PMID:26832443

  15. Structural basis for Mep2 ammonium transceptor activation by phosphorylation

    PubMed Central

    van den Berg, Bert; Chembath, Anupama; Jefferies, Damien; Basle, Arnaud; Khalid, Syma; Rutherford, Julian C.

    2016-01-01

    Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear. Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation. PMID:27088325

  16. Structural basis for Mep2 ammonium transceptor activation by phosphorylation.

    PubMed

    van den Berg, Bert; Chembath, Anupama; Jefferies, Damien; Basle, Arnaud; Khalid, Syma; Rutherford, Julian C

    2016-01-01

    Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear. Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation. PMID:27088325

  17. Phosphorylation of tyrosine residues of calmodulin in Rous sarcoma virus-transformed cells.

    PubMed Central

    Fukami, Y; Nakamura, T; Nakayama, A; Kanehisa, T

    1986-01-01

    Calmodulin, a wide-spread eukaryotic Ca2+-binding protein, was phosphorylated at its tyrosine residues in Rous sarcoma virus (RSV)-transformed chicken and rat cells but not in normal chicken embryo fibroblasts. In contrast, serine and threonine phosphorylation of calmodulin was found to occur in both normal and virus-transformed cells. In an in vitro system containing purified src kinase from RSV-transformed cells, tyrosine phosphorylation of calmodulin by the src kinase was inhibited by Ca2+. Furthermore, the tyrosine-phosphorylated calmodulin showed slower mobility than that of nonphosphorylated calmodulin in NaDodSO4/polyacrylamide gel electrophoresis when Ca2+ was present. These results suggest that the structure of calmodulin Ca2+ complex may be altered by tyrosine phosphorylation. It is thus inferred that Ca2+ may regulate the level of tyrosine phosphorylation of calmodulin in RSV-transformed cells, and phosphorylation in turn may attenuate the function of this protein in vivo. Images PMID:2424020

  18. Deciphering the Interplay among Multisite Phosphorylation, Interaction Dynamics, and Conformational Transitions in a Tripartite Protein System

    PubMed Central

    2016-01-01

    Multisite phosphorylation is a common pathway to regulate protein function, activity, and interaction pattern in vivo, but routine biochemical analysis is often insufficient to identify the number and order of individual phosphorylation reactions and their mechanistic impact on the protein behavior. Here, we integrate complementary mass spectrometry (MS)-based approaches to characterize a multisite phosphorylation-regulated protein system comprising Polo-like kinase 1 (Plk1) and its coactivators Aurora kinase A (Aur-A) and Bora, the interplay of which is essential for mitotic entry after DNA damage-induced cell cycle arrest. Native MS and cross-linking–MS revealed that Aur-A/Bora-mediated Plk1 activation is accompanied by the formation of Aur-A/Bora and Plk1/Bora heterodimers. We found that the Aur-A/Bora interaction is independent of the Bora phosphorylation state, whereas the Plk1/Bora interaction is dependent on extensive Bora multisite phosphorylation. Bottom-up and top-down proteomics analyses showed that Bora multisite phosphorylation proceeds via a well-ordered sequence of site-specific phosphorylation reactions, whereby we could reveal the involvement of up to 16 phosphorylated Bora residues. Ion mobility spectrometry–MS demonstrated that this multisite phosphorylation primes a substantial structural rearrangement of Bora, explaining the interdependence between extensive Bora multisite phosphorylation and Plk1/Bora complex formation. These results represent a first benchmark of our multipronged MS strategy, highlighting its potential to elucidate the mechanistic and structural implications of multisite protein phosphorylation. PMID:27504491

  19. The effect of phosphorylation on arrestin-rhodopsin interaction in the squid visual system.

    PubMed

    Robinson, Kelly A; Ou, Wei-Lin; Guan, Xinyu; Sugamori, Kim S; Bandyopadhyay, Abhishek; Ernst, Oliver P; Mitchell, Jane

    2015-12-01

    Invertebrate visual opsins are G protein-coupled receptors coupled to retinoid chromophores that isomerize reversibly between inactive rhodopsin and active metarhodopsin upon absorption of photons of light. The squid visual system has an arrestin protein that binds to metarhodopsin to block signaling to Gq and activation of phospholipase C. Squid rhodopsin kinase (SQRK) can phosphorylate both metarhodopsin and arrestin, a dual role that is unique among the G protein-coupled receptor kinases. The sites and role of arrestin phosphorylation by SQRK were investigated here using recombinant proteins. Arrestin was phosphorylated on serine 392 and serine 397 in the C-terminus. Unphosphorylated arrestin bound to metarhodopsin and phosphorylated metarhodopsin with similar high affinities (Kd 33 and 21 nM respectively), while phosphorylation of arrestin reduced the affinity 3- to 5-fold (Kd 104 nM). Phosphorylation of metarhodopsin slightly increased the dissociation of arrestin observed during a 1 hour incubation. Together these studies suggest a unique role for SQRK in phosphorylating both receptor and arrestin and inhibiting the binding of these two proteins in the squid visual system. Invertebrate visual systems are inactivated by arrestin binding to metarhodopsin that does not require receptor phosphorylation. Here we show that squid rhodopsin kinase phosphorylates arrestin on two serines (S392,S397) in the C-terminus and phosphorylation decreases the affinity of arrestin for squid metarhodopsin. Metarhodopsin phosphorylation has very little effect on arrestin binding but does increase arrestin dissociation. PMID:26375013

  20. Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues

    PubMed Central

    Shi, Lei; Pigeonneau, Nathalie; Ravikumar, Vaishnavi; Dobrinic, Paula; Macek, Boris; Franjevic, Damjan; Noirot-Gros, Marie-Francoise; Mijakovic, Ivan

    2014-01-01

    Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD, and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells. PMID:25278935

  1. SYMPOSIUM ON PLANT PROTEIN PHOSPHORYLATION

    SciTech Connect

    JOHN C WALKER

    2011-11-01

    Protein phosphorylation and dephosphorylation play key roles in many aspects of plant biology, including control of cell division, pathways of carbon and nitrogen metabolism, pattern formation, hormonal responses, and abiotic and biotic responses to environmental signals. A Symposium on Plant Protein Phosphorylation was hosted on the Columbia campus of the University of Missouri from May 26-28, 2010. The symposium provided an interdisciplinary venue at which scholars studying protein modification, as it relates to a broad range of biological questions and using a variety of plant species, presented their research. It also provided a forum where current international challenges in studies related to protein phosphorylation could be examined. The symposium also stimulated research collaborations through interactions and networking among those in the research community and engaged students and early career investigators in studying issues in plant biology from an interdisciplinary perspective. The proposed symposium, which drew 165 researchers from 13 countries and 21 States, facilitated a rapid dissemination of acquired knowledge and technical expertise regarding protein phosphorylation in plants to a broad range of plant biologists worldwide.

  2. Protein kinase C catalyses the phosphorylation and activation of rat liver phospholipid methyltransferase.

    PubMed Central

    Villalba, M; Pajares, M A; Renart, M F; Mato, J M

    1987-01-01

    When a partially purified rat liver phospholipid methyltransferase is incubated with [gamma-32P]ATP and rat brain protein kinase C, phospholipid methyltransferase (Mr 50,000, pI 4.75) becomes phosphorylated. Phosphorylation of the enzyme showed Ca2+/lipid-dependency. Protein kinase C-dependent phosphorylation of phospholipid methyltransferase was accompanied by an approx. 2-fold activation of the enzyme activity. Activity changes and enzyme phosphorylation showed the same time course. Activation of the enzyme also showed Ca2+/lipid-dependency. Protein kinase C mediates phosphorylation of predominantly serine residues of the methyltransferase. One major peak of phosphorylation was identified by analysis of tryptic phosphopeptides by isoelectrofocusing. This peak (pI 5.2) differs from that phosphorylated by the cyclic AMP-dependent protein kinase (pI 7.2), demonstrating the specificity of phosphorylation of protein kinase C. Tryptic-peptide mapping by h.p.l.c. of the methyltransferase phosphorylated by protein kinase C revealed one major peak of radioactivity, which could be resolved into two labelled phosphopeptides by t.l.c. The significance of protein kinase C-mediated phosphorylation of phospholipid methyltransferase is discussed. Images Fig. 1. Fig. 4. PMID:3593229

  3. Phosphorylation in halobacterial signal transduction.

    PubMed Central

    Rudolph, J; Tolliday, N; Schmitt, C; Schuster, S C; Oesterhelt, D

    1995-01-01

    Regulated phosphorylation of proteins has been shown to be a hallmark of signal transduction mechanisms in both Eubacteria and Eukarya. Here we demonstrate that phosphorylation and dephosphorylation are also the underlying mechanism of chemo- and phototactic signal transduction in Archaea, the third branch of the living world. Cloning and sequencing of the region upstream of the cheA gene, known to be required for chemo- and phototaxis in Halobacterium salinarium, has identified cheY and cheB analogs which appear to form part of an operon which also includes cheA and the following open reading frame of 585 nucleotides. The CheY and CheB proteins have 31.3 and 37.5% sequence identity compared with the known signal transduction proteins CheY and CheB from Escherichia coli, respectively. The biochemical activities of both CheA and CheY were investigated following their expression in E.coli, isolation and renaturation. Wild-type CheA could be phosphorylated in a time-dependent manner in the presence of [gamma-32P]ATP and Mg2+, whereas the mutant CheA(H44Q) remained unlabeled. Phosphorylated CheA was dephosphorylated rapidly by the addition of wild-type CheY. The mutant CheY(D53A) had no effect on phosphorylated CheA. The mechanism of chemo- and phototactic signal transduction in the Archaeon H.salinarium, therefore, is similar to the two-component signaling system known from chemotaxis in the eubacterium E.coli. Images PMID:7556066

  4. Identification of Phosphorylation Sites Regulating sst3 Somatostatin Receptor Trafficking.

    PubMed

    Lehmann, Andreas; Kliewer, Andrea; Günther, Thomas; Nagel, Falko; Schulz, Stefan

    2016-06-01

    The human somatostatin receptor 3 (sst3) is expressed in about 50% of all neuroendocrine tumors and hence a promising target for multireceptor somatostatin analogs. The sst3 receptor is unique among ssts in that it exhibits a very long intracellular C-terminal tail containing a huge number of potential phosphate acceptor sites. Consequently, our knowledge about the functional role of the C-terminal tail in sst3 receptor regulation is very limited. Here, we have generated a series of phosphorylation-deficient mutants that enabled us to determine crucial sites for its agonist-induced β-arrestin mobilization, internalization, and down-regulation. Based on this information, we generated phosphosite-specific antibodies for C-terminal Ser(337)/Thr(341), Thr(348), and Ser(361) that enabled us to investigate the temporal patterns of sst3 phosphorylation and dephosphorylation. We found that the endogenous ligand somatostatin induced a rapid and robust phosphorylation that was completely blocked by the sst3 antagonist NVP-ACQ090. The stable somatostatin analogs pasireotide and octreotide promoted clearly less phosphorylation compared with somatostatin. We also show that sst3 phosphorylation occurred within seconds to minutes, whereas dephosphorylation of the sst3 receptor occurred at a considerable slower rate. In addition, we also identified G protein-coupled receptor kinases 2 and 3 and protein phosphatase 1α and 1β as key regulators of sst3 phosphorylation and dephosphorylation, respectively. Thus, we here define the C-terminal phosphorylation motif of the human sst3 receptor that regulates its agonist-promoted phosphorylation, β-arrestin recruitment, and internalization of this clinically relevant receptor. PMID:27101376

  5. Phosphorylation of mouse melanopsin by protein kinase A.

    PubMed

    Blasic, Joseph R; Brown, R Lane; Robinson, Phyllis R

    2012-01-01

    The visual pigment melanopsin is expressed in intrinsically photosensitive retinal ganglion cells (ipRGCs) in the mammalian retina, where it is involved in non-image forming light responses including circadian photoentrainment, pupil constriction, suppression of pineal melatonin synthesis, and direct photic regulation of sleep. It has recently been shown that the melanopsin-based light response in ipRGCs is attenuated by the neurotransmitter dopamine. Here, we use a heterologous expression system to demonstrate that mouse melanopsin can be phosphorylated by protein kinase A, and that phosphorylation can inhibit melanopsin signaling in HEK cells. Site-directed mutagenesis experiments revealed that this inhibitory effect is primarily mediated by phosphorylation of sites T186 and S287 located in the second and third intracellular loops of melanopsin, respectively. Furthermore, we show that this phosphorylation can occur in vivo using an in situ proximity-dependent ligation assay (PLA). Based on these data, we suggest that the attenuation of the melanopsin-based light response by dopamine is mediated by direct PKA phosphorylation of melanopsin, rather than phosphorylation of a downstream component of the signaling cascade. PMID:23049792

  6. Bak apoptotic function is not directly regulated by phosphorylation.

    PubMed

    Tran, V H; Bartolo, R; Westphal, D; Alsop, A; Dewson, G; Kluck, R M

    2013-01-01

    During apoptosis, Bak and Bax permeabilize the mitochondrial outer membrane by undergoing major conformational change and oligomerization. This activation process in Bak is reported to require dephosphorylation of tyrosine-108 close to an activation trigger site. To investigate how dephosphorylation of Bak contributes to its activation and conformational change, one-dimensional isoelectric focusing (1D-IEF) and mutagenesis was used to monitor Bak phosphorylation. On 1D-IEF, Bak extracted from a range of cell types migrated as a single band near the predicted isoelectric point of 5.6 both before and after phosphatase treatment, indicating that Bak is not significantly phosphorylated at any residue. In contrast, three engineered 'phosphotagged' Bak variants showed a second band at lower pI, indicating phosphorylation. Apoptosis induced by several stimuli failed to alter Bak pI, indicating little change in phosphorylation status. In addition, alanine substitution of tyrosine-108 and other putative phosphorylation sites failed to enhance Bak activation or pro-apoptotic function. In summary, Bak is not significantly phosphorylated at any residue, and Bak activation during apoptosis does not require dephosphorylation. PMID:23303126

  7. Characterisation and properties of homo- and heterogenously phosphorylated nanocellulose.

    PubMed

    Kokol, Vanja; Božič, Mojca; Vogrinčič, Robert; Mathew, Aji P

    2015-07-10

    Nano-sized cellulose ester derivatives having phosphoryl side groups were synthesised by phosphorylation of nanofibrilated cellulose (NFC) and nanocrystaline cellulose (NCC), using different heterogeneous (in water) and homogeneous (in molten urea) processes with phosphoric acid as phosphoryl donor. The phosphorylation mechanism, efficacy, stability, as well as its influence on the NC crystallinity and thermal properties, were evaluated using ATR-FTIR and (13)C NMR spectroscopies, potentiometric titration, capillary electrophoresis, X-ray diffraction, colorimetry, thermogravimmetry and SEM. Phosphorylation under both processes created dibasic phosphate and monobasic tautomeric phosphite groups at C6 and C3 positioned hydroxyls of cellulose, yielded 60-fold (∼1,173 mmol/kg) and 2-fold (∼1.038 mmol/kg) higher surface charge density for p-NFC and p-NCC, respectively, under homogenous conditions. None of the phosphorylations affected neither the NC crystallinity degree nor the structure, and noticeably preventing the derivatives from weight loss during the pyrolysis process. The p-NC showed high hydrolytic stability to water at all pH mediums. Reusing of the treatment bath was examined after the heterogeneous process. PMID:25857987

  8. Tyrosine phosphorylation of clathrin heavy chain under oxidative stress.

    PubMed

    Ihara, Yoshito; Yasuoka, Chie; Kageyama, Kan; Wada, Yoshinao; Kondo, Takahito

    2002-09-20

    In mouse pancreatic insulin-producing betaTC cells, oxidative stress due to H(2)O(2) causes tyrosine phosphorylation in various proteins. To identify proteins bearing phosphotyrosine under stress, the proteins were affinity purified using an anti-phosphotyrosine antibody-conjugated agarose column. A protein of 180kDa was identified as clathrin heavy chain (CHC) by electrophoresis and mass spectrometry. Immunoprecipitated CHC showed tyrosine phosphorylation upon H(2)O(2) treatment and the phosphorylation was suppressed by the Src kinase inhibitor, PP2. The phosphorylation status of CHC affected the intracellular localization of CHC and the clathrin-dependent endocytosis of transferrin under oxidative stress. In conclusion, CHC is a protein that is phosphorylated at tyrosine by H(2)O(2) and this phosphorylation status is implicated in the intracellular localization and functions of CHC under oxidative stress. The present study demonstrates that oxidative stress affects intracellular vesicular trafficking via the alteration of clathrin-dependent vesicular trafficking. PMID:12237126

  9. Tonoplast-Bound Protein Kinase Phosphorylates Tonoplast Intrinsic Protein 1

    PubMed Central

    Johnson, Kenneth D.; Chrispeels, Maarten J.

    1992-01-01

    Tonoplast intrinsic protein (TIP) is a member of a family of putative membrane channels found in bacteria, animals, and plants. Plants have seed-specific, vegetative/reproductive organ-specific, and water-stress-induced forms of TIP. Here, we report that the seed-specific TIP is a phosphoprotein whose phosphorylation can be monitored in vivo by allowing bean cotyledons to take up [32P]orthophosphate and in vitro by incubating purified tonoplasts with γ-labeled [32P]ATP. Characterization of the in vitro phosphorylation of TIP indicates that a membrane-bound protein kinase phosphorylates TIP in a Ca2+-dependent manner. The capacity of the isolated tonoplast membranes to phosphorylate TIP declined markedly during seed germination, and this decline occurred well before the development-mediated decrease in TIP occurs. Phosphoamino acid analysis of purified, radiolabeled TIP showed that serine is the major, if not only, phosphorylated residue, and cyanogen bromide cleavage yielded a single radioactive peptide peak on a reverse-phase high-performance liquid chromatogram. Estimation of the molecular mass of the cyanogen bromide phosphopeptide by laser desorption mass spectroscopy led to its identification as the hydrophilic N-terminal domain of TIP. The putative phosphate-accepting serine residue occurs in a consensus phosphorylation site for serine/threonine protein kinases. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:16653198

  10. Protein phosphorylation in isolated hepatocytes of septic and endotoxemic rats

    SciTech Connect

    Deaciuc, I.V.; Spitzer, J.A. )

    1989-11-01

    The purpose of this study was to investigate possible alterations induced by sepsis and endotoxicosis in the late phase of Ca2+-dependent signaling in rat liver. Hepatocytes isolated from septic or chronically endotoxin (ET)-treated rats were labeled with (32P)H3PO4 and stimulated with various agents. Proteins were resolved by one-dimensional polyacrylamide gel electrophoresis and autoradiographed. Vasopressin (VP)- and phenylephrine (PE)-induced responses were attenuated in both septic and ET-treated rats for cytosolic and membrane proteins compared with their respective controls. Glucagon and 12-O-myristate phorbol-13-acetate (TPA) affected only the phosphorylation of membrane proteins. Glucagon-induced changes in the phosphorylation of membrane proteins were affected by both sepsis and endotoxicosis, whereas TPA-stimulated phosphorylation was lowered only in endotoxicosis. Response to the Ca2+ ionophore A23187 was depressed in septic rats for cytosolic proteins. The phosphorylation of two cytosolic proteins, i.e., 93 and 61 kDa (previously identified as glycogen phosphorylase and pyruvate kinase, respectively), in response to VP, PE, and A23187 was severely impaired by endotoxicosis and sepsis. TPA did not affect the phosphorylation state of these two proteins. The results show that sepsis and endotoxicosis produce perturbations of the phosphorylation step in Ca2+ transmembrane signaling. Such changes can explain alterations of glycogenolysis and gluconeogenesis associated with sepsis and endotoxicosis.

  11. Control of serotonin transporter phosphorylation by conformational state.

    PubMed

    Zhang, Yuan-Wei; Turk, Benjamin E; Rudnick, Gary

    2016-05-17

    Serotonin transporter (SERT) is responsible for reuptake and recycling of 5-hydroxytryptamine (5-HT; serotonin) after its exocytotic release during neurotransmission. Mutations in human SERT are associated with psychiatric disorders and autism. Some of these mutations affect the regulation of SERT activity by cGMP-dependent phosphorylation. Here we provide direct evidence that this phosphorylation occurs at Thr276, predicted to lie near the cytoplasmic end of transmembrane helix 5 (TM5). Using membranes from HeLa cells expressing SERT and intact rat basophilic leukemia cells, we show that agents such as Na(+) and cocaine that stabilize outward-open conformations of SERT decreased phosphorylation and agents that stabilize inward-open conformations (e.g., 5-HT, ibogaine) increased phosphorylation. The opposing effects of the inhibitors cocaine and ibogaine were each reversed by an excess of the other inhibitor. Inhibition of phosphorylation by Na(+) and stimulation by ibogaine occurred at concentrations that induced outward opening and inward opening, respectively, as measured by the accessibility of cysteine residues in the extracellular and cytoplasmic permeation pathways, respectively. The results are consistent with a mechanism of SERT regulation that is activated by the transport of 5-HT, which increases the level of inward-open SERT and may lead to unwinding of the TM5 helix to allow phosphorylation. PMID:27140629

  12. Phosphorylation of bovine rod photoreceptor cyclic GMP phosphodiesterase.

    PubMed Central

    Udovichenko, I P; Cunnick, J; Gonzales, K; Takemoto, D J

    1993-01-01

    The cyclic GMP phosphodiesterase (PDE) of retinal rods plays a key role in phototransduction and consists of two catalytic subunits (PDE alpha and PDE beta) and two identical inhibitory subunits (PDE gamma). Here we report that PDE alpha and PDE gamma are phosphorylated by protein kinase(s) C (PKC) from brain and rod outer segments (ROS). These same two types of PKC also phosphorylate PDE alpha in trypsin-activated PDE (without PDE gamma). In contrast, cyclic-AMP-dependent protein kinase catalytic subunit phosphorylates both PDE alpha and PDE beta, but not PDE gamma. This kinase does not phosphorylate trypsin-activated PDE. The synthetic peptides AKVISNLLGPREAAV (PDE alpha 30-44) and KQRQTRQFKSKPPKK (PDE gamma 31-45) inhibited phosphorylation of PDE by PKC from ROS. These data suggest that sites (at least one for each subunit) for phosphorylation of PDE by PKC are localized in these corresponding regions of PDE alpha and PDE gamma. Isoenzyme-specific PKC antibodies against peptides unique to the alpha, beta, gamma, delta, epsilon and zeta isoforms of protein kinase C were used to show that a major form of PKC in ROS is PKC alpha. However, other minor forms were also present. Images Figure 1 Figure 4 Figure 6 Figure 7 PMID:8216238

  13. PINK1-Mediated Phosphorylation of Parkin Boosts Parkin Activity in Drosophila

    PubMed Central

    Shiba-Fukushima, Kahori; Inoshita, Tsuyoshi; Hattori, Nobutaka; Imai, Yuzuru

    2014-01-01

    Two genes linked to early onset Parkinson's disease, PINK1 and Parkin, encode a protein kinase and a ubiquitin-ligase, respectively. Both enzymes have been suggested to support mitochondrial quality control. We have reported that Parkin is phosphorylated at Ser65 within the ubiquitin-like domain by PINK1 in mammalian cultured cells. However, it remains unclear whether Parkin phosphorylation is involved in mitochondrial maintenance and activity of dopaminergic neurons in vivo. Here, we examined the effects of Parkin phosphorylation in Drosophila, in which the phosphorylation residue is conserved at Ser94. Morphological changes of mitochondria caused by the ectopic expression of wild-type Parkin in muscle tissue and brain dopaminergic neurons disappeared in the absence of PINK1. In contrast, phosphomimetic Parkin accelerated mitochondrial fragmentation or aggregation and the degradation of mitochondrial proteins regardless of PINK1 activity, suggesting that the phosphorylation of Parkin boosts its ubiquitin-ligase activity. A non-phosphorylated form of Parkin fully rescued the muscular mitochondrial degeneration due to the loss of PINK1 activity, whereas the introduction of the non-phosphorylated Parkin mutant in Parkin-null flies led to the emergence of abnormally fused mitochondria in the muscle tissue. Manipulating the Parkin phosphorylation status affected spontaneous dopamine release in the nerve terminals of dopaminergic neurons, the survivability of dopaminergic neurons and flight activity. Our data reveal that Parkin phosphorylation regulates not only mitochondrial function but also the neuronal activity of dopaminergic neurons in vivo, suggesting that the appropriate regulation of Parkin phosphorylation is important for muscular and dopaminergic functions. PMID:24901221

  14. Eye movement abnormalities.

    PubMed

    Moncayo, Jorge; Bogousslavsky, Julien

    2012-01-01

    Generation and control of eye movements requires the participation of the cortex, basal ganglia, cerebellum and brainstem. The signals of this complex neural network finally converge on the ocular motoneurons of the brainstem. Infarct or hemorrhage at any level of the oculomotor system (though more frequent in the brain-stem) may give rise to a broad spectrum of eye movement abnormalities (EMAs). Consequently, neurologists and particularly stroke neurologists are routinely confronted with EMAs, some of which may be overlooked in the acute stroke setting and others that, when recognized, may have a high localizing value. The most complex EMAs are due to midbrain stroke. Horizontal gaze disorders, some of them manifesting unusual patterns, may occur in pontine stroke. Distinct varieties of nystagmus occur in cerebellar and medullary stroke. This review summarizes the most representative EMAs from the supratentorial level to the brainstem. PMID:22377853

  15. Cofilin/Twinstar Phosphorylation Levels Increase in Response to Impaired Coenzyme A Metabolism

    PubMed Central

    Siudeja, Katarzyna; Grzeschik, Nicola A.; Rana, Anil; de Jong, Jannie; Sibon, Ody C. M.

    2012-01-01

    Coenzyme A (CoA) is a pantothenic acid-derived metabolite essential for many fundamental cellular processes including energy, lipid and amino acid metabolism. Pantothenate kinase (PANK), which catalyses the first step in the conversion of pantothenic acid to CoA, has been associated with a rare neurodegenerative disorder PKAN. However, the consequences of impaired PANK activity are poorly understood. Here we use Drosophila and human neuronal cell cultures to show how PANK deficiency leads to abnormalities in F-actin organization. Cells with reduced PANK activity are characterized by abnormally high levels of phosphorylated cofilin, a conserved actin filament severing protein. The increased levels of phospho-cofilin coincide with morphological changes of PANK-deficient Drosophila S2 cells and human neuronal SHSY-5Y cells. The latter exhibit also markedly reduced ability to form neurites in culture – a process that is strongly dependent on actin remodeling. Our results reveal a novel and conserved link between a metabolic biosynthesis pathway, and regulation of cellular actin dynamics. PMID:22912811

  16. GSK-3β dysregulation contributes to parkinson's-like pathophysiology with associated region-specific phosphorylation and accumulation of tau and α-synuclein.

    PubMed

    Credle, J J; George, J L; Wills, J; Duka, V; Shah, K; Lee, Y-C; Rodriguez, O; Simkins, T; Winter, M; Moechars, D; Steckler, T; Goudreau, J; Finkelstein, D I; Sidhu, A

    2015-05-01

    Aberrant posttranslational modifications (PTMs) of proteins, namely phosphorylation, induce abnormalities in the biological properties of recipient proteins, underlying neurological diseases including Parkinson's disease (PD). Genome-wide studies link genes encoding α-synuclein (α-Syn) and Tau as two of the most important in the genesis of PD. Although several kinases are known to phosphorylate α-Syn and Tau, we focused our analysis on GSK-3β because of its accepted role in phosphorylating Tau and to increasing evidence supporting a strong biophysical relationship between α-Syn and Tau in PD. Therefore, we investigated transgenic mice, which express a point mutant (S9A) of human GSK-3β. GSK-3β-S9A is capable of activation through endogenous natural signaling events, yet is unable to become inactivated through phosphorylation at serine-9. We used behavioral, biochemical, and in vitro analysis to assess the contributions of GSK-3β to both α-Syn and Tau phosphorylation. Behavioral studies revealed progressive age-dependent impairment of motor function, accompanied by loss of tyrosine hydroxylase-positive (TH+ DA-neurons) neurons and dopamine production in the oldest age group. Magnetic resonance imaging revealed deterioration of the substantia nigra in aged mice, a characteristic feature of PD patients. At the molecular level, kinase-active p-GSK-3β-Y216 was seen at all ages throughout the brain, yet elevated levels of p-α-Syn-S129 and p-Tau (S396/404) were found to increase with age exclusively in TH+ DA-neurons of the midbrain. p-GSK-3β-Y216 colocalized with p-Tau and p-α-Syn-S129. In vitro kinase assays showed that recombinant human GSK-3β directly phosphorylated α-Syn at a single site, Ser129, in addition to its known ability to phosphorylate Tau. Moreover, α-Syn and Tau together cooperated with one another to increase the magnitude or rate of phosphorylation of the other by GSK-3β. Together, these data establish a novel upstream role for GSK-3

  17. γ-Aminobutyric Acid Type A (GABAA) Receptor Activation Modulates Tau Phosphorylation*

    PubMed Central

    Nykänen, Niko-Petteri; Kysenius, Kai; Sakha, Prasanna; Tammela, Päivi; Huttunen, Henri J.

    2012-01-01

    Abnormal phosphorylation and aggregation of the microtubule-associated protein Tau are hallmarks of various neurodegenerative diseases, such as Alzheimer disease. Molecular mechanisms that regulate Tau phosphorylation are complex and currently incompletely understood. We have developed a novel live cell reporter system based on protein-fragment complementation assay to study dynamic changes in Tau phosphorylation status. In this assay, fusion proteins of Tau and Pin1 (peptidyl-prolyl cis-trans-isomerase 1) carrying complementary fragments of a luciferase protein serve as a sensor of altered protein-protein interaction between Tau and Pin1, a critical regulator of Tau dephosphorylation at several disease-associated proline-directed phosphorylation sites. Using this system, we identified several structurally distinct GABAA receptor modulators as novel regulators of Tau phosphorylation in a chemical library screen. GABAA receptor activation promoted specific phosphorylation of Tau at the AT8 epitope (Ser-199/Ser-202/Thr-205) in cultures of mature cortical neurons. Increased Tau phosphorylation by GABAA receptor activity was associated with reduced Tau binding to protein phosphatase 2A and was dependent on Cdk5 but not GSK3β kinase activity. PMID:22235112

  18. Phospholipid transfer protein (PLTP) reduces phosphorylation of tau in human neuronal cells (HCN2)

    PubMed Central

    Dong, Weijiang; Albers, John J.; Vuletic, Simona

    2009-01-01

    Tau function is regulated by phosphorylation, and abnormal tau phosphorylation in neurons is one of the key processes associated with development of Alzheimer’s disease and other tauopathies. In this study we provide evidence that phospholipid transfer protein (PLTP), one of the main lipid transfer proteins in the brain, significantly reduces levels of phosphorylated tau, and increases levels of the inactive form of glycogen synthase kinase-3β (GSK3β) in HCN2 cells. Furthermore, inhibition of the phosphatidylinositol-3 kinase (PI3K) reversed the PLTP-induced increase in levels of GSK3β phosphorylated at serine 9 (pGSK3βSer9) and partially reversed the PLTP-induced reduction in tau phosphorylation. We provide evidence that the PLTP-induced changes are not due to activation of Disabled-1 (Dab1), since PLTP reduced levels of total and phosphorylated Dab1 in HCN2 cells. We have also shown that inhibition of tyrosine kinase activity of insulin receptor (IR) and/or insulin-like growth factor 1 (IGF1) receptor (IGFR) reverses PLTP-induced increase in levels of phosphorylated Akt (pAktThr308 and pAktSer473), suggesting that PLTP-mediated activation of the PI3K/Akt pathway is dependent on IR/IGFR receptor tyrosine kinase activity. Our study suggests that PLTP may be an important modulator of signal transduction pathways in human neurons. PMID:19472218

  19. In vitro phosphorylation does not influence the aggregation kinetics of WT α-synuclein in contrast to its phosphorylation mutants.

    PubMed

    Schreurs, Sarah; Gerard, Melanie; Derua, Rita; Waelkens, Etienne; Taymans, Jean-Marc; Baekelandt, Veerle; Engelborghs, Yves

    2014-01-01

    The aggregation of alpha-synuclein (α-SYN) into fibrils is characteristic for several neurodegenerative diseases, including Parkinson's disease (PD). Ninety percent of α-SYN deposited in Lewy Bodies, a pathological hallmark of PD, is phosphorylated on serine129. α-SYN can also be phosphorylated on tyrosine125, which is believed to regulate the membrane binding capacity and thus possibly its normal function. A better understanding of the effect of phosphorylation on the aggregation of α-SYN might shed light on its role in the pathogenesis of PD. In this study we compare the aggregation properties of WT α-SYN with the phospho-dead and phospho-mimic mutants S129A, S129D, Y125F and Y125E and in vitro phosphorylated α-SYN using turbidity, thioflavin T and circular dichroism measurements as well as transmission electron microscopy. We show that the mutants S129A and S129D behave similarly compared to wild type (WT) α-SYN, while the mutants Y125F and Y125E fibrillate significantly slower, although all mutants form fibrillar structures similar to the WT protein. In contrast, in vitro phosphorylation of α-SYN on either S129 or Y125 does not significantly affect the fibrillization kinetics. Moreover, FK506 binding proteins (FKBPs), enzymes with peptidyl-prolyl cis-trans isomerase activity, still accelerate the aggregation of phosphorylated α-SYN in vitro, as was shown previously for WT α-SYN. In conclusion, our results illustrate that phosphorylation mutants can display different aggregation properties compared to the more biologically relevant phosphorylated form of α-SYN. PMID:24434619

  20. Akt phosphorylation is essential for nuclear translocation and retention in NGF-stimulated PC12 cells

    SciTech Connect

    Truong Le Xuan Nguyen; Choi, Joung Woo; Lee, Sang Bae; Ye, Keqiang; Woo, Soo-Dong; Lee, Kyung-Hoon; Ahn, Jee-Yin . E-mail: jyahn@med.skku.ac.kr

    2006-10-20

    Nerve growth factor (NGF) elicits Akt translocation into the nucleus, where it phosphorylates nuclear targets. Here, we describe that Akt phosphorylation can promote the nuclear translocation of Akt and is necessary for its nuclear retention. Overexpression of Akt-K179A, T308A, S473A-mutant failed to show either nuclear translocation or nuclear Akt phosphorylation, whereas expression of wild-type counterpart elicited profound Akt phosphorylation and induced nuclear translocation under NGF stimulation. Employing the PI3K inhibitor and a variety of mutants PI3K, we showed that nuclear translocation of Akt was mediated by activation of PI3K, and Akt phosphorylation status in the nucleus required PI3K activity. Thus the activity of PI3K might contribute to the nuclear translocation of Akt, and that Akt phosphorylation is essential for its nuclear retention under NGF stimulation conditions.

  1. Soya protein attenuates abnormalities of the renin-angiotensin system in adipose tissue from obese rats.

    PubMed

    Frigolet, María E; Torres, Nimbe; Tovar, Armando R

    2012-01-01

    Several metabolic disturbances during obesity are associated with adipose tissue-altered functions. Adipocytes contain the renin-angiotensin system (RAS), which regulates signalling pathways that control angiogenesis via Akt in an autocrine fashion. Soya protein (Soy) consumption modifies the gene expression pattern in adipose tissue, resulting in an improved adipocyte function. Therefore, the aim of the present work is to study whether dietary Soy regulates the expression of RAS and angiogenesis-related genes and its association with the phosphorylated state of Akt in the adipose tissue of obese rats. Animals were fed a 30 % Soy or casein (Cas) diet containing 5 or 25 % fat for 160 d. mRNA abundance was studied in the adipose tissue, and Akt phosphorylation and hormone release were measured in the primary adipocyte culture. The present results show that Soy treatment in comparison with Cas consumption induces lower angiotensin release and increased insulin-stimulated Akt activation in adipocytes. Furthermore, Soy consumption varies the expression of RAS and angiogenesis-related genes, which maintain cell size and vascularity in the adipose tissue of rats fed a high-fat diet. Thus, adipocyte hypertrophy and impaired angiogenesis, which are frequently observed in dysfunctional adipose tissue, were avoided by consuming dietary Soy. Taken together, these findings suggest that Soy can be used as a dietary strategy to preserve adipocyte functionality and to prevent obesity abnormalities. PMID:21736766

  2. Effects of phosphorylation on function of the Rad GTPase.

    PubMed Central

    Moyers, J S; Zhu, J; Kahn, C R

    1998-01-01

    Rad, Gem and Kir possess unique structural features in comparison with other Ras-like GTPases, including a C-terminal 31-residue extension that lacks typical prenylation motifs. We have recently shown that Rad and Gem bind calmodulin in a Ca2+-dependent manner via this C-terminal extension, involving residues 278-297 in human Rad. This domain also contains several consensus sites for serine phosphorylation, and Rad is complexed with calmodulin-dependent protein kinase II (CaMKII) in C2C12 cells. Here we show that Rad serves as a substrate for phosphorylation by CaMKII, cAMP-dependent protein kinase (PKA), protein kinase C (PKC) and casein kinase II (CKII) with stoichiometries in vitro of 0.2-1.3 mol of phosphate/mol of Rad. By deletion and point mutation analysis we show that phosphorylation by CaMKII and PKA occurs on a single serine residue at position 273, whereas PKC and CKII phosphorylate multiple C-terminal serine residues, including Ser214, Ser257, Ser273, Ser290 and Ser299. Incubation of Rad with PKA decreases GTP binding by 60-70%, but this effect seems to be independent of phosphorylation, as it is observed with the Ser273-->Ala mutant of Rad containing a mutation at the site of PKA phosphorylation. The remainder of the serine kinases have no effect on Rad GTP binding, intrinsic GTP hydrolysis or GTP hydrolysis stimulated by the putative tumour metastasis suppressor nm23. However, phosphorylation of Rad by PKC and CKII abolishes the interaction of Rad with calmodulin. These findings suggest that the binding of Rad to calmodulin, as well as its ability to bind GTP, might be regulated by the activation of several serine kinases. PMID:9677319

  3. Effects of phosphorylation on function of the Rad GTPase.

    PubMed

    Moyers, J S; Zhu, J; Kahn, C R

    1998-08-01

    Rad, Gem and Kir possess unique structural features in comparison with other Ras-like GTPases, including a C-terminal 31-residue extension that lacks typical prenylation motifs. We have recently shown that Rad and Gem bind calmodulin in a Ca2+-dependent manner via this C-terminal extension, involving residues 278-297 in human Rad. This domain also contains several consensus sites for serine phosphorylation, and Rad is complexed with calmodulin-dependent protein kinase II (CaMKII) in C2C12 cells. Here we show that Rad serves as a substrate for phosphorylation by CaMKII, cAMP-dependent protein kinase (PKA), protein kinase C (PKC) and casein kinase II (CKII) with stoichiometries in vitro of 0.2-1.3 mol of phosphate/mol of Rad. By deletion and point mutation analysis we show that phosphorylation by CaMKII and PKA occurs on a single serine residue at position 273, whereas PKC and CKII phosphorylate multiple C-terminal serine residues, including Ser214, Ser257, Ser273, Ser290 and Ser299. Incubation of Rad with PKA decreases GTP binding by 60-70%, but this effect seems to be independent of phosphorylation, as it is observed with the Ser273-->Ala mutant of Rad containing a mutation at the site of PKA phosphorylation. The remainder of the serine kinases have no effect on Rad GTP binding, intrinsic GTP hydrolysis or GTP hydrolysis stimulated by the putative tumour metastasis suppressor nm23. However, phosphorylation of Rad by PKC and CKII abolishes the interaction of Rad with calmodulin. These findings suggest that the binding of Rad to calmodulin, as well as its ability to bind GTP, might be regulated by the activation of several serine kinases. PMID:9677319

  4. Is phosphorylated tau unique to chronic traumatic encephalopathy? Phosphorylated tau in epileptic brain and chronic traumatic encephalopathy.

    PubMed

    Puvenna, Vikram; Engeler, Madeline; Banjara, Manoj; Brennan, Chanda; Schreiber, Peter; Dadas, Aaron; Bahrami, Ashkon; Solanki, Jesal; Bandyopadhyay, Anasua; Morris, Jacqueline K; Bernick, Charles; Ghosh, Chaitali; Rapp, Edward; Bazarian, Jeffrey J; Janigro, Damir

    2016-01-01

    Repetitive traumatic brain injury (rTBI) is one of the major risk factors for the abnormal deposition of phosphorylated tau (PT) in the brain and chronic traumatic encephalopathy (CTE). CTE and temporal lobe epilepsy (TLE) affect the limbic system, but no comparative studies on PT distribution in TLE and CTE are available. It is also unclear whether PT pathology results from repeated head hits (rTBI). These gaps prevent a thorough understanding of the pathogenesis and clinical significance of PT, limiting our ability to develop preventative and therapeutic interventions. We quantified PT in TLE and CTE to unveil whether a history of rTBI is a prerequisite for PT accumulation in the brain. Six postmortem CTE (mean 73.3 years) and age matched control samples were compared to 19 surgically resected TLE brain specimens (4 months-58 years; mean 27.6 years). No history of TBI was present in TLE or control; all CTE patients had a history of rTBI. TLE and CTE brain displayed increased levels of PT as revealed by immunohistochemistry. No age-dependent changes were noted, as PT was present as early as 4 months after birth. In TLE and CTE, cortical neurons, perivascular regions around penetrating pial vessels and meninges were immunopositive for PT; white matter tracts also displayed robust expression of extracellular PT organized in bundles parallel to venules. Microscopically, there were extensive tau-immunoreactive neuronal, astrocytic and degenerating neurites throughout the brain. In CTE perivascular tangles were most prominent. Overall, significant differences in staining intensities were found between CTE and control (P<0.01) but not between CTE and TLE (P=0.08). pS199 tau analysis showed that CTE had the most high molecular weight tangle-associated tau, whereas epileptic brain contained low molecular weight tau. Tau deposition may not be specific to rTBI since TLE recapitulated most of the pathological features of CTE. PMID:26556772

  5. Ictal Cardiac Ryhthym Abnormalities

    PubMed Central

    Ali, Rushna

    2016-01-01

    Cardiac rhythm abnormalities in the context of epilepsy are a well-known phenomenon. However, they are under-recognized and often missed. The pathophysiology of these events is unclear. Bradycardia and asystole are preceded by seizure onset suggesting ictal propagation into the cortex impacting cardiac autonomic function, and the insula and amygdala being possible culprits. Sudden unexpected death in epilepsy (SUDEP) refers to the unanticipated death of a patient with epilepsy not related to status epilepticus, trauma, drowning, or suicide. Frequent refractory generalized tonic-clonic seizures, anti-epileptic polytherapy, and prolonged duration of epilepsy are some of the commonly identified risk factors for SUDEP. However, the most consistent risk factor out of these is an increased frequency of generalized tonic–clonic seizures (GTC). Prevention of SUDEP is extremely important in patients with chronic, generalized epilepsy. Since increased frequency of GTCS is the most consistently reported risk factor for SUDEP, effective seizure control is the most important preventive strategy. PMID:27347227

  6. Abnormal uterine bleeding.

    PubMed

    Whitaker, Lucy; Critchley, Hilary O D

    2016-07-01

    Abnormal uterine bleeding (AUB) is a common and debilitating condition with high direct and indirect costs. AUB frequently co-exists with fibroids, but the relationship between the two remains incompletely understood and in many women the identification of fibroids may be incidental to a menstrual bleeding complaint. A structured approach for establishing the cause using the Fédération International de Gynécologie et d'Obstétrique (FIGO) PALM-COEIN (Polyp, Adenomyosis, Leiomyoma, Malignancy (and hyperplasia), Coagulopathy, Ovulatory disorders, Endometrial, Iatrogenic and Not otherwise classified) classification system will facilitate accurate diagnosis and inform treatment options. Office hysteroscopy and increasing sophisticated imaging will assist provision of robust evidence for the underlying cause. Increased availability of medical options has expanded the choice for women and many will no longer need to recourse to potentially complicated surgery. Treatment must remain individualised and encompass the impact of pressure symptoms, desire for retention of fertility and contraceptive needs, as well as address the management of AUB in order to achieve improved quality of life. PMID:26803558

  7. Deletion of phenylalanine 508 causes attenuated phosphorylation-dependent activation of CFTR chloride channels.

    PubMed

    Wang, F; Zeltwanger, S; Hu, S; Hwang, T C

    2000-05-01

    In cell-attached patches stimulated with cAMP agonists, the single-channel open probability (Po) of the phenylalanine 508-deleted cystic fibrosis transmembrane conductance regulator (DeltaF508-CFTR) channel, the most common disease-associated mutation in cystic fibrosis, was abnormally low (a functional defect). To investigate the mechanism for the poor response of DeltaF508-CFTR to cAMP stimulation, we examined, in excised inside-out patches, protein kinase A (PKA)-dependent phosphorylation activation and ATP-dependent gating of wild-type (WT) and DeltaF508-CFTR channels expressed in NIH3T3 mouse fibroblasts. For WT-CFTR, the activation time course of CFTR channel current upon addition of PKA and ATP followed a sigmoidal function with time constants that decreased as [PKA] was increased. The curvilinear relationship between [PKA] and the apparent activation rate suggests an incremental phosphorylation-dependent activation of CFTR at multiple phosphorylation sites. The time course of PKA-dependent activation of DeltaF508-CFTR channel current also followed a sigmoidal function, but the rate of activation was at least 7-fold slower than that with WT channels. This result suggests that deletion of phenylalanine 508 causes attenuated PKA-dependent phosphorylation of the CFTR chloride channel. Once DeltaF508-CFTR channels were maximally activated with PKA, the mutant channel and WT channel had indistinguishable steady-state Po values, ATP dose-response relationships and single-channel kinetics, indicating that DeltaF508-CFTR is not defective in ATP-dependent gating. By measuring whole-cell current density, we compared the number of functional channels in WT- and DeltaF508-CFTR cell membrane. Our data showed that the estimated channel density for DeltaF508-CFTR was approximately 10-fold lower than that for WT-CFTR, but the cAMP-dependent whole-cell current density differed by approximately 200-fold. We thus conclude that the functional defect (a decrease in Po) of Delta

  8. Deletion of phenylalanine 508 causes attenuated phosphorylation-dependent activation of CFTR chloride channels

    PubMed Central

    Wang, Fei; Zeltwanger, Shawn; Hu, Shenghui; Hwang, Tzyh-Chang

    2000-01-01

    In cell-attached patches stimulated with cAMP agonists, the single-channel open probability (Po) of the phenylalanine 508-deleted cystic fibrosis transmembrane conductance regulator (ΔF508-CFTR) channel, the most common disease-associated mutation in cystic fibrosis, was abnormally low (a functional defect). To investigate the mechanism for the poor response of ΔF508-CFTR to cAMP stimulation, we examined, in excised inside-out patches, protein kinase A (PKA)-dependent phosphorylation activation and ATP-dependent gating of wild-type (WT) and ΔF508-CFTR channels expressed in NIH3T3 mouse fibroblasts.For WT-CFTR, the activation time course of CFTR channel current upon addition of PKA and ATP followed a sigmoidal function with time constants that decreased as [PKA] was increased. The curvilinear relationship between [PKA] and the apparent activation rate suggests an incremental phosphorylation-dependent activation of CFTR at multiple phosphorylation sites.The time course of PKA-dependent activation of ΔF508-CFTR channel current also followed a sigmoidal function, but the rate of activation was at least 7-fold slower than that with WT channels. This result suggests that deletion of phenylalanine 508 causes attenuated PKA-dependent phosphorylation of the CFTR chloride channel.Once ΔF508-CFTR channels were maximally activated with PKA, the mutant channel and WT channel had indistinguishable steady-state Po values, ATP dose-response relationships and single-channel kinetics, indicating that ΔF508-CFTR is not defective in ATP-dependent gating.By measuring whole-cell current density, we compared the number of functional channels in WT- and ΔF508-CFTR cell membrane. Our data showed that the estimated channel density for ΔF508-CFTR was ∼10-fold lower than that for WT-CFTR, but the cAMP-dependent whole-cell current density differed by ∼200-fold. We thus conclude that the functional defect (a decrease in Po) of ΔF508-CFTR is as important as the trafficking defect (a

  9. dimerization and DNA binding alter phosphorylation of Fos and Jun

    SciTech Connect

    Abate, C.; Baker, S.J.; Curran, T. ); Lees-Miller, S.P.; Anderson, C.W. ); Marshak, D.R. )

    1993-07-15

    Fos and Jun form dimeric complexes that bind to activator protein 1 (AP-1) DNA sequences and regulate gene expression. The levels of expression and activities of these proteins are regulated by a variety of extracellular stimuli. They are thought to function in nuclear signal transduction processes in many different cell types. The role of Fos and Jun in gene transcription is complex and may be regulated in several ways including association with different dimerization partners, interactions with other transcription factors, effects on DNA topology, and reduction/oxidation of a conserved cysteine residue in the DNA-binding domain. In addition, phosphorylation has been suggested to control the activity of Fos and Jun. Here the authors show that phosphorylation of Fos and Jun by several protein kinases is affected by dimerization and binding to DNA. Jun homodimers are phosphorylated efficiently by casein kinase II, whereas Fos-Jun heterodimers are not. DNA binding also reduces phosphorylation of Jun by casein kinase II, p34[sup cdc2] (cdc2) kinase, and protein kinase C. Phosphorylation of Fos by cAMP-dependent protein kinase and cdc2 is relatively insensitive to dimerization and DNA binding, whereas phosphorylation of Fos and Jun by DNA-dependent protein kinase is dramatically stimulated by binding to the AP-1 site. These results imply that different protein kinases can distinguish among Fos and Jun proteins in the form of monomers, homodimers, and heterodimers and between DNA-bound and non-DNA-bound proteins. Thus, potentially, these different states of Fos and Jun can be recognized and regulated independently by phosphorylation. 44 refs., 4 figs.

  10. Abundant protein phosphorylation potentially regulates Arabidopsis anther development

    PubMed Central

    Ye, Juanying; Zhang, Zaibao; You, Chenjiang; Zhang, Xumin; Lu, Jianan; Ma, Hong

    2016-01-01

    As the male reproductive organ of flowering plants, the stamen consists of the anther and filament. Previous studies on stamen development mainly focused on single gene functions by genetic methods or gene expression changes using comparative transcriptomic approaches, especially in model plants such as Arabidopsis thaliana. However, studies on Arabidopsis anther protein expression and post-translational modifications are still lacking. Here we report proteomic and phosphoproteomic studies on developing Arabidopsis anthers at stages 4–7 and 8–12. We identified 3908 high-confidence phosphorylation sites corresponding to 1637 phosphoproteins. Among the 1637 phosphoproteins, 493 were newly identified, with 952 phosphorylation sites. Phosphopeptide enrichment prior to LC-MS analysis facilitated the identification of low-abundance proteins and regulatory proteins, thereby increasing the coverage of proteomic analysis, and facilitated the analysis of more regulatory proteins. Thirty-nine serine and six threonine phosphorylation motifs were uncovered from the anther phosphoproteome and further analysis supports that phosphorylation of casein kinase II, mitogen-activated protein kinases, and 14-3-3 proteins is a key regulatory mechanism in anther development. Phosphorylated residues were preferentially located in variable protein regions among family members, but they were they were conserved across angiosperms in general. Moreover, phosphorylation might reduce activity of reactive oxygen species scavenging enzymes and hamper brassinosteroid signaling in early anther development. Most of the novel phosphoproteins showed tissue-specific expression in the anther according to previous microarray data. This study provides a community resource with information on the abundance and phosphorylation status of thousands of proteins in developing anthers, contributing to understanding post-translational regulatory mechanisms during anther development. PMID:27531888

  11. Integrin Ligation Results in Nephrin Tyrosine Phosphorylation In Vitro

    PubMed Central

    Verma, Rakesh; Venkatareddy, Madhusudan; Kalinowski, Anne; Patel, Sanjeevkumar R.; Garg, Puneet

    2016-01-01

    Nephrin is expressed at the basolateral aspect of podocytes and is an important signaling protein at the glomerular slit diaphragm. In vitro studies have demonstrated that Nephrin phosphorylation-dependent signaling is able to assemble a protein complex that is able to polymerize actin. However, proximal signaling events that result in nephrin tyrosine phosphorylation are not well understood. Nephrin deletion in mice and human nephrin mutations result in developmental failure of the podocyte intercellular junction resutling in proteinuria. This has been presumed to be due to a failure to respond to an external polarized cue in the absence of nephrin or a failure to transduce an outside-in signal in patients with nephrin mutations. The nephrin extracellular domain binds to itself or neph1 across the foot process intercellular junction. Nephrin is tyrosine phosphorylation-silent in healthy glomeruli when presumably the nephrin extracellular domain is in an engaged state. These observations raise the possibility of an alternate proximal signaling mechanism that might be responsible for nephrin tyrosine phosphorylation. Here we present data showing that integrin engagement at the basal aspect of cultured podocytes results in nephrin tyrosine phosphorylation. This is abrogated by incubating podocytes with an antibody that prevents integrin β1 ligation and activation in response to binding to extracellular matrix. Furthermore, nephrin tyrosine phosphorylation was observed in podocytes expressing a membrane-targeted nephrin construct that lacks the extracellular domain. We propose, integrin-activation based signaling might be responsible for nephrin phosphorylation rather than engagment of the nephrin extracellular domain by a ligand. PMID:26848974

  12. Abundant protein phosphorylation potentially regulates Arabidopsis anther development.

    PubMed

    Ye, Juanying; Zhang, Zaibao; You, Chenjiang; Zhang, Xumin; Lu, Jianan; Ma, Hong

    2016-09-01

    As the male reproductive organ of flowering plants, the stamen consists of the anther and filament. Previous studies on stamen development mainly focused on single gene functions by genetic methods or gene expression changes using comparative transcriptomic approaches, especially in model plants such as Arabidopsis thaliana However, studies on Arabidopsis anther protein expression and post-translational modifications are still lacking. Here we report proteomic and phosphoproteomic studies on developing Arabidopsis anthers at stages 4-7 and 8-12. We identified 3908 high-confidence phosphorylation sites corresponding to 1637 phosphoproteins. Among the 1637 phosphoproteins, 493 were newly identified, with 952 phosphorylation sites. Phosphopeptide enrichment prior to LC-MS analysis facilitated the identification of low-abundance proteins and regulatory proteins, thereby increasing the coverage of proteomic analysis, and facilitated the analysis of more regulatory proteins. Thirty-nine serine and six threonine phosphorylation motifs were uncovered from the anther phosphoproteome and further analysis supports that phosphorylation of casein kinase II, mitogen-activated protein kinases, and 14-3-3 proteins is a key regulatory mechanism in anther development. Phosphorylated residues were preferentially located in variable protein regions among family members, but they were they were conserved across angiosperms in general. Moreover, phosphorylation might reduce activity of reactive oxygen species scavenging enzymes and hamper brassinosteroid signaling in early anther development. Most of the novel phosphoproteins showed tissue-specific expression in the anther according to previous microarray data. This study provides a community resource with information on the abundance and phosphorylation status of thousands of proteins in developing anthers, contributing to understanding post-translational regulatory mechanisms during anther development. PMID:27531888

  13. Tyrosine Phosphorylation of SGEF Regulates RhoG Activity and Cell Migration

    PubMed Central

    Okuyama, Yusuke; Umeda, Kentaro; Negishi, Manabu; Katoh, Hironori

    2016-01-01

    SGEF and Ephexin4 are members of the Ephexin subfamily of RhoGEFs that specifically activate the small GTPase RhoG. It is reported that Ephexin1 and Ephexin5, two well-characterized Ephexin subfamily RhoGEFs, are tyrosine-phosphorylated by Src, and that their phosphorylation affect their activities and functions. In this study, we show that SGEF, but not Ephexin4, is tyrosine-phosphorylated by Src. Tyrosine phosphorylation of SGEF suppresses its interaction with RhoG, the elevation of RhoG activity, and SGEF-mediated promotion of cell migration. We identified tyrosine 530 (Y530), which is located within the Dbl homology domain, as a major phosphorylation site of SGEF by Src, and Y530F mutation blocked the inhibitory effect of Src on SGEF. Taken together, these results suggest that the activity of SGEF is negatively regulated by tyrosine phosphorylation of the DH domain. PMID:27437949

  14. Electrocardiograph abnormalities revealed during laparoscopy

    PubMed Central

    Nijjer, Sukhjinder; Dubrey, Simon William

    2010-01-01

    This brief case presents a well patient in whom an electrocardiograph abnormality consistent with an accessory pathway was found during a routine procedure. We present the electrocardiographs, explain the underlying condition, and consider why the abnormality was revealed in this manner. PMID:22419949

  15. Abnormal pressure in hydrocarbon environments

    USGS Publications Warehouse

    Law, B.E.; Spencer, C.W.

    1998-01-01

    Abnormal pressures, pressures above or below hydrostatic pressures, occur on all continents in a wide range of geological conditions. According to a survey of published literature on abnormal pressures, compaction disequilibrium and hydrocarbon generation are the two most commonly cited causes of abnormally high pressure in petroleum provinces. In young (Tertiary) deltaic sequences, compaction disequilibrium is the dominant cause of abnormal pressure. In older (pre-Tertiary) lithified rocks, hydrocarbon generation, aquathermal expansion, and tectonics are most often cited as the causes of abnormal pressure. The association of abnormal pressures with hydrocarbon accumulations is statistically significant. Within abnormally pressured reservoirs, empirical evidence indicates that the bulk of economically recoverable oil and gas occurs in reservoirs with pressure gradients less than 0.75 psi/ft (17.4 kPa/m) and there is very little production potential from reservoirs that exceed 0.85 psi/ft (19.6 kPa/m). Abnormally pressured rocks are also commonly associated with unconventional gas accumulations where the pressuring phase is gas of either a thermal or microbial origin. In underpressured, thermally mature rocks, the affected reservoirs have most often experienced a significant cooling history and probably evolved from an originally overpressured system.

  16. Haem degradation in abnormal haemoglobins.

    PubMed Central

    Brown, S B; Docherty, J C

    1978-01-01

    The coupled oxidation of certain abnormal haemoglobins leads to different bile-pigment isomer distributions from that of normal haemoglobin. The isomer pattern may be correlated with the structure of the abnormal haemoglobin in the neighbourhood of the haem pocket. This is support for haem degradation by an intramolecular reaction. PMID:708385

  17. Induction of protein tyrosine phosphorylation in macrophages incubated with tumor cells.

    PubMed

    Sodhi, A; Shrivastava, A; Kumar, R

    1995-03-01

    The cellular and molecular interaction between monocyte/macrophage and tumor cells leading to macrophage activation is not clearly understood. Since protein tyrosine phosphorylation appears to be a major intracellular signalling event, we checked whether the tumor cells alter tyrosine phosphorylation of proteins in macrophages. We found that both L929 and Yac-1 tumor cells induced increased tyrosine phosphorylation of several polypeptides in peritoneal as well as P388D-1 and IC-21 macrophages. Macrophages co-cultured with tumor cells also showed increased fluorescence with anti-phosphotyrosine-FITC antibody. These observations suggest that increased tyrosine phosphorylation plays a role in tumor cell-induced activation of macrophages. PMID:7539664

  18. Site-specific Proteasome Phosphorylation Controls Cell Proliferation and Tumorigenesis

    PubMed Central

    Guo, Xing; Wang, Xiaorong; Wang, Zhiping; Banerjee, Sourav; Yang, Jing; Huang, Lan; Dixon, Jack E.

    2015-01-01

    Despite the fundamental importance of proteasomal degradation in cells, little is known about whether and how the 26S proteasome itself is regulated in coordination with various physiological processes. Here we show that the proteasome is dynamically phosphorylated during cell cycle at Thr25 of the 19S subunit Rpt3. CRISPR/Cas9-mediated genome editing, RNA interference and biochemical studies demonstrate that blocking Rpt3-Thr25 phosphorylation markedly impairs proteasome activity and impedes cell proliferation. Through a kinome-wide screen, we have identified dual-specificity tyrosine-regulated kinase 2 (DYRK2) as the primary kinase that phosphorylates Rpt3-Thr25, leading to enhanced substrate translocation and degradation. Importantly, loss of the single phosphorylation of Rpt3-Thr25 or knockout of DYRK2 significantly inhibits tumor formation by proteasome-addicted human breast cancer cells in mice. These findings define an important mechanism for proteasome regulation and demonstrate the biological significance of proteasome phosphorylation in regulating cell proliferation and tumorigenesis. PMID:26655835

  19. Phosphorylation of Cysteine String Protein Triggers a Major Conformational Switch.

    PubMed

    Patel, Pryank; Prescott, Gerald R; Burgoyne, Robert D; Lian, Lu-Yun; Morgan, Alan

    2016-08-01

    Cysteine string protein (CSP) is a member of the DnaJ/Hsp40 chaperone family that localizes to neuronal synaptic vesicles. Impaired CSP function leads to neurodegeneration in humans and model organisms as a result of misfolding of client proteins involved in neurotransmission. Mammalian CSP is phosphorylated in vivo on Ser10, and this modulates its protein interactions and effects on neurotransmitter release. However, there are no data on the structural consequences of CSP phosphorylation to explain these functional effects. We show that Ser10 phosphorylation causes an order-to-disorder transition that disrupts CSP's extreme N-terminal α helix. This triggers the concomitant formation of a hairpin loop stabilized by ionic interactions between phosphoSer10 and the highly conserved J-domain residue, Lys58. These phosphorylation-induced effects result in significant changes to CSP conformation and surface charge distribution. The phospho-switch revealed here provides structural insight into how Ser10 phosphorylation modulates CSP function and also has potential implications for other DnaJ phosphoproteins. PMID:27452402

  20. Negative regulation of Vps34 by Cdk mediated phosphorylation

    PubMed Central

    Furuya, Tsuyoshi; Kim, Minsu; Lipinski, Marta; Li, Juying; Kim, Dohoon; Lu, Tao; Shen, Yong; Rameh, Lucia; Yankner, Bruce; Tsai, Li-Huei; Yuan, Junying

    2010-01-01

    Summary Vps34 (vacuolar protein sorting 34) complexes, the class III PtdIns3 kinase, specifically phosphorylate the D3-position of PtdIns to produce PtdIns3P. Vps34 is involved in the control of multiple key intracellular membrane trafficking pathways including endocytic sorting and autophagy. In mammalian cells, Vps34 interacts with Beclin 1, an orthologue of Atg6 in yeast, to regulate the production of PtdIns3P and autophagy. We show that Vps34 is phosphorylated on Thr159 by Cdk1, which negatively regulates its interaction with Beclin1 during mitosis. Cdk5/p25, a neuronal cdk shown to play a role in Alzheimer’s disease, can also phosphorylate Thr159 of Vps34. Phosphorylation of Vps34 on Thr159 inhibits its interaction with Beclin 1. We propose that phosphorylation of Thr159 in Vps34 is a key regulatory mechanism that controls the class III PtdIns3 kinase activity in cell cycle progression, development and human diseases including neurodegeneration and cancers. PMID:20513426

  1. Hyperactive mTOR pathway promotes lymphoproliferation and abnormal differentiation in autoimmune lymphoproliferative syndrome.

    PubMed

    Völkl, Simon; Rensing-Ehl, Anne; Allgäuer, Andrea; Schreiner, Elisabeth; Lorenz, Myriam Ricarda; Rohr, Jan; Klemann, Christian; Fuchs, Ilka; Schuster, Volker; von Bueren, André O; Naumann-Bartsch, Nora; Gambineri, Eleonora; Siepermann, Kathrin; Kobbe, Robin; Nathrath, Michaela; Arkwright, Peter D; Miano, Maurizio; Stachel, Klaus-Daniel; Metzler, Markus; Schwarz, Klaus; Kremer, Anita N; Speckmann, Carsten; Ehl, Stephan; Mackensen, Andreas

    2016-07-14

    Autoimmune lymphoproliferative syndrome (ALPS) is a human disorder characterized by defective Fas signaling, resulting in chronic benign lymphoproliferation and accumulation of TCRαβ(+) CD4(-) CD8(-) double-negative T (DNT) cells. Although their phenotype resembles that of terminally differentiated or exhausted T cells, lack of KLRG1, high eomesodermin, and marginal T-bet expression point instead to a long-lived memory state with potent proliferative capacity. Here we show that despite their terminally differentiated phenotype, human ALPS DNT cells exhibit substantial mitotic activity in vivo. Notably, hyperproliferation of ALPS DNT cells is associated with increased basal and activation-induced phosphorylation of serine-threonine kinases Akt and mechanistic target of rapamycin (mTOR). The mTOR inhibitor rapamycin abrogated survival and proliferation of ALPS DNT cells, but not of CD4(+) or CD8(+) T cells in vitro. In vivo, mTOR inhibition reduced proliferation and abnormal differentiation by DNT cells. Importantly, increased mitotic activity and hyperactive mTOR signaling was also observed in recently defined CD4(+) or CD8(+) precursor DNT cells, and mTOR inhibition specifically reduced these cells in vivo, indicating abnormal programming of Fas-deficient T cells before the DNT stage. Thus, our results identify the mTOR pathway as a major regulator of lymphoproliferation and aberrant differentiation in ALPS. PMID:27099149

  2. Crystal Structure of a Phosphorylation-coupled Saccharide Transporter

    SciTech Connect

    Y Cao; X Jin; E Levin; H Huang; Y Zong; W Hendrickson; J Javitch; K Rajashankar; M Zhou; et al.

    2011-12-31

    Saccharides have a central role in the nutrition of all living organisms. Whereas several saccharide uptake systems are shared between the different phylogenetic kingdoms, the phosphoenolpyruvate-dependent phosphotransferase system exists almost exclusively in bacteria. This multi-component system includes an integral membrane protein EIIC that transports saccharides and assists in their phosphorylation. Here we present the crystal structure of an EIIC from Bacillus cereus that transports diacetylchitobiose. The EIIC is a homodimer, with an expansive interface formed between the amino-terminal halves of the two protomers. The carboxy-terminal half of each protomer has a large binding pocket that contains a diacetylchitobiose, which is occluded from both sides of the membrane with its site of phosphorylation near the conserved His250 and Glu334 residues. The structure shows the architecture of this important class of transporters, identifies the determinants of substrate binding and phosphorylation, and provides a framework for understanding the mechanism of sugar translocation.

  3. Abnormal grain growth in Ni-5at.%W

    NASA Astrophysics Data System (ADS)

    Witte, M.; Belde, M.; Barrales Mora, L.; de Boer, N.; Gilges, S.; Klöwer, J.; Gottstein, G.

    2012-12-01

    The growth of abnormally large grains in textured Ni-5at.%W substrates for high-temperature superconductors deteriorates the sharp texture of these materials and thus has to be avoided. Therefore the growth of abnormal grains is investigated and how it is influenced by the grain orientation and the annealing atmosphere. Texture measurements and grain growth simulations show that the grain orientation only matters so far that a high-angle grain boundary exists between an abnormally growing grain and the Cube-orientated matrix grains. The annealing atmosphere has a large influence on abnormal grain growth which is attributed to the differences in oxygen partial pressure.

  4. Phosphorylation statuses at different residues of lamin B2, B1, and A/C dynamically and independently change throughout the cell cycle

    SciTech Connect

    Kuga, Takahisa; Nozaki, Naohito; Matsushita, Kazuyuki; Nomura, Fumio; Tomonaga, Takeshi

    2010-08-15

    Lamins, major components of the nuclear lamina, undergo phosphorylation at multiple residues during cell cycle progression, but their detailed phosphorylation kinetics remain largely undetermined. Here, we examined changes in the phosphorylation of major phosphorylation residues (Thr14, Ser17, Ser385, Ser387, and Ser401) of lamin B2 and the homologous residues of lamin B1, A/C during the cell cycle using novel antibodies to the site-specific phosphorylation. The phosphorylation levels of these residues independently changed during the cell cycle. Thr14 and Ser17 were phosphorylated during G{sub 2}/M phase to anaphase/telophase. Ser385 was persistently phosphorylated during mitosis to G{sub 1} phase, whereas Ser387 was phosphorylated discontinuously in prophase and G{sub 1} phase. Ser401 phosphorylation was enhanced in the G{sub 1}/S boundary. Immunoprecipitation using the phospho-antibodies suggested that metaphase-phosphorylation at Thr14, Ser17, and Ser385 of lamins occurred simultaneously, whereas G{sub 1}-phase phosphorylation at Ser385 and Ser387 occurred in distinct pools or with different timings. Additionally, we showed that lamin B2 phosphorylated at Ser17, but not Ser385, Ser387 and Ser401, was exclusively non-ionic detergent soluble, depolymerized forms in growing cells, implicating specific involvement of Ser17 phosphorylation in lamin depolymerization and nuclear envelope breakdown. These results suggest that the phosphorylations at different residues of lamins might play specific roles throughout the cell cycle.

  5. Chromosome missegregation during anaphase triggers p53 cell cycle arrest through histone H3.3 Ser31 phosphorylation.

    PubMed

    Hinchcliffe, Edward H; Day, Charles A; Karanjeet, Kul B; Fadness, Sela; Langfald, Alyssa; Vaughan, Kevin T; Dong, Zigang

    2016-06-01

    Maloriented chromosomes can evade the spindle assembly checkpoint and generate aneuploidy, a common feature of tumorigenesis. But chromosome missegregation in non-transformed cells triggers a p53-dependent fail-safe mechanism that blocks proliferation of normal cells that inadvertently become aneuploid. How this fail-safe is triggered is not known. Here we identify a conserved feedback mechanism that monitors missegregating chromosomes during anaphase through the differential phosphorylation of histone H3.3 at Ser31. We do this by inducing transient chromosome missegregation in diploid cells. During anaphase, H3.3 Ser31 is phosphorylated along the arms of lagging or misaligned chromosomes. Within minutes, Ser31 phosphorylation (Ser31P) spreads to all of the chromatids of both daughter cells, which persists into G1. Masking H3.3 Ser31P by antibody microinjection prevents nuclear p53 accumulation in the aneuploid daughters. Previous work demonstrated that prolonged prometaphase and DNA damage during abnormal mitosis can activate p53. We show that p53 activation in response to chromosome missegregation can occur without prolonged mitosis or DNA damage. Our study provides insight into how aneuploidy caused by chromosome missegregation is normally monitored and suppressed. PMID:27136267

  6. Abnormal interhemispheric connectivity in male psychopathic offenders

    PubMed Central

    Hoppenbrouwers, Sylco S.; De Jesus, Danilo R.; Sun, Yinming; Stirpe, Tania; Hofman, Dennis; McMaster, Jeff; Hughes, Ginny; Daskalakis, Zafiris J.; Schutter, Dennis J.L.G.

    2014-01-01

    Background Psychopathic offenders inevitably violate interpersonal norms and frequently resort to aggressive and criminal behaviour. The affective and cognitive deficits underlying these behaviours have been linked to abnormalities in functional interhemispheric connectivity. However, direct neurophysiological evidence for dysfunctional connectivity in psychopathic offenders is lacking. Methods We used transcranial magnetic stimulation combined with electroencephalography to examine interhemispheric connectivity in the dorsolateral and motor cortex in a sample of psychopathic offenders and healthy controls. We also measured intracortical inhibition and facilitation over the left and right motor cortex to investigate the effects of local cortical processes on interhemispheric connectivity. Results We enrolled 17 psychopathic offenders and 14 controls in our study. Global abnormalities in right to left functional connectivity were observed in psychopathic offenders compared with controls. Furthermore, in contrast to controls, psychopathic offenders showed increased intracortical inhibition in the right, but not the left, hemisphere. Limitations The relatively small sample size limited the sensitivity to show that the abnormalities in interhemispheric connectivity were specifically related to the dorsolateral prefrontal cortex in psychopathic offenders. Conclusion To our knowledge, this study provides the first neurophysiological evidence for abnormal interhemispheric connectivity in psychopathic offenders and may further our understanding of the disruptive antisocial behaviour of these offenders. PMID:23937798

  7. Stabilization of Microtubule-Unbound Tau via Tau Phosphorylation at Ser262/356 by Par-1/MARK Contributes to Augmentation of AD-Related Phosphorylation and Aβ42-Induced Tau Toxicity

    PubMed Central

    Ando, Kanae; Maruko-Otake, Akiko; Ohtake, Yosuke; Hayashishita, Motoki; Sekiya, Michiko; Iijima, Koichi M.

    2016-01-01

    Abnormal accumulation of the microtubule-interacting protein tau is associated with neurodegenerative diseases including Alzheimer’s disease (AD). β-amyloid (Aβ) lies upstream of abnormal tau behavior, including detachment from microtubules, phosphorylation at several disease-specific sites, and self-aggregation into toxic tau species in AD brains. To prevent the cascade of events leading to neurodegeneration in AD, it is essential to elucidate the mechanisms underlying the initial events of tau mismetabolism. Currently, however, these mechanisms remain unclear. In this study, using transgenic Drosophila co-expressing human tau and Aβ, we found that tau phosphorylation at AD-related Ser262/356 stabilized microtubule-unbound tau in the early phase of tau mismetabolism, leading to neurodegeneration. Aβ increased the level of tau detached from microtubules, independent of the phosphorylation status at GSK3-targeted SP/TP sites. Such mislocalized tau proteins, especially the less phosphorylated species, were stabilized by phosphorylation at Ser262/356 via PAR-1/MARK. Levels of Ser262 phosphorylation were increased by Aβ42, and blocking this stabilization of tau suppressed Aβ42-mediated augmentation of tau toxicity and an increase in the levels of tau phosphorylation at the SP/TP site Thr231, suggesting that this process may be involved in AD pathogenesis. In contrast to PAR-1/MARK, blocking tau phosphorylation at SP/TP sites by knockdown of Sgg/GSK3 did not reduce tau levels, suppress tau mislocalization to the cytosol, or diminish Aβ-mediated augmentation of tau toxicity. These results suggest that stabilization of microtubule-unbound tau by phosphorylation at Ser262/356 via the PAR-1/MARK may act in the initial steps of tau mismetabolism in AD pathogenesis, and that such tau species may represent a potential therapeutic target for AD. PMID:27023670

  8. Starch phosphorylation: insights and perspectives.

    PubMed

    Mahlow, Sebastian; Orzechowski, Sławomir; Fettke, Joerg

    2016-07-01

    During starch metabolism, the phosphorylation of glucosyl residues of starch, to be more precise of amylopectin, is a repeatedly observed process. This phosphorylation is mediated by dikinases, the glucan, water dikinase (GWD) and the phosphoglucan, water dikinase (PWD). The starch-related dikinases utilize ATP as dual phosphate donor transferring the terminal γ-phosphate group to water and the β-phosphate group selectively to either C6 position or C3 position of a glucosyl residue within amylopectin. By the collaborative action of both enzymes, the initiation of a transition of α-glucans from highly ordered, water-insoluble state to a less order state is realized and thus the initial process of starch degradation. Consequently, mutants lacking either GWD or PWD reveal a starch excess phenotype as well as growth retardation. In this review, we focus on the increased knowledge collected over the last years related to enzymatic properties, the precise definition of the substrates, the physiological implications, and discuss ongoing questions. PMID:27147464

  9. Chromosomal abnormalities in human sperm

    SciTech Connect

    Martin, R.H.

    1985-01-01

    The ability to analyze human sperm chromosome complements after penetration of zona pellucida-free hamster eggs provides the first opportunity to study the frequency and type of chromosomal abnormalities in human gametes. Two large-scale studies have provided information on normal men. We have studied 1,426 sperm complements from 45 normal men and found an abnormality rate of 8.9%. Brandriff et al. (5) found 8.1% abnormal complements in 909 sperm from 4 men. The distribution of numerical and structural abnormalities was markedly dissimilar in the 2 studies. The frequency of aneuploidy was 5% in our sample and only 1.6% in Brandriff's, perhaps reflecting individual variability among donors. The frequency of 24,YY sperm was low: 0/1,426 and 1/909. This suggests that the estimates of nondisjunction based on fluorescent Y body data (1% to 5%) are not accurate. We have also studied men at increased risk of sperm chromosomal abnormalities. The frequency of chromosomally unbalanced sperm in 6 men heterozygous for structural abnormalities varied dramatically: 77% for t11;22, 32% for t6;14, 19% for t5;18, 13% for t14;21, and 0% for inv 3 and 7. We have also studied 13 cancer patients before and after radiotherapy and demonstrated a significant dose-dependent increase of sperm chromosome abnormalities (numerical and structural) 36 months after radiation treatment.

  10. QSAR studies of hydrazone uncouplers of oxidative phosphorylation.

    PubMed

    Winkler, D A; Holan, G; Smith, D R; Middleton, E J; Hart, N K; Rihs, K; Smith, K W

    1988-07-01

    Semiempirical molecular orbital calculations have been performed on a series of hydrazone uncouplers of mitochondrial oxidative phosphorylation which show insecticidal activity. Regression analysis yielded significant correlations between uncoupling activity, insecticidal potency and such physicochemical or theoretically-derived parameters as lipophilicity, pKa and atom charges. PMID:3255329

  11. Anxiolytic action of pterostilbene: involvement of hippocampal ERK phosphorylation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pterostilbene, a natural analog of resveratrol, has diverse health-beneficial properties. However, the neurological activities of this compound are largely unexplored. Here we report that pterostilbene shows anxiolytic action by downregulating phosphorylated levels of ERKs in the hippocampus of mice...

  12. Stem rust spores elicit rapid RPG1 phosphorylation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Stem rust threatens cereal production worldwide. Understanding the mechanism by which durable resistance genes, such as Rpg1, function is critical. We show that the RPG1 protein is phosphorylated within 5 min by exposure to spores from avirulent but not virulent races of stem rust. Transgenic mutant...

  13. Haematological abnormalities in mitochondrial disorders

    PubMed Central

    Finsterer, Josef; Frank, Marlies

    2015-01-01

    INTRODUCTION This study aimed to assess the kind of haematological abnormalities that are present in patients with mitochondrial disorders (MIDs) and the frequency of their occurrence. METHODS The blood cell counts of a cohort of patients with syndromic and non-syndromic MIDs were retrospectively reviewed. MIDs were classified as ‘definite’, ‘probable’ or ‘possible’ according to clinical presentation, instrumental findings, immunohistological findings on muscle biopsy, biochemical abnormalities of the respiratory chain and/or the results of genetic studies. Patients who had medical conditions other than MID that account for the haematological abnormalities were excluded. RESULTS A total of 46 patients (‘definite’ = 5; ‘probable’ = 9; ‘possible’ = 32) had haematological abnormalities attributable to MIDs. The most frequent haematological abnormality in patients with MIDs was anaemia. 27 patients had anaemia as their sole haematological problem. Anaemia was associated with thrombopenia (n = 4), thrombocytosis (n = 2), leucopenia (n = 2), and eosinophilia (n = 1). Anaemia was hypochromic and normocytic in 27 patients, hypochromic and microcytic in six patients, hyperchromic and macrocytic in two patients, and normochromic and microcytic in one patient. Among the 46 patients with a mitochondrial haematological abnormality, 78.3% had anaemia, 13.0% had thrombopenia, 8.7% had leucopenia and 8.7% had eosinophilia, alone or in combination with other haematological abnormalities. CONCLUSION MID should be considered if a patient’s abnormal blood cell counts (particularly those associated with anaemia, thrombopenia, leucopenia or eosinophilia) cannot be explained by established causes. Abnormal blood cell counts may be the sole manifestation of MID or a collateral feature of a multisystem problem. PMID:26243978

  14. Histone tyrosine phosphorylation comes of age

    PubMed Central

    Singh, Rakesh Kumar

    2011-01-01

    Histones were discovered over a century ago and have since been found to be the most extensively post-translationally modified proteins, although tyrosine phosphorylation of histones had remained elusive until recently. The year 2009 proved to be a landmark year for histone tyrosine (Y) phosphorylation as five research groups independently discovered this modification. Three groups describe phosphorylation of Y142 in the variant histone H2A.X, where it may be involved in the cellular decision making process to either undergo DNA repair or apoptosis in response to DNA damage. Further, one group suggests that phosphorylation of histone H3 on Y99 is crucial for its regulated proteolysis in yeast, while another found that Y41 phosphorylation modulates chromatin architecture and oncogenesis in mammalian cells. These pioneering studies provide the initial conceptual framework for further analyses of the diverse roles of tyrosine phosphorylation on different histones, with far reaching implications for human health and disease. PMID:20935492

  15. Prebiotic phosphorylation of nucleosides in formamide

    NASA Technical Reports Server (NTRS)

    Schoffstall, A. M.

    1976-01-01

    Results are presented for an experimental study intended to assess phosphorylation under neither aqueous nor dry thermal conditions. Instead, phosphorylations were attempted in possible nonaqueous prebiotic solvents. Formamide appeared to be the most obvious candidate for phosphorylation studies. Three main classes of phosphorylated products were formed in formamide solution: adenosine monophosphates, cyclic adenosine phosphate, and adenosine diphosphates. Experiments were designed to investigate the extent of phosphorylation of nucleosides in formamide, the relative amounts of nucleoside monophosphate, diphosphates and cyclic phosphate formed and the relative effectiveness of different sources of phosphate as phosphorylating agents in formamide. Reaction variables were temperature, nature of the phosphate or condensed phosphate, nucleoside, concentration of reactants and possible effects of additives. Product identification was based on qualitative and quantitative thin layer chromatography.

  16. Parsing abnormal grain growth in specialty aluminas

    NASA Astrophysics Data System (ADS)

    Lawrence, Abigail Kremer

    multivariate statistical tool called canonical correlation analysis was adopted to seek out relationships between a set of input variables and the abnormal character values. The input variables include the MgO, CaO, Na 2O, and SiO2 contents, the ratio of MgO:(CaO+SiO2), and the annealing time and temperature. The analysis was applied to 33 different samples and showed that the composition ratio and MgO content were the strongest processing variables. These variables are most closely related to the correlation between grain size and aspect ratio, the average magnitude of abnormality, and the variance in grain size. The physical implications of these relationships are explored for a number of samples with different abnormal grain growth behaviors. Several of the samples contained a beta"-alumina phase that is shown to have a dampening effect on abnormal grain growth. TEM investigation provides evidence that there is a grain boundary complexion with a different composition and structure than the second phase. A series of samples are compared after annealing for different times and are shown to have very different behaviors as a result of the second phase competing with complexions for control over the microstructure.

  17. Mitotic phosphorylation of VCIP135 blocks p97ATPase-mediated Golgi membrane fusion

    SciTech Connect

    Totsukawa, Go; Matsuo, Ayaka; Kubota, Ayano; Taguchi, Yuya; Kondo, Hisao

    2013-04-05

    Highlights: •VCIP135 is mitotically phosphorylated on Threonine-760 and Serine-767 by Cdc2. •Phosphorylated VCIP135 does not bind to p97ATPase. •The phosphorylation of VCIP135 inhibits p97ATPase-mediated Golgi membrane fusion. -- Abstract: In mammals, the Golgi apparatus is disassembled early mitosis and reassembled at the end of mitosis. For Golgi disassembly, membrane fusion needs to be blocked. Golgi biogenesis requires two distinct p97ATPase-mediated membrane fusion, the p97/p47 and p97/p37 pathways. We previously reported that p47 phosphorylation on Serine-140 and p37 phosphorylation on Serine-56 and Threonine-59 result in mitotic inhibition of the p97/p47 and the p97/p37 pathways, respectively [11,14]. In this study, we show another mechanism of mitotic inhibition of p97-mediated Golgi membrane fusion. We clarified that VCIP135, an essential factor in both p97 membrane fusion pathways, is phosphorylated on Threonine-760 and Serine-767 by Cdc2 at mitosis and that this phosphorylated VCIP135 does not bind to p97. An in vitro Golgi reassembly assay revealed that VCIP135(T760E, S767E), which mimics mitotic phosphorylation, caused no cisternal regrowth. Our results indicate that the phosphorylation of VCIP135 on Threonine-760 and Serine-767 inhibits p97-mediated Golgi membrane fusion at mitosis.

  18. Crosstalk between signaling pathways provided by single and multiple protein phosphorylation sites

    PubMed Central

    Nishi, Hafumi; Demir, Emek; Panchenko, Anna R.

    2014-01-01

    Cellular fate depends on the spatio-temporal separation and integration of signaling processes which can be provided by phosphorylation events. In this study we identify the crucial points in signaling crosstalk which can be triggered by discrete phosphorylation events on a single target protein. We integrated the data on individual human phosphosites with the evidence on their corresponding kinases, the functional consequences on phosphorylation on activity of the target protein and corresponding pathways. Our results show that there is a substantial fraction of phosphosites that can play critical roles in crosstalk between alternative or redundant pathways and regulatory outcome of phosphorylation can be linked to a type of phosphorylated residue. These regulatory phosphosites can serve as hubs in the signal flow and their functional roles are directly connected to their specific properties. Namely, phosphosites with similar regulatory functions are phosphorylated by the same kinases and participate in regulation of similar biochemical pathways. Such sites are more likely to cluster in sequence and space unlike sites with antagonistic outcomes of their phosphorylation on a target protein. In addition we found that in silico phosphorylation of sites with similar functional consequences have comparable outcomes on a target protein stability. An important role of phosphorylation sites in biological crosstalk is evident from the analysis of their evolutionary conservation. PMID:25451034

  19. Describing the Sensory Abnormalities of Children and Adults with Autism

    ERIC Educational Resources Information Center

    Leekam, Susan R.; Nieto, Carmen; Libby, Sarah J.; Wing, Lorna; Gould, Judith

    2007-01-01

    Patterns of sensory abnormalities in children and adults with autism were examined using the Diagnostic Interview for Social and Communication Disorders (DISCO). This interview elicits detailed information about responsiveness to a wide range of sensory stimuli. Study 1 showed that over 90% of children with autism had sensory abnormalities and had…

  20. Cytoplasmic Retention of Protein Phosphatase 2A Inhibitor 2 (I2PP2A) Induces Alzheimer-like Abnormal Hyperphosphorylation of Tau*

    PubMed Central

    Arif, Mohammad; Wei, Jianshe; Zhang, Qi; Liu, Fei; Basurto-Islas, Gustavo; Grundke-Iqbal, Inge; Iqbal, Khalid

    2014-01-01

    Abnormal hyperphosphorylation of Tau leads to the formation of neurofibrillary tangles, a hallmark of Alzheimer disease (AD), and related tauopathies. The phosphorylation of Tau is regulated by protein phosphatase 2A (PP2A), which in turn is modulated by endogenous inhibitor 2 (I2PP2A). In AD brain, I2PP2A is translocated from neuronal nucleus to cytoplasm, where it inhibits PP2A activity and promotes abnormal phosphorylation of Tau. Here we describe the identification of a potential nuclear localization signal (NLS) in the C-terminal region of I2PP2A containing a conserved basic motif, 179RKR181, which is sufficient for directing its nuclear localization. The current study further presents an inducible cell model (Tet-Off system) of AD-type abnormal hyperphosphorylation of Tau by expressing I2PP2A in which the NLS was inactivated by 179RKR181 → AAA along with 168KR169 → AA mutations. In this model, the mutant NLS (mNLS)-I2PP2A (I2PP2AAA-AAA) was retained in the cell cytoplasm, where it physically interacted with PP2A and inhibited its activity. Inhibition of PP2A was associated with the abnormal hyperphosphorylation of Tau, which resulted in microtubule network instability and neurite outgrowth impairment. Expression of mNLS-I2PP2A activated CAMKII and GSK-3β, which are Tau kinases regulated by PP2A. The immunoprecipitation experiments showed the direct interaction of I2PP2A with PP2A and GSK-3β but not with CAMKII. Thus, the cell model provides insights into the nature of the potential NLS and the mechanistic relationship between I2PP2A-induced inhibition of PP2A and hyperphosphorylation of Tau that can be utilized to develop drugs preventing Tau pathology. PMID:25128526

  1. [Sugar phosphorylation activities in acetogenic bacteria].

    PubMed

    Jiang, W; Patterson, J A

    1999-12-01

    Seven acetogenic bacteria (Acetitomaculum ruminis, Acetobacterium woodii, Eubacterium limosum as well as isolates A2, A4, A10 and H3HH) were tested for PEP- and ATP-dependent phosphorylation of glucose and 2-deoxyglucose. Although all organisms had detectable phosphorylation activity, substantial variation existed in the rates of both PEP- and ATP-dependent phosphorylation. Isolate Alo had the highest rate of PEP-dependent phosphorylation of 11.62 nmol.L-1.mg-1.min-1. Isolate A10, H3HH as well as E. limosum most likely have a glucose phosphotransferase system(PTS). In contrast, A ruminis, A. woodii and isolate A2, A4 had PEP-dependent glucose phosphorylation rates very similar to control rates, suggesting the lack of PTS activity. The rates of ATP-dependent glucose phosphorylation were higher than PEP-dependent phosphorylation in all organisms surveyed. However, substantial variation existed in the rates of ATP-dependent glucose phosphorylation. The glucose PTS of isolates A10 and H3HH were induced by the presence of extracellular glucose. Moreover, the specific activity of the glucose PTS of both isolates increased as cultures progressed from the early log to late log phase of growth. ATP- and PEP-dependent maltose and sucrose phosphorylation was detected in isolates A10 and H3HH. Although activity was detected in both isolates(A10 and H3HH), the rate of activity varied considerably, depending on the sugar and organism tested. PMID:12555560

  2. Eph-mediated tyrosine phosphorylation of citron kinase controls abscission.

    PubMed

    Jungas, Thomas; Perchey, Renaud T; Fawal, Mohamad; Callot, Caroline; Froment, Carine; Burlet-Schiltz, Odile; Besson, Arnaud; Davy, Alice

    2016-08-29

    Cytokinesis is the last step of cell division, culminating in the physical separation of daughter cells at the end of mitosis. Cytokinesis is a tightly regulated process that until recently was mostly viewed as a cell-autonomous event. Here, we investigated the role of Ephrin/Eph signaling, a well-known local cell-to-cell communication pathway, in cell division. We show that activation of Eph signaling in vitro leads to multinucleation and polyploidy, and we demonstrate that this is caused by alteration of the ultimate step of cytokinesis, abscission. Control of abscission requires Eph kinase activity, and Src and citron kinase (CitK) are downstream effectors in the Eph-induced signal transduction cascade. CitK is phosphorylated on tyrosines in neural progenitors in vivo, and Src kinase directly phosphorylates CitK. We have identified the specific tyrosine residues of CitK that are phosphorylated and show that tyrosine phosphorylation of CitK impairs cytokinesis. Finally, we show that, similar to CitK, Ephrin/Eph signaling controls neuronal ploidy in the developing neocortex. Our study indicates that CitK integrates intracellular and extracellular signals provided by the local environment to coordinate completion of cytokinesis. PMID:27551053

  3. Semen abnormalities with SSRI antidepressants.

    PubMed

    2015-01-01

    Despite decades of widespread use, the adverse effect profile of "selective" serotonin reuptake inhibitor (SSRI) antidepressants has still not been fully elucidated. Studies in male animals have shown delayed sexual development and reduced fertility. Three prospective cohort studies conducted in over one hundred patients exposed to an SSRI for periods ranging from 5 weeks to 24 months found altered semen param-eters after as little as 3 months of exposure: reduced sperm concentration, reduced sperm motility, a higher percentage of abnormal spermatozoa, and increased levels of sperm DNA fragmentation. One clinical trial showed growth retardation in children considered depressed who were exposed to SSRls. SSRls may have endocrine disrupting properties. Dapoxetine is a short-acting serotonin reuptake inhibitor that is chemically related to fluoxetine and marketed in the European Union for men complaining of premature ejaculation. But the corresponding European summary of product characteristics does not mention any effects on fertility. In practice, based on the data available as of mid-2014, the effects of SSRI exposure on male fertility are unclear. However, it is a risk that should be taken into account and pointed out to male patients who would like to father a child or who are experiencing fertility problems. PMID:25729824

  4. Abnormal behaviors detection using particle motion model

    NASA Astrophysics Data System (ADS)

    Chen, Yutao; Zhang, Hong; Cheng, Feiyang; Yuan, Ding; You, Yuhu

    2015-03-01

    Human abnormal behaviors detection is one of the most challenging tasks in the video surveillance for the public security control. Interaction Energy Potential model is an effective and competitive method published recently to detect abnormal behaviors, but their model of abnormal behaviors is not accurate enough, so it has some limitations. In order to solve this problem, we propose a novel Particle Motion model. Firstly, we extract the foreground to improve the accuracy of interest points detection since the complex background usually degrade the effectiveness of interest points detection largely. Secondly, we detect the interest points using the graphics features. Here, the movement of each human target can be represented by the movements of detected interest points of the target. Then, we track these interest points in videos to record their positions and velocities. In this way, the velocity angles, position angles and distance between each two points can be calculated. Finally, we proposed a Particle Motion model to calculate the eigenvalue of each frame. An adaptive threshold method is proposed to detect abnormal behaviors. Experimental results on the BEHAVE dataset and online videos show that our method could detect fight and robbery events effectively and has a promising performance.

  5. Familial Precocious Fetal Abnormal Cortical Sulcation.

    PubMed

    Frassoni, Carolina; Avagliano, Laura; Inverardi, Francesca; Spaccini, Luigina; Parazzini, Cecilia; Rustico, Maria Angela; Bulfamante, Gaetano; Righini, Andrea

    2016-08-01

    The development of the human cerebral cortex is a complex and precisely programmed process by which alterations may lead to morphological and functional neurological abnormalities. We report familial cases of prenatally diagnosed abnormal brain, characterized by aberrant symmetrical mesial oversulcation of the parietooccipital lobes, in fetuses affected by abnormal skeletal features. Fetal brain anomalies were characterized by prenatal magnetic resonance imaging at 21 weeks of gestation and histologically evaluated at 22 weeks. Histological examination added relevant information showing some focal cortical areas of micropoligyria and heterotopic extension of the cortical plate into the marginal zone beneath the cortical surface. Genetic analysis of the fetuses excluded FGFR3 mutations known to be related to skeletal dysplasia and aberrant symmetrical oversulcation in other brain areas (temporal lobes). Hence, the present report suggests the existence of a class of rare syndromes of skeleton and brain development abnormality unrelated to FGFR3 mutations or related to other not described FGFR3 gene defects. Using magnetic resonance imaging, histopathology and molecular characterization we provide an example of a translational study of a rare and unreported brain congenital malformation. PMID:27177044

  6. Behavioral abnormalities in captive nonhuman primates.

    PubMed

    Mallapur, Avanti; Choudhury, B C

    2003-01-01

    In this study, we dealt with 11 species of nonhuman primates across 10 zoos in India. We recorded behavior as instantaneous scans between 9 a.m. and 5 p.m. In the study, we segregated behaviors for analyses into abnormal, undesirable, active, and resting. The 4 types of abnormal behavior exhibited included floating limb, self-biting, self-clasping, and stereotypic pacing. In the study, we recorded 2 types of undesirable behavior: autoerotic stimulation and begging. Langurs and group-housed macaques did not exhibit undesirable behaviors. A male lion-tailed macaque and a male gibbon exhibited begging behavior. autoerotic stimulation and self-biting occurred rarely. Males exhibited higher levels of undesirable behavior than did females. Animals confiscated from touring zoos, circuses, and animal traders exhibited higher levels of abnormal behaviors than did animals reared in larger, recognized zoos. The stump-tailed macaque was the only species to exhibit floating limb, autoerotic stimulation, self-biting, and self-clasping. Our results show that rearing experience and group composition influence the proportions of abnormal behavior exhibited by nonhuman primates in captivity. The history of early social and environmental deprivation in these species of captive nonhuman primates probably is critical in the development of behavioral pathologies. Establishing this will require further research. PMID:14965782

  7. Phosphorylation of human link proteins

    SciTech Connect

    Oester, D.A.; Caterson, B.; Schwartz, E.R.

    1986-06-13

    Three link proteins of 48, 44 and 40 kDa were purified from human articular cartilage and identified with monoclonal anti-link protein antibody 8-A-4. Two sets of lower molecular weight proteins of 30-31 kDa and 24-26 kDa also contained link protein epitopes recognized by the monoclonal antibody and were most likely degradative products of the intact link proteins. The link proteins of 48 and 40 kDa were identified as phosphoproteins while the 44 kDa link protein did not contain /sup 32/P. The phosphorylated 48 and 40 kDa link proteins contained approximately 2 moles PO/sub 4//mole link protein.

  8. Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion.

    PubMed

    Malmersjö, Seth; Di Palma, Serena; Diao, Jiajie; Lai, Ying; Pfuetzner, Richard A; Wang, Austin L; McMahon, Moira A; Hayer, Arnold; Porteus, Matthew; Bodenmiller, Bernd; Brunger, Axel T; Meyer, Tobias

    2016-08-15

    Membrane fusion is essential for eukaryotic life, requiring SNARE proteins to zipper up in an α-helical bundle to pull two membranes together. Here, we show that vesicle fusion can be suppressed by phosphorylation of core conserved residues inside the SNARE domain. We took a proteomics approach using a PKCB knockout mast cell model and found that the key mast cell secretory protein VAMP8 becomes phosphorylated by PKC at multiple residues in the SNARE domain. Our data suggest that VAMP8 phosphorylation reduces vesicle fusion in vitro and suppresses secretion in living cells, allowing vesicles to dock but preventing fusion with the plasma membrane. Markedly, we show that the phosphorylation motif is absent in all eukaryotic neuronal VAMPs, but present in all other VAMPs. Thus, phosphorylation of SNARE domains is a general mechanism to restrict how much cells secrete, opening the door for new therapeutic strategies for suppression of secretion. PMID:27402227

  9. B lymphocytes from patients with chronic lymphocytic leukemia contain signal transducer and activator of transcription (STAT) 1 and STAT3 constitutively phosphorylated on serine residues.

    PubMed

    Frank, D A; Mahajan, S; Ritz, J

    1997-12-15

    To explore the pathogenesis of chronic lymphocytic leukemia (CLL), we examined whether phosphorylation of one or more signal transducer and activator of transcription (STAT) factors was abnormal in cells from CLL patients. No constitutive tyrosine phosphorylation was detected on any STAT in CLL cells. To assess the phosphorylation of serine residues of STAT1 and STAT3 in CLL cells, we raised antibodies that specifically recognize the form of STAT1 phosphorylated on ser-727 and the form of STAT3 phosphorylated on ser-727. We found that in 100% of patients with CLL (n = 32), STAT1 and STAT3 were constitutively phosphorylated on serine. This was in contrast to normal peripheral blood B lymphocytes or CD5+) B cells isolated from tonsils, in which this phosphorylation was absent. Serine phosphorylation of STAT1 and STAT3 was seen occasionally in other leukemias, but it was a universal finding only in CLL. The serine phosphorylation of these STATs was a continuous process, as incubation of CLL cells with the kinase inhibitor H7 led to the dephosphorylation of these serine residues. The STAT serine kinase in CLL cells has not been identified, and appears to be neither mitogen-activated protein kinase nor pp70(s6k). In summary, the constitutive serine phosphorylation of STAT1 and STAT3 is present in all CLL samples tested to date, although the physiologic significance of this modification remains to be determined. PMID:9399961

  10. Protein phosphorylation in Bradyrhizobium japonicum bacteroids and cultures.

    PubMed Central

    Karr, D B; Emerich, D W

    1989-01-01

    Protein phosphorylation was demonstrated in Bradyrhizobium japonicum bacteroids in vivo and in cultures in vivo and in vitro. Comparison of in vivo-labeled phosphoproteins of bacteroids and of cultured cells showed differences in both the pattern and intensity of labeling. In cultured cells, comparison of the labeling patterns and intensities of in vivo- and in vitro-labeled phosphoproteins showed a number of similarities; however, several phosphoproteins were found only after one of the two labeling conditions. The labeling intensity was time dependent in both in vivo and in vitro assays and was dependent on the presence of magnesium in in vitro assays. Differences in the rates of phosphorylation and dephosphorylation were noted for a number of proteins. The level of incorporation of 32P into protein was only 2% or less of the total phosphate accumulated during the in vivo labeling period. Several isolation and sample preparation procedures resulted in differences in labeling patterns. Phosphatase inhibitors and several potential metabolic effectors had negligible effects on the phosphorylation pattern. There were no significant changes in the phosphorylation patterns of cells cultured on mannitol, acetate, and succinate, although the intensity of the labeling did vary with the carbon source. Images PMID:2498290

  11. Mechanism of Ribonuclease III Catalytic Regulation by Serine Phosphorylation

    PubMed Central

    Gone, Swapna; Alfonso-Prieto, Mercedes; Paudyal, Samridhdi; Nicholson, Allen W.

    2016-01-01

    Ribonuclease III (RNase III) is a conserved, gene-regulatory bacterial endonuclease that cleaves double-helical structures in diverse coding and noncoding RNAs. RNase III is subject to multiple levels of control, reflective of its global regulatory functions. Escherichia coli (Ec) RNase III catalytic activity is known to increase during bacteriophage T7 infection, reflecting the expression of the phage-encoded protein kinase, T7PK. However, the mechanism of catalytic enhancement is unknown. This study shows that Ec-RNase III is phosphorylated on serine in vitro by purified T7PK, and identifies the targets as Ser33 and Ser34 in the N-terminal catalytic domain. Kinetic experiments reveal a 5-fold increase in kcat and a 1.4-fold decrease in Km following phosphorylation, providing a 7.4–fold increase in catalytic efficiency. Phosphorylation does not change the rate of substrate cleavage under single-turnover conditions, indicating that phosphorylation enhances product release, which also is the rate-limiting step in the steady-state. Molecular dynamics simulations provide a mechanism for facilitated product release, in which the Ser33 phosphomonoester forms a salt bridge with the Arg95 guanidinium group, thereby weakening RNase III engagement of product. The simulations also show why glutamic acid substitution at either serine does not confer enhancement, thus underscoring the specific requirement for a phosphomonoester. PMID:27150669

  12. Protein phosphorylation in Bradyrhizobium japonicum bacteroids and cultures

    SciTech Connect

    Karr, D.B.; Emerich, D.W. )

    1989-06-01

    Protein phosphorylation was demonstrated in Bradyrhizobium japonicum bacteroids in vivo and in cultures in vivo and in vitro. Comparison of in vivo-labeled phosphoproteins of bacteroids and of cultured cells showed differences in both the pattern and intensity of labeling. In cultured cells, comparison of the labeling patterns and intensities of in vivo- and in vitro-labeled phosphoproteins showed a number of similarities; however, several phosphoproteins were found only after one of the two labeling conditions. The labeling intensity was time dependent in both in vivo and in vitro assays and was dependent on the presence of magnesium in in vitro assays. Differences in the rates of phosphorylation and dephosphorylation were noted for a number of proteins. The level of incorporation of {sup 32}P into protein was only 2% or less of the total phosphate accumulated during the in vivo labeling period. Several isolation and sample preparation procedures resulted in differences in labeling patterns. Phosphatase inhibitors and several potential metabolic effectors had negligible effects on the phosphorylation pattern. There were no significant changes in the phosphorylation patterns of cells cultured on mannitol, acetate, and succinate, although the intensity of the labeling did vary with the carbon source.

  13. Mechanism of Ribonuclease III Catalytic Regulation by Serine Phosphorylation

    NASA Astrophysics Data System (ADS)

    Gone, Swapna; Alfonso-Prieto, Mercedes; Paudyal, Samridhdi; Nicholson, Allen W.

    2016-05-01

    Ribonuclease III (RNase III) is a conserved, gene-regulatory bacterial endonuclease that cleaves double-helical structures in diverse coding and noncoding RNAs. RNase III is subject to multiple levels of control, reflective of its global regulatory functions. Escherichia coli (Ec) RNase III catalytic activity is known to increase during bacteriophage T7 infection, reflecting the expression of the phage-encoded protein kinase, T7PK. However, the mechanism of catalytic enhancement is unknown. This study shows that Ec-RNase III is phosphorylated on serine in vitro by purified T7PK, and identifies the targets as Ser33 and Ser34 in the N-terminal catalytic domain. Kinetic experiments reveal a 5-fold increase in kcat and a 1.4-fold decrease in Km following phosphorylation, providing a 7.4–fold increase in catalytic efficiency. Phosphorylation does not change the rate of substrate cleavage under single-turnover conditions, indicating that phosphorylation enhances product release, which also is the rate-limiting step in the steady-state. Molecular dynamics simulations provide a mechanism for facilitated product release, in which the Ser33 phosphomonoester forms a salt bridge with the Arg95 guanidinium group, thereby weakening RNase III engagement of product. The simulations also show why glutamic acid substitution at either serine does not confer enhancement, thus underscoring the specific requirement for a phosphomonoester.

  14. Bakers' cyst and tibiofemoral abnormalities are more distinctive MRI features of symptomatic osteoarthritis than patellofemoral abnormalities

    PubMed Central

    Visser, A W; Mertens, B; Reijnierse, M; Bloem, J L; de Mutsert, R; le Cessie, S; Rosendaal, F R; Kloppenburg, M

    2016-01-01

    Objective To investigate which structural MR abnormalities discriminate symptomatic knee osteoarthritis (OA), taking co-occurrence of abnormalities in all compartments into account. Methods The Netherlands Epidemiology of Obesity (NEO) study is a population-based cohort aged 45–65 years. In 1285 participants (median age 56 years, 55% women, median body mass index (BMI) 30 kg/m2), MRI of the right knee were obtained. Structural abnormalities (osteophytes, cartilage loss, bone marrow lesions (BMLs), subchondral cysts, meniscal abnormalities, effusion, Baker's cyst) at 9 patellofemoral and tibiofemoral locations were scored following the knee OA scoring system. Symptomatic OA in the imaged knee was defined following the American College of Rheumatology criteria. Logistic ridge regression analyses were used to investigate which structural abnormalities discriminate best between individuals with and without symptomatic OA, crude and adjusted for age, sex and BMI. Results Symptomatic knee OA was present in 177 individuals. Structural MR abnormalities were highly frequent both in individuals with OA and in those without. Baker's cysts showed the highest adjusted regression coefficient (0.293) for presence of symptomatic OA, followed by osteophytes and BMLs in the medial tibiofemoral compartment (0.185–0.279), osteophytes in the medial trochlear facet (0.262) and effusion (0.197). Conclusions Baker's cysts discriminate best between individuals with and without symptomatic knee OA. Structural MR abnormalities, especially in the medial side of the tibiofemoral joint and effusion, add further in discriminating symptomatic OA. Baker's cysts may present as a target for treatment. PMID:27252896

  15. Phosphorylation acts positively and negatively to regulate MRTF-A subcellular localisation and activity.

    PubMed

    Panayiotou, Richard; Miralles, Francesc; Pawlowski, Rafal; Diring, Jessica; Flynn, Helen R; Skehel, Mark; Treisman, Richard

    2016-01-01

    The myocardin-related transcription factors (MRTF-A and MRTF-B) regulate cytoskeletal genes through their partner transcription factor SRF. The MRTFs bind G-actin, and signal-regulated changes in cellular G-actin concentration control their nuclear accumulation. The MRTFs also undergo Rho- and ERK-dependent phosphorylation, but the function of MRTF phosphorylation, and the elements and signals involved in MRTF-A nuclear export are largely unexplored. We show that Rho-dependent MRTF-A phosphorylation reflects relief from an inhibitory function of nuclear actin. We map multiple sites of serum-induced phosphorylation, most of which are S/T-P motifs and show that S/T-P phosphorylation is required for transcriptional activation. ERK-mediated S98 phosphorylation inhibits assembly of G-actin complexes on the MRTF-A regulatory RPEL domain, promoting nuclear import. In contrast, S33 phosphorylation potentiates the activity of an autonomous Crm1-dependent N-terminal NES, which cooperates with five other NES elements to exclude MRTF-A from the nucleus. Phosphorylation thus plays positive and negative roles in the regulation of MRTF-A. PMID:27304076

  16. Phosphorylation acts positively and negatively to regulate MRTF-A subcellular localisation and activity

    PubMed Central

    Panayiotou, Richard; Miralles, Francesc; Pawlowski, Rafal; Diring, Jessica; Flynn, Helen R; Skehel, Mark; Treisman, Richard

    2016-01-01

    The myocardin-related transcription factors (MRTF-A and MRTF-B) regulate cytoskeletal genes through their partner transcription factor SRF. The MRTFs bind G-actin, and signal-regulated changes in cellular G-actin concentration control their nuclear accumulation. The MRTFs also undergo Rho- and ERK-dependent phosphorylation, but the function of MRTF phosphorylation, and the elements and signals involved in MRTF-A nuclear export are largely unexplored. We show that Rho-dependent MRTF-A phosphorylation reflects relief from an inhibitory function of nuclear actin. We map multiple sites of serum-induced phosphorylation, most of which are S/T-P motifs and show that S/T-P phosphorylation is required for transcriptional activation. ERK-mediated S98 phosphorylation inhibits assembly of G-actin complexes on the MRTF-A regulatory RPEL domain, promoting nuclear import. In contrast, S33 phosphorylation potentiates the activity of an autonomous Crm1-dependent N-terminal NES, which cooperates with five other NES elements to exclude MRTF-A from the nucleus. Phosphorylation thus plays positive and negative roles in the regulation of MRTF-A. DOI: http://dx.doi.org/10.7554/eLife.15460.001 PMID:27304076

  17. Understanding the influence of phosphorylation and polysialylation of gelatin on mineralization and osteogenic differentiation.

    PubMed

    Arora, Aditya; Katti, Dhirendra S

    2016-08-01

    Post-translational modifications such as phosphorylation and sialylation impart crucial functions such as mineral deposition and osteogenic differentiation to non-collagenous bone matrix proteins. In this work, the influence of phosphorylation and polysialylation of gelatin on mineralization in simulated body fluid (SBF) and on osteogenic differentiation of mesenchymal stem cells (MSC) was studied. It was observed that increase in phosphorylation could be directly correlated with the mineralization ability of phosphorylated gelatin in SBF. The total calcium and phosphate deposited increased with increase in degree of phosphorylation and was >3 fold higher on the highest degree of phosphorylation. Whereas, polysialylation did not have any significant influence on mineral deposition in SBF. On the other hand, when MSCs were cultured on polysialylated surfaces they showed relatively higher cell elongation with 1.5 fold higher cell aspect ratio, higher alkaline phosphatase activity and 3 fold higher mineral deposition when compared to control and phosphorylated gelatin surfaces. In conclusion, phosphorylation and polysialylation of gelatin show a significant influence on mineralization and osteogenic differentiation respectively which can be advantageously used for bone tissue engineering. PMID:27157722

  18. Phosphorylation of the multidrug resistance associated glycoprotein

    SciTech Connect

    Mellado, W.; Horwitz, S.B.

    1987-11-03

    Drug-resistant cell lines derived from the mouse macrophage-like cell line J774.2 express the multidrug resistant phenotype which includes the overexpression of a membrane glycoprotein (130-140 kilodaltons). Phosphorylation of this resistant-specific glycoprotein (P-glycoprotein) in intact cells and in cell-free membrane fractions has been studied. The phosphorylated glycoprotein can be immunoprecipitated by a rabbit polyclonal antibody specific for the glycoprotein. Phosphorylation studies done with partially purified membrane fractions derived from colchicine-resistant cells indicated that (a) phosphorylation of the glycoprotein in 1 mM MgCl/sub 2/ was enhanced a minimum of 2-fold by 10 ..mu..M cAMP and (b) the purified catalytic subunit of the cAMP-dependent protein kinase (protein kinase A) phosphorylated partially purified glycoprotein that was not phosphorylated by (..gamma..-/sup 32/P)ATP alone, suggesting that autophosphorylation was not involved. These results indicate that the glycoprotein is a phosphoprotein and that at least one of the kinases responsible for its phosphorylation is a membrane-associated protein kinase A. The state of phosphorylation of the glycoprotein, which is a major component of the multidrug resistance phenotype, may be related to the role of the glycoprotein in maintaining drug resistance.

  19. Phosphorylation of the multidrug resistance associated glycoprotein.

    PubMed

    Mellado, W; Horwitz, S B

    1987-11-01

    Drug-resistant cell lines derived from the mouse macrophage-like cell line J774.2 express the multidrug resistance phenotype which includes the overexpression of a membrane glycoprotein (130-140 kilodaltons). Phosphorylation of this resistant-specific glycoprotein (P-glycoprotein) in intact cells and in cell-free membrane fractions has been studied. The phosphorylated glycoprotein can be immunoprecipitated by a rabbit polyclonal antibody specific for the glycoprotein. Phosphorylation studies done with partially purified membrane fractions derived from colchicine-resistant cells indicated that (a) phosphorylation of the glycoprotein in 1 mM MgCl2 was enhanced a minimum of 2-fold by 10 microM cAMP and (b) the purified catalytic subunit of the cAMP-dependent protein kinase (protein kinase A) phosphorylated partially purified glycoprotein that was not phosphorylated by [gamma-32P]ATP alone, suggesting that autophosphorylation was not involved. These results indicate that the glycoprotein is a phosphoprotein and that at least one of the kinases responsible for its phosphorylation is a membrane-associated protein kinase A. The state of phosphorylation of the glycoprotein, which is a major component of the multidrug resistance phenotype, may be related to the role of the glycoprotein in maintaining drug resistance. PMID:3427052

  20. Evaluation of Phosphorylated Psyllium Seed Polysaccharide as a Release Retardant.

    PubMed

    Rao, Monica R P; Warrier, Deepa U; Rao, Shivani H

    2015-01-01

    The aim of the present study was to modify psyllium seed polysaccharide and evaluate the modified polysaccharide as release retardant in tablets employing ciprofloxacin hydrochloride as model drug. Studies on polysaccharide from psyllium husk has been reported but no work has been reported on characterization and modification of the polysaccharide present in the psyllium (Plantago ovata) seed and the use of the modified polysaccharide as a release retardant in tablets. In this study, the seed gum was modified using sodium trimetaphosphate as crosslinking agent. Sustained release matrix tablets of ciprofloxacin hydrochloride were prepared by wet granulation using various drug-polymer ratios. The polymers investigated were psyllium polysaccharide, phosphorylated psyllium polysaccharide and widely used release retardant hydroxypropyl methylcellulose K100M. The tablets were evaluated for hardness, friability, drug content, swelling profile and in vitro dissolution studies. The matrix tablets containing 1:3 proportion of drug-phosphorylated psyllium polysaccharide was found to have higher hardness as compared to tablets containing 1:1 and 1:2 proportions. The results of swelling behavior in water showed that the tablets containing 1:3 drug:phosphorylated psyllium polysaccharide ratio had swelling comparable to that of tablets containing 1:3 drug:hydroxypropyl methylcellulose ratio. The in vitro dissolution studies shows that the dissolution rate was retarded from 98.41 to 37.6% in 6 h with increase in concentration of phosphorylated psyllium polysaccharide from 100 to 300 mg. Formulations containing psyllium polysaccharide showed complete drug release in 8 h whereas those formulated with phosphorylated psyllium polysaccharide exhibited extended drug release over the 12 h period. Drug release kinetic studies revealed that drug release followed Korsmeyer-Peppas model. PMID:26798177

  1. Evaluation of Phosphorylated Psyllium Seed Polysaccharide as a Release Retardant

    PubMed Central

    Rao, Monica R. P.; Warrier, Deepa U.; Rao, Shivani H.

    2015-01-01

    The aim of the present study was to modify psyllium seed polysaccharide and evaluate the modified polysaccharide as release retardant in tablets employing ciprofloxacin hydrochloride as model drug. Studies on polysaccharide from psyllium husk has been reported but no work has been reported on characterization and modification of the polysaccharide present in the psyllium (Plantago ovata) seed and the use of the modified polysaccharide as a release retardant in tablets. In this study, the seed gum was modified using sodium trimetaphosphate as crosslinking agent. Sustained release matrix tablets of ciprofloxacin hydrochloride were prepared by wet granulation using various drug-polymer ratios. The polymers investigated were psyllium polysaccharide, phosphorylated psyllium polysaccharide and widely used release retardant hydroxypropyl methylcellulose K100M. The tablets were evaluated for hardness, friability, drug content, swelling profile and in vitro dissolution studies. The matrix tablets containing 1:3 proportion of drug-phosphorylated psyllium polysaccharide was found to have higher hardness as compared to tablets containing 1:1 and 1:2 proportions. The results of swelling behavior in water showed that the tablets containing 1:3 drug:phosphorylated psyllium polysaccharide ratio had swelling comparable to that of tablets containing 1:3 drug:hydroxypropyl methylcellulose ratio. The in vitro dissolution studies shows that the dissolution rate was retarded from 98.41 to 37.6% in 6 h with increase in concentration of phosphorylated psyllium polysaccharide from 100 to 300 mg. Formulations containing psyllium polysaccharide showed complete drug release in 8 h whereas those formulated with phosphorylated psyllium polysaccharide exhibited extended drug release over the 12 h period. Drug release kinetic studies revealed that drug release followed Korsmeyer-Peppas model. PMID:26798177

  2. Cisplatin stimulates protein tyrosine phosphorylation in macrophages.

    PubMed

    Kumar, R; Shrivastava, A; Sodhi, A

    1995-03-01

    Cisplatin [cis-dichlorodiamine platinum (II)], a potent anti-tumor compound, stimulates immune responses by activating monocyte-macrophages and other cells of the immune system. The mechanism by which cisplatin activates these cells is poorly characterized. Since protein tyrosine phosphorylation appears to be a major intracellular signalling event that mediates cellular responses, we examined whether cisplatin alters tyrosine phosphorylation in macrophages. We found that cisplatin increased tyrosine phosphorylation of several proteins in peritoneal macrophages and in P388D1 and IC-21 macrophage cell lines. Treatment of macrophages with tyrosine kinase inhibitors, genestein and lavendustin A, inhibited cisplatin-stimulated protein tyrosine phosphorylation in macrophages. Macrophages treated with cisplatin also exhibit increased fluorescence with anti-phosphotyrosine-FITC antibody. These data indicate that protein tyrosine phosphorylation plays a role in cisplatin-induced activation of macrophages. PMID:7539662

  3. Functional phosphoproteomic profiling of phosphorylation sites in membrane fractions of salt-stressed Arabidopsis thaliana

    PubMed Central

    2009-01-01

    Background Under conditions of salt stress, plants respond by initiating phosphorylation cascades. Many key phosphorylation events occur at the membrane. However, to date only limited sites have been identified that are phosphorylated in response to salt stress in plants. Results Membrane fractions from three-day and 200 mM salt-treated Arabidopsis suspension plants were isolated, followed by protease shaving and enrichment using Zirconium ion-charged magnetic beads, and tandem mass spectrometry analyses. From this isolation, 18 phosphorylation sites from 15 Arabidopsis proteins were identified. A unique phosphorylation site in 14-3-3-interacting protein AHA1 was predominately identified in 200 mM salt-treated plants. We also identified some phosphorylation sites in aquaporins. A doubly phosphorylated peptide of PIP2;1 as well as a phosphopeptide containing a single phosphorylation site (Ser-283) and a phosphopeptide containing another site (Ser-286) of aquaporin PIP2;4 were identified respectively. These two sites appeared to be novel of which were not reported before. In addition, quantitative analyses of protein phosphorylation with either label-free or stable-isotope labeling were also employed in this study. The results indicated that level of phosphopeptides on five membrane proteins such as AHA1, STP1, Patellin-2, probable inactive receptor kinase (At3g02880), and probable purine permease 18 showed at least two-fold increase in comparison to control in response to 200 mM salt-stress. Conclusion In this study, we successfully identified novel salt stress-responsive protein phosphorylation sites from membrane isolates of abiotic-stressed plants by membrane shaving followed by Zr4+-IMAC enrichment. The identified phosphorylation sites can be important in the salt stress response in plants. PMID:19900291

  4. TNF-α Suppresses α-Smooth Muscle Actin Expression in Human Dermal Fibroblasts: An Implication for Abnormal Wound Healing

    PubMed Central

    Goldberg, Mytien T.; Han, Yuan-Ping; Yan, Chunli; Shaw, Michael C.; Garner, Warren L.

    2008-01-01

    Abnormal wound healing encompasses a wide spectrum, from chronic wounds to hypertrophic scars. Both conditions are associated with an abnormal cytokine profile in the wound bed. In this study, we sought to understand the dynamic relationships between myofibroblast differentiation and mechanical performance of the collagen matrix under tissue growth factor–β (TGF-β) and tumor necrosis factor–α (TNF-α) stimulation. We found TGF-β increased α-smooth muscle actin (α-SMA) and TNF-α alone decreased the basal α-SMA expression. When TGF-β1 and TNF-α were both added, the α-SMA expression was suppressed below the baseline. Real-time PCR showed that TNF-α suppresses TGF-β1-induced myofibroblast (fibroproliferative) phenotypic genes, for example, α-SMA, collagen type 1A, and fibronectin at the mRNA level. TNF-α suppresses TGF-β1-induced gene expression by affecting its mRNA stability. Our results further showed that TNF-α inhibits TGF-β1-induced Smad-3 phosphorylation via Jun N-terminal kinase signaling. Mechanical testing showed that TNF-α decreases the stiffness and contraction of the lattices after 5 days in culture. We proposed that changes in α-SMA, collagen, and fibronectin expression result in decreased contraction and stiffness of collagen matrices. Therefore, the balance of cytokines in a wound defines the mechanical properties of the extracellular matrix and optimal wound healing. PMID:17554369

  5. PKD2 and RSK1 Regulate Integrin β4 Phosphorylation at Threonine 1736

    PubMed Central

    te Molder, Lisa; Sonnenberg, Arnoud

    2015-01-01

    The integrin α6β4, a major component of hemidesmosomes (HDs), stabilizes keratinocyte cell adhesion to the epidermal basement membrane through binding to the cytoskeletal linker protein plectin and association with keratin filaments. Disruption of the α6β4-plectin interaction through phosphorylation of the β4 subunit results in a reduction in adhesive strength of keratinocytes to laminin-332 and the dissolution of HDs. Previously, we have demonstrated that phosphorylation of T1736 in the C-terminal end of the β4 cytoplasmic domain disrupts the interaction of β4 with the plakin domain of plectin. Furthermore, we showed that β4-T1736 can be phosphorylated by PKD1 in vitro, and although both PMA and EGF induced T1736 phosphorylation, only PMA was able to activate PKD1. Here, we show that depletion of [Ca2+]i augments PMA- and EGF-induced phosphorylation of β4-T1736 and that this is caused by inhibition of the calcium-sensitive protein phosphatase calcineurin and augmentation of ERK1/2 activation. We also show that in keratinocytes the PMA-stimulated phosphorylation of β4-T1736 primarily is mediated by PKD2 activation downstream of PKCδ. On the other hand, both the EGF-stimulated phosphorylation of T1736 and the EGF-induced dissolution of HDs are dependent on a functional MAPK signaling pathway, and treatment with the RSK inhibitor BI-D1870 prevented EGF-stimulated phosphorylation of β4-T1736. Moreover, phosphorylation of β4-T1736 is enhanced by overexpression of wild-type RSK1, while it is reduced by the expression of kinase-inactive RSK1 or by siRNA-mediated depletion of RSK1. In summary, our data indicate that different stimuli can lead to the phosphorylation of β4-T1736 by either PKD2 or RSK1. PMID:26580203

  6. Chronic restraint stress induces sperm acrosome reaction and changes in testicular tyrosine phosphorylated proteins in rats

    PubMed Central

    Arun, Supatcharee; Burawat, Jaturon; Sukhorum, Wannisa; Sampannang, Apichakan; Maneenin, Chanwit; Iamsaard, Sitthichai

    2016-01-01

    Background: Stress is a cause of male infertility. Although sex hormones and sperm quality have been shown to be low in stress, sperm physiology and testicular functional proteins, such as phosphotyrosine proteins, have not been documented. Objective: To investigate the acrosome status and alterations of testicular proteins involved in spermatogenesis and testosterone synthesis in chronic stress in rats. Materials and Methods: In this experimental study, male rats were divided into 2 groups (control and chronic stress (CS), n=7). CS rats were immobilized (4 hr/day) for 42 consecutive days. The blood glucose level (BGL), corticosterone, testosterone, acrosome status, and histopathology were examined. The expressions of testicular steroidogenic acute regulatory (StAR), cytochrome P450 side chain cleavage (CYP11A1), and phosphorylated proteins were analyzed. Results: Results showed that BGL (71.25±2.22 vs. 95.60±3.36 mg/dl), corticosterone level (24.33±4.23 vs. 36.9±2.01 ng/ml), acrosome reacted sperm (3.25±1.55 vs. 17.71±5.03%), and sperm head abnormality (3.29±0.71 vs. 6.21±1.18%) were significantly higher in CS group in comparison with control. In contrast, seminal vesicle (0.41±0.05 vs. 0.24±0.07 g/100g), testosterone level (3.37±0.79 vs. 0.61±0.29 ng/ml), and sperm concentration (115.33±7.70 vs. 79.13±3.65×106 cells/ml) of CS were significantly lower (p<0.05) than controls. Some atrophic seminiferous tubules and low sperm mass were apparent in CS rats. The expression of CYP11A1 except StAR protein was markedly decreased in CS rats. In contrast, a 55 kDa phosphorylated protein was higher in CS testes. Conclusion: CS decreased the expression of CYP11A, resulting in decreased testosterone, and increased acrosome-reacted sperm, assumed to be the result of an increase of 55 kDa phosphorylated protein. PMID:27525328

  7. Coagulation abnormalities in sepsis.

    PubMed

    Tsao, Cheng-Ming; Ho, Shung-Tai; Wu, Chin-Chen

    2015-03-01

    Although the pathophysiology of sepsis has been elucidated with the passage of time, sepsis may be regarded as an uncontrolled inflammatory and procoagulant response to infection. The hemostatic changes in sepsis range from subclinical activation of blood coagulation to acute disseminated intravascular coagulation (DIC). DIC is characterized by widespread microvascular thrombosis, which contributes to multiple organ dysfunction/failure, and subsequent consumption of platelets and coagulation factors, eventually causing bleeding manifestations. The diagnosis of DIC can be made using routinely available laboratory tests, scoring algorithms, and thromboelastography. In this cascade of events, the inhibition of coagulation activation and platelet function is conjectured as a useful tool for attenuating inflammatory response and improving outcomes in sepsis. A number of clinical trials of anticoagulants were performed, but none of them have been recognized as a standard therapy because recombinant activated protein C was withdrawn from the market owing to its insufficient efficacy in a randomized controlled trial. However, these subgroup analyses of activated protein C, antithrombin, and thrombomodulin trials show that overt coagulation activation is strongly associated with the best therapeutic effect of the inhibitor. In addition, antiplatelet drugs, including acetylsalicylic acid, P2Y12 inhibitors, and glycoprotein IIb/IIIa antagonists, may reduce organ failure and mortality in the experimental model of sepsis without a concomitant increased bleeding risk, which should be supported by solid clinical data. For a state-of-the-art treatment of sepsis, the efficacy of anticoagulant and antiplatelet agents needs to be proved in further large-scale prospective, interventional, randomized validation trials. PMID:25544351

  8. Myofibrillar myopathy with abnormal foci of desmin positivity. II. Immunocytochemical analysis reveals accumulation of multiple other proteins.

    PubMed

    De Bleecker, J L; Engel, A G; Ertl, B B

    1996-05-01

    The two major types of lesions in myofibrillar myopathy consist of hyaline spheroidal structures composed of compacted myofibrillar residues, and nonhyaline lesions that comprise foci of myofibrillar destruction. We employed immunocytochemical analysis to further characterize these abnormalities. The nonhyaline lesions are depleted of actin, alpha-actinin, myosin, and, less consistently, of titin and nebulin. Thus, each major component of the myofibrils is lost or decreased. These lesions also react strongly for both NCAM and desmin. By contrast, the hyaline structures are highly enriched in actin, are immunoreactive for fast and slow myosin, and show increased expression of titin, nebulin, and alpha-actinin. They fail to react for NCAM and react variably for desmin. Both types of lesion react, but with differing intensities, for gelsolin, dystrophin, beta-amyloid precursor protein (beta APP) epitopes amino-terminal to the alpha-secretase site, alpha 1-antichymotrypsin, and ubiquitin, and both can be congophilic. The increased expressions of desmin, dystrophin and gelsolin in muscle are also confirmed by immunoblot studies. The results, in harmony with the ultrastructural findings described in the companion paper, suggest that myofibrillar myopathy is conditioned by abnormal activation of a degradative process that primarily affects the myofibrils. A structural abnormality of desmin alone may not be sufficient to disrupt the myofibrillar architecture, but abnormal activation of a phosphorylating process could account for dissolution of the myofibrils. The cause and significance of the ectopic overexpression of desmin, dystrophin, NCAM, and beta APP components, and the chemical basis of the congophilia remain unknown. PMID:8627347

  9. Phosphorylation of influenza A virus NS1 protein at threonine 49 suppresses its interferon antagonistic activity.

    PubMed

    Kathum, Omer Abid; Schräder, Tobias; Anhlan, Darisuren; Nordhoff, Carolin; Liedmann, Swantje; Pande, Amit; Mellmann, Alexander; Ehrhardt, Christina; Wixler, Viktor; Ludwig, Stephan

    2016-06-01

    Phosphorylation and dephosphorylation acts as a fundamental molecular switch that alters protein function and thereby regulates many cellular processes. The non-structural protein 1 (NS1) of influenza A virus is an important factor regulating virulence by counteracting cellular immune responses against viral infection. NS1 was shown to be phosphorylated at several sites; however, so far, no function has been conclusively assigned to these post-translational events yet. Here, we show that the newly identified phospho-site threonine 49 of NS1 is differentially phosphorylated in the viral replication cycle. Phosphorylation impairs binding of NS1 to double-stranded RNA and TRIM25 as well as complex formation with RIG-I, thereby switching off its interferon antagonistic activity. Because phosphorylation was shown to occur at later stages of infection, we hypothesize that at this stage other functions of the multifunctional NS1 beyond its interferon-antagonistic activity are needed. PMID:26687707

  10. [Walking abnormalities in children].

    PubMed

    Segawa, Masaya

    2010-11-01

    Walking is a spontaneous movement termed locomotion that is promoted by activation of antigravity muscles by serotonergic (5HT) neurons. Development of antigravity activity follows 3 developmental epochs of the sleep-wake (S-W) cycle and is modulated by particular 5HT neurons in each epoch. Activation of antigravity activities occurs in the first epoch (around the age of 3 to 4 months) as restriction of atonia in rapid eye movement (REM) stage and development of circadian S-W cycle. These activities strengthen in the second epoch, with modulation of day-time sleep and induction of crawling around the age of 8 months and induction of walking by 1 year. Around the age of 1 year 6 months, absence of guarded walking and interlimb cordination is observed along with modulation of day-time sleep to once in the afternoon. Bipedal walking in upright position occurs in the third epoch, with development of a biphasic S-W cycle by the age of 4-5 years. Patients with infantile autism (IA), Rett syndrome (RTT), or Tourette syndrome (TS) show failure in the development of the first, second, or third epoch, respectively. Patients with IA fail to develop interlimb coordination; those with RTT, crawling and walking; and those with TS, walking in upright posture. Basic pathophysiology underlying these condition is failure in restricting atonia in REM stage; this induces dysfunction of the pedunculopontine nucleus and consequently dys- or hypofunction of the dopamine (DA) neurons. DA hypofunction in the developing brain, associated with compensatory upward regulation of the DA receptors causes psychobehavioral disorders in infancy (IA), failure in synaptogenesis in the frontal cortex and functional development of the motor and associate cortexes in late infancy through the basal ganglia (RTT), and failure in functional development of the prefrontal cortex through the basal ganglia (TS). Further, locomotion failure in early childhood causes failure in development of functional

  11. Detection of dominant flow and abnormal events in surveillance video

    NASA Astrophysics Data System (ADS)

    Kwak, Sooyeong; Byun, Hyeran

    2011-02-01

    We propose an algorithm for abnormal event detection in surveillance video. The proposed algorithm is based on a semi-unsupervised learning method, a kind of feature-based approach so that it does not detect the moving object individually. The proposed algorithm identifies dominant flow without individual object tracking using a latent Dirichlet allocation model in crowded environments. It can also automatically detect and localize an abnormally moving object in real-life video. The performance tests are taken with several real-life databases, and their results show that the proposed algorithm can efficiently detect abnormally moving objects in real time. The proposed algorithm can be applied to any situation in which abnormal directions or abnormal speeds are detected regardless of direction.

  12. Molecular abnormalities in Ewing's sarcoma.

    PubMed

    Burchill, Susan Ann

    2008-10-01

    Ewing's sarcoma is one of the few solid tumors for which the underlying molecular genetic abnormality has been described: rearrangement of the EWS gene on chromosome 22q12 with an ETS gene family member. These translocations define the Ewing's sarcoma family of tumors (ESFT) and provide a valuable tool for their accurate and unequivocal diagnosis. They also represent ideal targets for the development of tumor-specific therapeutics. Although secondary abnormalities occur in over 80% of primary ESFT the clinical utility of these is currently unclear. However, abnormalities in genes that regulate the G(1)/S checkpoint are frequently described and may be important in predicting outcome and response. Increased understanding of the molecular events that arise in ESFT and their role in the development and maintenance of the malignant phenotype will inform the improved stratification of patients for therapy and identify targets and pathways for the design of more effective cancer therapeutics. PMID:18925858

  13. Complex patterns of abnormal heartbeats

    NASA Technical Reports Server (NTRS)

    Schulte-Frohlinde, Verena; Ashkenazy, Yosef; Goldberger, Ary L.; Ivanov, Plamen Ch; Costa, Madalena; Morley-Davies, Adrian; Stanley, H. Eugene; Glass, Leon

    2002-01-01

    Individuals having frequent abnormal heartbeats interspersed with normal heartbeats may be at an increased risk of sudden cardiac death. However, mechanistic understanding of such cardiac arrhythmias is limited. We present a visual and qualitative method to display statistical properties of abnormal heartbeats. We introduce dynamical "heartprints" which reveal characteristic patterns in long clinical records encompassing approximately 10(5) heartbeats and may provide information about underlying mechanisms. We test if these dynamics can be reproduced by model simulations in which abnormal heartbeats are generated (i) randomly, (ii) at a fixed time interval following a preceding normal heartbeat, or (iii) by an independent oscillator that may or may not interact with the normal heartbeat. We compare the results of these three models and test their limitations to comprehensively simulate the statistical features of selected clinical records. This work introduces methods that can be used to test mathematical models of arrhythmogenesis and to develop a new understanding of underlying electrophysiologic mechanisms of cardiac arrhythmia.

  14. Phosphorylation of AKT and abdominal aortic aneurysm formation.

    PubMed

    Ghosh, Abhijit; Lu, Guanyi; Su, Gang; McEvoy, Brendan; Sadiq, Omar; DiMusto, Paul D; Laser, Adriana; Futchko, John S; Henke, Peter K; Eliason, Jonathan L; Upchurch, Gilbert R

    2014-01-01

    It is hypothesized that differential AKT phosphorylation between sexes is important in abdominal aortic aneurysm (AAA) formation. Male C57BL/6 mice undergoing elastase treatment showed a typical AAA phenotype (80% over baseline, P < 0.001) and significantly increased phosphorylated AKT-308 (p308) and total-AKT (T-AKT) at day 14 compared with female mice. Elastase-treated Raw cells produced increased p308 and significant amounts of matrix metalloproteinase 9 (MMP-9), and these effects were suppressed by LY294002 treatment, a known AKT inhibitor. Male and female rat aortic smooth muscle cells treated with elastase for 1, 6, or 24 hours demonstrated that the p308/T-AKT and AKT-Ser-473/T-AKT ratios peaked at 6 hours and were significantly higher in the elastase-treated cells compared with controls. Similarly, male cells had higher phosphorylated AKT/T-AKT levels than female cells. LY294002 also inhibited elastase-induced p308 formation more in female smooth muscle cells than in males, and the corresponding cell media had less pro-MMP-9. AKT siRNA significantly decreased secretion of pro-MMP-9, as well as pro-MMP-2 and active MMP-2 from elastase-treated male rat aortic smooth muscle cells. IHC of male mice AAA aortas showed increased p308, AKT-Ser-473, and T-AKT compared with female mice. Aortas from male AAA patients had a significantly higher p308/T-AKT ratio than female AAA tissues. These data suggest that AKT phosphorylation is important in the upstream regulation of MMP activity, and that differential phosphorylation may be important in sex differences in AAA. PMID:24332015

  15. Alterations of Histone H1 Phosphorylation During Bladder Carcinogenesis

    PubMed Central

    Telu, Kelly H.; Abbaoui, Besma; Thomas-Ahner, Jennifer M.; Zynger, Debra L.; Clinton, Steven K.

    2013-01-01

    There is a crucial need for development of prognostic and predictive biomarkers in human bladder carcinogenesis in order to personalize preventive and therapeutic strategies and improve outcomes. Epigenetic alterations, such as histone modifications, are implicated in the genetic dysregulation that is fundamental to carcinogenesis. Here we focus on profiling the histone modifications during the progression of bladder cancer. Histones were extracted from normal human bladder epithelial cells, an immortalized human bladder epithelial cell line (hTERT), and four human bladder cancer cell lines (RT4, J82, T24, and UMUC3) ranging from superficial low-grade to invasive high-grade cancers. Liquid Chromatography-Mass Spectrometry (LC-MS) profiling revealed a statistically significant increase in phosphorylation of H1 linker histones from normal human bladder epithelial cells to low-grade superficial to high-grade invasive bladder cancer cells. This finding was further validated by immunohistochemical staining of the normal epithelium and transitional cell cancer from human bladders. Cell cycle analysis of histone H1 phosphorylation by western blotting showed an increase of phosphorylation from G0/G1 phase to M phase, again supporting this as a proliferative marker. Changes in histone H1 phosphorylation status may further clarify epigenetic changes during bladder carcinogenesis and provide diagnostic and prognostic biomarkers or targets for future therapeutic interventions. PMID:23675690

  16. Stress Induces Pain Transition by Potentiation of AMPA Receptor Phosphorylation

    PubMed Central

    Li, Changsheng; Yang, Ya; Liu, Sufang; Fang, Huaqiang; Zhang, Yong; Furmanski, Orion; Skinner, John; Xing, Ying; Johns, Roger A.

    2014-01-01

    Chronic postsurgical pain is a serious issue in clinical practice. After surgery, patients experience ongoing pain or become sensitive to incident, normally nonpainful stimulation. The intensity and duration of postsurgical pain vary. However, it is unclear how the transition from acute to chronic pain occurs. Here we showed that social defeat stress enhanced plantar incision-induced AMPA receptor GluA1 phosphorylation at the Ser831 site in the spinal cord and greatly prolonged plantar incision-induced pain. Interestingly, targeted mutation of the GluA1 phosphorylation site Ser831 significantly inhibited stress-induced prolongation of incisional pain. In addition, stress hormones enhanced GluA1 phosphorylation and AMPA receptor-mediated electrical activity in the spinal cord. Subthreshold stimulation induced spinal long-term potentiation in GluA1 phosphomimetic mutant mice, but not in wild-type mice. Therefore, spinal AMPA receptor phosphorylation contributes to the mechanisms underlying stress-induced pain transition. PMID:25297100

  17. A secretory kinase complex regulates extracellular protein phosphorylation

    PubMed Central

    Cui, Jixin; Xiao, Junyu; Tagliabracci, Vincent S; Wen, Jianzhong; Rahdar, Meghdad; Dixon, Jack E

    2015-01-01

    Although numerous extracellular phosphoproteins have been identified, the protein kinases within the secretory pathway have only recently been discovered, and their regulation is virtually unexplored. Fam20C is the physiological Golgi casein kinase, which phosphorylates many secreted proteins and is critical for proper biomineralization. Fam20A, a Fam20C paralog, is essential for enamel formation, but the biochemical function of Fam20A is unknown. Here we show that Fam20A potentiates Fam20C kinase activity and promotes the phosphorylation of enamel matrix proteins in vitro and in cells. Mechanistically, Fam20A is a pseudokinase that forms a functional complex with Fam20C, and this complex enhances extracellular protein phosphorylation within the secretory pathway. Our findings shed light on the molecular mechanism by which Fam20C and Fam20A collaborate to control enamel formation, and provide the first insight into the regulation of secretory pathway phosphorylation. DOI: http://dx.doi.org/10.7554/eLife.06120.001 PMID:25789606

  18. Histone deacetylase inhibitors block IFNγ-induced STAT1 phosphorylation.

    PubMed

    Ginter, Torsten; Bier, Carolin; Knauer, Shirley K; Sughra, Kalsoom; Hildebrand, Dagmar; Münz, Tobias; Liebe, Theresa; Heller, Regine; Henke, Andreas; Stauber, Roland H; Reichardt, Werner; Schmid, Johannes A; Kubatzky, Katharina F; Heinzel, Thorsten; Krämer, Oliver H

    2012-07-01

    Signal transducer and activator of transcription 1 (STAT1) is important for innate and adaptive immunity. Histone deacetylase inhibitors (HDACi) antagonize unbalanced immune functions causing chronic inflammation and cancer. Phosphorylation and acetylation regulate STAT1 and different IFNs induce phosphorylated STAT1 homo-/heterodimers, e.g. IFNα activates several STATs whereas IFNγ only induces phosphorylated STAT1 homodimers. In transformed cells HDACi trigger STAT1 acetylation linked to dephosphorylation by the phosphatase TCP45. It is unclear whether acetylation differentially affects STAT1 activated by IFNα or IFNγ, and if cellular responses to both cytokines depend on a phosphatase-dependent inactivation of acetylated STAT1. Here, we report that HDACi counteract IFN-induced phosphorylation of a critical tyrosine residue in the STAT1 C-terminus in primary cells and hematopoietic cells. STAT1 mutants mimicking a functionally inactive DNA binding domain (DBD) reveal that the number of acetylation-mimicking sites in STAT1 determines whether STAT1 is recruited to response elements after stimulation with IFNγ. Furthermore, we show that IFNα-induced STAT1 heterodimers carrying STAT1 molecules mimicking acetylation bind cognate DNA and provide innate anti-viral immunity. IFNγ-induced acetylated STAT1 homodimers are though inactive, suggesting that heterodimerization and complex formation can rescue STAT1 lacking a functional DBD. Apparently, the type of cytokine determines how acetylation affects the nuclear entry and DNA binding of STAT1. Our data contribute to a better understanding of STAT1 regulation by acetylation. PMID:22425562

  19. CGGBP1 phosphorylation constitutes a telomere-protection signal

    PubMed Central

    Singh, Umashankar; Maturi, Varun; Jones, Rhiannon E; Paulsson, Ylva; Baird, Duncan M; Westermark, Bengt

    2014-01-01

    The shelterin proteins are required for telomere integrity. Shelterin dysfunction can lead to initiation of unwarranted DNA damage and repair pathways at chromosomal termini. Interestingly, many shelterin accessory proteins are involved in DNA damage signaling and repair. We demonstrate here that in normal human fibroblasts, telomeric ends are protected by phosphorylation of CGG triplet repeat-binding protein 1 (CGGBP1) at serine 164 (S164). We show that serine 164 is a major phosphorylation site on CGGBP1 with important functions. We provide evidence that one of the kinases that can phosphorylate S164 CGGBP1 is ATR. Overexpression of S164A phospho-deficient CGGBP1 exerted a dominant-negative effect, causing telomeric dysfunction, accelerated telomere shortening, enhanced fusion of telomeres, and crisis. However, overexpression of wild-type or phospho-mimicking S164E CGGBP1 did not cause these effects. This telomere damage was associated with reduced binding of the shelterin protein POT1 to telomeric DNA. Our results suggest that CGGBP1 phosphorylation at S164 is a novel telomere protection signal, which can affect telomere-protective function of the shelterin complex. PMID:24196442

  20. CDC7 inhibition blocks pathological TDP-43 phosphorylation and neurodegeneration

    PubMed Central

    Liachko, Nicole F.; McMillan, Pamela J.; Guthrie, Chris R.; Bird, Thomas D.; Leverenz, James B.; Kraemer, Brian C.

    2013-01-01

    Objective Kinase hyperactivity occurs in both neurodegenerative disease and cancer. Lesions containing hyperphosphorylated aggregated TDP-43 characterize amyotrophic lateral sclerosis and frontotemporal lobar degeneration with TDP-43 inclusions. Dual phosphorylation of TDP-43 at serines 409/410 drives neurotoxicity in disease models; therefore, TDP-43 specific kinases are candidate targets for intervention. Methods To find therapeutic targets for the prevention of TDP-43 phosphorylation, we assembled and screened a comprehensive RNA interference library targeting kinases in TDP-43 transgenic C. elegans. Results We show CDC7 robustly phosphorylates TDP-43 at pathological residues S409/410 in C. elegans, in vitro, and in human cell culture. In FTLD-TDP cases, CDC7 immunostaining overlaps with the phospho-TDP-43 pathology found in frontal cortex. Furthermore PHA767491, a small molecule inhibitor of CDC7, reduces TDP-43 phosphorylation and prevents TDP-43 dependent neurodegeneration in TDP-43 transgenic animals. Interpretation Taken together these data support CDC7 as a novel therapeutic target for TDP-43 proteinopathies including FTLD-TDP and ALS. PMID:23424178

  1. Phosphorylation sites in BubR1 that regulate kinetochore attachment, tension, and mitotic exit

    PubMed Central

    Huang, Haomin; Hittle, James; Zappacosta, Francesca; Annan, Roland S.; Hershko, Avram; Yen, Timothy J.

    2008-01-01

    BubR1 kinase is essential for the mitotic checkpoint and also for kinetochores to establish microtubule attachments. In this study, we report that BubR1 is phosphorylated in mitosis on four residues that differ from sites recently reported to be phosphorylated by Plk1 (Elowe, S., S. Hummer, A. Uldschmid, X. Li, and E.A. Nigg. 2007. Genes Dev. 21:2205–2219; Matsumura, S., F. Toyoshima, and E. Nishida. 2007. J. Biol. Chem. 282:15217–15227). S670, the most conserved residue, is phosphorylated at kinetochores at the onset of mitosis and dephosphorylated before anaphase onset. Unlike the Plk1-dependent S676 phosphorylation, S670 phosphorylation is sensitive to microtubule attachments but not to kinetochore tension. Functionally, phosphorylation of S670 is essential for error correction and for kinetochores with end-on attachments to establish tension. Furthermore, in vitro data suggest that the phosphorylation status of BubR1 is important for checkpoint inhibition of the anaphase-promoting complex/cyclosome. Finally, RNA interference experiments show that Mps1 is a major but not the exclusive kinase that specifies BubR1 phosphorylation in vivo. The combined data suggest that BubR1 may be an effector of multiple kinases that are involved in discrete aspects of kinetochore attachments and checkpoint regulation. PMID:19015317

  2. Conserved mechanism for coordinating replication fork helicase assembly with phosphorylation of the helicase

    PubMed Central

    Bruck, Irina; Kaplan, Daniel L.

    2015-01-01

    Dbf4-dependent kinase (DDK) phosphorylates minichromosome maintenance 2 (Mcm2) during S phase in yeast, and Sld3 recruits cell division cycle 45 (Cdc45) to minichromosome maintenance 2-7 (Mcm2-7). We show here DDK-phosphoryled Mcm2 preferentially interacts with Cdc45 in vivo, and that Sld3 stimulates DDK phosphorylation of Mcm2 by 11-fold. We identified a mutation of the replication initiation factor Sld3, Sld3-m16, that is specifically defective in stimulating DDK phosphorylation of Mcm2. Wild-type expression levels of sld3-m16 result in severe growth and DNA replication defects. Cells expressing sld3-m16 exhibit no detectable Mcm2 phosphorylation in vivo, reduced replication protein A-ChIP signal at an origin, and diminished Go, Ichi, Ni, and San association with Mcm2-7. Treslin, the human homolog of Sld3, stimulates human DDK phosphorylation of human Mcm2 by 15-fold. DDK phosphorylation of human Mcm2 decreases the affinity of Mcm5 for Mcm2, suggesting a potential mechanism for helicase ring opening. These data suggest a conserved mechanism for replication initiation: Sld3/Treslin coordinates Cdc45 recruitment to Mcm2-7 with DDK phosphorylation of Mcm2 during S phase. PMID:26305950

  3. ERK5 pathway regulates the phosphorylation of tumour suppressor hDlg during mitosis

    SciTech Connect

    Inesta-Vaquera, Francisco A.; Campbell, David G.; Arthur, J. Simon C.; Cuenda, Ana

    2010-08-13

    Research highlights: {yields} hDlg is phosphorylated during mitosis in multiple residues. {yields} Prospho-hDlg is excluded from the midbody during mitosis. {yields} hDlg is not phosphorylated by p38{gamma} or JNK1/2 during mitosis. {yields} ERK5 pathway mediates hDlg phosphorylation in mitosis. -- Abstract: Human disc-large (hDlg) is a scaffold protein critical for the maintenance of cell polarity and adhesion. hDlg is thought to be a tumour suppressor that regulates the cell cycle and proliferation. However, the mechanism and pathways involved in hDlg regulation during these processes is still unclear. Here we report that hDlg is phosphorylated during mitosis, and we establish the identity of at least three residues phosphorylated in hDlg; some are previously unreported. Phosphorylation affects hDlg localisation excluding it from the contact point between the two daughter cells. Our results reveal a previously unreported pathway for hDlg phosphorylation in mitosis and show that ERK5 pathway mediates hDlg cell cycle dependent phosphorylation. This is likely to have important implications in the correct timely mitotic entry and mitosis progression.

  4. Flagellin-induced NLRC4 phosphorylation primes the inflammasome for activation by NAIP5.

    PubMed

    Matusiak, Magdalena; Van Opdenbosch, Nina; Vande Walle, Lieselotte; Sirard, Jean-Claude; Kanneganti, Thirumala-Devi; Lamkanfi, Mohamed

    2015-02-01

    The Nlrc4 inflammasome contributes to immunity against intracellular pathogens that express flagellin and type III secretion systems, and activating mutations in NLRC4 cause autoinflammation in patients. Both Naip5 and phosphorylation of Nlrc4 at Ser533 are required for flagellin-induced inflammasome activation, but how these events converge upon inflammasome activation is not known. Here, we showed that Nlrc4 phosphorylation occurs independently of Naip5 detection of flagellin because Naip5 deletion in macrophages abolished caspase-1 activation, interleukin (IL)-1β secretion, and pyroptosis, but not Nlrc4 phosphorylation by cytosolic flagellin of Salmonella Typhimurium and Yersinia enterocolitica. ASC speck formation and caspase-1 expression also were dispensable for Nlrc4 phosphorylation. Interestingly, Helicobacter pylori flagellin triggered robust Nlrc4 phosphorylation, but failed to elicit caspase-1 maturation, IL-1β secretion, and pyroptosis, suggesting that it retained Nlrc4 Ser533 phosphorylating-activity despite escaping Naip5 detection. In agreement, the flagellin D0 domain was required and sufficient for Nlrc4 phosphorylation, whereas deletion of the S. Typhimurium flagellin carboxy-terminus prevented caspase-1 maturation only. Collectively, this work suggests a biphasic activation mechanism for the Nlrc4 inflammasome in which Ser533 phosphorylation prepares Nlrc4 for subsequent activation by the flagellin sensor Naip5. PMID:25605939

  5. Identification of a novel mitotic phosphorylation motif associated with protein localization to the mitotic apparatus

    SciTech Connect

    Yang, Feng; Camp, David G.; Gritsenko, Marina A.; Luo, Quanzhou; Kelly, Ryan T.; Clauss, Therese RW; Brinkley, William R.; Smith, Richard D.; Stenoien, David L.

    2007-11-16

    The chromosomal passenger complex (CPC) is a critical regulator of chromosome, cytoskeleton and membrane dynamics during mitosis. Here, we identified phosphopeptides and phosphoprotein complexes recognized by a phosphorylation specific antibody that labels the CPC using liquid chromatography coupled to mass spectrometry. A mitotic phosphorylation motif (PX{G/T/S}{L/M}[pS]P or WGL[pS]P) was identified in 11 proteins including Fzr/Cdh1 and RIC-8, two proteins with potential links to the CPC. Phosphoprotein complexes contained known CPC components INCENP, Aurora-B and TD-60, as well as SMAD2, 14-3-3 proteins, PP2A, and Cdk1, a likely kinase for this motif. Protein sequence analysis identified phosphorylation motifs in additional proteins including SMAD2, Plk3 and INCENP. Mitotic SMAD2 and Plk3 phosphorylation was confirmed using phosphorylation specific antibodies, and in the case of Plk3, phosphorylation correlates with its localization to the mitotic apparatus. A mutagenesis approach was used to show INCENP phosphorylation is required for midbody localization. These results provide evidence for a shared phosphorylation event that regulates localization of critical proteins during mitosis.

  6. XIAP is essential for shear stress-enhanced Tyr-576 phosphorylation of FAK

    SciTech Connect

    Ahn, Sunyoung; Park, Heonyong

    2010-08-20

    Research highlights: {yields} Laminar shear stress phosphorylates Tyr-576 in FAK. {yields} XIAP is essential for shear stress-induced phosphorylation of Tyr-576. {yields} XIAP knockdown induces shear stress-triggered translocation of FAK into nucleus. {yields} XIAP regulates ERK activation by maintaining the Src-accessible location of FAK. -- Abstract: In endothelial cells, X-chromosome linked inhibitor of apoptosis protein (XIAP) regulates cell survival, migration and adhesion. We have recently found that XIAP recruits focal adhesion kinase (FAK) into integrin-associated focal adhesions, controlling cell migration. However, little is understood about the molecular mechanisms by which FAK modulation is controlled by XIAP. In this study, we show that XIAP modulates FAK activity through the control of FAK phosphorylation. In bovine aortic endothelial cells (BAEC), phosphorylation of Tyr-576 in FAK is elevated by laminar shear stress. This elevated phosphorylation appears to be responsible for shear stress-stimulated ERK activation. We found that XIAP knockdown reduces shear stress-enhanced phosphorylation of Tyr-576 and induces shear stress-triggered translocation of FAK into nucleus. Nuclear translocation of FAK reduces contact between FAK and Src, a kinase which phosphorylates Tyr-576. This spatial segregation of FAK from Src decreases Tyr-576 phosphorylation and thus shear-stimulated ERK activation. Taken together, our results demonstrate that XIAP plays a key role in shear stress-stimulated ERK activation by maintaining the Src-accessible location of FAK.

  7. Postsynaptic density protein 95-regulated NR2B tyrosine phosphorylation and interactions of Fyn with NR2B in levodopa-induced dyskinesia rat models

    PubMed Central

    Ba, Maowen; Kong, Min; Ma, Guozhao

    2015-01-01

    Context Abnormality in interactions between N-methyl-d-aspartate (NMDA) receptor and its signaling molecules occurs in the lesioned striatum in Parkinson’s disease (PD) and levodopa-induced dyskinesia (LID). It was reported that Fyn-mediated NR2B tyrosine phosphorylation, can enhance NMDA receptor function. Postsynaptic density protein 95 (PSD-95), one of the synapse-associated proteins, regulates interactions between receptor and downstream-signaling molecules. In light of the relationship between PSD-95, NR2B, and Fyn kinases, does PSD-95 contribute to the overactivity of NMDA receptor function induced by dopaminergic treatment? To further prove the possibility, the effects of regulating the PSD-95 expression on the augmented NR2B tyrosine phosphorylation and on the interactions of Fyn and NR2B in LID rat models were evaluated. Methods In the present study, parkinsonian rat models were established by injecting 6-hydroxydopamine. Subsequently, valid PD rats were treated with levodopa (50 mg/kg/day with benserazide 12.5 mg/kg/day, twice daily) intraperitoneally for 22 days to create LID rat models. Then, the effect of pretreatment with an intrastriatal injection of the PSD-95mRNA antisense oligonucleotides (PSD-95 ASO) on the rotational response to levodopa challenge was assessed. The effects of pretreatment with an intrastriatal injection of PSD-95 ASO on the augmented NR2B tyrosine phosphorylation and interactions of Fyn with NR2B in the LID rat models were detected by immunoblotting and immunoprecipitation. Results Levodopa administration twice daily for 22 days to parkinsonian rats shortened the rotational duration and increased the peak turning responses. The altered rotational responses were attenuated by PSD-95 ASO pretreatment. Meanwhile, PSD-95 ASO pretreatment decreased the level of PSD-95 protein expression and reduced both the augmented NR2B tyrosine phosphorylation and interactions of Fyn with NR2B triggered during the levodopa administration in the

  8. Myosin regulatory light chain phosphorylation enhances cardiac β-myosin in vitro motility under load.

    PubMed

    Karabina, Anastasia; Kazmierczak, Katarzyna; Szczesna-Cordary, Danuta; Moore, Jeffrey R

    2015-08-15

    Familial hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy and myofibrillar disarray, and often results in sudden cardiac death. Two HCM mutations, N47K and R58Q, are located in the myosin regulatory light chain (RLC). The RLC mechanically stabilizes the myosin lever arm, which is crucial to myosin's ability to transmit contractile force. The N47K and R58Q mutations have previously been shown to reduce actin filament velocity under load, stemming from a more compliant lever arm (Greenberg, 2010). In contrast, RLC phosphorylation was shown to impart stiffness to the myosin lever arm (Greenberg, 2009). We hypothesized that phosphorylation of the mutant HCM-RLC may mitigate distinct mutation-induced structural and functional abnormalities. In vitro motility assays were utilized to investigate the effects of RLC phosphorylation on the HCM-RLC mutant phenotype in the presence of an α-actinin frictional load. Porcine cardiac β-myosin was depleted of its native RLC and reconstituted with mutant or wild-type human RLC in phosphorylated or non-phosphorylated form. Consistent with previous findings, in the presence of load, myosin bearing the HCM mutations reduced actin sliding velocity compared to WT resulting in 31-41% reductions in force production. Myosin containing phosphorylated RLC (WT or mutant) increased sliding velocity and also restored mutant myosin force production to near WT unphosphorylated values. These results point to RLC phosphorylation as a general mechanism to increase force production of the individual myosin motor and as a potential target to ameliorate the HCM-induced phenotype at the molecular level. PMID:26116789

  9. Myosin regulatory light chain phosphorylation enhances cardiac β-myosin in vitro motility under load

    PubMed Central

    Karabina, Anastasia; Kazmierczak, Katarzyna; Szczesna-Cordary, Danuta; Moore, Jeffrey R.

    2016-01-01

    Familial hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy and myofibrillar disarray, and often results in sudden cardiac death. Two HCM mutations, N47K and R58Q, are located in the myosin regulatory light chain (RLC). The RLC mechanically stabilizes the myosin lever arm, which is crucial to myosin’s ability to transmit contractile force. The N47K and R58Q mutations have previously been shown to reduce actin filament velocity under load, stemming from a more compliant lever arm (Greenberg, 2010). In contrast, RLC phosphorylation was shown to impart stiffness to the myosin lever arm (Greenberg, 2009). We hypothesized that phosphorylation of the mutant HCM-RLC may mitigate distinct mutation-induced structural and functional abnormalities. In vitro motility assays were utilized to investigate the effects of RLC phosphorylation on the HCM-RLC mutant phenotype in the presence of an α-actinin frictional load. Porcine cardiac β-myosin was depleted of its native RLC and reconstituted with mutant or wild-type human RLC in phosphorylated or non-phosphorylated form. Consistent with previous findings, in the presence of load, myosin bearing the HCM mutations reduced actin sliding velocity compared to WT resulting in 31–41% reductions in force production. Myosin containing phosphorylated RLC (WT or mutant) increased sliding velocity and also restored mutant myosin force production to near WT unphosphorylated values. These results point to RLC phosphorylation as a general mechanism to increase force production of the individual myosin motor and as a potential target to ameliorate the HCM-induced phenotype at the molecular level. PMID:26116789

  10. Affibody-mediated retention of the epidermal growth factor receptor in the secretory compartments leads to inhibition of phosphorylation in the kinase domain.

    PubMed

    Vernet, Erik; Lundberg, Emma; Friedman, Mikaela; Rigamonti, Nicolò; Klausing, Sandra; Nygren, Per-Ake; Gräslund, Torbjörn

    2009-09-01

    Abnormal activity of the epidermal growth factor receptor (EGFR) is associated with various cancer-related processes and motivates the search for strategies that can selectively block EGFR signalling. In this study, functional knockdown of EGFR was achieved through expression of an affibody construct, (ZEGFR:1907)(2-)KDEL, with high affinity for EGFR and extended with the amino acids KDEL to make it resident in the secretory compartments. Expression of (ZEGFR:1907)(2-)KDEL resulted in 80% reduction ofthe cell surface level of EGFR, and fluorescent staining for EGFR and the (ZEGFR:1907)(2-)KDEL construct showed overlapping intracellular localisation. Immunocapture of EGFR from cell lysates showed that an intracellular complex between EGFR and the affibody construct had been formed, further indicating aspecific interaction between the affibody construct and EGFR. Surface depletion of EGFR led to a dramatic decrease in the amount of kinase domain phosphorylated EGFR, coincident with a significant decrease in the proliferation rate. PMID:19552886

  11. [Emotion Disorders and Abnormal Perspiration].

    PubMed

    Umeda, Satoshi

    2016-08-01

    This article reviewed the relationship between emotional disorders and abnormal perspiration. First, I focused on local brain areas related to emotional processing, and summarized the functions of the emotional network involving those local areas. Functional disorders followed by the damage in the amygdala, orbitofrontal cortex, and insular cortex were reviewed, including related abnormal perspiration. I then addressed the mechanisms of how autonomic disorders influence emotional processing. Finally, possible future directions for integrated understanding of the connection between neural activities and bodily reactions were discussed. PMID:27503817

  12. Ultrasonographic assessment of abnormal pregnancy.

    PubMed

    England, G C

    1998-07-01

    Ultrasonographic imaging is widely used in small animal practice for the diagnosis of pregnancy and the determination of fetal number. Ultrasonography can also be used to monitor abnormal pregnancies, for example, conceptuses that are poorly developed for their gestational age (and therefore are likely to fail), and pregnancies in which there is embryonic resorption or fetal abortion. An ultrasound examination may reveal fetal abnormalities and therefore alter the management of the pregnant bitch or queen prior to parturition. There are, however, a number of ultrasonographic features of normal pregnancies that may mimic disease, and these must be recognized. PMID:9698618

  13. Molecular profiling of activated neurons by phosphorylated ribosome capture.

    PubMed

    Knight, Zachary A; Tan, Keith; Birsoy, Kivanc; Schmidt, Sarah; Garrison, Jennifer L; Wysocki, Robert W; Emiliano, Ana; Ekstrand, Mats I; Friedman, Jeffrey M

    2012-11-21

    The mammalian brain is composed of thousands of interacting neural cell types. Systematic approaches to establish the molecular identity of functional populations of neurons would advance our understanding of neural mechanisms controlling behavior. Here, we show that ribosomal protein S6, a structural component of the ribosome, becomes phosphorylated in neurons activated by a wide range of stimuli. We show that these phosphorylated ribosomes can be captured from mouse brain homogenates, thereby enriching directly for the mRNAs expressed in discrete subpopulations of activated cells. We use this approach to identify neurons in the hypothalamus regulated by changes in salt balance or food availability. We show that galanin neurons are activated by fasting and that prodynorphin neurons restrain food intake during scheduled feeding. These studies identify elements of the neural circuit that controls food intake and illustrate how the activity-dependent capture of cell-type-specific transcripts can elucidate the functional organization of a complex tissue. PMID:23178128

  14. Fyn is required for oxidative- and hyperosmotic-stress-induced tyrosine phosphorylation of caveolin-1.

    PubMed Central

    Sanguinetti, Amy R; Cao, Haiming; Corley Mastick, Cynthia

    2003-01-01

    Caveolin-1 is phosphorylated on Tyr(14) in response to both oxidative and hyperosmotic stress. In the present paper, we show that this phosphorylation requires activation of the Src family kinase Fyn. Stress-induced caveolin phosphorylation was abolished by three Src kinase inhibitors, SU6656, PP2 and PD180970, and was not observed in fibroblasts derived from a Src, Yes and Fyn triple-knockout mouse (SYF-/-). Using cell lines derived from single-kinase-knockout mice (Src-/-, Yes-/- and Fyn-/-), we show that expression of Fyn, but not Src or Yes, is required for stress-induced caveolin phosphorylation. Heterologous expression of Fyn in the SYF-/- and Fyn-/- cells was sufficient to reconstitute stress-induced caveolin phosphorylation, and overexpression of Fyn in wild-type cells induced hyperphosphorylation of caveolin. Fyn was autophosphorylated following oxidative stress, verifying activation of this kinase. Interestingly, there was a concomitant increase in the phosphorylation of Fyn on its Csk (C-terminal Src kinase) site, indicating feedback inhibition. Csk binds to phosphocaveolin [Cao, Courchesne and Mastick (2002) J. Biol. Chem. 277, 8771-8774] and should phosphorylate any co-localized Src-family kinases. Oxidative-stress-induced phosphorylation of caveolin-1 also requires expression of Abl [Sanguinetti and Mastick (2003) Cell Signal. 15, 289-298]. Using inhibitors and cells derived from knockout mice, we verified a requirement for both Abl and Fyn in stress-induced caveolin phosphorylation in a single cell type. Our data suggest a novel mechanism for attenuation of Src-kinase activity by Abl: stable tyrosine phosphorylation of a scaffolding protein, caveolin, and recruitment of Csk. Paxillin, a substrate of both Abl and Src, organizes a similar regulatory complex. PMID:12921535

  15. Television Quiz Show Simulation

    ERIC Educational Resources Information Center

    Hill, Jonnie Lynn

    2007-01-01

    This article explores the simulation of four television quiz shows for students in China studying English as a foreign language (EFL). It discusses the adaptation and implementation of television quiz shows and how the students reacted to them.

  16. Phosphorylation at serine 52 and 635 does not alter the transport properties of glucosinolate transporter AtGTR1

    PubMed Central

    Jørgensen, Morten Egevang; Olsen, Carl Erik; Halkier, Barbara Ann; Nour-Eldin, Hussam Hassan

    2016-01-01

    Little is known about how plants regulate transporters of defense compounds. In A. thaliana, glucosinolates are transported between tissues by NPF2.10 (AtGTR1) and NPF2.11 (AtGTR2). Mining of the PhosPhat4.0 database showed two cytosol exposed phosphorylation sites for AtGTR1 and one membrane-buried phosphorylation site for AtGTR2. In this study, we investigate whether mutation of the two potential regulatory sites of AtGTR1 affected transport of glucosinolates in Xenopus oocytes. Characterization of AtGTR1 phosphorylation mutants showed that phosphorylation of AtGTR1 - at the two reported phosphorylation sites - is not directly involved in regulating AtGTR1 transport activity. We hypothesize a role for AtGTR1-phosphorylation in regulating protein-protein interactions. PMID:26340317

  17. In the Beginning, There Was Protein Phosphorylation

    PubMed Central

    Kyriakis, John M.

    2014-01-01

    The importance of reversible protein phosphorylation to cellular regulation cannot be overstated. In eukaryotic cells, protein kinase/phosphatase signaling pathways regulate a staggering number of cellular processes, including cell proliferation, cell death (apoptosis, necroptosis, necrosis), metabolism (at both the cellular and organismal levels), behavior and neurological function, development, and pathogen resistance. Although protein phosphorylation as a mode of eukaryotic cell regulation is familiar to most biochemists, many are less familiar with protein kinase/phosphatase signaling networks that function in prokaryotes. In this thematic minireview series, we present four minireviews that cover the important field of prokaryotic protein phosphorylation. PMID:24554697

  18. Rapid changes in plasma membrane protein phosphorylation during initiation of cell wall digestion

    SciTech Connect

    Blowers, D.P.; Boss, W.F.; Trewavas, A.J. )

    1988-02-01

    Plasma membrane vesicles from wild carrot cells grown in suspension culture were isolated by aqueous two-phase partitioning, and ATP-dependent phosphorylation was measured with ({gamma}-{sup 32}P)ATP in the presence and absence of calcium. Treatment of the carrot cells with the cell wall digestion enzymes, driselase, in a sorbitol osmoticum for 1.5 min altered the protein phosphorylation pattern compared to that of cells treated with sorbitol alone. Driselase treatment resulted in decreased phosphorylation of a band of M{sub r} 80,000 which showed almost complete calcium dependence in the osmoticum treated cells; decreased phosphorylation of a band of M{sub r} 15,000 which showed little calcium activation, and appearance of a new band of calcium-dependent phosphorylation at M{sub r} 22,000. However, protein phosphorylation was decreased. Adding driselase to the in vitro reaction mixture caused a general decrease in the membrane protein phosphorylation either in the presence or absence of calcium which did not mimic the in vivo response. Cells labeled in vivo with inorganic {sup 32}P also showed a response to the Driselase treatment. An enzymically active driselas preparation was required for the observed responses.

  19. Cytoplasmic cyclin D1 regulates cell invasion and metastasis through the phosphorylation of paxillin.

    PubMed

    Fusté, Noel P; Fernández-Hernández, Rita; Cemeli, Tània; Mirantes, Cristina; Pedraza, Neus; Rafel, Marta; Torres-Rosell, Jordi; Colomina, Neus; Ferrezuelo, Francisco; Dolcet, Xavier; Garí, Eloi

    2016-01-01

    Cyclin D1 (Ccnd1) together with its binding partner Cdk4 act as a transcriptional regulator to control cell proliferation and migration, and abnormal Ccnd1·Cdk4 expression promotes tumour growth and metastasis. While different nuclear Ccnd1·Cdk4 targets participating in cell proliferation and tissue development have been identified, little is known about how Ccnd1·Cdk4 controls cell adherence and invasion. Here, we show that the focal adhesion component paxillin is a cytoplasmic substrate of Ccnd1·Cdk4. This complex phosphorylates a fraction of paxillin specifically associated to the cell membrane, and promotes Rac1 activation, thereby triggering membrane ruffling and cell invasion in both normal fibroblasts and tumour cells. Our results demonstrate that localization of Ccnd1·Cdk4 to the cytoplasm does not simply act to restrain cell proliferation, but constitutes a functionally relevant mechanism operating under normal and pathological conditions to control cell adhesion, migration and metastasis through activation of a Ccnd1·Cdk4-paxillin-Rac1 axis. PMID:27181366

  20. PINK1-phosphorylated mitofusin 2 is a Parkin receptor for culling damaged mitochondria.

    PubMed

    Chen, Yun; Dorn, Gerald W

    2013-04-26

    Senescent and damaged mitochondria undergo selective mitophagic elimination through mechanisms requiring two Parkinson's disease factors, the mitochondrial kinase PINK1 (PTEN-induced putative kinase protein 1; PTEN is phosphatase and tensin homolog) and the cytosolic ubiquitin ligase Parkin. The nature of the PINK-Parkin interaction and the identity of key factors directing Parkin to damaged mitochondria are unknown. We show that the mitochondrial outer membrane guanosine triphosphatase mitofusin (Mfn) 2 mediates Parkin recruitment to damaged mitochondria. Parkin bound to Mfn2 in a PINK1-dependent manner; PINK1 phosphorylated Mfn2 and promoted its Parkin-mediated ubiqitination. Ablation of Mfn2 in mouse cardiac myocytes prevented depolarization-induced translocation of Parkin to the mitochondria and suppressed mitophagy. Accumulation of morphologically and functionally abnormal mitochondria induced respiratory dysfunction in Mfn2-deficient mouse embryonic fibroblasts and cardiomyocytes and in Parkin-deficient Drosophila heart tubes, causing dilated cardiomyopathy. Thus, Mfn2 functions as a mitochondrial receptor for Parkin and is required for quality control of cardiac mitochondria. PMID:23620051

  1. Cytoplasmic cyclin D1 regulates cell invasion and metastasis through the phosphorylation of paxillin

    PubMed Central

    Fusté, Noel P.; Fernández-Hernández, Rita; Cemeli, Tània; Mirantes, Cristina; Pedraza, Neus; Rafel, Marta; Torres-Rosell, Jordi; Colomina, Neus; Ferrezuelo, Francisco; Dolcet, Xavier; Garí, Eloi

    2016-01-01

    Cyclin D1 (Ccnd1) together with its binding partner Cdk4 act as a transcriptional regulator to control cell proliferation and migration, and abnormal Ccnd1·Cdk4 expression promotes tumour growth and metastasis. While different nuclear Ccnd1·Cdk4 targets participating in cell proliferation and tissue development have been identified, little is known about how Ccnd1·Cdk4 controls cell adherence and invasion. Here, we show that the focal adhesion component paxillin is a cytoplasmic substrate of Ccnd1·Cdk4. This complex phosphorylates a fraction of paxillin specifically associated to the cell membrane, and promotes Rac1 activation, thereby triggering membrane ruffling and cell invasion in both normal fibroblasts and tumour cells. Our results demonstrate that localization of Ccnd1·Cdk4 to the cytoplasm does not simply act to restrain cell proliferation, but constitutes a functionally relevant mechanism operating under normal and pathological conditions to control cell adhesion, migration and metastasis through activation of a Ccnd1·Cdk4-paxillin-Rac1 axis. PMID:27181366

  2. Extracellular Matrix Abnormalities in Schizophrenia

    PubMed Central

    Berretta, Sabina

    2011-01-01

    Emerging evidence points to the involvement of the brain extracellular matrix (ECM) in the pathophysiology of schizophrenia (SZ). Abnormalities affecting several ECM components, including Reelin and chondroitin sulfate proteoglycans (CSPGs), have been described in subjects with this disease. Solid evidence supports the involvement of Reelin, an ECM glycoprotein involved in corticogenesis, synaptic functions and glutamate NMDA receptor regulation, expressed prevalently in distinct populations of GABAergic neurons, which secrete it into the ECM. Marked changes of Reelin expression in SZ have typically been reported in association with GABA-related abnormalities in subjects with SZ and bipolar disorder. Recent findings from our group point to substantial abnormalities affecting CSPGs, a main ECM component, in the amygdala and entorhinal cortex of subjects with schizophrenia, but not bipolar disorder. Striking increases of glial cells expressing CSPGs were accompanied by reductions of perineuronal nets, CSPG- and Reelin-enriched ECM aggregates enveloping distinct neuronal populations. CSPGs developmental and adult functions, including neuronal migration, axon guidance, synaptic and neurotransmission regulation are highly relevant to the pathophysiology of SZ. Together with reports of anomalies affecting several other ECM components, these findings point to the ECM as a key component of the pathology of SZ. We propose that ECM abnormalities may contribute to several aspects of the pathophysiology of this disease, including disrupted connectivity and neuronal migration, synaptic anomalies and altered GABAergic, glutamatergic and dopaminergic neurotransmission. PMID:21856318

  3. A trans acting ribozyme that phosphorylates exogenous RNA.

    PubMed

    Saran, Dayal; Nickens, David G; Burke, Donald H

    2005-11-15

    The structural complexity required for substrate recognition within an active site constrains the evolution of novel catalytic functions. To evaluate those constraints within populations of incipient ribozymes, we performed a selection for kinase ribozymes under conditions that allowed competition for phosphorylation at nine candidate sites. Two candidate sites are the hydroxyl groups on a "quasi-diffusible" chloramphenicol (Cam) moiety tethered to the evolving library through an inert, flexible linker. A subtractive step was included to allow only seven ribose 2' hydroxyls to compete with the two Cam hydroxyls for phosphorylation. After the library was incubated with gamma-thio-ATP (ATPgammaS), active species were recovered from a polyacrylamide gel containing [(N-acryloylamino)phenyl] mercury (APM) and amplified for further cycles of selection. Activity assays on selected isolates and truncated derivatives identified the essential secondary structure of the dominant RNA motif. Phosphorylation was independent of the Cam moiety, indicating ribose 2' phosphorylation. The dominant motif was separated into catalytic "ribozyme" and "substrate" strands. Partial alkaline digestion of the substrate strand before and after phosphorylation identified the precise modification site as the first purine (R) within the required sequence 5'-RAAAANCG-3'. The reaction shows approximately 10-fold preference for ATPgammaS over ATP and is independent of pH over a wide range (5.5-8.9), consistent with a dissociative reaction mechanism that is rate-limited by formation of a metaphosphate transition state. Divalent metal ions are required, with a slight preference of Mn(2+) > Mg(2+) > Ca(2+). Lack of reactivity in [Co(NH(3))(6)](3+) indicates a requirement for inner sphere contact with the metal ion, either for structural stabilization, catalysis, or both. PMID:16274247

  4. Palmitoyl Serotonin Inhibits L-dopa-induced Abnormal Involuntary Movements in the Mouse Parkinson Model.

    PubMed

    Park, Hye-Yeon; Ryu, Young-Kyoung; Go, Jun; Son, Eunjung; Kim, Kyoung-Shim; Kim, Mee Ree

    2016-08-01

    L-3,4-dihydroxyphenylalanine (L-DOPA) is the most common treatment for patients with Parkinson's disease (PD). However, long term use of L-DOPA for PD therapy lead to abnormal involuntary movements (AIMs) known as dyskinesia. Fatty acid amide hydrolase (FAAH) is enriched protein in basal ganglia, and inhibition of the protein reduces dyskinetic behavior of mice. Palmitoyl serotonin (PA-5HT) is a hybrid molecule patterned after arachidonoyl serotonin, antagonist of FAAH. However, the effect of PA-5HT on L-DOPA-induced dyskinesia (LID) in PD have not yet been elucidated. To investigate whether PA-5HT relieve LID in PD and decrease hyperactivation of dopamine D1 receptors, we used the 6-hydroxydopomine (6-OHDA)-lesioned mouse model of PD and treated the L-DOPA (20 mg/kg) for 10 days with PA-5HT (0.3 mg/kg/day). The number of wall contacts with the forelimb in the cylinder test was significantly decreased by 6-OHDA lesion in mice and the pharmacotherapeutic effect of L-DOPA was also revealed in PA-5HT-treated mice. Moreover, in AIMs test, PA-5HT-treated mice showed significant reduction of locomotive, axial, limb, and orofacial AIMs score compared to the vehicle-treated mice. LID-induced hyper-phosphorylation of ERK1/2 and overexpression of FosB/ΔFosB was markedly decreased in 6-OHDA-lesioned striatum of PA-5HT-treated mice, indicating that PA-5HT decreased the dopamine D1 receptor-hyperactivation induced by chronic treatment of L-DOPA in dopamine-denervated striatum. These results suggest that PA-5HT effectively attenuates the development of LID and enhance of ERK1/2 phosphorylation and FosB/ΔFosB expression in the hemi-parkinsonian mouse model. PA-5HT may have beneficial effect on the LID in PD. PMID:27574484

  5. Palmitoyl Serotonin Inhibits L-dopa-induced Abnormal Involuntary Movements in the Mouse Parkinson Model

    PubMed Central

    Park, Hye-Yeon; Ryu, Young-Kyoung; Go, Jun; Son, Eunjung

    2016-01-01

    L-3,4-dihydroxyphenylalanine (L-DOPA) is the most common treatment for patients with Parkinson's disease (PD). However, long term use of L-DOPA for PD therapy lead to abnormal involuntary movements (AIMs) known as dyskinesia. Fatty acid amide hydrolase (FAAH) is enriched protein in basal ganglia, and inhibition of the protein reduces dyskinetic behavior of mice. Palmitoyl serotonin (PA-5HT) is a hybrid molecule patterned after arachidonoyl serotonin, antagonist of FAAH. However, the effect of PA-5HT on L-DOPA-induced dyskinesia (LID) in PD have not yet been elucidated. To investigate whether PA-5HT relieve LID in PD and decrease hyperactivation of dopamine D1 receptors, we used the 6-hydroxydopomine (6-OHDA)-lesioned mouse model of PD and treated the L-DOPA (20 mg/kg) for 10 days with PA-5HT (0.3 mg/kg/day). The number of wall contacts with the forelimb in the cylinder test was significantly decreased by 6-OHDA lesion in mice and the pharmacotherapeutic effect of L-DOPA was also revealed in PA-5HT-treated mice. Moreover, in AIMs test, PA-5HT-treated mice showed significant reduction of locomotive, axial, limb, and orofacial AIMs score compared to the vehicle-treated mice. LID-induced hyper-phosphorylation of ERK1/2 and overexpression of FosB/ΔFosB was markedly decreased in 6-OHDA-lesioned striatum of PA-5HT-treated mice, indicating that PA-5HT decreased the dopamine D1 receptor-hyperactivation induced by chronic treatment of L-DOPA in dopamine-denervated striatum. These results suggest that PA-5HT effectively attenuates the development of LID and enhance of ERK1/2 phosphorylation and FosB/ΔFosB expression in the hemi-parkinsonian mouse model. PA-5HT may have beneficial effect on the LID in PD. PMID:27574484

  6. Band 3 Erythrocyte Membrane Protein Acts as Redox Stress Sensor Leading to Its Phosphorylation by p72 Syk

    PubMed Central

    Pantaleo, Antonella; Ferru, Emanuela; Pau, Maria Carmina; Khadjavi, Amina; Mandili, Giorgia; Mattè, Alessandro; Spano, Alessandra; De Franceschi, Lucia; Pippia, Proto; Turrini, Francesco

    2016-01-01

    In erythrocytes, the regulation of the redox sensitive Tyr phosphorylation of band 3 and its functions are still partially defined. A role of band 3 oxidation in regulating its own phosphorylation has been previously suggested. The current study provides evidences to support this hypothesis: (i) in intact erythrocytes, at 2 mM concentration of GSH, band 3 oxidation, and phosphorylation, Syk translocation to the membrane and Syk phosphorylation responded to the same micromolar concentrations of oxidants showing identical temporal variations; (ii) the Cys residues located in the band 3 cytoplasmic domain are 20-fold more reactive than GSH; (iii) disulfide linked band 3 cytoplasmic domain docks Syk kinase; (iv) protein Tyr phosphatases are poorly inhibited at oxidant concentrations leading to massive band 3 oxidation and phosphorylation. We also observed that hemichromes binding to band 3 determined its irreversible oxidation and phosphorylation, progressive hemolysis, and serine hyperphosphorylation of different cytoskeleton proteins. Syk inhibitor suppressed the phosphorylation of band 3 also preventing serine phosphorylation changes and hemolysis. Our data suggest that band 3 acts as redox sensor regulating its own phosphorylation and that hemichromes leading to the protracted phosphorylation of band 3 may trigger a cascade of events finally leading to hemolysis. PMID:27034738

  7. ADIPOCYTES FROM WOMEN WITH POLYCYSTIC OVARY SYNDROME DEMONSTRATE ALTERED PHOSPHORYLATION AND ACTIVITY OF GLYCOGEN SYNTHASE KINASE 3

    PubMed Central

    Chang, Wendy; Goodarzi, Mark O.; Williams, Heith; Magoffin, Denis A.; Pall, Marita; Azziz, Ricardo

    2009-01-01

    Objective To test the hypothesis that an abnormality in glycogen synthase kinase-3 (GSK3) is a pathogenic factor in PCOS. Design Prospective experimental study (adipocytes). Setting Tertiary care academic medical center and teaching hospital Patients Patients with PCOS and healthy controls. Interventions Blood sampling, physical exam, biopsy of subcutaneous lower abdominal fat. Main Outcome Measure(s) Glucose transport and protein levels and phosphorylation state of GSK3α and GSK3β in adipocytes, assessment of GSK3β activity. Results Basal protein levels of glycogen synthase kinase (GSK3α and GSK3β) did not differ between controls and women with PCOS, nor did basal or insulin-stimulated levels of serine phosphorylated GSK3α. However, in adipocytes of PCOS women insulin stimulation was not associated with increased serine phosphorylation of GSK3β, in contrast to controls. Tyrosine phosphorylation of GSK3β was also higher in PCOS compared to controls. Consistent with the phosphorylation data, GSK3β activity was elevated in PCOS adipocytes. Conclusions These data suggest GSK3β is hyperactivated and resistant to downregulation by insulin in PCOS. Using physiologic approaches, we demonstrated that abnormal GSK3β regulation is a potential mechanism for the insulin resistance seen in some women with PCOS, which may contribute to their development of the syndrome. PMID:18178198

  8. An Extensive Survey of Tyrosine Phosphorylation Revealing New Sites in Human Mammary Epithelial Cells

    SciTech Connect

    Heibeck, Tyler H.; Ding, Shi-Jian; Opresko, Lee K.; Zhao, Rui; Schepmoes, Athena A.; Yang, Feng; Tolmachev, Aleksey V.; Monroe, Matthew E.; Camp, David G.; Smith, Richard D.; Wiley, H. S.; Qian, Weijun

    2009-08-01

    Protein tyrosine phosphorylation is a central regulatory mechanism in cell signaling. To extensively characterize the site-specific tyrosine phosphorylation in human cells, we present here a global survey of tyrosine phosphorylation sites in a normal-derived human mammary epithelial cell (HMEC) line by applying anti-phosphotyrosine (pTyr) peptide immunoaffinity purification (IP) coupled with high sensitivity LC-MS/MS. A total of 481 tyrosine phosphorylation sites (covered by 716 unique peptides) from 285 proteins were confidently identified in HMEC following the analysis of both the basal condition and an acute stimulated condition with epidermal growth factor (EGF). The estimated false discovery rate is 1.0% as measured by comparison against a scrambled database search. Comparison of these data to the literature showed significant agreement in site matches. Additionally 281 sites were not previously observed in HMEC culture were found. Twenty-nine of these sites have not been reported in any human cell or tissue system. The global profiling also allowed us to examine the phosphorylation stoichiometry differences based on spectral count information. Comparison of the data to a previous global proteome profiling study illustrates that most of the highly phoshorylated proteins are of relatively low-abundance. Large differences in phosphorylation stoichiometry for sites within the same protein were also observed for many of the identified proteins, suggesting potentially more important functional roles for those highly phosphorylated pTyr sites within a given protein. By mapping to major signaling networks such as EGF receptor and insulin growth factor-1 receptor signaling pathways, many known proteins involved in these pathways were revealed to be tyrosine phosphorylated, which should allow us to select interesting targeted involved in a given pathway for more directed studies. This extensive HMEC tyrosine phosphorylation dataset represents an important database

  9. Abnormal Activity Detection Using Pyroelectric Infrared Sensors

    PubMed Central

    Luo, Xiaomu; Tan, Huoyuan; Guan, Qiuju; Liu, Tong; Zhuo, Hankz Hankui; Shen, Baihua

    2016-01-01

    Healthy aging is one of the most important social issues. In this paper, we propose a method for abnormal activity detection without any manual labeling of the training samples. By leveraging the Field of View (FOV) modulation, the spatio-temporal characteristic of human activity is encoded into low-dimension data stream generated by the ceiling-mounted Pyroelectric Infrared (PIR) sensors. The similarity between normal training samples are measured based on Kullback-Leibler (KL) divergence of each pair of them. The natural clustering of normal activities is discovered through a self-tuning spectral clustering algorithm with unsupervised model selection on the eigenvectors of a modified similarity matrix. Hidden Markov Models (HMMs) are employed to model each cluster of normal activities and form feature vectors. One-Class Support Vector Machines (OSVMs) are used to profile the normal activities and detect abnormal activities. To validate the efficacy of our method, we conducted experiments in real indoor environments. The encouraging results show that our method is able to detect abnormal activities given only the normal training samples, which aims to avoid the laborious and inconsistent data labeling process. PMID:27271632

  10. Abnormal Activity Detection Using Pyroelectric Infrared Sensors.

    PubMed

    Luo, Xiaomu; Tan, Huoyuan; Guan, Qiuju; Liu, Tong; Zhuo, Hankz Hankui; Shen, Baihua

    2016-01-01

    Healthy aging is one of the most important social issues. In this paper, we propose a method for abnormal activity detection without any manual labeling of the training samples. By leveraging the Field of View (FOV) modulation, the spatio-temporal characteristic of human activity is encoded into low-dimension data stream generated by the ceiling-mounted Pyroelectric Infrared (PIR) sensors. The similarity between normal training samples are measured based on Kullback-Leibler (KL) divergence of each pair of them. The natural clustering of normal activities is discovered through a self-tuning spectral clustering algorithm with unsupervised model selection on the eigenvectors of a modified similarity matrix. Hidden Markov Models (HMMs) are employed to model each cluster of normal activities and form feature vectors. One-Class Support Vector Machines (OSVMs) are used to profile the normal activities and detect abnormal activities. To validate the efficacy of our method, we conducted experiments in real indoor environments. The encouraging results show that our method is able to detect abnormal activities given only the normal training samples, which aims to avoid the laborious and inconsistent data labeling process. PMID:27271632

  11. The Chemical Biology of Protein Phosphorylation

    PubMed Central

    Tarrant, Mary Katherine; Cole, Philip A.

    2011-01-01

    The explosion of scientific interest in protein kinase-mediated signaling networks has led to the infusion of new chemical methods and their applications related to the analysis of phosphorylation pathways. We highlight some of these chemical biology approaches across three areas. First, we discuss the development of chemical tools to modulate the activity of protein kinases to explore kinase mechanisms and their contributions to phosphorylation events and cellular processes. Second, we describe chemical techniques developed in the past few years to dissect the structural and functional effects of phosphate modifications at specific sites in proteins. Third, we cover newly developed molecular imaging approaches to elucidate the spatiotemporal aspects of phosphorylation cascades in live cells. Exciting advances in our understanding of protein phosphorylation have been obtained with these chemical biology approaches, but continuing opportunities for technological innovation remain. PMID:19489734

  12. Varenicline and Abnormal Sleep Related Events

    PubMed Central

    Savage, Ruth L.; Zekarias, Alem; Caduff-Janosa, Pia

    2015-01-01

    Study Objectives: To assess adverse drug reaction reports of “abnormal sleep related events” associated with varenicline, a partial agonist to the α4β2 subtype of nicotinic acetylcholine receptors on neurones, indicated for smoking cessation. Design: Twenty-seven reports of “abnormal sleep related events” often associated with abnormal dreams, nightmares, or somnambulism, which are known to be associated with varenicline use, were identified in the World Health Organisation (WHO) Global Individual Case Safety Reports Database. Original anonymous reports were obtained from the four national pharmacovigilance centers that submitted these reports and assessed for reaction description and causality. Measurements and Results: These 27 reports include 10 of aggressive activity occurring during sleep and seven of other sleep related harmful or potentially harmful activities, such as apparently deliberate self-harm, moving a child or a car, or lighting a stove or a cigarette. Assessment of these 17 reports of aggression or other actual or potential harm showed that nine patients recovered or were recovering on varenicline withdrawal and there were no consistent alternative explanations. Thirteen patients experienced single events, and two had multiple events. Frequency was not stated for the remaining two patients. Conclusions: The descriptions of the reports of aggression during sleep with violent dreaming are similar to those of rapid eye movement sleep behavior disorder and also nonrapid eye movement (NREM) sleep parasomnias in some adults. Patients who experience somnambulism or dreams of a violent nature while taking varenicline should be advised to consult their health providers. Consideration should be given to clarifying the term sleep disorders in varenicline product information and including sleep related harmful and potentially harmful events. Citation: Savage RL, Zekarias A, Caduff-Janosa P. Varenicline and abnormal sleep related events. SLEEP 2015

  13. The Wordpath Show.

    ERIC Educational Resources Information Center

    Anderton, Alice

    The Intertribal Wordpath Society is a nonprofit educational corporation formed to promote the teaching, status, awareness, and use of Oklahoma Indian languages. The Society produces "Wordpath," a weekly 30-minute public access television show about Oklahoma Indian languages and the people who are teaching and preserving them. The show aims to…

  14. Stress-induced inhibition of translation independently of eIF2α phosphorylation.

    PubMed

    Knutsen, Jon Halvor Jonsrud; Rødland, Gro Elise; Bøe, Cathrine Arnason; Håland, Tine Weise; Sunnerhagen, Per; Grallert, Beáta; Boye, Erik

    2015-12-01

    Exposure of fission yeast cells to ultraviolet (UV) light leads to inhibition of translation and phosphorylation of the eukaryotic initiation factor-2α (eIF2α). This phosphorylation is a common response to stress in all eukaryotes. It leads to inhibition of translation at the initiation stage and is thought to be the main reason why stressed cells dramatically reduce protein synthesis. Phosphorylation of eIF2α has been taken as a readout for downregulation of translation, but the role of eIF2α phosphorylation in the downregulation of general translation has not been much investigated. We show here that UV-induced global inhibition of translation in fission yeast cells is independent of eIF2α phosphorylation and the eIF2α kinase general control nonderepressible-2 protein (Gcn2). Also, in budding yeast and mammalian cells, the UV-induced translational depression is largely independent of GCN2 and eIF2α phosphorylation. Furthermore, exposure of fission yeast cells to oxidative stress generated by hydrogen peroxide induced an inhibition of translation that is also independent of Gcn2 and of eIF2α phosphorylation. Our findings show that stress-induced translational inhibition occurs through an unknown mechanism that is likely to be conserved through evolution. PMID:26493332

  15. Stress-induced inhibition of translation independently of eIF2α phosphorylation

    PubMed Central

    Knutsen, Jon Halvor Jonsrud; Rødland, Gro Elise; Bøe, Cathrine Arnason; Håland, Tine Weise; Sunnerhagen, Per; Grallert, Beáta; Boye, Erik

    2015-01-01

    ABSTRACT Exposure of fission yeast cells to ultraviolet (UV) light leads to inhibition of translation and phosphorylation of the eukaryotic initiation factor-2α (eIF2α). This phosphorylation is a common response to stress in all eukaryotes. It leads to inhibition of translation at the initiation stage and is thought to be the main reason why stressed cells dramatically reduce protein synthesis. Phosphorylation of eIF2α has been taken as a readout for downregulation of translation, but the role of eIF2α phosphorylation in the downregulation of general translation has not been much investigated. We show here that UV-induced global inhibition of translation in fission yeast cells is independent of eIF2α phosphorylation and the eIF2α kinase general control nonderepressible-2 protein (Gcn2). Also, in budding yeast and mammalian cells, the UV-induced translational depression is largely independent of GCN2 and eIF2α phosphorylation. Furthermore, exposure of fission yeast cells to oxidative stress generated by hydrogen peroxide induced an inhibition of translation that is also independent of Gcn2 and of eIF2α phosphorylation. Our findings show that stress-induced translational inhibition occurs through an unknown mechanism that is likely to be conserved through evolution. PMID:26493332

  16. Disease Mutations in the Ryanodine Receptor Central Region: Crystal Structures of a Phosphorylation Hot Spot Domain

    SciTech Connect

    Yuchi, Zhiguang; Lau, Kelvin; Van Petegem, Filip

    2015-02-09

    Ryanodine Receptors (RyRs) are huge Ca{sup 2+} release channels in the endoplasmic reticulum membrane and form targets for phosphorylation and disease mutations. We present crystal structures of a domain in three RyR isoforms, containing the Ser2843 (RyR1) and Ser2808/Ser2814 (RyR2) phosphorylation sites. The RyR1 domain is the target for 11 disease mutations. Several of these are clustered near the phosphorylation sites, suggesting that phosphorylation and disease mutations may affect the same interface. The L2867G mutation causes a drastic thermal destabilization and aggregation at room temperature. Crystal structures for other disease mutants show that they affect surface properties and intradomain salt bridges. In vitro phosphorylation experiments show that up to five residues in one long loop of RyR2 can be phosphorylated by PKA or CaMKII. Docking into cryo-electron microscopy maps suggests a putative location in the clamp region, implying that mutations and phosphorylation may affect the allosteric motions within this area.

  17. Intrinsic disorder and multiple phosphorylations constrain the evolution of the flightin N-terminal region.

    PubMed

    Lemas, Dominick; Lekkas, Panagiotis; Ballif, Bryan A; Vigoreaux, Jim O

    2016-03-01

    Flightin is a myosin binding phosphoprotein that originated in the ancestor to Pancrustacea ~500 MYA. In Drosophila melanogaster, flightin is essential for length determination and flexural rigidity of thick filaments. Here, we show that among 12 Drosophila species, the N-terminal region is characterized by low sequence conservation, low pI, a cluster of phosphorylation sites, and a high propensity to intrinsic disorder (ID) that is augmented by phosphorylation. Using mass spectrometry, we identified eight phosphorylation sites within a 29 amino acid segment in the N-terminal region of D. melanogaster flightin. We show that phosphorylation of D. melanogaster flightin is modulated during flight and, through a comparative analysis to orthologs from other Drosophila species, we found phosphorylation sites that remain invariant, sites that retain the charge character, and sites that are clade-specific. While the number of predicted phosphorylation sites differs across species, we uncovered a conserved pattern that relates the number of phosphorylation sites to pI and ID. Extending the analysis to orthologs of other insects, we found additional conserved features in flightin despite the near absence of sequence identity. Collectively, our results demonstrate that structural constraints demarcate the evolution of the highly variable N-terminal region. PMID:26691840

  18. Synaptic activation of ribosomal protein S6 phosphorylation occurs locally in activated dendritic domains.

    PubMed

    Pirbhoy, Patricia Salgado; Farris, Shannon; Steward, Oswald

    2016-06-01

    Previous studies have shown that induction of long-term potentiation (LTP) induces phosphorylation of ribosomal protein S6 (rpS6) in postsynaptic neurons, but the functional significance of rpS6 phosphorylation is poorly understood. Here, we show that synaptic stimulation that induces perforant path LTP triggers phosphorylation of rpS6 (p-rpS6) locally near active synapses. Using antibodies specific for phosphorylation at different sites (ser235/236 versus ser240/244), we show that strong synaptic activation led to dramatic increases in immunostaining throughout postsynaptic neurons with selectively higher staining for p-ser235/236 in the activated dendritic lamina. Following LTP induction, phosphorylation at ser235/236 was detectable by 5 min, peaked at 30 min, and was maintained for hours. Phosphorylation at both sites was completely blocked by local infusion of the NMDA receptor antagonist, APV. Despite robust induction of p-rpS6 following high frequency stimulation, assessment of protein synthesis by autoradiography revealed no detectable increases. Exploration of a novel environment led to increases in the number of p-rpS6-positive neurons throughout the forebrain in a pattern reminiscent of immediate early gene induction and many individual neurons that were p-rpS6-positive coexpressed Arc protein. Our results constrain hypotheses about the possible role of rpS6 phosphorylation in regulating postsynaptic protein synthesis during induction of synaptic plasticity. PMID:27194793

  19. Protein phosphorylation: Localization in regenerating optic axons

    SciTech Connect

    Larrivee, D. )

    1990-09-01

    A number of axonal proteins display changes in phosphorylation during goldfish optic nerve regeneration. (1) To determine whether the phosphorylation of these proteins was closely linked to their synthesis in the retinal ganglion cell body, cycloheximide was injected intraocularly into goldfish whose optic nerves had been regenerating for 3 weeks. Cycloheximide reduced the incorporation of (3H)proline and 32P orthophosphate into total nerve protein by 84% and 46%, respectively. Of the 20 individual proteins examined, 17 contained less than 15% of the (3H)proline label measured in corresponding controls, whereas 18 proteins contained 50% or more of the 32P label, suggesting that phosphorylation was largely independent of synthesis. (2) To determine whether the proteins were phosphorylated in the ganglion cell axons, axonal transport of proteins was blocked by intraocular injection of vincristine. Vincristine reduced (3H)proline labeling of total protein by 88% and 32P labeling by 49%. Among the individual proteins (3H)proline labeling was reduced by 90% or more in 18 cases but 32P labeling was reduced only by 50% or less. (3) When 32P was injected into the cranial cavity near the ends of the optic axons, all of the phosphoproteins were labeled more intensely in the optic tract than in the optic nerve. These results suggest that most of the major phosphoproteins that undergo changes in phosphorylation in the course of regeneration are phosphorylated in the optic axons.

  20. Protein phosphorylation in neurodegeneration: friend or foe?

    PubMed Central

    Tenreiro, Sandra; Eckermann, Katrin; Outeiro, Tiago F.

    2014-01-01

    Protein misfolding and aggregation is a common hallmark in neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), and fronto-temporal dementia (FTD). In these disorders, the misfolding and aggregation of specific proteins occurs alongside neuronal degeneration in somewhat specific brain areas, depending on the disorder and the stage of the disease. However, we still do not fully understand the mechanisms governing protein aggregation, and whether this constitutes a protective or detrimental process. In PD, alpha-synuclein (aSyn) forms protein aggregates, known as Lewy bodies, and is phosphorylated at serine 129. Other residues have also been shown to be phosphorylated, but the significance of phosphorylation in the biology and pathophysiology of the protein is still controversial. In AD and in FTD, hyperphosphorylation of tau protein causes its misfolding and aggregation. Again, our understanding of the precise consequences of tau phosphorylation in the biology and pathophysiology of the protein is still limited. Through the use of a variety of model organisms and technical approaches, we are now gaining stronger insight into the effects of phosphorylation in the behavior of these proteins. In this review, we cover recent findings in the field and discuss how targeting phosphorylation events might be used for therapeutic intervention in these devastating diseases of the nervous system. PMID:24860424

  1. Protein phosphorylation during Plasmodium berghei gametogenesis.

    PubMed

    Alonso-Morales, Alberto; González-López, Lorena; Cázares-Raga, Febe Elena; Cortés-Martínez, Leticia; Torres-Monzón, Jorge Aurelio; Gallegos-Pérez, José Luis; Rodríguez, Mario Henry; James, Anthony A; Hernández-Hernández, Fidel de la Cruz

    2015-09-01

    Plasmodium gametogenesis within the mosquito midgut is a complex differentiation process involving signaling mediated by phosphorylation, which modulate metabolic routes and protein synthesis required to complete this development. However, the mechanisms leading to gametogenesis activation are poorly understood. We analyzed protein phosphorylation during Plasmodium berghei gametogenesis in vitro in serum-free medium using bidimensional electrophoresis (2-DE) combined with immunoblotting (IB) and antibodies specific to phosphorylated serine, threonine and tyrosine. Approximately 75 protein exhibited phosphorylation changes, of which 23 were identified by mass spectrometry. These included components of the cytoskeleton, heat shock proteins, and proteins involved in DNA synthesis and signaling pathways among others. Novel phosphorylation events support a role for these proteins during gametogenesis. The phosphorylation sites of six of the identified proteins, HSP70, WD40 repeat protein msi1, enolase, actin-1 and two isoforms of large subunit of ribonucleoside reductase were investigated using TiO2 phosphopeptides enrichment and tandem mass spectrometry. In addition, transient exposure to hydroxyurea, an inhibitor of ribonucleoside reductase, impaired male gametocytes exflagellation in a dose-dependent manner, and provides a resource for functional studies. PMID:26008612

  2. Fibronectin phosphorylation by ecto-protein kinase

    SciTech Connect

    Imada, Sumi; Sugiyama, Yayoi; Imada, Masaru )

    1988-12-01

    The presence of membrane-associated, extracellular protein kinase (ecto-protein kinase) and its substrate proteins was examined with serum-free cultures of Swiss 3T3 fibroblast. When cells were incubated with ({gamma}-{sup 32})ATP for 10 min at 37{degree}C, four proteins with apparent molecular weights between 150 and 220 kDa were prominently phosphorylated. These proteins were also radiolabeled by lactoperoxidase catalyzed iodination and were sensitive to mild tryptic digestion, suggesting that they localized on the cell surface or in the extracellular matrix. Phosphorylation of extracellular proteins with ({gamma}-{sup 32}P)ATP in intact cell culture is consistent with the existence of ecto-protein kinase. Anti-fibronectin antibody immunoprecipitated one of the phosphoproteins which comigrated with a monomer and a dimer form of fibronectin under reducing and nonreducing conditions of electrophoresis, respectively. The protein had affinity for gelatin as demonstrated by retention with gelatin-conjugated agarose. This protein substrate of ecto-protein kinase was thus concluded to be fibronectin. The sites of phosphorylation by ecto-protein kinase were compared with those of intracellularly phosphorylated fibronectin by the analysis of radiolabeled amino acids and peptides. Ecto-protein kinase phosphorylated fibronectin at serine and threonine residues which were distinct from the sites of intracellular fibronectin phosphorylation.

  3. Long-term dynamics of multisite phosphorylation.

    PubMed

    Rubinstein, Boris Y; Mattingly, Henry H; Berezhkovskii, Alexander M; Shvartsman, Stanislav Y

    2016-07-15

    Multisite phosphorylation cycles are ubiquitous in cell regulation systems and are studied at multiple levels of complexity, from molecules to organisms, with the ultimate goal of establishing predictive understanding of the effects of genetic and pharmacological perturbations of protein phosphorylation in vivo. Achieving this goal is essentially impossible without mathematical models, which provide a systematic framework for exploring dynamic interactions of multiple network components. Most of the models studied to date do not discriminate between the distinct partially phosphorylated forms and focus on two limiting reaction regimes, distributive and processive, which differ in the number of enzyme-substrate binding events needed for complete phosphorylation or dephosphorylation. Here we use a minimal model of extracellular signal-related kinase regulation to explore the dynamics of a reaction network that includes all essential phosphorylation forms and arbitrary levels of reaction processivity. In addition to bistability, which has been studied extensively in distributive mechanisms, this network can generate periodic oscillations. Both bistability and oscillations can be realized at high levels of reaction processivity. Our work provides a general framework for systematic analysis of dynamics in multisite phosphorylation systems. PMID:27226482

  4. Compartment-Specific Phosphorylation of Squid Neurofilaments.

    PubMed

    Grant, Philip; Pant, Harish C

    2016-01-01

    Studies of the giant axon and synapse of third-order neurons in the squid stellate ganglion have provided a vast literature on neuronal physiology and axon transport. Large neuronal size also lends itself to comparative biochemical studies of cell body versus axon. These have focused on the regulation of synthesis, assembly, posttranslational modification and function of neuronal cytoskeletal proteins (microtubules (MTs) and neurofilaments (NFs)), the predominant proteins in axoplasm. These contribute to axonal organization, stability, transport, and impulse transmission responsible for rapid contractions of mantle muscles underlying jet propulsion. Studies of vertebrate NFs have established an extensive literature on NF structure, organization, and function; studies of squid NFs, however, have made it possible to compare compartment-specific regulation of NF synthesis, assembly, and function in soma versus axoplasm. Since NFs contain over 100 eligible sites for phosphorylation by protein kinases, the compartment-specific patterns of phosphorylation have been a primary focus of biochemical studies. We have learned that NF phosphorylation is tightly compartmentalized; extensive phosphorylation occurs only in the axonal compartment in squid and in vertebrate neurons. This extensive phosphorylation plays a key role in organizing NFs, in association with microtubules (MTs), into a stable, dynamic functional lattice that supports axon growth, diameter, impulse transmission, and synaptic activity. To understand how cytoskeletal phosphorylation is topographically regulated, the kinases and phosphatases, bound to NFs isolated from cell bodies and axoplasm, have also been studied. PMID:26795486

  5. Phosphorylation meets nuclear import: a review

    PubMed Central

    2010-01-01

    Phosphorylation is the most common and pleiotropic modification in biology, which plays a vital role in regulating and finely tuning a multitude of biological pathways. Transport across the nuclear envelope is also an essential cellular function and is intimately linked to many degeneration processes that lead to disease. It is therefore not surprising that phosphorylation of cargos trafficking between the cytoplasm and nucleus is emerging as an important step to regulate nuclear availability, which directly affects gene expression, cell growth and proliferation. However, the literature on phosphorylation of nucleocytoplasmic trafficking cargos is often confusing. Phosphorylation, and its mirror process dephosphorylation, has been shown to have opposite and often contradictory effects on the ability of cargos to be transported across the nuclear envelope. Without a clear connection between attachment of a phosphate moiety and biological response, it is difficult to fully understand and predict how phosphorylation regulates nucleocytoplasmic trafficking. In this review, we will recapitulate clue findings in the field and provide some general rules on how reversible phosphorylation can affect the nuclear-cytoplasmic localization of substrates. This is only now beginning to emerge as a key regulatory step in biology. PMID:21182795

  6. Chromosome abnormalities in Indonesian patients with short stature

    PubMed Central

    2012-01-01

    Background Short stature is associated with several disorders including wide variations of chromosomal disorders and single gene disorders. The objective of this report is to present the cytogenetic findings in Indonesian patients with short stature. Methods G-banding and interphase/metaphase FISH were performed on short stature patients with and without other clinical features who were referred by clinicians all over Indonesia to our laboratory during the year 2003–2009. Results The results of chromosomal analysis of ninety seven patients (mean age: 10.7 years old) were collected. The group of patients with other clinical features showed sex chromosome abnormalities in 45% (18/40) and autosomal abnormalities in 10% (4/40), whereas those with short stature only, 42.1% (24/57) had sex chromosome abnormalities and 1.75% (1/57) had autosomal abnormalities. The autosomal chromosomal abnormalities involved mostly subtelomeric regions. Results discrepancies between karyotype and FISH were found in 10 patients, including detection of low-level monosomy X mosaicism in 6 patients with normal karyotype, and detection of mosaic aneuploidy chromosome 18 in 1 patient with 45,XX,rob(13;14)(q10;q10). Statistical analysis showed no significant association between the groups and the type of chromosomal abnormalities. Conclusion Chromosome abnormalities account for about 50% of the short stature patients. Wide variations of both sex and autosomal chromosomes abnormalities were detected in the study. Since three out of five patients had autosomal structural abnormalities involving the subtelomeric regions, thus in the future, subtelomeric FISH or even a more sensitive method such as genomic/SNP microarray is needed to confirm deletions of subtelomeric regions of chromosome 9, 11 and 18. Low-level mosaicism in normal karyotype patients indicates interphase FISH need to be routinely carried out in short stature patients as an adjunct to karyotyping. PMID:22863325

  7. A Holographic Road Show.

    ERIC Educational Resources Information Center

    Kirkpatrick, Larry D.; Rugheimer, Mac

    1979-01-01

    Describes the viewing sessions and the holograms of a holographic road show. The traveling exhibits, believed to stimulate interest in physics, include a wide variety of holograms and demonstrate several physical principles. (GA)

  8. PEBP2-beta/CBF-beta-dependent phosphorylation of RUNX1 and p300 by HIPK2: implications for leukemogenesis.

    PubMed

    Wee, Hee-Jun; Voon, Dominic Chih-Cheng; Bae, Suk-Chul; Ito, Yoshiaki

    2008-11-01

    The heterodimeric transcription factor RUNX1/PEBP2-beta (also known as AML1/CBF-beta) is essential for definitive hematopoiesis. Here, we show that interaction with PEBP2-beta leads to the phosphorylation of RUNX1, which in turn induces p300 phosphorylation. This is mediated by homeodomain interacting kinase 2 (HIPK2), targeting Ser(249), Ser(273), and Thr(276) in RUNX1, in a manner that is also dependent on the RUNX1 PY motif. Importantly, we observed the in vitro disruption of this phosphorylation cascade by multiple leukemogenic genetic defects targeting RUNX1/CBFB. In particular, the oncogenic protein PEBP2-beta-SMMHC prevents RUNX1/p300 phosphorylation by sequestering HIPK2 to mislocalized RUNX1/beta-SMMHC complexes. Therefore, phosphorylation of RUNX1 appears a critical step in its association with and phosphorylation of p300, and its disruption may be a common theme in RUNX1-associated leukemogenesis. PMID:18695000

  9. Phosphorylation of the beta-subunit of CD11/CD18 integrins by protein kinase C correlates with leukocyte adhesion.

    PubMed

    Valmu, L; Autero, M; Siljander, P; Patarroyo, M; Gahmberg, C G

    1991-11-01

    Adhesion of activated leukocytes to cells is of critical functional importance. The adhesion is known to be mediated mainly by the CD11/CD18 integrins, also known as leukocytic cell adhesion molecules, or Leu-CAM. We have now studied the phosphorylation of Leu-CAM by protein kinase C and the correlation of phosphorylation with the generation of the adhesive phenotype among human peripheral blood mononuclear leukocytes during cell activation. We here show that a good correlation exists between the phosphorylation of the beta subunit of Leu-CAM (CD18), and the extent of cell-to-cell adhesion. The phosphorylated CD18 subunit was associated with both CD11a and CD11b. Purified protein kinase C was able to phosphorylate the beta subunit of isolated Leu-CAM in vitro. The phosphorylation occurred mainly on serine residues. PMID:1682156

  10. Phosphorylation of Sox9 is required for neural crest delamination and is regulated downstream of BMP and canonical Wnt signaling.

    PubMed

    Liu, Jessica A J; Wu, Ming-Hoi; Yan, Carol H; Chau, Bolton K H; So, Henry; Ng, Alvis; Chan, Alan; Cheah, Kathryn S E; Briscoe, James; Cheung, Martin

    2013-02-19

    Coordination of neural crest cell (NCC) induction and delamination is orchestrated by several transcription factors. Among these, Sry-related HMG box-9 (Sox9) and Snail2 have been implicated in both the induction of NCC identity and, together with phoshorylation, NCC delamination. How phosphorylation effects this function has not been clear. Here we show, in the developing chick neural tube, that phosphorylation of Sox9 on S64 and S181 facilitates its SUMOylation, and the phosphorylated forms of Sox9 are essential for trunk neural crest delamination. Both phosphorylation and to a lesser extent SUMOylation, of Sox9 are required to cooperate with Snail2 to promote delamination. Moreover, bone morphogenetic protein and canonical Wnt signaling induce phosphorylation of Sox9, thereby connecting extracellular signals with the delamination of NCCs. Together the data suggest a model in which extracellular signals initiate phosphorylation of Sox9 and its cooperation with Snail2 to induce NCC delamination. PMID:23382206

  11. GLIAL ABNORMALITIES IN MOOD DISORDERS

    PubMed Central

    Öngür, Dost; Bechtholt, Anita J.; Carlezon, William A.; Cohen, Bruce M.

    2015-01-01

    Multiple lines of evidence indicate that mood disorders are associated with abnormalities in the brain's cellular composition, especially in glial cells. Considered inert support cells in the past, glial cells are now known to be important for brain function. Treatments for mood disorders enhance glial cell proliferation, and experimental stimulation of cell growth has antidepressant effects in animal models of mood disorders. These findings suggest that the proliferation and survival of glial cells may be important in the pathogenesis of mood disorders and may be possible targets for the development of new treatments. In this chapter, we will review the evidence for glial abnormalities in mood disorders. We will discuss glial cell biology and evidence from postmortem studies of mood disorders. This is not carry out a comprehensive review; rather we selectively discuss existing evidence in building an argument for the role of glial cells in mood disorders. PMID:25377605

  12. Capillary Isoelectric Focusing of Akt Isoforms Identifies Highly Dynamic Phosphorylation in Neuronal Cells and Brain Tissue.

    PubMed

    Schrötter, Sandra; Leondaritis, George; Eickholt, Britta J

    2016-05-01

    The PI3K/PTEN/Akt pathway has been established as a core signaling pathway that is crucial for the integration of neurons into neuronal circuits and the maintenance of the architecture and function of neurons in the adult brain. Akt1-3 kinases are specifically activated by two phosphorylation events on residues Thr(308) and Ser(473) upon growth factor signaling, which subsequently phosphorylate a vast cohort of downstream targets. However, we still lack a clear understanding of the complexity and regulation of isoform specificity within the PI3K/PTEN/Akt pathway. We utilized a capillary-based isoelectric focusing method to study dynamics of Akt phosphorylation in neuronal cells and the developing brain and identify previously undescribed features of Akt phosphorylation and activation. First, we show that the accumulation of multiple phosphorylation events on Akt forms occur concurrently with Ser(473) and Thr(308) phosphorylation upon acute PI3K activation and provide evidence for uncoupling of Ser(473) and Thr(308) phosphorylation, as well as differential sensitivities of Akt1 forms upon PI3K inhibition. Second, we detect a transient shift in Akt isoform phosphorylation and activation pattern during early postnatal brain development, at stages corresponding to synapse development and maturation. Third, we show differential sensitivities of Ser(473)-Akt species to PTEN deletion in mature neurons, which suggests inherent differences in the Akt pools that are accessible to growth factors as compared with the pools that are controlled by PTEN. Our study demonstrates the presence of complex phosphorylation events of Akt in a time- and signal-dependent manner in neurons. PMID:26945062

  13. Capillary Isoelectric Focusing of Akt Isoforms Identifies Highly Dynamic Phosphorylation in Neuronal Cells and Brain Tissue*

    PubMed Central

    Schrötter, Sandra; Leondaritis, George; Eickholt, Britta J.

    2016-01-01

    The PI3K/PTEN/Akt pathway has been established as a core signaling pathway that is crucial for the integration of neurons into neuronal circuits and the maintenance of the architecture and function of neurons in the adult brain. Akt1–3 kinases are specifically activated by two phosphorylation events on residues Thr308 and Ser473 upon growth factor signaling, which subsequently phosphorylate a vast cohort of downstream targets. However, we still lack a clear understanding of the complexity and regulation of isoform specificity within the PI3K/PTEN/Akt pathway. We utilized a capillary-based isoelectric focusing method to study dynamics of Akt phosphorylation in neuronal cells and the developing brain and identify previously undescribed features of Akt phosphorylation and activation. First, we show that the accumulation of multiple phosphorylation events on Akt forms occur concurrently with Ser473 and Thr308 phosphorylation upon acute PI3K activation and provide evidence for uncoupling of Ser473 and Thr308 phosphorylation, as well as differential sensitivities of Akt1 forms upon PI3K inhibition. Second, we detect a transient shift in Akt isoform phosphorylation and activation pattern during early postnatal brain development, at stages corresponding to synapse development and maturation. Third, we show differential sensitivities of Ser473-Akt species to PTEN deletion in mature neurons, which suggests inherent differences in the Akt pools that are accessible to growth factors as compared with the pools that are controlled by PTEN. Our study demonstrates the presence of complex phosphorylation events of Akt in a time- and signal-dependent manner in neurons. PMID:26945062

  14. Tyrosine Phosphorylation of Caspase-8 Abrogates Its Apoptotic Activity and Promotes Activation of c-Src

    PubMed Central

    Tsang, Jennifer LY; Jia, Song Hui; Parodo, Jean; Plant, Pamela; Lodyga, Monika; Charbonney, Emmanuel; Szaszi, Katalin; Kapus, Andras; Marshall, John C.

    2016-01-01

    Src family tyrosine kinases (SFKs) phosphorylate caspase-8A at tyrosine (Y) 397 resulting in suppression of apoptosis. In addition, the phosphorylation of caspase-8A at other sites including Y465 has been implicated in the regulation of caspase-8 activity. However, the functional consequences of these modifications on caspase-8 processing/activity have not been elucidated. Moreover, various Src substrates are known to act as potent Src regulators, but no such role has been explored for caspase-8. We asked whether the newly identified caspase-8 phosphorylation sites might regulate caspase-8 activation and conversely, whether caspase-8 phosphorylation might affect Src activity. Here we show that Src phosphorylates caspase-8A at multiple tyrosine sites; of these, we have focused on Y397 within the linker region and Y465 within the p12 subunit of caspase-8A. We show that phosphomimetic mutation of caspase-8A at Y465 prevents its cleavage and the subsequent activation of caspase-3 and suppresses apoptosis. Furthermore, simultaneous phosphomimetic mutation of caspase-8A at Y397 and Y465 promotes the phosphorylation of c-Src at Y416 and increases c-Src activity. Finally, we demonstrate that caspase-8 activity prevents its own tyrosine phosphorylation by Src. Together these data reveal that dual phosphorylation converts caspase-8 from a pro-apoptotic to a pro-survival mediator. Specifically, tyrosine phosphorylation by Src renders caspase-8 uncleavable and thereby inactive, and at the same time converts it to a Src activator. This novel dynamic interplay between Src and caspase-8 likely acts as a potent signal-integrating switch directing the cell towards apoptosis or survival. PMID:27101103

  15. Ouabain-induced changes in MAP kinase phosphorylation in primary culture of rat cerebellar cells.

    PubMed

    Lopachev, Alexander V; Lopacheva, Olga M; Osipova, Ekaterina A; Vladychenskaya, Elizaveta A; Smolyaninova, Larisa V; Fedorova, Tatiana N; Koroleva, Olga V; Akkuratov, Evgeny E

    2016-07-01

    Cardiotonic steroid (CTS) ouabain is a well-established inhibitor of Na,K-ATPase capable of inducing signalling processes including changes in the activity of the mitogen activated protein kinases (MAPK) in various cell types. With increasing evidence of endogenous CTS in the blood and cerebrospinal fluid, it is of particular interest to study ouabain-induced signalling in neurons, especially the activation of MAPK, because they are the key kinases activated in response to extracellular signals and regulating cell survival, proliferation and apoptosis. In this study we investigated the effect of ouabain on the level of phosphorylation of three MAPK (ERK1/2, JNK and p38) and on cell survival in the primary culture of rat cerebellar cells. Using Western blotting we described the time course and concentration dependence of phosphorylation for ERK1/2, JNK and p38 in response to ouabain. We discovered that ouabain at a concentration of 1 μM does not cause cell death in cultured neurons while it changes the phosphorylation level of the three MAPK: ERK1/2 is phosphorylated transiently, p38 shows sustained phosphorylation, and JNK is dephosphorylated after a long-term incubation. We showed that ERK1/2 phosphorylation increase does not depend on ouabain-induced calcium increase and p38 activation. Changes in p38 phosphorylation, which is independent from ERK1/2 activation, are calcium dependent. Changes in JNK phosphorylation are calcium dependent and also depend on ERK1/2 and p38 activation. Ten-micromolar ouabain leads to cell death, and we conclude that different effects of 1-μM and 10-μM ouabain depend on different ERK1/2 and p38 phosphorylation profiles. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27338714

  16. Show What You Know

    ERIC Educational Resources Information Center

    Eccleston, Jeff

    2007-01-01

    Big things come in small packages. This saying came to the mind of the author after he created a simple math review activity for his fourth grade students. Though simple, it has proven to be extremely advantageous in reinforcing math concepts. He uses this activity, which he calls "Show What You Know," often. This activity provides the perfect…

  17. The Ozone Show.

    ERIC Educational Resources Information Center

    Mathieu, Aaron

    2000-01-01

    Uses a talk show activity for a final assessment tool for students to debate about the ozone hole. Students are assessed on five areas: (1) cooperative learning; (2) the written component; (3) content; (4) self-evaluation; and (5) peer evaluation. (SAH)

  18. Honored Teacher Shows Commitment.

    ERIC Educational Resources Information Center

    Ratte, Kathy

    1987-01-01

    Part of the acceptance speech of the 1985 National Council for the Social Studies Teacher of the Year, this article describes the censorship experience of this honored social studies teacher. The incident involved the showing of a videotape version of the feature film entitled "The Seduction of Joe Tynan." (JDH)

  19. Talk Show Science.

    ERIC Educational Resources Information Center

    Moore, Mitzi Ruth

    1992-01-01

    Proposes having students perform skits in which they play the roles of the science concepts they are trying to understand. Provides the dialog for a skit in which hot and cold gas molecules are interviewed on a talk show to study how these properties affect wind, rain, and other weather phenomena. (MDH)

  20. Stage a Water Show

    ERIC Educational Resources Information Center

    Frasier, Debra

    2008-01-01

    In the author's book titled "The Incredible Water Show," the characters from "Miss Alaineus: A Vocabulary Disaster" used an ocean of information to stage an inventive performance about the water cycle. In this article, the author relates how she turned the story into hands-on science teaching for real-life fifth-grade students. The author also…

  1. Showing What They Know

    ERIC Educational Resources Information Center

    Cech, Scott J.

    2008-01-01

    Having students show their skills in three dimensions, known as performance-based assessment, dates back at least to Socrates. Individual schools such as Barrington High School--located just outside of Providence--have been requiring students to actively demonstrate their knowledge for years. The Rhode Island's high school graduating class became…

  2. Tyrosine 370 phosphorylation of ATM positively regulates DNA damage response

    PubMed Central

    Lee, Hong-Jen; Lan, Li; Peng, Guang; Chang, Wei-Chao; Hsu, Ming-Chuan; Wang, Ying-Nai; Cheng, Chien-Chia; Wei, Leizhen; Nakajima, Satoshi; Chang, Shih-Shin; Liao, Hsin-Wei; Chen, Chung-Hsuan; Lavin, Martin; Ang, K Kian; Lin, Shiaw-Yih; Hung, Mien-Chie

    2015-01-01

    Ataxia telangiectasia mutated (ATM) mediates DNA damage response by controling irradiation-induced foci formation, cell cycle checkpoint, and apoptosis. However, how upstream signaling regulates ATM is not completely understood. Here, we show that upon irradiation stimulation, ATM associates with and is phosphorylated by epidermal growth factor receptor (EGFR) at Tyr370 (Y370) at the site of DNA double-strand breaks. Depletion of endogenous EGFR impairs ATM-mediated foci formation, homologous recombination, and DNA repair. Moreover, pretreatment with an EGFR kinase inhibitor, gefitinib, blocks EGFR and ATM association, hinders CHK2 activation and subsequent foci formation, and increases radiosensitivity. Thus, we reveal a critical mechanism by which EGFR directly regulates ATM activation in DNA damage response, and our results suggest that the status of ATM Y370 phosphorylation has the potential to serve as a biomarker to stratify patients for either radiotherapy alone or in combination with EGFR inhibition. PMID:25601159

  3. Interplay between phosphorylation and palmitoylation mediates plasma membrane targeting and sorting of GAP43

    PubMed Central

    Gauthier-Kemper, Anne; Igaev, Maxim; Sündermann, Frederik; Janning, Dennis; Brühmann, Jörg; Moschner, Katharina; Reyher, Hans-Jürgen; Junge, Wolfgang; Glebov, Konstantin; Walter, Jochen; Bakota, Lidia; Brandt, Roland

    2014-01-01

    Phosphorylation and lipidation provide posttranslational mechanisms that contribute to the distribution of cytosolic proteins in growing nerve cells. The growth-associated protein GAP43 is susceptible to both phosphorylation and S-palmitoylation and is enriched in the tips of extending neurites. However, how phosphorylation and lipidation interplay to mediate sorting of GAP43 is unclear. Using a combination of biochemical, genetic, and imaging approaches, we show that palmitoylation is required for membrane association and that phosphorylation at Ser-41 directs palmitoylated GAP43 to the plasma membrane. Plasma membrane association decreased the diffusion constant fourfold in neuritic shafts. Sorting to the neuritic tip required palmitoylation and active transport and was increased by phosphorylation-mediated plasma membrane interaction. Vesicle tracking revealed transient association of a fraction of GAP43 with exocytic vesicles and motion at a fast axonal transport rate. Simulations confirmed that a combination of diffusion, dynamic plasma membrane interaction and active transport of a small fraction of GAP43 suffices for efficient sorting to growth cones. Our data demonstrate a complex interplay between phosphorylation and lipidation in mediating the localization of GAP43 in neuronal cells. Palmitoylation tags GAP43 for global sorting by piggybacking on exocytic vesicles, whereas phosphorylation locally regulates protein mobility and plasma membrane targeting of palmitoylated GAP43. PMID:25165142

  4. Mimicking phosphorylation of alphaB-crystallin affects its chaperone activity.

    PubMed

    Ecroyd, Heath; Meehan, Sarah; Horwitz, Joseph; Aquilina, J Andrew; Benesch, Justin L P; Robinson, Carol V; Macphee, Cait E; Carver, John A

    2007-01-01

    AlphaB-crystallin is a member of the sHsp (small heat-shock protein) family that prevents misfolded target proteins from aggregating and precipitating. Phosphorylation at three serine residues (Ser19, Ser45 and Ser59) is a major post-translational modification that occurs to alphaB-crystallin. In the present study, we produced recombinant proteins designed to mimic phosphorylation of alphaB-crystallin by incorporating a negative charge at these sites. We employed these mimics to undertake a mechanistic and structural investigation of the effect of phosphorylation on the chaperone activity of alphaB-crystallin to protect against two types of protein misfolding, i.e. amorphous aggregation and amyloid fibril assembly. We show that mimicking phosphorylation of alphaB-crystallin results in more efficient chaperone activity against both heat-induced and reduction-induced amorphous aggregation of target proteins. Mimick-ing phosphorylation increased the chaperone activity of alphaB-crystallin against one amyloid-forming target protein (kappa-casein), but decreased it against another (ccbeta-Trp peptide). We observed that both target protein identity and solution (buffer) conditions are critical factors in determining the relative chaperone ability of wild-type and phosphorylated alphaB-crystallins. The present study provides evidence for the regulation of the chaperone activity of alphaB-crystallin by phosphorylation and indicates that this may play an important role in alleviating the pathogenic effects associated with protein conformational diseases. PMID:16928191

  5. Monitoring protein phosphorylation by acrylamide pendant Phos-Tag™ in various plants

    PubMed Central

    Bekešová, Slávka; Komis, George; Křenek, Pavel; Vyplelová, Petra; Ovečka, Miroslav; Luptovčiak, Ivan; Illés, Peter; Kuchařová, Anna; Šamaj, Jozef

    2015-01-01

    The aim of the present study is to rationalize acrylamide pendant Phos-Tag™ in-gel discrimination of phosphorylated and non-phosphorylated plant protein species with standard immunoblot analysis, and optimize sample preparation, efficient electrophoretic separation and transfer. We tested variants of the method including extraction buffers suitable for preservation of phosphorylated protein species in crude extracts from plants and we addressed the importance of the cation (Mn2+ or Zn2+) used in the gel recipe for efficient transfer to PVDF membranes for further immunoblot analysis. We demonstrate the monitoring of Medicago sativa stress-induced mitogen activated protein kinase (SIMK) in stress-treated wild type plants and transgenic SIMKK RNAi line. We further show the hyperosmotically-induced phosphorylation of the previously uncharacterized HvMPK4 of barley. The method is validated using inducible phosphorylation of barley and wheat α-tubulin and of Arabidopsis MPK6. Acrylamide pendant Phos-Tag™offers a flexible tool for studying protein phosphorylation in crops and Arabidopsis circumventing radioactive labeling and the use of phosphorylation specific antibodies. PMID:26029234

  6. Akt mediated phosphorylation of LARP6; critical step in biosynthesis of type I collagen

    PubMed Central

    Zhang, Yujie; Stefanovic, Branko

    2016-01-01

    La ribonucleoprotein domain family, member 6 (LARP6) is the RNA binding protein, which regulates translation of collagen mRNAs and synthesis of type I collagen. Posttranslational modifications of LARP6 and how they affect type I collagen synthesis have not been studied. We show that in lung fibroblasts LARP6 is phosphorylated at 8 serines, 6 of which are located within C-terminal domain. Phosphorylation of LARP6 follows a hierarchical order; S451 phosphorylation being a prerequisite for phosphorylations of other serines. Inhibition of PI3K/Akt pathway reduced the phosphorylation of LARP6, but had no effect on the S451A mutant, suggesting that PI3K/Akt pathway targets S451 and we have identified Akt as the responsible kinase. Overexpression of S451A mutant had dominant negative effect on collagen biosynthesis; drastically reduced secretion of collagen and induced hyper-modifications of collagen α2 (I) polypeptides. This indicates that LARP6 phosphorylation at S451 is critical for regulating translation and folding of collagen polypeptides. Akt inhibitor, GSK-2141795, which is in clinical trials for treatment of solid tumors, reduced collagen production by human lung fibroblasts with EC50 of 150 nM. This effect can be explained by inhibition of LARP6 phosphorylation and suggests that Akt inhibitors may be effective in treatment of various forms of fibrosis. PMID:26932461

  7. Definition of smad3 phosphorylation events that affect malignant and metastatic behaviors in breast cancer cells.

    PubMed

    Bae, Eunjin; Sato, Misako; Kim, Ran-Ju; Kwak, Mi-Kyung; Naka, Kazuhito; Gim, Jungsoo; Kadota, Mitsutaka; Tang, Binwu; Flanders, Kathleen C; Kim, Tae-Aug; Leem, Sun-Hee; Park, Taesung; Liu, Fang; Wakefield, Lalage M; Kim, Seong-Jin; Ooshima, Akira

    2014-11-01

    Smad3, a major intracellular mediator of TGFβ signaling, functions as both a positive and negative regulator in carcinogenesis. In response to TGFβ, the TGFβ receptor phosphorylates serine residues at the Smad3 C-tail. Cancer cells often contain high levels of the MAPK and CDK activities, which can lead to the Smad3 linker region becoming highly phosphorylated. Here, we report, for the first time, that mutation of the Smad3 linker phosphorylation sites markedly inhibited primary tumor growth, but significantly increased lung metastasis of breast cancer cell lines. In contrast, mutation of the Smad3 C-tail phosphorylation sites had the opposite effect. We show that mutation of the Smad3 linker phosphorylation sites greatly intensifies all TGFβ-induced responses, including growth arrest, apoptosis, reduction in the size of putative cancer stem cell population, epithelial-mesenchymal transition, and invasive activity. Moreover, all TGFβ responses were completely lost on mutation of the Smad3 C-tail phosphorylation sites. Our results demonstrate a critical role of the counterbalance between the Smad3 C-tail and linker phosphorylation in tumorigenesis and metastasis. Our findings have important implications for therapeutic intervention of breast cancer. PMID:25205100

  8. SUMOylation of Rb enhances its binding with CDK2 and phosphorylation at early G1 phase.

    PubMed

    Meng, Fengxi; Qian, Jiang; Yue, Han; Li, Xiaofeng; Xue, Kang

    2016-07-01

    Retinoblastoma protein (Rb) is a prototypical tumor suppressor that is vital to the negative regulation of the cell cycle and tumor progression. Hypo-phosphorylated Rb is associated with G0/G1 arrest by suppressing E2F transcription factor activity, whereas Rb hyper-phosphorylation allows E2F release and cell cycle progression from G0/G1 to S phase. However, the factors that regulate cyclin-dependent protein kinase (CDK)-dependent hyper-phosphorylation of Rb during the cell cycle remain obscure. In this study, we show that throughout the cell cycle, Rb is specifically small ubiquitin-like modifier (SUMO)ylated at early G1 phase. SUMOylation of Rb stimulates its phosphorylation level by recruiting a SUMO-interaction motif (SIM)-containing kinase CDK2, leading to Rb hyper-phosphorylation and E2F-1 release. In contrast, a SUMO-deficient Rb mutant results in reduced SUMOylation and phosphorylation, weakened CDK2 binding, and attenuated E2F-1 sequestration. Furthermore, we reveal that Rb SUMOylation is required for cell proliferation. Therefore, our study describes a novel mechanism that regulates Rb phosphorylation during cell cycle progression. PMID:27163259

  9. Lectin-induced activation of platelets may require only limited phosphorylation of the 47K protein

    SciTech Connect

    Ganguly, C.; Chelladurai, M.; Ganguly, P.

    1986-05-01

    Wheat germ agglutinin (WGA) is an N-acetylglucosamine (Glc-NAc) specific lectin which can activate platelets. Like thrombin, stimulation of platelets by WGA is accompanied by enhanced phosphorylation of two polypeptides of M/sub r/ 47K and 20K. Addition of GlcNAc at different time intervals arrested that aggregation of platelets by WGA and paralleled the modification of phosphorylation of the 47K polypeptide. So, the phosphorylation of the 47K polypeptide may regulate the WGA-receptor mediated stimulation of platelets. However, the ratio of phosphoserine to phosphothreonine in the 47K protein was markedly different in WGA-activated than thrombin-stimulated platelets. Thus, the molecular mechanism of action of thrombin and WGA could be different. To explore this idea, /sup 32/P/sub i/-labeled platelets were stimulated with WGA and the activation arrested with N-acetyl-glucosamine at different times. Two-dimensional gel electrophoresis of total protein at 5s showed only two phosphorylated species of 47K protein. At 60s, maximally four phosphorylated species were noted. In contrast, with thrombin using the same technique, seven to nine phosphorylated components have been reported. These results suggest that the different activators of platelets may act by different mechanisms. In addition, activation of platelets may require only limited levels of phosphorylation of the 47K polypeptide.

  10. Identification and functional analysis of phosphorylation in Newcastle disease virus phosphoprotein.

    PubMed

    Qiu, Xusheng; Zhan, Yuan; Meng, Chunchun; Wang, Junqing; Dong, LuNa; Sun, Yingjie; Tan, Lei; Song, Cuiping; Yu, Shengqing; Ding, Chan

    2016-08-01

    Newcastle disease virus (NDV) encodes a highly phosphorylated P protein; however, the phosphorylation sites have not been identified, and the relationship between phosphorylation and protein function is still unclear. In this study, we bioinformatically predicted 26 amino acid residues in the P protein as potential phosphorylation sites. Furthermore, we treated infected cells with kinase inhibitors to investigate NDV propagation and found that protein kinase C (PKC) is involved in the NDV life cycle and that PKC-activated phosphorylation functions in NDV replication. Using an NDV minigenome assay, we found that expression of a reporter protein decreased when the minigenome system contained P mutants lacking T44, S48, T271, S373 and especially T111. The phosphorylation status of S48, T111, S125 and T271 was determined by Phos-tag SDS-PAGE analysis. Coimmunoprecipitation assays showed that the binding activity of NP and the P-T111A mutant was stronger than that of NP and the wild-type P, suggesting that P-T111 is involved in NP-P interaction. This study sheds light on the mechanism by which P protein phosphorylation affects NDV replication and transcription. PMID:27160999

  11. SUMOylation of Rb enhances its binding with CDK2 and phosphorylation at early G1 phase

    PubMed Central

    Meng, Fengxi; Qian, Jiang; Yue, Han; Li, Xiaofeng; Xue, Kang

    2016-01-01

    ABSTRACT Retinoblastoma protein (Rb) is a prototypical tumor suppressor that is vital to the negative regulation of the cell cycle and tumor progression. Hypo-phosphorylated Rb is associated with G0/G1 arrest by suppressing E2F transcription factor activity, whereas Rb hyper-phosphorylation allows E2F release and cell cycle progression from G0/G1 to S phase. However, the factors that regulate cyclin-dependent protein kinase (CDK)-dependent hyper-phosphorylation of Rb during the cell cycle remain obscure. In this study, we show that throughout the cell cycle, Rb is specifically small ubiquitin-like modifier (SUMO)ylated at early G1 phase. SUMOylation of Rb stimulates its phosphorylation level by recruiting a SUMO-interaction motif (SIM)-containing kinase CDK2, leading to Rb hyper-phosphorylation and E2F-1 release. In contrast, a SUMO-deficient Rb mutant results in reduced SUMOylation and phosphorylation, weakened CDK2 binding, and attenuated E2F-1 sequestration. Furthermore, we reveal that Rb SUMOylation is required for cell proliferation. Therefore, our study describes a novel mechanism that regulates Rb phosphorylation during cell cycle progression. PMID:27163259

  12. ROS-dependent phosphorylation of Bax by wortmannin sensitizes melanoma cells for TRAIL-induced apoptosis

    PubMed Central

    Quast, S-A; Berger, A; Eberle, J

    2013-01-01

    The pathways of reactive oxygen species (ROS)-mediated apoptosis induction, of Bax activation and the sensitization of tumor cells for TRAIL (TNF-related apoptosis-inducing ligand)-induced apoptosis are still largely elusive. Here, sensitization of melanoma cells for TRAIL by the PI3-kinase inhibitor wortmannin correlated to the activation of mitochondrial apoptosis pathways. Apoptosis was dependent on Bax and abrogated by Bcl-2 overexpression. The synergistic enhancement was explained by Bax activation through wortmannin, which tightly correlated to the characteristic Bax phosphorylation patterns. Thus, wortmannin resulted in early reduction of the Bax-inactivating phosphorylation at serine-184, whereas the Bax-activating phosphorylation at threonine-167 was enhanced. Proving the responsibility of the pathway, comparable effects were obtained with an Akt inhibitor (MK-2206); while suppressed phosphorylation of serine-184 may be attributed to reduced Akt activity itself, the causes of enhanced threonine-167 phosphorylation were addressed here. Characteristically, production of ROS was seen early in response to wortmannin and MK-2206. Providing the link between ROS and Bax, we show that abrogated ROS production by α-tocopherol or by NADPH oxidase 4 (NOX4) siRNA suppressed apoptosis and Bax activation. This correlated with reduced Bax phosphorylation at threonine-167. The data unraveled a mechanism by which NOX4-dependent ROS production controls apoptosis via Bax phosphorylation. The pathway may be considered for proapoptotic, anticancer strategies. PMID:24113173

  13. Effect of sialylation on EGFR phosphorylation and resistance to tyrosine kinase inhibition.

    PubMed

    Yen, Hsin-Yung; Liu, Ying-Chih; Chen, Nai-Yu; Tsai, Chia-Feng; Wang, Yi-Ting; Chen, Yu-Ju; Hsu, Tsui-Ling; Yang, Pan-Chyr; Wong, Chi-Huey

    2015-06-01

    Epidermal growth factor receptor (EGFR) is a heavily glycosylated transmembrane receptor tyrosine kinase. Upon EGF-binding, EGFR undergoes conformational changes to dimerize, resulting in kinase activation and autophosphorylation and downstream signaling. Tyrosine kinase inhibitors (TKIs) have been used to treat lung cancer by inhibiting EGFR phosphorylation. Previously, we demonstrated that EGFR sialylation suppresses its dimerization and phosphorylation. In this report, we further investigated the effect of sialylation on the phosphorylation profile of EGFR in TKI-sensitive and TKI-resistant cells. Sialylation was induced in cancer progression to inhibit the association of EGFR with EGF and the subsequent autophosphorylation. In the absence of EGF the TKI-resistant EGFR mutant (L858R/T790M) had a higher degree of sialylation and phosphorylation at Y1068, Y1086, and Y1173 than the TKI-sensitive EGFR. In addition, although sialylation in the TKI-resistant mutants suppresses EGFR tyrosine phosphorylation, with the most significant effect on the Y1173 site, the sialylation effect is not strong enough to stop cancer progression by inhibiting the phosphorylation of these three sites. These findings were supported further by the observation that the L858R/T790M EGFR mutant, when treated with sialidase or sialyltransferase inhibitor, showed an increase in tyrosine phosphorylation, and the sensitivity of the corresponding resistant lung cancer cells to gefitinib was reduced by desialylation and was enhanced by sialylation. PMID:25971727

  14. Plk1 phosphorylation of IRS2 prevents premature mitotic exit via AKT inactivation

    PubMed Central

    Chen, Long; Li, Zhiguo; Ahmad, Nihal; Liu, Xiaoqi

    2016-01-01

    Insulin receptor substrate (IRS) proteins play important roles by acting as a platform in transducing signals from transmembrane receptors upon growth factor stimulation. Although tyrosine phosphorylation on IRS proteins plays critical roles in signal transduction, phosphorylation of IRS proteins on serine/threonine residues are believed to play various regulatory roles on IRS protein function. However, studies on serine/threonine phosphorylation of IRS proteins are very limited, especially for insulin receptor substrate 2 (IRS2), one member of the IRS protein family. In this study, we identify Polo-like kinase 1 (Plk1) as the responsible kinase for phosphorylation of IRS2 on two serine residues, Ser 556 and Ser 1098. Phosphorylation of IRS2 on these two serine residues by Plk1 prevents the activation of the PI3K pathway upon growth factor stimulation by inhibiting the binding between IRS2 and the PI3K pathway components and increasing IRS2 protein degradation. Of significance, we show that IRS2 phosphorylation is cell cycle regulated and that Plk1 phosphorylation of IRS2 prevents premature mitotic exit via AKT inactivation. PMID:25830382

  15. Metabolic abnormalities of the heart in type II diabetes.

    PubMed

    Amaral, Nelson; Okonko, Darlington O

    2015-07-01

    Type 2 diabetes mellitus escalates the risk of heart failure partly via its ability to induce a cardiomyopathic state that is independent of coronary artery disease and hypertension. Although the pathogenesis of diabetic cardiomyopathy has yet to be fully elucidated, aberrations in cardiac substrate metabolism and energetics are thought to be key drivers. These aberrations include excessive fatty acid utilisation and storage, suppressed glucose oxidation and impaired mitochondrial oxidative phosphorylation. An appreciation of how these abnormalities arise and synergise to promote adverse cardiac remodelling is critical to their effective amelioration. This review focuses on disturbances in myocardial fuel (fatty acids and glucose) flux and energetics in type 2 diabetes, how these disturbances relate to the development of diabetic cardiomyopathy and the potential therapeutic agents that could be used to correct them. PMID:25941161

  16. Accumulation of phosphorylated α-synuclein in subpial and periventricular astrocytes in multiple system atrophy of long duration.

    PubMed

    Nakamura, Keiko; Mori, Fumiaki; Kon, Tomoya; Tanji, Kunikazu; Miki, Yasuo; Tomiyama, Masahiko; Kurotaki, Hidekachi; Toyoshima, Yasuko; Kakita, Akiyoshi; Takahashi, Hitoshi; Yamada, Masahito; Wakabayashi, Koichi

    2016-04-01

    The histological hallmark of multiple system atrophy (MSA) is accumulation of phosphorylated α-synuclein in oligodendrocytes. However, it is uncertain whether phosphorylated α-synuclein accumulates in astrocytes of MSA patients. We immunohistochemically examined the frontal and temporal lobes, basal ganglia, cerebellum, brainstem and spinal cord of patients with MSA (n = 15) and Lewy body disease (n = 20), and also in control subjects (n = 20). Accumulation of abnormally phosphorylated and aggregated α-synuclein was found in subpial and periventricular astrocytes in six of the 15 patients with MSA (40%). The structures were confined to the subpial surface of the ventro-lateral part of the spinal cord and brainstem, as well as the subependymal region of the lateral ventricles. They were not visualized by Gallyas-Braak staining, and were immunonegative for ubiquitin and p62. Immunoelectron microscopy revealed that the phosphorylated α-synuclein-immunoreactive structures in astrocytes were non-fibrillar and associated with granular and vesicular structures. The extent of phosphorylated α-synuclein-immunoreactive astrocytes was correlated with disease duration. No such structures were found in Lewy body disease or controls. Accumulation of phosphorylated α-synuclein can occur in subpial and periventricular astrocytes in patients with MSA, especially in those with a long disease duration. PMID:26331967

  17. Prevalence of chromosomal abnormalities in infertile couples in romania.

    PubMed

    Mierla, D; Malageanu, M; Tulin, R; Albu, D

    2015-06-01

    The purpose of this study was to establish a correlation between the presence of chromosomal abnormalities in one of the partners and infertility. This retrospective study was performed at the Department of Reproductive Medicine, Life Memorial Hospital, Bucharest, Romania, between August 2007 to December 2011. Two thousand, one hundred and ninety-five patients with reproductive problems were investigated, and the frequency of chromosomal abnormalities was calculated. The control group consisting of 87 fertile persons who had two or more children, was investigated in this retrospective study. All the patients of this study were investigated by cytogenetic techniques and the results of the two groups were compared by a two-tailed Fisher's exact test. In this study, 94.99% patients had a normal karyotype and 5.01% had chromosomal abnormalities (numerical and structural chromosomal abnormalities). In the study group, numerical chromosomal abnormalities were detected in 1.14% of infertile men and 0.62% of infertile women, and structural chromosomal abnormalities were detected in 1.38% of infertile men and 1.87% of infertile women, respectively. The correlation between the incidence of chromosomal anomalies in the two sexes in couple with reproductive problems was not statistically significant. Recently, a possible association between infertility and chromosomal abnormalities with a significant statistical association has been reported. Our study shows that there is no association between chromosomal abnormalities and infertility, but this study needs to be confirmed with further investigations and a larger control group to establish the role of chromosomal abnormalities in the etiology of infertility. PMID:26929902

  18. Prevalence of chromosomal abnormalities in infertile couples in romania

    PubMed Central

    Mierla, D; Malageanu, M; Tulin, R; Albu, D

    2015-01-01

    The purpose of this study was to establish a correlation between the presence of chromosomal abnormalities in one of the partners and infertility. This retrospective study was performed at the Department of Reproductive Medicine, Life Memorial Hospital, Bucharest, Romania, between August 2007 to December 2011. Two thousand, one hundred and ninety-five patients with reproductive problems were investigated, and the frequency of chromosomal abnormalities was calculated. The control group consisting of 87 fertile persons who had two or more children, was investigated in this retrospective study. All the patients of this study were investigated by cytogenetic techniques and the results of the two groups were compared by a two-tailed Fisher’s exact test. In this study, 94.99% patients had a normal karyotype and 5.01% had chromosomal abnormalities (numerical and structural chromosomal abnormalities). In the study group, numerical chromosomal abnormalities were detected in 1.14% of infertile men and 0.62% of infertile women, and structural chromosomal abnormalities were detected in 1.38% of infertile men and 1.87% of infertile women, respectively. The correlation between the incidence of chromosomal anomalies in the two sexes in couple with reproductive problems was not statistically significant. Recently, a possible association between infertility and chromosomal abnormalities with a significant statistical association has been reported. Our study shows that there is no association between chromosomal abnormalities and infertility, but this study needs to be confirmed with further investigations and a larger control group to establish the role of chromosomal abnormalities in the etiology of infertility. PMID:26929902

  19. Thermodynamic study of the native and phosphorylated regulatory domain of the CFTR

    SciTech Connect

    Marasini, Carlotta; Galeno, Lauretta; Moran, Oscar

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer CFTR mutations produce cystic fibrosis. Black-Right-Pointing-Pointer Chloride transport depends on the regulatory domain phosphorylation. Black-Right-Pointing-Pointer Regulatory domain is intrinsically disordered. Black-Right-Pointing-Pointer Secondary structure and protein stability change upon phosphorylation. -- Abstract: The regulatory domain (RD) of the cystic fibrosis transmembrane conductance regulator (CFTR), the defective protein in cystic fibrosis, is the region of the channel that regulates the CFTR activity with multiple phosphorylation sites. This domain is an intrinsically disordered protein, characterized by lack of stable or unique tertiary structure. The disordered character of a protein is directly correlated with its function. The flexibility of RD may be important for its regulatory role: the continuous conformational change may be necessary for the progressive phosphorylation, and thus activation, of the channel. However, the lack of a defined and stable structure results in a considerable limitation when trying to in build a unique molecular model for the RD. Moreover, several evidences indicate significant structural differences between the native, non-phosphorylated state, and the multiple phosphorylated state of the protein. The aim of our work is to provide data to describe the conformations and the thermodynamic properties in these two functional states of RD. We have done the circular dichroism (CD) spectra in samples with a different degree of phosphorylation, from the non-phosphorylated state to a bona fide completely phosphorylated state. Analysis of CD spectra showed that the random coil and {beta}-sheets secondary structure decreased with the polypeptide phosphorylation, at expenses of an increase of {alpha}-helix. This observation lead to interpret phosphorylation as a mechanism favoring a more structured state. We also studied the thermal denaturation curves of the protein in the two

  20. Phosphorylation Modulates Catalytic Activity of Mycobacterial Sirtuins

    PubMed Central

    Yadav, Ghanshyam S.; Ravala, Sandeep K.; Malhotra, Neha; Chakraborti, Pradip K.

    2016-01-01

    Sirtuins are NAD+-dependent deacetylases involved in the regulation of diverse cellular processes and are conserved throughout phylogeny. Here we report about in vitro transphosphorylation of the only NAD+-dependent deacetylase (mDAC) present in the genome of Mycobacterium tuberculosis by eukaryotic-type Ser/Thr kinases, particularly PknA. The phosphorylated mDAC displayed decreased deacetylase activity compared to its unphosphorylated counterpart. Mass-spectrometric study identified seven phosphosites in mDAC; however, mutational analysis highlighted major contribution of Thr-214 for phosphorylation of the protein. In concordance to this observation, variants of mDAC substituting Thr-214 with either Ala (phospho-ablated) or Glu (phosphomimic) exhibited significantly reduced deacetylase activity suggesting phosphorylation mediated control of enzymatic activity. To assess the role of phosphorylation towards functionality of mDAC, we opted for a sirtuin knock-out strain of Escherichia coli (Δdac), where interference of endogenous mycobacterial kinases could be excluded. The Δdac strain in nutrient deprived acetate medium exhibited compromised growth and complementation with mDAC reversed this phenotype. The phospho-ablated or phosphomimic variant, on the other hand, was unable to restore the functionality of mDAC indicating the role of phosphorylation per se in the process. We further over-expressed mDAC or mDAC-T214A as His-tagged protein in M. smegmatis, where endogenous eukaryotic-type Ser/Thr kinases are present. Anti-phosphothreonine antibody recognized both mDAC and mDAC-T214A proteins in western blotting. However, the extent of phosphorylation as adjudged by scanning the band intensity, was significantly low in the mutant protein (mDAC-T214A) compared to that of the wild-type (mDAC). Furthermore, expression of PknA in the mDAC complemented Δdac strain was able to phosphorylate M. tuberculosis sirtuin. The growth profile of this culture in acetate medium was

  1. Inhibitory effect of ethanol on AMPK phosphorylation is mediated in part through elevated ceramide levels

    PubMed Central

    Sozio, Margaret S.; Shin, Eric; Zhao, Zhenwen; Xu, Yan; Ross, Ruth A.; Zeng, Yan; Crabb, David W.

    2010-01-01

    Ethanol treatment of cultured hepatoma cells and of mice inhibited the activity of AMP-activated protein kinase (AMPK). This study shows that the inhibitory effect of ethanol on AMPK phosphorylation is exerted through the inhibition of the phosphorylation of upstream kinases and the activation of protein phosphatase 2A (PP2A).Inhibition of AMPK phosphorylation by palmitate was attributed to ceramide-dependent PP2A activation. We hypothesized that the inhibitory effect of ethanol on AMPK phosphorylation was mediated partly through the generation of ceramide. The effect of ethanol and inhibitors of ceramide synthesis on AMPK phosphorylation, ceramide levels, and PP2A activity were assessed in rat hepatoma cells (H4IIEC3). The effect of ethanol on hepatic ceramide levels was also studied in C57BL/6J mice fed the Lieber-DeCarli diet. In H4IIEC3 cells, ceramide reduced AMPK phosphorylation when they were treated for between 4 and 12 h. The basal level of AMPK phosphorylation in hepatoma cells was increased with the treatment of ceramide synthase inhibitor, fumonisin B1. Ethanol treatment significantly increased cellular ceramide content and PP2A activity by ∼18–23%, when the cells were treated with ethanol for between 4 and 12 h. These changes in intracellular ceramide concentrations and PP2A activity correlated with the time course over which ethanol inhibited AMPK phosphorylation. The activation of PP2A and inhibition of AMPK phosphorylation caused by ethanol was attenuated by fumonisin B1 and imipramine, an acid sphingomyelinase (SMase) inhibitor. There was a significant increase in the levels of ceramide and acid SMase mRNA in the livers of ethanol-fed mice compared with controls. We concluded that the effect of ethanol on AMPK appears to be mediated in part through increased cellular levels of ceramide and activation of PP2A. PMID:20224005

  2. Phosphorylation regulates the water channel activity of the seed-specific aquaporin alpha-TIP.

    PubMed

    Maurel, C; Kado, R T; Guern, J; Chrispeels, M J

    1995-07-01

    The vacuolar membrane protein alpha-TIP is a seed-specific protein of the Major Intrinsic Protein family. Expression of alpha-TIP in Xenopus oocytes conferred a 4- to 8-fold increase in the osmotic water permeability (Pf) of the oocyte plasma membrane, showing that alpha-TIP forms water channels and is thus a new aquaporin. alpha-TIP has three putative phosphorylation sites on the cytoplasmic side of the membrane (Ser7, Ser23 and Ser99), one of which (Ser7) has been shown to be phosphorylated. We present several lines of evidence that the activity of this aquaporin is regulated by phosphorylation. First, mutation of the putative phosphorylation sites in alpha-TIP (Ser7Ala, Ser23Ala and Ser99Ala) reduced the apparent water transport activity of alpha-TIP in oocytes, suggesting that phosphorylation of alpha-TIP occurs in the oocytes and participates in the control of water channel activity. Second, exposure of oocytes to the cAMP agonists 8-bromoadenosine 3',5'-cyclic monophosphate, forskolin and 3-isobutyl-1-methylxanthine, which stimulate endogenous protein kinase A (PKA), increased the water transport activity of alpha-TIP by 80-100% after 60 min. That the protein can be phosphorylated by PKA was demonstrated by phosphorylating alpha-TIP in isolated oocyte membranes with the bovine PKA catalytic subunit. Third, the integrity of the three sites at positions 7, 23 and 99 was necessary for the cAMP-dependent increase in the Pf of oocytes expressing alpha-TIP, as well as for in vitro phosphorylation of alpha-TIP. These findings demonstrate that the alpha-TIP water channel can be modulated via phosphorylation of Ser7, Ser23 and Ser99.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7542585

  3. Taking in a Show.

    PubMed

    Boden, Timothy W

    2016-01-01

    Many medical practices have cut back on education and staff development expenses, especially those costs associated with conventions and conferences. But there are hard-to-value returns on your investment in these live events--beyond the obvious benefits of acquired knowledge and skills. Major vendors still exhibit their services and wares at many events, and the exhibit hall is a treasure-house of information and resources for the savvy physician or administrator. Make and stick to a purposeful plan to exploit the trade show. You can compare products, gain new insights and ideas, and even negotiate better deals with representatives anxious to realize returns on their exhibition investments. PMID:27249887

  4. Profilin-1 Phosphorylation Directs Angiocrine Expression and Glioblastoma Progression through HIF-1α Accumulation

    PubMed Central

    Fan, Yi; Potdar, Alka A.; Gong, Yanqing; Eswarappa, Sandeepa M.; Donnola, Shannon; Lathia, Justin D.; Hambardzumyan, Dolores; Rich, Jeremy N.; Fox, Paul L.

    2014-01-01

    SUMMARY The tumor vascular microenvironment supports tumorigenesis by supplying not only oxygen and diffusible nutrients but also by secreting soluble factors that promote tumorigenesis. Here we identify a feed-forward mechanism in which endothelial cells (EC), in response to tumor-derived mediators, release angiocrines driving aberrant vascularization and glioblastoma multiforme (GBM) progression through a hypoxia-independent induction of hypoxia-inducible factor (HIF)-1α. Phosphorylation of profilin-1 (Pfn-1) at Tyr129 in EC induces binding to tumor suppressor protein von Hippel-Lindau (VHL), prevents VHL-mediated degradation of prolyl-hydroxylated HIF-1α, culminating in HIF-1α accumulation even in normoxia. Elevated HIF-1α induces expression of multiple angiogenic factors, leading to vascular abnormality and tumor progression. In a genetic model of GBM, mice with an EC-specific defect in Pfn-1 phosphorylation exhibit reduced tumor angiogenesis, normalized vasculature, and improved survival. Moreover, EC-specific Pfn-1 phosphorylation is associated with tumor aggressiveness in human glioma. These findings suggest that targeting Pfn-1 phosphorylation may offer a selective strategy for therapeutic intervention of malignant solid tumors. PMID:24747440

  5. Phosphorylated tau and the neurodegenerative foldopathies.

    PubMed

    Kosik, Kenneth S; Shimura, Hideki

    2005-01-01

    Many studies have implicated phosphorylated tau in the Alzheimer disease process. However, the cellular fate of phosphorylated tau has only recently been described. Recent work has shown that tau phosphorylation at substrate sites for the kinases Cdk5 and GSK3-beta can trigger the binding of tau to the chaperones Hsc70 and Hsp27. The binding of phosphorylated tau to Hsc70 implied that the complex may be a substrate for the E3 ligase CHIP and this possibility was experimentally verified. The presence of this system in cells suggests that phosphorylated tau may hold toxic dangers for cell viability, and the response of the cell is to harness a variety of protective mechanisms. These include binding to chaperones, which may prevent more toxic conformations of the protein, ubiquitination which will direct the protein to the proteasome, segregation of tau aggregates from the cellular machinery, and recruitment of Hsp27 which will confer anti-apoptotic properties to the cell. PMID:15615647

  6. Extensive phosphorylation of AMPA receptors in neurons.

    PubMed

    Diering, Graham H; Heo, Seok; Hussain, Natasha K; Liu, Bian; Huganir, Richard L

    2016-08-16

    Regulation of AMPA receptor (AMPAR) function is a fundamental mechanism controlling synaptic strength during long-term potentiation/depression and homeostatic scaling. AMPAR function and membrane trafficking is controlled by protein-protein interactions, as well as by posttranslational modifications. Phosphorylation of the GluA1 AMPAR subunit at S845 and S831 play especially important roles during synaptic plasticity. Recent controversy has emerged regarding the extent to which GluA1 phosphorylation may contribute to synaptic plasticity. Here we used a variety of methods to measure the population of phosphorylated GluA1-containing AMPARs in cultured primary neurons and mouse forebrain. Phosphorylated GluA1 represents large fractions from 12% to 50% of the total population under basal and stimulated conditions in vitro and in vivo. Furthermore, a large fraction of synapses are positive for phospho-GluA1-containing AMPARs. Our results support the large body of research indicating a prominent role of GluA1 phosphorylation in synaptic plasticity. PMID:27482106

  7. Protein phosphorylation systems in postmortem human brain

    SciTech Connect

    Walaas, S.I.; Perdahl-Wallace, E.; Winblad, B.; Greengard, P. )

    1989-01-01

    Protein phosphorylation systems regulated by cyclic adenosine 3',5'-monophosphate (cyclic AMP), or calcium in conjunction with calmodulin or phospholipid/diacylglycerol, have been studied by phosphorylation in vitro of particulate and soluble fractions from human postmortem brain samples. One-dimensional or two-dimensional gel electrophoretic protein separations were used for analysis. Protein phosphorylation catalyzed by cyclic AMP-dependent protein kinase was found to be highly active in both particulate and soluble preparations throughout the human CNS, with groups of both widely distributed and region-specific substrates being observed in different brain nuclei. Dopamine-innervated parts of the basal ganglia and cerebral cortex contained the phosphoproteins previously observed in rodent basal ganglia. In contrast, calcium/phospholipid-dependent and calcium/calmodulin-dependent protein phosphorylation systems were less prominent in human postmortem brain than in rodent brain, and only a few widely distributed substrates for these protein kinases were found. Protein staining indicated that postmortem proteolysis, particularly of high-molecular-mass proteins, was prominent in deeply located, subcortical regions in the human brain. Our results indicate that it is feasible to use human postmortem brain samples, when obtained under carefully controlled conditions, for qualitative studies on brain protein phosphorylation. Such studies should be of value in studies on human neurological and/or psychiatric disorders.

  8. Sugar phosphorylation activity in ruminal acetogens.

    PubMed

    Jiang, W; Pinder, R S; Patterson, J A; Ricke, S C

    2012-01-01

    Acetogenic bacteria Acetitomaculum ruminis, Acetobacterium woodii, and Eubacterium limosum were compared for phosphoenolpyruvate (PEP) and ATP-dependent phosphorylation of glucose and 2-deoxy-glucose. Rate of phosphorylation activity was measured in toluene-treated acetogenic cells using PEP and ATP and radiolabled glucose or 2-deoxy glucose. Eubacterium limosum, most likely has a glucose phosphotransferase system (PTS). In contrast, A. ruminis, and A. woodii had PEP-dependent glucose phosphorylation rates very similar to control rates, suggesting the lack of PTS activity. These results were confirmed by PEP dependent 2-deoxyglucose phosphorylation data. The rates of ATP-dependent glucose phosphorylation were higher than PEP-dependent glucose dependent in all organisms surveyed. Only E. limosum appeared to have PTS. The presence of PTS in E. limosum could explain why it is not capable of utilizing sugars and H(2)/CO(2) simultaneously and why acetogenesis is not as prominant in the rumen because of the availability of carbohydrates as alternative energy substrates. PMID:22423990

  9. Prognostic Impact of Cytogenetic Abnormalities in Multiple Myeloma

    PubMed Central

    Jian, Yuan; Chen, Xiaolei; Zhou, Huixing; Zhu, Wanqiu; Liu, Nian; Geng, Chuanying; Chen, Wenming

    2016-01-01

    Abstract The identification of specific cytogenetic abnormalities by interphase fluorescence in situ hybridization (i-FISH) has become a routine procedure for prognostic stratification of multiple myeloma (MM) patients. In this study, the prognostic significance of cytogenetic abnormalities detected by interphase fluorescence in situ hybridization (iFISH) in 229 newly diagnosed multiple myeloma patients was retrospectively analyzed. Results showed that del (17p), t(4;14), and 1q21 gain were adverse predictors of progression-free survival (PFS). Patients who carried these cytogenetic abnormalities were more likely to have more adverse biological parameters and lower response rate. Multivariate analysis showed that del (17p), t(4;14), and 1q21 gain were statistically independent predictors of PFS, whereas del (17p) was also adverse predictor of overall survival. Multiple coexisting cytogenetic abnormalities also had a negative correlation with PFS. Bortezomib-based therapy could improve the rate and depth of response in patients with t(4;14) translocation and 1q21 gain. Autologous stem cell transplantation could improve, but not overcome the adverse prognostic effect of high-risk cytogenetic abnormalities. These results demonstrate that MM patients with iFISH abnormalities, especially del (17p), are more likely to have a poor prognosis. PMID:27175647

  10. Phosphorylation is an on/off switch for 5-hydroxyconiferaldehyde O-methyltransferase activity in poplar monolignol biosynthesis

    PubMed Central

    Wang, Jack P.; Chuang, Ling; Loziuk, Philip L.; Chen, Hao; Lin, Ying-Chung; Shi, Rui; Qu, Guan-Zheng; Muddiman, David C.; Sederoff, Ronald R.; Chiang, Vincent L.

    2015-01-01

    Although phosphorylation has long been known to be an important regulatory modification of proteins, no unequivocal evidence has been presented to show functional control by phosphorylation for the plant monolignol biosynthetic pathway. Here, we present the discovery of phosphorylation-mediated on/off regulation of enzyme activity for 5-hydroxyconiferaldehyde O-methyltransferase 2 (PtrAldOMT2), an enzyme central to monolignol biosynthesis for lignification in stem-differentiating xylem (SDX) of Populus trichocarpa. Phosphorylation turned off the PtrAldOMT2 activity, as demonstrated in vitro by using purified phosphorylated and unphosphorylated recombinant PtrAldOMT2. Protein extracts of P. trichocarpa SDX, which contains endogenous kinases, also phosphorylated recombinant PtrAldOMT2 and turned off the recombinant protein activity. Similarly, ATP/Mn2+-activated phosphorylation of SDX protein extracts reduced the endogenous SDX PtrAldOMT2 activity by ∼60%, and dephosphorylation fully restored the activity. Global shotgun proteomic analysis of phosphopeptide-enriched P. trichocarpa SDX protein fractions identified PtrAldOMT2 monophosphorylation at Ser123 or Ser125 in vivo. Phosphorylation-site mutagenesis verified the PtrAldOMT2 phosphorylation at Ser123 or Ser125 and confirmed the functional importance of these phosphorylation sites for O-methyltransferase activity. The PtrAldOMT2 Ser123 phosphorylation site is conserved across 93% of AldOMTs from 46 diverse plant species, and 98% of the AldOMTs have either Ser123 or Ser125. PtrAldOMT2 is a homodimeric cytosolic enzyme expressed more abundantly in syringyl lignin-rich fiber cells than in guaiacyl lignin-rich vessel cells. The reversible phosphorylation of PtrAldOMT2 is likely to have an important role in regulating syringyl monolignol biosynthesis of P. trichocarpa. PMID:26109572

  11. Muscle-specific calpain-3 is phosphorylated in its unique insertion region for enrichment in a myofibril fraction.

    PubMed

    Ojima, Koichi; Ono, Yasuko; Hata, Shoji; Noguchi, Satoru; Nishino, Ichizo; Sorimachi, Hiroyuki

    2014-11-01

    CAPN3 (also called p94/calpain-3) is a skeletal muscle-specific calpain, an intracellular cysteine protease. Loss of CAPN3 protease activity and/or structural functions cause limb-girdle muscular dystrophy type 2A (LGMD2A). However, the precise mechanism of action of CAPN3 in skeletal muscles in vivo remains largely elusive. By studying the protein modifications that regulate CAPN3 activity, we found that CAPN3 was phosphorylated. By performing mutagenesis and mass spectrometry analyses, we identified two Ser residues at positions 629 and 636 in human CAPN3 that are phosphorylated and showed that S629 is a major phosphorylation site. Intriguingly, rapid and exhaustive autolysis of CAPN3 was slightly attenuated by the substitution of S629. In skeletal muscles, phosphorylated CAPN3 was enriched in the myofibril fraction. These results imply that phosphorylated CAPN3 is a myofibril structural component and/or participates in myofibril-based signaling pathways, rather than functions as a protease. We evaluated the relationship between phosphorylated CAPN3 and the pathology of LGMD2A. The level of phosphorylated CAPN3 was greatly reduced in LGMD2A muscles. Our findings suggest that phosphorylated CAPN3 is involved in the pathology of LGMD2A through defects in myofibril integrity and/or signaling pathways. This is the first report that phosphorylation of CAPN3 may be involved in its physiological function. PMID:25252031

  12. Phosphorylation of varicella-zoster virus glycoprotein gpI by mammalian casein kinase II and casein kinase I

    SciTech Connect

    Grose, C.; Jackson, W. ); Traugh, J.A. )

    1989-09-01

    Varicella-zoster virus (VZV) glycoprotein gpI is the predominant viral glycoprotein within the plasma membranes of infected cells. This viral glycoprotein is phosphorylated on its polypeptide backbone during biosynthesis. In this report, the authors investigated the protein kinases which participate in the phosphorylation events. Under in vivo conditions, VZV gpI was phosphorylated on its serine and threonine residues by protein kinases present within lysates of either VZV-infected or uninfected cells. Because this activity was diminished by heparin, a known inhibitor of casein kinase II, isolated gpI was incubated with purified casein kinase II and shown to be phosphorylated in an in vitro assay containing ({gamma}-{sup 32}P)ATP. The same glycoprotein was phosphorylated when ({sup 32}P)GTP was substituted for ({sup 32}P)ATP in the protein kinase assay. They also tested whether VZV gpI was phosphorylated by two other ubiquitous mammalian protein kinases--casein kinase I and cyclic AMP-dependent kinase--and found that only casein kinase I modified gpI. When the predicted 623-amino-acid sequence of gpI was examined, two phosphorylation sites known to be optimal for casein kinase II were observed. In summary, this study showed that VZV gpI was phosphorylated by each of two mammalian protein kinases (casein kinase I and casein kinase II) and that potential serine-threonine phosphorylation sites for each of these two kinases were present in the viral glycoprotein.

  13. Regulation and Function of Phosphorylation on VP8, the Major Tegument Protein of Bovine Herpesvirus 1

    PubMed Central

    Zhang, Kuan; Afroz, Sharmin; Brownlie, Robert; Snider, Marlene

    2015-01-01

    ABSTRACT The major tegument protein of bovine herpesvirus 1 (BoHV-1), VP8, is essential for virus replication in cattle. VP8 is phosphorylated in vitro by casein kinase 2 (CK2) and BoHV-1 unique short protein 3 (US3). In this study, VP8 was found to be phosphorylated in both transfected and infected cells but was detected as a nonphosphorylated form in mature virions. This suggests that phosphorylation of VP8 is strictly controlled during different stages of the viral life cycle. The regulation and function of VP8 phosphorylation by US3 and CK2 were further analyzed. An in vitro kinase assay, site-directed mutagenesis, and liquid chromatography-mass spectrometry were used to identify the active sites for US3 and CK2. The two kinases phosphorylate VP8 at different sites, resulting in distinct phosphopeptide patterns. S16 is a primary phosphoreceptor for US3, and it subsequently triggers phosphorylation at S32. CK2 has multiple active sites, among which T107 appears to be the preferred residue. Additionally, CK2 consensus motifs in the N terminus of VP8 are essential for phosphorylation. Based on these results, a nonphosphorylated VP8 mutant was constructed and used for further studies. In transfected cells phosphorylation was not required for nuclear localization of VP8. Phosphorylated VP8 appeared to recruit promyelocytic leukemia (PML) protein and to remodel the distribution of PML in the nucleus; however, PML protein did not show an association with nonphosphorylated VP8. This suggests that VP8 plays a role in resisting PML-related host antiviral defenses by redistributing PML protein and that this function depends on the phosphorylation of VP8. IMPORTANCE The progression of VP8 phosphorylation over time and its function in BoHV-1 replication have not been characterized. This study demonstrates that activation of S16 initiates further phosphorylation at S32 by US3. Additionally, VP8 is phosphorylated by CK2 at several residues, with T107 having the highest level

  14. Mice lacking FABP9/PERF15 develop sperm head abnormalities but are fertile

    PubMed Central

    Selvaraj, Vimal; Asano, Atsushi; Page, Jennifer L.; Nelson, Jacquelyn L.; Kothapalli, Kumar S. D.; Foster, James A.; Brenna, J. Thomas; Weiss, Robert S.; Travis, Alexander J.

    2010-01-01

    The male germ cell-specific fatty acid binding protein 9 (FABP9/PERF15) is the major component of the murine sperm perforatorium and perinuclear theca. Based on its cytoskeletal association and sequence homology to myelin P2 (FABP8), it has been suggested that FABP9 tethers sperm membranes to the underlying cytoskeleton. Furthermore, its upregulation in apoptotic testicular germ cells and its increased phosphorylation status during capacitation suggested multiple important functions for FABP9. Therefore, we investigated specific functions for FABP9 by means of targeted gene disruption in mice. FABP9−/− mice were viable and fertile. Phenotypic analysis showed that FABP9−/− mice had significant increases in sperm head abnormalities (~8% greater than their WT cohorts); in particular, we observed the reduction or absence of the characteristic structural element known as the “ventral spur” in ~10% of FABP9−/− sperm. However, deficiency of FABP9 neither affected membrane tethering to the perinuclear theca nor the fatty acid composition of sperm. Moreover, epididymal sperm numbers were not affected in FABP9−/− mice. Therefore, we conclude that FABP9 plays only a minor role in providing the murine sperm head its characteristic shape and is not absolutely required for spermatogenesis or sperm function. PMID:20920498

  15. Not a "reality" show.

    PubMed

    Wrong, Terence; Baumgart, Erica

    2013-01-01

    The authors of the preceding articles raise legitimate questions about patient and staff rights and the unintended consequences of allowing ABC News to film inside teaching hospitals. We explain why we regard their fears as baseless and not supported by what we heard from individuals portrayed in the filming, our decade-long experience making medical documentaries, and the full un-aired context of the scenes shown in the broadcast. The authors don't and can't know what conversations we had, what documents we reviewed, and what protections we put in place in each televised scene. Finally, we hope to correct several misleading examples cited by the authors as well as their offhand mischaracterization of our program as a "reality" show. PMID:23631336

  16. Dopamine Pathology in Schizophrenia: Analysis of Total and Phosphorylated Tyrosine Hydroxylase in the Substantia Nigra

    PubMed Central

    Perez-Costas, Emma; Melendez-Ferro, Miguel; Rice, Matthew W.; Conley, Robert R.; Roberts, Rosalinda C.

    2012-01-01

    Introduction: Despite the importance of dopamine neurotransmission in schizophrenia, very few studies have addressed anomalies in the mesencephalic dopaminergic neurons of the substantia nigra/ventral tegmental area (SN/VTA). Tyrosine hydroxylase (TH) is the rate-limiting enzyme for the production of dopamine, and a possible contributor to the anomalies in the dopaminergic neurotransmission observed in schizophrenia. Objectives: In this study, we had three objectives: (1) Compare TH expression (mRNA and protein) in the SN/VTA of schizophrenia and control postmortem samples. (2) Assess the effect of antipsychotic medications on the expression of TH in the SN/VTA. (3) Examine possible regional differences in TH expression anomalies within the SN/VTA. Methods: To achieve these objectives three independent studies were conducted: (1) A pilot study to compare TH mRNA and TH protein levels in the SN/VTA of postmortem samples from schizophrenia and controls. (2) A chronic treatment study was performed in rodents to assess the effect of antipsychotic medications in TH protein levels in the SN/VTA. (3) A second postmortem study was performed to assess TH and phosphorylated TH protein levels in two types of samples: schizophrenia and control samples containing the entire rostro-caudal extent of the SN/VTA, and schizophrenia and control samples containing only mid-caudal regions of the SN/VTA. Results and Conclusion: Our studies showed impairment in the dopaminergic system in schizophrenia that could be mainly (or exclusively) located in the rostral region of the SN/VTA. Our studies also showed that TH protein levels were significantly abnormal in schizophrenia, while mRNA expression levels were not affected, indicating that TH pathology in this region may occur posttranscriptionally. Lastly, our antipsychotic animal treatment study showed that TH protein levels were not significantly affected by antipsychotic treatment, indicating that these anomalies are an intrinsic

  17. Making chromosome abnormalities treatable conditions.

    PubMed

    Cody, Jannine DeMars; Hale, Daniel Esten

    2015-09-01

    Individuals affected by the classic chromosome deletion syndromes which were first identified at the beginning of the genetic age, are now positioned to benefit from genomic advances. This issue highlights five of these conditions (4p-, 5p-, 11q-, 18p-, and 18q-). It focuses on the increased in understanding of the molecular underpinnings and envisions how these can be transformed into effective treatments. While it is scientifically exciting to see the phenotypic manifestations of hemizygosity being increasingly understood at the molecular and cellular level, it is even more amazing to consider that we are now on the road to making chromosome abnormalities treatable conditions. PMID:26351122

  18. Foot abnormalities of wild birds

    USGS Publications Warehouse

    Herman, C.M.; Locke, L.N.; Clark, G.M.

    1962-01-01

    The various foot abnormalities that occur in birds, including pox, scaly-leg, bumble-foot, ergotism and freezing are reviewed. In addition, our findings at the Patuxent Wildlife Research Center include pox from dove, mockingbird, cowbird, grackle and several species of sparrows. Scaly-leg has been particularly prevalent on icterids. Bumble foot has been observed in a whistling swan and in a group of captive woodcock. Ergotism is reported from a series of captive Canada geese from North Dakota. Several drug treatments recommended by others are presented.

  19. Regulation of the autophagy protein LC3 by phosphorylation

    PubMed Central

    Cherra, Salvatore J.; Kulich, Scott M.; Uechi, Guy; Balasubramani, Manimalha; Mountzouris, John; Day, Billy W.

    2010-01-01

    Macroautophagy is a major catabolic pathway that impacts cell survival, differentiation, tumorigenesis, and neurodegeneration. Although bulk degradation sustains carbon sources during starvation, autophagy contributes to shrinkage of differentiated neuronal processes. Identification of autophagy-related genes has spurred rapid advances in understanding the recruitment of microtubule-associated protein 1 light chain 3 (LC3) in autophagy induction, although braking mechanisms remain less understood. Using mass spectrometry, we identified a direct protein kinase A (PKA) phosphorylation site on LC3 that regulates its participation in autophagy. Both metabolic (rapamycin) and pathological (MPP+) inducers of autophagy caused dephosphorylation of endogenous LC3. The pseudophosphorylated LC3 mutant showed reduced recruitment to autophagosomes, whereas the nonphosphorylatable mutant exhibited enhanced puncta formation. Finally, autophagy-dependent neurite shortening induced by expression of a Parkinson disease–associated G2019S mutation in leucine-rich repeat kinase 2 was inhibited by dibutyryl–cyclic adenosine monophosphate, cytoplasmic expression of the PKA catalytic subunit, or the LC3 phosphorylation mimic. These data demonstrate a role for phosphorylation in regulating LC3 activity. PMID:20713600

  20. Protein tyrosine phosphorylation during meiotic divisions of starfish oocytes

    SciTech Connect

    Peaucellier, G.; Andersen, A.C.; Kinsey, W.H. )

    1990-04-01

    We have used an antibody specific for phosphotyrosine to investigate protein phosphorylation on tyrosine during hormone-induced maturation of starfish oocytes. Analysis of immunoprecipitates from cortices of in vivo labeled Marthasterias glacialis oocytes revealed the presence of labeled phosphotyrosine-containing proteins only after hormone addition. Six major phosphoproteins of 195, 155, 100, 85, 45, and 35 kDa were detected. Total activity in immunoprecipitates increased until first polar body emission and was greatly reduced upon completion of meiosis but some proteins exhibited different kinetics. The labeling of the 155-kDa protein reached a maximum at germinal vesicle breakdown, while the 35-kDa appeared later and disappeared after polar body emission. Similar results were obtained with Asterias rubens oocytes. In vitro phosphorylation of cortices showed that tyrosine kinase activity is a major protein kinase activity in this fraction, the main endogenous substrate being a 68-kDa protein. The proteins phosphorylated on tyrosine in vitro were almost similar in extracts from oocytes treated or not with the hormone.

  1. Phosphorylated silk fibroin matrix for methotrexate release.

    PubMed

    Volkov, Vadim; Sárria, Marisa P; Gomes, Andreia C; Cavaco-Paulo, Artur

    2015-01-01

    Silk-based matrix was produced for delivery of a model anticancer drug, methotrexate (MTX). The calculation of net charge of silk fibroin and MTX was performed to better understand the electrostatic interactions during matrix formation upon casting. Silk fibroin films were cast at pH 7.2 and pH 3.5. Protein kinase A was used to prepare phosphorylated silk fibroin. The phosphorylation content of matrix was controlled by mixing at specific ratios the phosphorylated and unphosphorylated solutions. In vitro release profiling data suggest that the observed interactions are mainly structural and not electrostatical. The release of MTX is facilitated by use of proteolytic enzymes and higher pHs. The elevated β-sheet content and crystallinity of the acidified-cast fibroin solution seem not to favor drug retention. All the acquired data underline the prevalence of structural interactions over electrostatical interactions between methotrexate and silk fibroin. PMID:25435334

  2. Phosphorylation of RACK1 in plants

    SciTech Connect

    Chen, Jay -Gui

    2015-08-31

    Receptor for Activated C Kinase 1 (RACK1) is a versatile scaffold protein that interacts with a large, diverse group of proteins to regulate various signaling cascades. RACK1 has been shown to regulate hormonal signaling, stress responses and multiple processes of growth and development in plants. However, little is known about the molecular mechanism underlying these regulations. Recently, it has been demonstrated that Arabidopsis RACK1 is phosphorylated by an atypical serine/threonine protein kinase, WITH NO LYSINE 8 (WNK8). Furthermore, RACK1 phosphorylation by WNK8 negatively regulates RACK1 function by influencing its protein stability. In conclusion, these findings promote a new regulatory system in which the action of RACK1 is controlled by phosphorylation and subsequent protein degradation.

  3. Phosphorylation of RACK1 in plants

    DOE PAGESBeta

    Chen, Jay -Gui

    2015-08-31

    Receptor for Activated C Kinase 1 (RACK1) is a versatile scaffold protein that interacts with a large, diverse group of proteins to regulate various signaling cascades. RACK1 has been shown to regulate hormonal signaling, stress responses and multiple processes of growth and development in plants. However, little is known about the molecular mechanism underlying these regulations. Recently, it has been demonstrated that Arabidopsis RACK1 is phosphorylated by an atypical serine/threonine protein kinase, WITH NO LYSINE 8 (WNK8). Furthermore, RACK1 phosphorylation by WNK8 negatively regulates RACK1 function by influencing its protein stability. In conclusion, these findings promote a new regulatory systemmore » in which the action of RACK1 is controlled by phosphorylation and subsequent protein degradation.« less

  4. Study of sperm cell phosphorylating systems using nucleotide photoaffinity probes

    SciTech Connect

    Khatoon, S.

    1983-01-01

    The major thrust of the research presented in this thesis was to identify specific nucleotide binding proteins and phosphoproteins of rat caput and cauda sperm. Also, the differences in these proteins between caput and cauda sperm were investigated as well as determination of the membrane sidedness of the proteins and their location in either the head or tail/mid-piece region. In addition, the effects of small molecular weight modifers such as cGMP, cAMP and Ca/sup 2 +/ on the detection of binding proteins and phosphorylated proteins was studied. The technique used to identify and locate nucleotide binding proteins was photoaffinity labeling using the proven 8-azidopurine nucleotide analogs of cAMP, ATP and GTP in radioactive form. The first study presented involved the use of (/sup 32/P)8-N /sub 3/cAMP which showed that both caput and cauda sperm contained both type I and type II regulatory subunits (R/sub I/ and R/sub II/, respectively) of the cAMP dependent kinases and that the great majority of the regulatory subunits were located in the tail/mid-piece section and not in the sperm head. The second phase of this study involved the use of (..gamma../sup 32/P)8-azidoadensosine triphosphate ((..gamma../sup 32/P)8-N/sub 3/ATP) and (..gamma../sup 32/P)8-azidoguanosine triphosphate ((..gamma../sup 32/P)8-N/sub 3/GTP) to photolable specific ATP and GTP binding proteins and to phosphorylate specific phosphoproteins. Again, this was done on caput versus cauda sperm and the location of the majority of the photolabeled or phosphorylated proteins was shown to be in the tail/mid-piece fraction. In addition, considerable differences were found in both the phosphorylated and photolabeled proteins of caput versus cauda sperm.

  5. Public medical shows.

    PubMed

    Walusinski, Olivier

    2014-01-01

    In the second half of the 19th century, Jean-Martin Charcot (1825-1893) became famous for the quality of his teaching and his innovative neurological discoveries, bringing many French and foreign students to Paris. A hunger for recognition, together with progressive and anticlerical ideals, led Charcot to invite writers, journalists, and politicians to his lessons, during which he presented the results of his work on hysteria. These events became public performances, for which physicians and patients were transformed into actors. Major newspapers ran accounts of these consultations, more like theatrical shows in some respects. The resultant enthusiasm prompted other physicians in Paris and throughout France to try and imitate them. We will compare the form and substance of Charcot's lessons with those given by Jules-Bernard Luys (1828-1897), Victor Dumontpallier (1826-1899), Ambroise-Auguste Liébault (1823-1904), Hippolyte Bernheim (1840-1919), Joseph Grasset (1849-1918), and Albert Pitres (1848-1928). We will also note their impact on contemporary cinema and theatre. PMID:25273491

  6. Intrinsically active variants of Erk oncogenically transform cells and disclose unexpected autophosphorylation capability that is independent of TEY phosphorylation

    PubMed Central

    Smorodinsky-Atias, Karina; Goshen-Lago, Tal; Goldberg-Carp, Anat; Melamed, Dganit; Shir, Alexei; Mooshayef, Navit; Beenstock, Jonah; Karamansha, Yael; Darlyuk-Saadon, Ilona; Livnah, Oded; Ahn, Natalie G.; Admon, Arie; Engelberg, David

    2016-01-01

    The receptor-tyrosine kinase (RTK)/Ras/Raf pathway is an essential cascade for mediating growth factor signaling. It is abnormally overactive in almost all human cancers. The downstream targets of the pathway are members of the extracellular regulated kinases (Erk1/2) family, suggesting that this family is a mediator of the oncogenic capability of the cascade. Although all oncogenic mutations in the pathway result in strong activation of Erks, activating mutations in Erks themselves were not reported in cancers. Here we used spontaneously active Erk variants to check whether Erk’s activity per se is sufficient for oncogenic transformation. We show that Erk1(R84S) is an oncoprotein, as NIH3T3 cells that express it form foci in tissue culture plates, colonies in soft agar, and tumors in nude mice. We further show that Erk1(R84S) and Erk2(R65S) are intrinsically active due to an unusual autophosphorylation activity they acquire. They autophosphorylate the activatory TEY motif and also other residues, including the critical residue Thr-207 (in Erk1)/Thr-188 (in Erk2). Strikingly, Erk2(R65S) efficiently autophosphorylates its Thr-188 even when dually mutated in the TEY motif. Thus this study shows that Erk1 can be considered a proto-oncogene and that Erk molecules possess unusual autoregulatory properties, some of them independent of TEY phosphorylation. PMID:26658610

  7. Src kinase regulation by phosphorylation and dephosphorylation

    SciTech Connect

    Roskoski, Robert . E-mail: biocrr@lsuhsc.edu

    2005-05-27

    Src and Src-family protein-tyrosine kinases are regulatory proteins that play key roles in cell differentiation, motility, proliferation, and survival. The initially described phosphorylation sites of Src include an activating phosphotyrosine 416 that results from autophosphorylation, and an inhibiting phosphotyrosine 527 that results from phosphorylation by C-terminal Src kinase (Csk) and Csk homologous kinase. Dephosphorylation of phosphotyrosine 527 increases Src kinase activity. Candidate phosphotyrosine 527 phosphatases include cytoplasmic PTP1B, Shp1 and Shp2, and transmembrane enzymes include CD45, PTP{alpha}, PTP{epsilon}, and PTP{lambda}. Dephosphorylation of phosphotyrosine 416 decreases Src kinase activity. Thus far PTP-BL, the mouse homologue of human PTP-BAS, has been shown to dephosphorylate phosphotyrosine 416 in a regulatory fashion. The platelet-derived growth factor receptor protein-tyrosine kinase mediates the phosphorylation of Src Tyr138; this phosphorylation has no direct effect on Src kinase activity. The platelet-derived growth factor receptor and the ErbB2/HER2 growth factor receptor protein-tyrosine kinases mediate the phosphorylation of Src Tyr213 and activation of Src kinase activity. Src kinase is also a substrate for protein-serine/threonine kinases including protein kinase C (Ser12), protein kinase A (Ser17), and CDK1/cdc2 (Thr34, Thr46, and Ser72). Of the three protein-serine/threonine kinases, only phosphorylation by CDK1/cdc2 has been demonstrated to increase Src kinase activity. Although considerable information on the phosphoprotein phosphatases that catalyze the hydrolysis of Src phosphotyrosine 527 is at hand, the nature of the phosphatases that mediate the hydrolysis of phosphotyrosine 138 and 213, and phosphoserine and phosphothreonine residues has not been determined.

  8. Chromosome abnormalities in primary ovarian cancer

    SciTech Connect

    Yonescu, R.; Currie, J.; Griffin, C.A.

    1994-09-01

    Chromosome abnormalities that are specific and recurrent may occur in regions of the genome that are involved in the conversion of normal cells to those with tumorigenic potential. Ovarian cancer is the primary cause of death among patients with gynecological malignancies. We have performed cytogenetic analysis of 16 ovarian tumors from women age 28-82. Three tumors of low malignant potential and three granulosa cell tumors had normal karyotypes. To look for the presence of trisomy 12, which has been suggested to be a common aberration in this group of tumors, interphase fluorescence in situ hybridization was performed on direct preparations from three of these tumors using a probe for alpha satellite sequences of chromosome 12. In the 3 preparations, 92-98 percent of the cells contained two copies of chromosome 12, indicating that trisomy 12 is not a universal finding in low grade ovarian tumors. Endometrioid carcinoma of the ovary is histologically indistinguishable from endometial carcinoma of the uterus. We studied 10 endometrioid tumors to determine the degree of genetic similarity between these two carcinomas. Six out of ten endometrioid tumors showed a near-triploid modal number, and one presented with a tetraploid modal number. Eight of the ten contained structural chromosome abnormalities, of which the most frequent were 1p- (5 tumors), 19q+ (3 tumors), 6q- or ins(6) (4 tumors), 3q- or 3q+ (4 tumors). These cytogenetic results resemble those reported for papillary ovarian tumors and differ from those of endometrial carcinoma of the uterus. We conclude that despite the histologic similarities between the endometrioid and endometrial carcinomas, the genetic abnormalities in the genesis of these tumors differ significantly.

  9. PML tumor suppressor is regulated by HIPK2-mediated phosphorylation in response to DNA damage.

    PubMed

    Gresko, E; Ritterhoff, S; Sevilla-Perez, J; Roscic, A; Fröbius, K; Kotevic, I; Vichalkovski, A; Hess, D; Hemmings, B A; Schmitz, M L

    2009-02-01

    The promyelocytic leukemia (PML) tumor suppressor protein, a central regulator of cell proliferation and apoptosis, is frequently fused to the retinoic acid receptor-alpha (RARalpha) in acute PML. Here we show the interaction of PML with another tumor suppressor protein, the serine/threonine kinase homeodomain-interacting protein kinase (HIPK2). In response to DNA damage, HIPK2 phosphorylates PML at serines 8 and 38. Although HIPK2-mediated phosphorylation of PML occurs early during the DNA damage response, the oncogenic PML-RARalpha fusion protein is phosphorylated with significantly delayed kinetics. DNA damage or HIPK2 expression leads to the stabilization of PML and PML-RARalpha proteins. The N-terminal phosphorylation sites contribute to the DNA damage-induced PML SUMOylation and are required for the ability of PML to cooperate with HIPK2 for the induction of cell death. PMID:19015637

  10. A Cholesterol-Based Allostery Model of T Cell Receptor Phosphorylation.

    PubMed

    Swamy, Mahima; Beck-Garcia, Katharina; Beck-Garcia, Esmeralda; Hartl, Frederike A; Morath, Anna; Yousefi, O Sascha; Dopfer, Elaine Pashupati; Molnár, Eszter; Schulze, Anna K; Blanco, Raquel; Borroto, Aldo; Martín-Blanco, Nadia; Alarcon, Balbino; Höfer, Thomas; Minguet, Susana; Schamel, Wolfgang W A

    2016-05-17

    Signaling through the T cell receptor (TCR) controls adaptive immune responses. Antigen binding to TCRαβ transmits signals through the plasma membrane to induce phosphorylation of the CD3 cytoplasmic tails by incompletely understood mechanisms. Here we show that cholesterol bound to the TCRβ transmembrane region keeps the TCR in a resting, inactive conformation that cannot be phosphorylated by active kinases. Only TCRs that spontaneously detached from cholesterol could switch to the active conformation (termed primed TCRs) and then be phosphorylated. Indeed, by modulating cholesterol binding genetically or enzymatically, we could switch the TCR between the resting and primed states. The active conformation was stabilized by binding to peptide-MHC, which thus controlled TCR signaling. These data are explained by a model of reciprocal allosteric regulation of TCR phosphorylation by cholesterol and ligand binding. Our results provide both a molecular mechanism and a conceptual framework for how lipid-receptor interactions regulate signal transduction. PMID:27192576

  11. Cyclic AMP-dependent phosphorylation of neuronal nitric oxide synthase mediates penile erection

    PubMed Central

    Hurt, K. Joseph; Sezen, Sena F.; Lagoda, Gwen F.; Musicki, Biljana; Rameau, Gerald A.; Snyder, Solomon H.; Burnett, Arthur L.

    2012-01-01

    Nitric oxide (NO) generated by neuronal NO synthase (nNOS) initiates penile erection, but has not been thought to participate in the sustained erection required for normal sexual performance. We now show that cAMP-dependent phosphorylation of nNOS mediates erectile physiology, including sustained erection. nNOS is phosphorylated by cAMP-dependent protein kinase (PKA) at serine(S)1412. Electrical stimulation of the penile innervation increases S1412 phosphorylation that is blocked by PKA inhibitors but not by PI3-kinase/Akt inhibitors. Stimulation of cAMP formation by forskolin also activates nNOS phosphorylation. Sustained penile erection elicited by either intracavernous forskolin injection, or augmented by forskolin during cavernous nerve electrical stimulation, is prevented by the NOS inhibitor l-NAME or in nNOS-deleted mice. Thus, nNOS mediates both initiation and maintenance of penile erection, implying unique approaches for treating erectile dysfunction. PMID:23012472

  12. Phosphorylation of the proapoptotic BH3-only protein bid primes mitochondria for apoptosis during mitotic arrest.

    PubMed

    Wang, Pengbo; Lindsay, Jennefer; Owens, Thomas W; Mularczyk, Ewa J; Warwood, Stacey; Foster, Fiona; Streuli, Charles H; Brennan, Keith; Gilmore, Andrew P

    2014-05-01

    Mitosis is a moment of exquisite vulnerability for a metazoan cell. Failure to complete mitosis accurately can lead to aneuploidy and cancer initiation. Therefore, if the exit from mitosis is delayed, normal cells are usually removed by apoptosis. However, how failure to complete mitosis activates apoptosis is still unclear. Here, we demonstrate that a phosphorylated form of the BH3-only protein Bid regulates apoptosis if mitotic exit is delayed. Bid is phosphorylated on serine 66 as cells enter mitosis, and this phosphorylation is lost during the metaphase-to-anaphase transition. Cells expressing a nonphosphorylatable version of Bid or a BH3-domain mutant were resistant to mitotic-arrest-induced apoptosis. Thus, we show that Bid phosphorylation primes cells to undergo mitochondrial apoptosis if mitotic exit is delayed. Avoidance of this mechanism may explain the selective pressure for cancer cells to undergo mitotic slippage. PMID:24767991

  13. The Great Cometary Show

    NASA Astrophysics Data System (ADS)

    2007-01-01

    its high spatial and spectral resolution, it was possible to zoom into the very heart of this very massive star. In this innermost region, the observations are dominated by the extremely dense stellar wind that totally obscures the underlying central star. The AMBER observations show that this dense stellar wind is not spherically symmetric, but exhibits a clearly elongated structure. Overall, the AMBER observations confirm that the extremely high mass loss of Eta Carinae's massive central star is non-spherical and much stronger along the poles than in the equatorial plane. This is in agreement with theoretical models that predict such an enhanced polar mass-loss in the case of rapidly rotating stars. ESO PR Photo 06c/07 ESO PR Photo 06c/07 RS Ophiuchi in Outburst Several papers from this special feature focus on the later stages in a star's life. One looks at the binary system Gamma 2 Velorum, which contains the closest example of a star known as a Wolf-Rayet. A single AMBER observation allowed the astronomers to separate the spectra of the two components, offering new insights in the modeling of Wolf-Rayet stars, but made it also possible to measure the separation between the two stars. This led to a new determination of the distance of the system, showing that previous estimates were incorrect. The observations also revealed information on the region where the winds from the two stars collide. The famous binary system RS Ophiuchi, an example of a recurrent nova, was observed just 5 days after it was discovered to be in outburst on 12 February 2006, an event that has been expected for 21 years. AMBER was able to detect the extension of the expanding nova emission. These observations show a complex geometry and kinematics, far from the simple interpretation of a spherical fireball in extension. AMBER has detected a high velocity jet probably perpendicular to the orbital plane of the binary system, and allowed a precise and careful study of the wind and the shockwave

  14. Abnormality on Liver Function Test

    PubMed Central

    2013-01-01

    Children with abnormal liver function can often be seen in outpatient clinics or inpatients wards. Most of them have respiratory disease, or gastroenteritis by virus infection, accompanying fever. Occasionally, hepatitis by the viruses causing systemic infection may occur, and screening tests are required. In patients with jaundice, the tests for differential diagnosis and appropriate treatment are important. In the case of a child with hepatitis B virus infection vertically from a hepatitis B surface antigen positive mother, the importance of the recognition of immune clearance can't be overstressed, for the decision of time to begin treatment. Early diagnosis changes the fate of a child with Wilson disease. So, screening test for the disease should not be omitted. Non-alcoholic fatty liver disease, which is mainly discovered in obese children, is a new strong candidate triggering abnormal liver function. Muscular dystrophy is a representative disease mimicking liver dysfunction. Although muscular dystrophy is a progressive disorder, and early diagnosis can't change the fate of patients, it will be better to avoid parent's blame for delayed diagnosis. PMID:24511518

  15. Medical management of abnormal pregnancy.

    PubMed

    Ratnam, S S; Prasad, R N

    1990-06-01

    Medical termination of abnormal pregnancy requires specific techniques since some conditions make therapy more effective, e.g., missed abortion intrauterine death and molar pregnancy, and others less so, e.g. anencephalic pregnancy. In all cases it is best to terminate the pregnancy as soon as possible to reduce anguish and risks of complications such as consumptive coagulopathy. Oxytocin is not consistently effective, but intraamniotic rivanol has oxytocic properties, and prostaglandins (PGs) are effective by several routes. Surgical methods are more popular in Japan and the US. A diagnostic flow chart is included and described. For missed abortion and fetal death vacuum aspiration or dilatation and evacuation are appropriate for early pregnancy, or PGs are used for later pregnancy, unless there are medical contraindications. Anencephalic pregnancy, usually diagnoses in 2nd or 3rd trimester, is resistant to medical therapy and must often be terminated by cesarean section. Molar pregnancy can be managed with vacuum aspiration at any length of gestation, but must be completed by curettage. Intraamniotic PGs are not advised for mole or fetal death. PG analogs can be administered intramuscularly, or vaginally in gel form. Other types of abnormal pregnancy that can be managed with PGs are spina bifida, hydrocephalus, hydrops fetalis, Dandy-Walker syndrome and Down's syndrome. Tubal pregnancy can be evacuated with intratubally administered PGs under laparoscopic control, thereby preserving tubal integrity. PMID:2225605

  16. Abnormalities of AMPK Activation and Glucose Uptake in Cultured Skeletal Muscle Cells from Individuals with Chronic Fatigue Syndrome

    PubMed Central

    Brown, Audrey E.; Jones, David E.; Walker, Mark; Newton, Julia L.

    2015-01-01

    Background Post exertional muscle fatigue is a key feature in Chronic Fatigue Syndrome (CFS). Abnormalities of skeletal muscle function have been identified in some but not all patients with CFS. To try to limit potential confounders that might contribute to this clinical heterogeneity, we developed a novel in vitro system that allows comparison of AMP kinase (AMPK) activation and metabolic responses to exercise in cultured skeletal muscle cells from CFS patients and control subjects. Methods Skeletal muscle cell cultures were established from 10 subjects with CFS and 7 age-matched controls, subjected to electrical pulse stimulation (EPS) for up to 24h and examined for changes associated with exercise. Results In the basal state, CFS cultures showed increased myogenin expression but decreased IL6 secretion during differentiation compared with control cultures. Control cultures subjected to 16h EPS showed a significant increase in both AMPK phosphorylation and glucose uptake compared with unstimulated cells. In contrast, CFS cultures showed no increase in AMPK phosphorylation or glucose uptake after 16h EPS. However, glucose uptake remained responsive to insulin in the CFS cells pointing to an exercise-related defect. IL6 secretion in response to EPS was significantly reduced in CFS compared with control cultures at all time points measured. Conclusion EPS is an effective model for eliciting muscle contraction and the metabolic changes associated with exercise in cultured skeletal muscle cells. We found four main differences in cultured skeletal muscle cells from subjects with CFS; increased myogenin expression in the basal state, impaired activation of AMPK, impaired stimulation of glucose uptake and diminished release of IL6. The retention of these differences in cultured muscle cells from CFS subjects points to a genetic/epigenetic mechanism, and provides a system to identify novel therapeutic targets. PMID:25836975

  17. Changes in phosphoinositide turnover, Ca sup 2+ mobilization, and protein phosphorylation in platelets from NIDDM patients

    SciTech Connect

    Ishii, H.; Umeda, F.; Hashimoto, T.; Nawata, H. )

    1990-12-01

    Enhanced platelet functions have been demonstrated in patients with non-insulin-dependent diabetes mellitus (NIDDM). This study evaluated abnormalities in platelet signal transduction in diabetic patients, including turnover of phosphoinositides, mobilization of intracellular Ca2+, and phosphorylation of 20,000- and 47,000-Mr proteins (P20 and P47). Washed platelets were obtained from 6 patients with NIDDM whose platelet aggregation rates were abnormally elevated (DM-A group), 11 NIDDM patients with normal platelet aggregation rates (DM-B group), and 8 age-matched healthy control subjects. The mass and specific radioactivity of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol (PI), and phosphatidic acid (PA) in 32P-labeled platelets were not different among the three groups. Hydrolysis of PIP2, PIP, and PI; accumulation of PA; and phosphorylation of P20 in platelets stimulated by 0.05 U/ml thrombin were significantly increased in the DM-A group compared with the control or DM-B group. There was no difference in P47 phosphorylation among the three groups. On the contrary, P20 and P47 phosphorylation induced by 50 nM of 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C, was significantly decreased in the DM-A group. Additionally, the intracellular free Ca2+ concentration (( Ca2+)i) was measured with the fluorescent Ca2+ indicator fura 2. Although the basal (Ca2+)i value was similar in the three groups, the rise in (Ca2+)i induced by 0.05 U/ml thrombin in the presence and the absence of extracellular Ca2+ was significantly higher in the DM-A group than the other groups.

  18. Wild-type human TDP-43 expression causes TDP-43 phosphorylation, mitochondrial aggregation, motor deficits and early mortality in transgenic mice

    PubMed Central

    Xu, Ya-Fei; Gendron, Tania F.; Zhang, Yong-Jie; Lin, Wen-Lang; D’Alton, Simon; Sheng, Hong; Casey, Monica Castanedes; Tong, Jimei; Knight, Joshua; Yu, Xin; Rademakers, Rosa; Boylan, Kevin; Hutton, Mike; McGowan, Eileen; Dickson, Dennis W.; Lewis, Jada; Petrucelli, Leonard

    2011-01-01

    Transactivation response DNA-binding protein 43 (TDP-43) is a principal component of ubiquitinated inclusions in frontotemporal lobar degeneration with ubiquitin-positive inclusions and in amyotrophic lateral sclerosis (ALS). Mutations in TARDBP, the gene encoding TDP-43, are associated with sporadic and familial ALS, yet multiple neurodegenerative diseases exhibit TDP-43 pathology without known TARDBP mutations. While TDP-43 has been ascribed a number of roles in normal biology, including mRNA splicing and transcription regulation, elucidating disease mechanisms associated with this protein is hindered by the lack of models to dissect such functions. We have generated transgenic (TDP-43PrP) mice expressing full-length human TDP-43 (hTDP-43) driven by the mouse prion promoter to provide a tool to analyze the role of wild-type hTDP-43 in the brain and spinal cord. Expression of hTDP-43 caused a dose dependant down regulation of mouse TDP-43 RNA and protein. Moderate overexpression of hTDP-43 resulted in TDP-43 truncation, increased cytoplasmic and nuclear ubiquitin levels, as well as intranuclear and cytoplasmic aggregates that were immunopositive for phosphorylated TDP-43. Of note, abnormal juxtanuclear aggregates of mitochondria were observed, accompanied by enhanced levels of Fis1 and phosphorylated DLP1, key components of the mitochondrial fission machinery. Conversely, a marked reduction in mitofusin 1 expression, which plays an essential role in mitochondrial fusion, was observed in TDP-43PrP mice. Finally, TDP-43PrP mice showed reactive gliosis, axonal and myelin degeneration, gait abnormalities and early lethality. This TDP-43 transgenic line provides a valuable tool for identifying potential roles of wild-type TDP-43 within the central nervous system and for studying TDP-43-associated neurotoxicity. PMID:20702714

  19. Attenuated Levels of Hippocampal Connexin 43 and its Phosphorylation Correlate with Antidepressant- and Anxiolytic-Like Activities in Mice

    PubMed Central

    Quesseveur, Gaël; Portal, Benjamin; Basile, Jean-Arnaud; Ezan, Pascal; Mathou, Alexia; Halley, Hélène; Leloup, Corinne; Fioramonti, Xavier; Déglon, Nicole; Giaume, Christian; Rampon, Claire; Guiard, Bruno P.

    2015-01-01

    Clinical and preclinical studies have implicated glial anomalies in major depression. Conversely, evidence suggests that the activity of antidepressant drugs is based, at least in part, on their ability to stimulate density and/or activity of astrocytes, a major glial cell population. Despite this recent evidence, little is known about the mechanism(s) by which astrocytes regulate emotionality. Glial cells communicate with each other through gap junction channels (GJCs), while they can also directly interact with neurons by releasing gliotransmitters in the extracellular compartment via an hemichannels (HCs)-dependent process. Both GJCs and HCs are formed by two main protein subunits: connexins (Cx) 30 and 43 (Cx30 and Cx43). Here we investigate the role of hippocampal Cx43 in the regulation of depression-like symptoms using genetic and pharmacological approaches. The first aim of this study was to evaluate the impact of the constitutive knock-down of Cx43 on a set of behaviors known to be affected in depression. Conversely, the expression of Cx43 was assessed in the hippocampus of mice subjected to prolonged corticosterone (CORT) exposure, given either alone or in combination with an antidepressant drug, the selective serotonin reuptake inhibitor fluoxetine. Our results indicate that the constitutive deficiency of Cx43 resulted in the expression of some characteristic hallmarks of antidepressant-/anxiolytic-like behavioral activities along with an improvement of cognitive performances. Moreover, in a new cohort of wild-type mice, we showed that CORT exposure elicited anxiety and depression-like abnormalities that were reversed by chronic administration of fluoxetine. Remarkably, CORT also increased hippocampal amounts of phosphorylated form of Cx43 whereas fluoxetine treatment normalized this parameter. From these results, we envision that antidepressant drugs may exert their therapeutic activity by decreasing the expression and/or activity of Cx43 resulting from

  20. The role of phosphorylation in dentin phosphoprotein peptide absorption to hydroxyapatite surfaces: a molecular dynamics study

    PubMed Central

    Villarreal-Ramirez, Eduardo; Garduño-Juarez, Ramon; Gericke, Arne; Boskey, Adele

    2015-01-01

    Dentin phosphoprotein (DPP) is a protein expressed mainly in dentin and to a lesser extent in bone. DPP has a disordered structure, rich in glutamic acid, aspartic acid and phosphorylated serine/threonine residues. It has a high capacity for binding to calcium ions and to hydroxyapatite (HA) crystal surfaces. We used molecular dynamics (MD) simulations as a method for virtually screening interactions between DPP motifs and HA. The goal was to determine which motifs are absorbed to HA surfaces. For these simulations, we considered five peptides from the human DPP sequence. All-atom MD simulations were performed using GROMACS, the peptides were oriented parallel to the {100} HA crystal surface, the distance between the HA and the peptide was 3 nm. The system was simulated for 20 ns. Preliminary results show that for the unphosphorylated peptides, the acidic amino acids present an electrostatic attraction where their side chains are oriented towards HA. This attraction, however, is slow to facilitate bulk transport to the crystal surface. On the other hand, the phosphorylated (PP) peptides are rapidly absorbed on the surface of the HA with their centers of mass closer to the HA surface. More importantly, the root mean square fluctuation (RMSF) indicates that the average structures of the phosphorylated peptides are very inflexible and elongate, while that of the unphosphorylated peptides are flexible. Radius of gyration (Rg) analysis showed the compactness of un-phosphorylated peptides is lower than phosphorylated peptides. Phosphorylation of the DPP peptides is necessary for binding to HA surfaces. PMID:25158198

  1. Phosphorylation regulates the Star-PAP-PIPKIα interaction and directs specificity toward mRNA targets

    PubMed Central

    Mohan, Nimmy; AP, Sudheesh; Francis, Nimmy; Anderson, Richard; Laishram, Rakesh S.

    2015-01-01

    Star-PAP is a nuclear non-canonical poly(A) polymerase (PAP) that shows specificity toward mRNA targets. Star-PAP activity is stimulated by lipid messenger phosphatidyl inositol 4,5 bisphoshate (PI4,5P2) and is regulated by the associated Type I phosphatidylinositol-4-phosphate 5-kinase that synthesizes PI4,5P2 as well as protein kinases. These associated kinases act as coactivators of Star-PAP that regulates its activity and specificity toward mRNAs, yet the mechanism of control of these interactions are not defined. We identified a phosphorylated residue (serine 6, S6) on Star-PAP in the zinc finger region, the domain required for PIPKIα interaction. We show that S6 is phosphorylated by CKIα within the nucleus which is required for Star-PAP nuclear retention and interaction with PIPKIα. Unlike the CKIα mediated phosphorylation at the catalytic domain, Star-PAP S6 phosphorylation is insensitive to oxidative stress suggesting a signal mediated regulation of CKIα activity. S6 phosphorylation together with coactivator PIPKIα controlled select subset of Star-PAP target messages by regulating Star-PAP-mRNA association. Our results establish a novel role for phosphorylation in determining Star-PAP target mRNA specificity and regulation of 3′-end processing. PMID:26138484

  2. Mixtures of opposing phosphorylations within hexamers precisely time feedback in the cyanobacterial circadian clock

    PubMed Central

    Lin, Jenny; Chew, Justin; Chockanathan, Udaysankar; Rust, Michael J.

    2014-01-01

    Circadian oscillations are generated by the purified cyanobacterial clock proteins, KaiA, KaiB, and KaiC, through rhythmic interactions that depend on multisite phosphorylation of KaiC. However, the mechanisms that allow these phosphorylation reactions to robustly control the timing of oscillations over a range of protein stoichiometries are not clear. We show that when KaiC hexamers consist of a mixture of differentially phosphorylated subunits, the two phosphorylation sites have opposing effects on the ability of each hexamer to bind to the negative regulator KaiB. We likewise show that the ability of the positive regulator KaiA to act on KaiC depends on the phosphorylation state of the hexamer and that KaiA and KaiB recognize alternative allosteric states of the KaiC ring. Using mathematical models with kinetic parameters taken from experimental data, we find that antagonism of the two KaiC phosphorylation sites generates an ultrasensitive switch in negative feedback strength necessary for stable circadian oscillations over a range of component concentrations. Similar strategies based on opposing modifications may be used to support robustness in other timing systems and in cellular signaling more generally. PMID:25197081

  3. Transforming Growth Factor {beta} Can Stimulate Smad1 Phosphorylation Independently of Bone Morphogenic Protein Receptors.

    PubMed

    Wrighton, Katharine H; Lin, Xia; Yu, Paul B; Feng, Xin-Hua

    2009-04-10

    Transforming growth factor-beta (TGFbeta) superfamily ligands control a diverse set of cellular processes by activating type I and type II serine-threonine receptor kinases. Canonical TGFbeta signaling is mediated via the TbetaRI/ALK5 type I receptor that phosphorylates Smad2 and Smad3 in their SXS motif to facilitate their activation and subsequent role in transcriptional regulation. Canonical bone morphogenic protein (BMP) signaling is mediated via the ALK1/2/3/6 type I receptors that phosphorylate Smad1, Smad5, and Smad8 in their SXS motif. However, studies in endothelial cells have shown that TGFbeta can also lead to the phosphorylation of Smad1, dependent on ALK1 receptor activity. Here we present data showing that TGFbeta can significantly induce Smad1 phosphorylation in several non-endothelial cell lineages. Additionally, by using chemical inhibitors specific for the TGFbeta/activin/nodal (ALK4/5/7) and BMP (ALK1/2/3/6) type I receptors, we show that in some cell types TGFbeta induces Smad1 phosphorylation independently of the BMP type I receptors. Thus, TGFbeta-mediated Smad1 phosphorylation appears to occur via different receptor complexes in a cell type-specific manner. PMID:19224917

  4. Differential regulation of the histone chaperone HIRA during muscle cell differentiation by a phosphorylation switch

    PubMed Central

    Yang, Jae-Hyun; Song, Tae-Yang; Jo, Chanhee; Park, Jinyoung; Lee, Han-Young; Song, Ilang; Hong, Suji; Jung, Kwan Young; Kim, Jaehoon; Han, Jeung-Whan; Youn, Hong-Duk; Cho, Eun-Jung

    2016-01-01

    Replication-independent incorporation of variant histone H3.3 has a profound impact on chromatin function and numerous cellular processes, including the differentiation of muscle cells. The histone chaperone HIRA and H3.3 have essential roles in MyoD regulation during myoblast differentiation. However, the precise mechanism that determines the onset of H3.3 deposition in response to differentiation signals is unclear. Here we show that HIRA is phosphorylated by Akt kinase, an important signaling modulator in muscle cells. By generating a phosphospecific antibody, we found that a significant amount of HIRA was phosphorylated in myoblasts. The phosphorylation level of HIRA and the occupancy of phosphorylated protein on muscle genes gradually decreased during cellular differentiation. Remarkably, the forced expression of the phosphomimic form of HIRA resulted in reduced H3.3 deposition and suppressed the activation of muscle genes in myotubes. Our data show that HIRA phosphorylation limits the expression of myogenic genes, while the dephosphorylation of HIRA is required for proficient H3.3 deposition and gene activation, demonstrating that the phosphorylation switch is exploited to modulate HIRA/H3.3-mediated muscle gene regulation during myogenesis. PMID:27515126

  5. Conformational selection in the recognition of phosphorylated substrates by the catalytic domain of human Pin1.

    PubMed

    Velazquez, Hector A; Hamelberg, Donald

    2011-11-01

    Post-translational phosphorylation and the related conformational changes in signaling proteins are responsible for regulating a wide range of subcellular processes. Human Pin1 is central to many of these cell signaling pathways in normal and aberrant subcellular processes, catalyzing cis-trans isomerization of the peptide ω-bond in phosphorylated serine/threonine-proline motifs in many proteins. Pin1 has therefore been identified as a possible drug target in many diseases, including cancer and Alzheimer's. The effects of phosphorylation on Pin1 substrates, and the atomistic basis for Pin1 recognition and catalysis, are not well understood. Here, we determine the conformational consequences of phosphorylation on Pin1 substrate analogues and the mechanism of recognition by the catalytic domain of Pin1 using all-atom molecular dynamics simulations. We show that phosphorylation induces backbone conformational changes on the peptide substrate analogues. We also show that Pin1 recognizes specific conformations of its substrate by conformational selection. Furthermore, dynamical correlated motions in the free Pin1 enzyme are present in the enzyme of the enzyme-substrate complex when the substrate is in the transition state configuration, suggesting that these motions play significant roles during catalytic turnover. These results provide a detailed atomistic picture of the mechanism of Pin1 recognition that can be exploited for drug design purposes and further our understanding of the synergistic complexities of post-translational phosphorylation and cis-trans isomerization. PMID:21967280

  6. A New Subfamily of Polyphosphate Kinase 2 (Class III PPK2) Catalyzes both Nucleoside Monophosphate Phosphorylation and Nucleoside Diphosphate Phosphorylation

    PubMed Central

    Motomura, Kei; Hirota, Ryuichi; Okada, Mai; Ikeda, Takeshi; Ishida, Takenori

    2014-01-01

    Inorganic polyphosphate (polyP) is a linear polymer of tens to hundreds of phosphate (Pi) residues linked by “high-energy” phosphoanhydride bonds as in ATP. PolyP kinases, responsible for the synthesis and utilization of polyP, are divided into two families (PPK1 and PPK2) due to differences in amino acid sequence and kinetic properties. PPK2 catalyzes preferentially polyP-driven nucleotide phosphorylation (utilization of polyP), which is important for the survival of microbial cells under conditions of stress or pathogenesis. Phylogenetic analysis suggested that the PPK2 family could be divided into three subfamilies (classes I, II, and III). Class I and II PPK2s catalyze nucleoside diphosphate and nucleoside monophosphate phosphorylation, respectively. Here, we demonstrated that class III PPK2 catalyzes both nucleoside monophosphate and nucleoside diphosphate phosphorylation, thereby enabling us to synthesize ATP from AMP by a single enzyme. Moreover, class III PPK2 showed broad substrate specificity over purine and pyrimidine bases. This is the first demonstration that class III PPK2 possesses both class I and II activities. PMID:24532069

  7. Rapid alteration of protein phosphorylation during postmortem: implication in the study of protein phosphorylation.

    PubMed

    Wang, Yifan; Zhang, Yanchong; Hu, Wen; Xie, Shutao; Gong, Cheng-Xin; Iqbal, Khalid; Liu, Fei

    2015-01-01

    Protein phosphorylation is an important post-translational modification of proteins. Postmortem tissues are widely being utilized in the biomedical studies, but the effects of postmortem on protein phosphorylation have not been received enough attention. In the present study, we found here that most proteins in mouse brain, heart, liver, and kidney were rapidly dephosphorylated to various degrees during 20 sec to 10 min postmortem. Phosphorylation of tau at Thr212 and glycogen synthase kinase 3β (GSK-3β) at Ser9 was reduced by 50% in the brain with 40 sec postmortem, a regular time for tissue processing. During postmortem, phosphorylation of cAMP-dependent protein kinase (PKA) and AMP activated kinase (AMPK) was increased in the brain, but not in other organs. Perfusion of the brain with cold or room temperature phosphate-buffered saline (PBS) also caused significant alteration of protein phosphorylation. Cooling down and maintaining mouse brains in the ice-cold buffer prevented the alteration effectively. This study suggests that phosphorylation of proteins is rapidly changed during postmortem. Thus, immediate processing of tissues followed by cooling down in ice-cold buffer is vitally important and perfusion has to be avoided when protein phosphorylation is to be studied. PMID:26511732

  8. Rapid alteration of protein phosphorylation during postmortem: implication in the study of protein phosphorylation

    PubMed Central

    Wang, Yifan; Zhang, Yanchong; Hu, Wen; Xie, Shutao; Gong, Cheng-Xin; Iqbal, Khalid; Liu, Fei

    2015-01-01

    Protein phosphorylation is an important post-translational modification of proteins. Postmortem tissues are widely being utilized in the biomedical studies, but the effects of postmortem on protein phosphorylation have not been received enough attention. In the present study, we found here that most proteins in mouse brain, heart, liver, and kidney were rapidly dephosphorylated to various degrees during 20 sec to 10 min postmortem. Phosphorylation of tau at Thr212 and glycogen synthase kinase 3β (GSK-3β) at Ser9 was reduced by 50% in the brain with 40 sec postmortem, a regular time for tissue processing. During postmortem, phosphorylation of cAMP-dependent protein kinase (PKA) and AMP activated kinase (AMPK) was increased in the brain, but not in other organs. Perfusion of the brain with cold or room temperature phosphate-buffered saline (PBS) also caused significant alteration of protein phosphorylation. Cooling down and maintaining mouse brains in the ice-cold buffer prevented the alteration effectively. This study suggests that phosphorylation of proteins is rapidly changed during postmortem. Thus, immediate processing of tissues followed by cooling down in ice-cold buffer is vitally important and perfusion has to be avoided when protein phosphorylation is to be studied. PMID:26511732

  9. Tyrosine phosphorylation of CD19 in pre-B and mature B cells.

    PubMed Central

    Chalupny, N J; Kanner, S B; Schieven, G L; Wee, S F; Gilliland, L K; Aruffo, A; Ledbetter, J A

    1993-01-01

    Cross-linking of B cell surface immunoglobulins (sIg) results in activation of mature B cells and stimulates a molecular signaling mechanism for antigen-specific B cell expansion and differentiation. This signaling pathway is dependent on tyrosine (Tyr) phosphorylation and results in the activation of sIg-associated src family kinases and p72SYK. Rapid Tyr phosphorylation occurs on multiple protein substrates. Here we show that activation of B cells by cross-linking sIg results in an increase in Tyr phosphorylation of the lineage-restricted B cell surface antigen CD19, and show that it is a major substrate of activated Tyr kinase following sIg stimulation. Lower levels of constitutive CD19 Tyr phosphorylation occurred in most sIg+ mature B cell lines examined and in normal dense tonsillar B cells. We also find that when CD19 is Tyr-phosphorylated it becomes competent to interact with SH2 domains suggesting a mechanism whereby, following B cell activation, CD19 could be linked to intracellular signaling pathways. In sIg- pre-B cell lines, CD19 was expressed but was not constitutively phosphorylated on tyrosine. Upon CD19 cross-linking, Tyr phosphorylation of CD19 was induced in sIg- pre-B cell lines. CD19 cross-linking also directly induced Tyr phosphorylation of CD19 and other substrates in mature B cells. The ability of CD19 to signal in the absence of sIg expression may provide important stimulation in pre-B cell development. Images PMID:7687539

  10. Morphometric Brain Abnormalities in Boys with Conduct Disorder

    ERIC Educational Resources Information Center

    Huebner, Thomas; Vloet, Timo D.; Marx, Ivo; Konrad, Kerstin; Fink, Gereon R.; Herpertz, Sabine C.; Herpertz-Dahlmann, Beate

    2008-01-01

    Conduct disorder (CD) is associated with antisocial personality behavior that violates the basic rights of others. Results, on examining the structural brain aberrations in boys' CD, show that boys with CD and cormobid attention-deficit/hyperactivity disorder showed abnormalities in frontolimbic areas that could contribute to antisocial…

  11. BRCC36 is essential for ionizing radiation-induced BRCA1 phosphorylation and nuclear foci formation.

    PubMed

    Chen, Xiaowei; Arciero, Cletus A; Wang, Chunrong; Broccoli, Dominique; Godwin, Andrew K

    2006-05-15

    We have previously reported the identification and characterization of a novel BRCA1/2 interacting protein complex, BRCC (BRCA1/2-containing complex). BRCC36, one of the proteins in BRCC, directly interacts with BRCA1, and regulates the ubiquitin E3 ligase activity of BRCC. Importantly, BRCC36 is aberrantly expressed in the vast majority of breast tumors, indicating a potential role in the pathogenesis of this disease. To further elucidate the functional consequence of abnormal BRCC36 expression in breast cancer, we have done in vivo silencing studies using small interfering RNAs targeting BRCC36 in breast cancer cell lines, i.e., MCF-7, ZR-75-1, and T47D. Knock-down of BRCC36 alone does not affect cell growth, but when combined with ionizing radiation (IR) exposure, it leads to an increase in the percentage of cells undergoing apoptosis when compared with the small interfering RNA control group in breast cancer cells. Immunoblot analysis shows that inhibition of BRCC36 has no effect on the activation of ATM, expression of p21 and p53, or BRCA1-BARD1 interaction following IR exposure. Importantly, BRCC36 depletion disrupts IR-induced phosphorylation of BRCA1. Immunofluorescent staining of BRCA1 and gamma-H2AX indicates that BRCC36 depletion prevents the formation of BRCA1 nuclear foci in response to DNA damage in breast cancer cells. These results show that down-regulation of BRCC36 expression impairs the DNA repair pathway activated in response to IR by inhibiting BRCA1 activation, thereby sensitizing breast cancer cells to IR-induced apoptosis. PMID:16707425

  12. Heat-shock protein-25/27 phosphorylation by the delta isoform of protein kinase C.

    PubMed Central

    Maizels, E T; Peters, C A; Kline, M; Cutler, R E; Shanmugam, M; Hunzicker-Dunn, M

    1998-01-01

    Small heat-shock proteins (sHSPs) are widely expressed 25-28 kDa proteins whose functions are dynamically regulated by phosphorylation. While recent efforts have clearly delineated a stress-responsive p38 mitogen-activated protein-kinase (MAPK)-dependent kinase pathway culminating in activation of the heat-shock (HSP)-kinases, mitogen-activated protein-kinase-activated protein kinase-2 and -3, not all sHSP phosphorylation events can be explained by the p38 MAPK-dependent pathway. The contribution of protein kinase C (PKC) to sHSP phosphorylation was suggested by early studies but later questioned on the basis of the reported poor ability of purified PKC to phosphorylate sHSP in vitro. The current study re-evaluates the role of PKC in sHSP phosphorylation in the light of the isoform complexity of the PKC family. We evaluated the sHSP phosphorylation status in rat corpora lutea obtained from two stages of pregnancy, mid-pregnancy and late-pregnancy, which express different levels of the novel PKC isoform, PKC-delta. Two-dimensional Western blot analysis showed that HSP-27 was more highly phosphorylated in vivo in corpora lutea of late pregnancy, corresponding to the developmental stage in which PKC-delta is abundant and active. Late-pregnant luteal extracts contained a lipid-sensitive HSP-kinase activity which exactly co-purified with PKC-delta using hydroxyapatite and S-Sepharose column chromatography. To determine whether there might be preferential phosphorylation of sHSP by a particular PKC isoform, purified recombinant PKC isoforms corresponding to those PKC isoforms detected in rat corpora lutea were evaluated for HSP-kinase activity in vitro. Recombinant PKC-delta effectively catalysed the phosphorylation of sHSP in vitro, and PKC-alpha was 30-50% as effective as an HSP-kinase; other PKCs tested (beta1, beta2, epsilon and zeta) were poor HSP-kinases. These results show that select PKC family members can function as direct HSP-kinases in vitro. Moreover, the

  13. Native fluorescence characterization of human liver abnormalities

    NASA Astrophysics Data System (ADS)

    Ganesan, Singaravelu; Madhuri, S.; Aruna, Prakasa R.; Suchitra, S.; Srinivasan, T. G.

    1999-05-01

    Fluorescence spectroscopy of intrinsic biomolecules has been extensively used in biology and medicine for the past several decades. In the present study, we report the native fluorescence characteristics of blood plasma from normal human subjects and patients with different liver abnormalities such as hepatitis, leptospirosis, jaundice, cirrhosis and liver cell failure. Native fluorescence spectra of blood plasma -- acetone extract were measured at 405 nm excitation. The average spectrum of normal blood plasma has a prominent emission peak around 464 nm whereas in the case of liver diseased subjects, the primary peak is red shifted with respect to normal. In addition, liver diseased cases show distinct secondary emission peak around 615 nm, which may be attributed to the presence of endogenous porphyrins. The red shift of the prominent emission peak with respect to normal is found to be maximum for hepatitis and minimum for cirrhosis whereas the secondary emission peak around 615 nm was found to be more prominent in the case of cirrhosis than the rest. The ratio parameter I465/I615 is found to be statistically significant (p less than 0.001) in discriminating liver abnormalities from normal.

  14. Ligand-induced IFN gamma receptor tyrosine phosphorylation couples the receptor to its signal transduction system (p91).

    PubMed Central

    Greenlund, A C; Farrar, M A; Viviano, B L; Schreiber, R D

    1994-01-01

    Herein we report that interferon-gamma (IFN gamma) induces the rapid and reversible tyrosine phosphorylation of the IFN gamma receptor. Using a panel of receptor intracellular domain mutants, we show that a membrane-proximal LPKS sequence (residues 266-269) is required for ligand-induced tyrosine kinase activation and/or kinase-receptor association and biological responsiveness, and a functionally critical membrane-distal tyrosine residue (Y440) is a target of the activated enzyme. The biological significance of Y440 phosphorylation was demonstrated by showing that a receptor-derived nonapeptide corresponding to receptor residues 436-444 and containing phosphorylated Y440 bound specifically to p91, blocked p91 phosphorylation and inhibited the generation of an active p91-containing transcription factor complex. In contrast, nonphosphorylated wild-type, phosphorylated mutant, or phosphorylated irrelevant peptides did not. Moreover, the phosphorylated Y440-containing peptide did not interact with a related but distinct latent transcription factor (p113) which is activatible by IFN alpha but not IFN gamma. These results thus document the specific and inducible association of p91 with the phosphorylated IFN gamma receptor and thereby elucidate the mechanism by which ligand couples the IFN gamma receptor to its signal transduction system. Images PMID:8156998

  15. White Matter Abnormalities in Schizophrenia and Schizotypal Personality Disorder

    PubMed Central

    Lener, Marc S.; Wong, Edmund; Tang, Cheuk Y.; Byne, William; Goldstein, Kim E.; Blair, Nicholas J.; Haznedar, M. Mehmet; New, Antonia S.; Chemerinski, Eran; Chu, King-Wai; Rimsky, Liza S.; Siever, Larry J.; Koenigsberg, Harold W.; Hazlett, Erin A.

    2015-01-01

    Prior diffusion tensor imaging (DTI) studies examining schizotypal personality disorder (SPD) and schizophrenia, separately have shown that compared with healthy controls (HCs), patients show frontotemporal white matter (WM) abnormalities. This is the first DTI study to directly compare WM tract coherence with tractography and fractional anisotropy (FA) across the schizophrenia spectrum in a large sample of demographically matched HCs (n = 55), medication-naive SPD patients (n = 49), and unmedicated/never-medicated schizophrenia patients (n = 22) to determine whether (a) frontal-striatal-temporal WM tract abnormalities in schizophrenia are similar to, or distinct from those observed in SPD; and (b) WM tract abnormalities are associated with clinical symptom severity indicating a common underlying pathology across the spectrum. Compared with both the HC and SPD groups, schizophrenia patients showed WM abnormalities, as indexed by lower FA in the temporal lobe (inferior longitudinal fasciculus) and cingulum regions. SPD patients showed lower FA in the corpus callosum genu compared with the HC group, but this regional abnormality was more widespread in schizophrenia patients. Across the schizophrenia spectrum, greater WM disruptions were associated with greater symptom severity. Overall, frontal-striatal-temporal WM dysconnectivity is attenuated in SPD compared with schizophrenia patients and may mitigate the emergence of psychosis. PMID:24962608

  16. Trifluoromethanesulfonamide anthelmintics. Protonophoric uncouplers of oxidative phosphorylation.

    PubMed

    McCracken, R O; Carr, A W; Stillwell, W H; Lipkowitz, K B; Boisvenue, R; O'Doherty, G O; Wickiser, D I

    1993-05-01

    A series of trifluoromethanesulfonamides (TFMS) was synthesized and tested for uncoupling activity in rat liver mitochondria. With succinate as the mitochondrial substrate, and the respiratory control index (RCI) as an indicator of their uncoupling ability, we found that all of the TFMS tested were uncouplers of oxidative phosphorylation; the effective concentration (RCI I50) ranged from less than 1 microM to greater than 1000 microM. Correlation techniques were used to assess the strength of the relationship between the ability of a TFMS to uncouple oxidative phosphorylation and its ability to lower the electrical resistance of planar bimolecular lipid membranes. There was a highly significant (P < 0.001) positive linear relationship (r = 0.97) between the ability of a TFMS to uncouple oxidative phosphorylation and its ability to lower electrical resistance. These findings are consistent with the view that the TFMS are lipophilic protonophoric uncouplers of mitochondrial oxidative phosphorylation. Quantitative structure-activity relationship studies using experiment and semiempirical molecular orbital theory revealed that the hydrophobicity of a TFMS and its molecular dipole moment were the principal determinants of mitochondrial uncoupling activity within the pKa range examined. PMID:8388210

  17. Identification of extracellularly phosphorylated membrane proteins.

    PubMed

    Burghoff, Sandra; Willberg, Wibke; Schrader, Jürgen

    2015-10-01

    Ecto-protein kinases phosphorylate extracellular membrane proteins and exhibit similarities to casein kinases and protein kinases A and C. However, the identification of their protein substrates still remains a challenge because a clear separation from intracellular phosphoproteins is difficult. Here, we describe a straightforward method for the identification of extracellularly phosphorylated membrane proteins in human umbilical vein endothelial cells (HUVECs) and K562 cells which used the protease bromelain to selectively remove ectoproteins from intact cells and combined this with the subsequent analysis using IMAC and LC-MS/MS. A "false-positive" strategy in which cells without protease treatment served as controls was applied. Using this approach we identified novel phosphorylation sites on five ectophosphoproteins (NOTCH1, otopetrin 1, regulator of G-protein signalling 13 (RGS13), protein tyrosine phosphatase receptor type D isoform 3 (PTPRD), usherin isoform B (USH2A)). Use of bromelain appears to be a reliable technique for the further identification of phosphorylated surface-exposed peptides when extracellular adenosine-5'-triphosphate is elevated during purinergic signalling. PMID:26152529

  18. Regulation of protein phosphorylation in oat mitochondria

    SciTech Connect

    Pike, C.; Kopeck, K.; Sceppa, E. )

    1989-04-01

    We sought to identify phosphorylated proteins in isolated oat mitocchondria and to characterize the enzymatic and regulatory properties of the protein kinase(s). Mitochondria from oats (Avena sativa L. cv. Garry) were purified on Percoll gradients. Mitochondria were incubated with {sup 32}P-{gamma}-ATP; proteins were separated by SDS-PAGE. A small number of bands was detected on autoradiograms, most prominently at 70 kD and 42 kD; the latter band has been tentatively identified as a subunit of the pyruvate dehydrogenase complex, a well-known phosphoprotein. The protein kinase(s) could also phosphorylate casein, but not histone. Spermine enhanced the phosphorylation of casein and inhibited the phosphorylation of the 42 kD band. These studies were carried out on both intact and burst mitochondria. Control by calcium and other ions was investigated. The question of the action of regulators on protein kinase or protein phosphatase was studied by the use of {sup 35}S-adenosine thiotriphosphate.

  19. Metaphase protein phosphorylation in Xenopus laevis eggs.

    PubMed Central

    Lohka, M J; Kyes, J L; Maller, J L

    1987-01-01

    Cytoplasmic extracts of metaphase (M-phase)-arrested Xenopus laevis eggs support nuclear envelope breakdown and chromosome condensation in vitro. Induction of nuclear breakdown is inhibited by AMPP(NH)P, a nonhydrolyzable ATP analog, but not by ATP or gamma-S-ATP, a hydrolyzable ATP analog, suggesting that protein phosphorylation may be required for M-phase nuclear events in vitro. By addition of [gamma-32P]ATP, we have identified in cytoplasmic extracts and in intact eggs at least six phosphoproteins that are present during M-phase but absent in G1/S-phase. These phosphoproteins also appear in response to partially purified preparations of maturation-promoting factor. A subset of these proteins are thiophosphorylated by gamma-S-ATP under conditions that promote nuclear envelope breakdown and chromosome condensation. Each of these proteins is phosphorylated on serine and threonine, and one, a 42-kilodalton protein, is also phosphorylated on tyrosine both in extracts and in intact eggs. These results indicate that activation of protein kinases accounts for at least part of the increased phosphorylation in M-phase and that both protein-serine-threonine kinases and protein-tyrosine kinases may play a role in controlling M-phase nuclear behavior. Images PMID:3821728

  20. Nucleoside phosphorylation by the mineral schreibersite

    PubMed Central

    Gull, Maheen; Mojica, Mike A.; Fernández, Facundo M.; Gaul, David A.; Orlando, Thomas M.; Liotta, Charles L.; Pasek, Matthew A.

    2015-01-01

    Phosphorylation of the nucleosides adenosine and uridine by the simple mixing and mild heating of aqueous solutions of the organic compounds with synthetic analogs of the meteoritic mineral schreibersite, (Fe,Ni)3P under slightly basic conditions (pH ~9) is reported. These results suggest a potential role for meteoritic phosphorus in the origin and development of early life. PMID:26606901

  1. Ion channels, phosphorylation and mammalian sperm capacitation

    PubMed Central

    Visconti, Pablo E; Krapf, Dario; de la Vega-Beltrán, José Luis; Acevedo, Juan José; Darszon, Alberto

    2011-01-01

    Sexually reproducing animals require an orchestrated communication between spermatozoa and the egg to generate a new individual. Capacitation, a maturational complex phenomenon that occurs in the female reproductive tract, renders spermatozoa capable of binding and fusing with the oocyte, and it is a requirement for mammalian fertilization. Capacitation encompasses plasma membrane reorganization, ion permeability regulation, cholesterol loss and changes in the phosphorylation state of many proteins. Novel tools to study sperm ion channels, image intracellular ionic changes and proteins with better spatial and temporal resolution, are unraveling how modifications in sperm ion transport and phosphorylation states lead to capacitation. Recent evidence indicates that two parallel pathways regulate phosphorylation events leading to capacitation, one of them requiring activation of protein kinase A and the second one involving inactivation of ser/thr phosphatases. This review examines the involvement of ion transporters and phosphorylation signaling processes needed for spermatozoa to achieve capacitation. Understanding the molecular mechanisms leading to fertilization is central for societies to deal with rising male infertility rates, to develop safe male gamete-based contraceptives and to preserve biodiversity through better assisted fertilization strategies. PMID:21540868

  2. Phosphorylation of plastoglobular proteins in Arabidopsis thaliana.

    PubMed

    Lohscheider, Jens N; Friso, Giulia; van Wijk, Klaas J

    2016-06-01

    Plastoglobules (PGs) are plastid lipid-protein particles with a small specialized proteome and metabolome. Among the 30 core PG proteins are six proteins of the ancient ABC1 atypical kinase (ABC1K) family and their locations in an Arabidopsis mRNA-based co-expression network suggested central regulatory roles. To identify candidate ABC1K targets and a possible ABC1K hierarchical phosphorylation network within the chloroplast PG proteome, we searched Arabidopsis phosphoproteomics data from publicly available sources. Evaluation of underlying spectra and/or associated information was challenging for a variety of reasons, but supported pSer sites and a few pThr sites in nine PG proteins, including five FIBRILLINS. PG phosphorylation motifs are discussed in the context of possible responsible kinases. The challenges of collection and evaluation of published Arabidopsis phosphorylation data are discussed, illustrating the importance of deposition of all mass spectrometry data in well-organized repositories such as PRIDE and ProteomeXchange. This study provides a starting point for experimental testing of phosho-sites in PG proteins and also suggests that phosphoproteomics studies specifically designed toward the PG proteome and its ABC1K are needed to understand phosphorylation networks in these specialized particles. PMID:26962209

  3. Phosphorylation of plastoglobular proteins in Arabidopsis thaliana

    PubMed Central

    Lohscheider, Jens N.; Friso, Giulia; van Wijk, Klaas J.

    2016-01-01

    Plastoglobules (PGs) are plastid lipid–protein particles with a small specialized proteome and metabolome. Among the 30 core PG proteins are six proteins of the ancient ABC1 atypical kinase (ABC1K) family and their locations in an Arabidopsis mRNA-based co-expression network suggested central regulatory roles. To identify candidate ABC1K targets and a possible ABC1K hierarchical phosphorylation network within the chloroplast PG proteome, we searched Arabidopsis phosphoproteomics data from publicly available sources. Evaluation of underlying spectra and/or associated information was challenging for a variety of reasons, but supported pSer sites and a few pThr sites in nine PG proteins, including five FIBRILLINS. PG phosphorylation motifs are discussed in the context of possible responsible kinases. The challenges of collection and evaluation of published Arabidopsis phosphorylation data are discussed, illustrating the importance of deposition of all mass spectrometry data in well-organized repositories such as PRIDE and ProteomeXchange. This study provides a starting point for experimental testing of phosho-sites in PG proteins and also suggests that phosphoproteomics studies specifically designed toward the PG proteome and its ABC1K are needed to understand phosphorylation networks in these specialized particles. PMID:26962209

  4. Abnormalities of the Erythrocyte Membrane

    PubMed Central

    Gallagher, Patrick G.

    2014-01-01

    Synopsis Primary abnormalities of the erythrocyte membrane, including the hereditary spherocytosis and hereditary elliptocytosis syndromes, are an important group of inherited hemolytic anemias. Classified by distinctive morphology on peripheral blood smear, these disorders are characterized by clinical, laboratory, and genetic heterogeneity. Among this group, hereditary spherocytosis patients are more likely to experience symptomatic anemia. Treatment of hereditary spherocytosis with splenectomy is curative in most patients. Once considered routine, growing recognition of the longterm risks of splenectomy, including cardiovascular disease, thrombotic disorders, and pulmonary hypertension, as well as the emergence of penicillin-resistant pneumococci, a concern for infection in overwhelming postsplenectomy infection, have led to re-evaluation of the role of splenectomy. Current management guidelines acknowledge these important considerations when entertaining splenectomy and recommend detailed discussion between health care providers, patient, and family. The hereditary elliptocytosis syndromes are the most common primary disorders of erythrocyte membrane proteins. However, most elliptocytosis patients are asymptomatic and do not require therapy. PMID:24237975

  5. The time of onset of abnormal calcification in spondylometaepiphyseal dysplasia, short limb-abnormal calcification type.

    PubMed

    Tüysüz, Beyhan; Gazioğlu, Nurperi; Ungür, Savaş; Aji, Dolly Yafet; Türkmen, Seval

    2009-01-01

    A 1-month-old boy with shortness of extremities on prenatal US was referred to our department with a provisional diagnosis of achondroplasia. His height was normal but he had short extremities and platyspondyly, premature carpal epiphyses on both hands, and short tubular bones with irregular metaphyses on radiographs. Re-evaluation of the patient at the age of 1 year revealed very short height and premature calcification of the costal cartilages and epiphyses. Spondylometaepiphyseal dysplasia (SMED), short limb-abnormal calcification type was diagnosed. This condition is a very rare autosomal recessively inherited disorder, and most of the patients die in early childhood due to neurological involvement. At the age of 2 years and 5 months, a CT scan showed narrowing of the cervical spinal canal. One month later he died suddenly because of spinal cord injury. In conclusion early diagnosis is very important because the recurrence risk is high and patients may die due to early neurological complications. The time of onset of abnormal calcifications, a diagnostic finding of the disease, is at the age of around 1 year in most patients. When abnormal calcifications are not yet present, but radiological changes associated with SMED are present, this rare disease must be considered. PMID:19002453

  6. Adults with Chromosome 18 Abnormalities.

    PubMed

    Soileau, Bridgette; Hasi, Minire; Sebold, Courtney; Hill, Annice; O'Donnell, Louise; Hale, Daniel E; Cody, Jannine D

    2015-08-01

    The identification of an underlying chromosome abnormality frequently marks the endpoint of a diagnostic odyssey. However, families are frequently left with more questions than answers as they consider their child's future. In the case of rare chromosome conditions, a lack of longitudinal data often makes it difficult to provide anticipatory guidance to these families. The objective of this study is to describe the lifespan, educational attainment, living situation, and behavioral phenotype of adults with chromosome 18 abnormalities. The Chromosome 18 Clinical Research Center has enrolled 483 individuals with one of the following conditions: 18q-, 18p-, Tetrasomy 18p, and Ring 18. As a part of the ongoing longitudinal study, we collect data on living arrangements, educational level attained, and employment status as well as data on executive functioning and behavioral skills on an annual basis. Within our cohort, 28 of the 483 participants have died, the majority of whom have deletions encompassing the TCF4 gene or who have unbalanced rearrangement involving other chromosomes. Data regarding the cause of and age at death are presented. We also report on the living situation, educational attainment, and behavioral phenotype of the 151 participants over the age of 18. In general, educational level is higher for people with all these conditions than implied by the early literature, including some that received post-high school education. In addition, some individuals are able to live independently, though at this point they represent a minority of patients. Data on executive function and behavioral phenotype are also presented. Taken together, these data provide insight into the long-term outcome for individuals with a chromosome 18 condition. This information is critical in counseling families on the range of potential outcomes for their child. PMID:25403900

  7. Abnormal pupillary light reflex with chromatic pupillometry in Gaucher disease.

    PubMed

    Narita, Aya; Shirai, Kentarou; Kubota, Norika; Takayama, Rumiko; Takahashi, Yukitoshi; Onuki, Takanori; Numakura, Chikahiko; Kato, Mitsuhiro; Hamada, Yusuke; Sakai, Norio; Ohno, Atsuko; Asami, Maya; Matsushita, Shoko; Hayashi, Anri; Kumada, Tomohiro; Fujii, Tatsuya; Horino, Asako; Inoue, Takeshi; Kuki, Ichiro; Asakawa, Ken; Ishikawa, Hitoshi; Ohno, Koyo; Nishimura, Yoko; Tamasaki, Akiko; Maegaki, Yoshihiro; Ohno, Kousaku

    2014-02-01

    The hallmark of neuronopathic Gaucher disease (GD) is oculomotor abnormalities, but ophthalmological assessment is difficult in uncooperative patients. Chromatic pupillometry is a quantitative method to assess the pupillary light reflex (PLR) with minimal patient cooperation. Thus, we investigated whether chromatic pupillometry could be useful for neurological evaluations in GD. In our neuronopathic GD patients, red light-induced PLR was markedly impaired, whereas blue light-induced PLR was relatively spared. In addition, patients with non-neuronopathic GD showed no abnormalities. These novel findings show that chromatic pupillometry is a convenient method to detect neurological signs and monitor the course of disease in neuronopathic GD. PMID:25356393

  8. Abnormal pupillary light reflex with chromatic pupillometry in Gaucher disease

    PubMed Central

    Narita, Aya; Shirai, Kentarou; Kubota, Norika; Takayama, Rumiko; Takahashi, Yukitoshi; Onuki, Takanori; Numakura, Chikahiko; Kato, Mitsuhiro; Hamada, Yusuke; Sakai, Norio; Ohno, Atsuko; Asami, Maya; Matsushita, Shoko; Hayashi, Anri; Kumada, Tomohiro; Fujii, Tatsuya; Horino, Asako; Inoue, Takeshi; Kuki, Ichiro; Asakawa, Ken; Ishikawa, Hitoshi; Ohno, Koyo; Nishimura, Yoko; Tamasaki, Akiko; Maegaki, Yoshihiro; Ohno, Kousaku

    2014-01-01

    The hallmark of neuronopathic Gaucher disease (GD) is oculomotor abnormalities, but ophthalmological assessment is difficult in uncooperative patients. Chromatic pupillometry is a quantitative method to assess the pupillary light reflex (PLR) with minimal patient cooperation. Thus, we investigated whether chromatic pupillometry could be useful for neurological evaluations in GD. In our neuronopathic GD patients, red light-induced PLR was markedly impaired, whereas blue light-induced PLR was relatively spared. In addition, patients with non-neuronopathic GD showed no abnormalities. These novel findings show that chromatic pupillometry is a convenient method to detect neurological signs and monitor the course of disease in neuronopathic GD. PMID:25356393

  9. Enhanced prediction of conformational flexibility and phosphorylation in proteins.

    PubMed

    Swaminathan, Karthikeyan; Adamczak, Rafal; Porollo, Aleksey; Meller, Jarosław

    2010-01-01

    Many sequence-based predictors of structural and functional properties of proteins have been developed in the past. In this study, we developed new methods for predicting measures of conformational flexibility in proteins, including X-ray structure-derived temperature (B-) factors and the variance within NMR structural ensemble, as effectively measured by the solvent accessibility standard deviations (SASDs). We further tested whether these predicted measures of conformational flexibility in crystal lattices and solution, respectively, can be used to improve the prediction of phosphorylation in proteins. The latter is an example of a common post-translational modification that modulates protein function, e.g., by affecting interactions and conformational flexibility of phosphorylated sites. Using robust epsilon-insensitive support vector regression (ε-SVR) models, we assessed two specific representations of protein sequences: one based on the position-specific scoring matrices (PSSMs) derived from multiple sequence alignments, and an augmented representation that incorporates real-valued solvent accessibility and secondary structure predictions (RSA/SS) as additional measures of local structural propensities. We showed that a combination of PSSMs and real-valued SS/RSA predictions provides systematic improvements in the accuracy of both B-factors and SASD prediction. These intermediate predictions were subsequently combined into an enhanced predictor of phosphorylation that was shown to significantly outperform methods based on PSSM alone. We would like to stress that to the best of our knowledge, this is the first example of using predicted from sequence NMR structure-based measures of conformational flexibility in solution for the prediction of other properties of proteins. Phosphorylation prediction methods typically employ a two-class classification approach with the limitation that the set of negative examples used for training may include some sites that are

  10. Phosphorylation of the Aspergillus oryzae Woronin body protein, AoHex1, by protein kinase C: evidence for its role in the multimerization and proper localization of the Woronin body protein

    PubMed Central

    Juvvadi, Praveen Rao; Maruyama, Jun-ichi; Kitamoto, Katsuhiko

    2007-01-01

    Woronin body, a specialized peroxisome, is a unique organelle involved in septal pore sealing and protecting filamentous fungus from excessive cytoplasmic bleeding. We recently characterized the Aohex1 gene encoding the major protein of the Woronin body in the fungus Aspergillus oryzae. Although three-dimensional microscopy revealed plugging of the septal pore by Woronin body, the mechanism of its formation remains unknown. We report here a reduction in the oligomeric forms (dimeric and tetrameric) of AoHex1 upon λ-phosphatase treatment, which indicated that AoHex1 phosphorylation in vivo facilitates its oligomerization. Concomitant with the presence of a highly conserved predicted PKC (protein kinase C)-phosphorylatable site (Ser151), the recombinant AoHex1 was phosphorylated by PKC in vitro and the administration of the PKC inhibitors, bisindolylmaleimide I and chelerythrine, resulted in the reduction of the oligomeric forms of AoHex1 in vivo. While spherical dot-like Woronin bodies were visualized by expressing the dsred2–Aohex1 and egfp (enhanced green fluorescent protein)–Aohex1 constructs in A. oryzae, treatment with the PKC inhibitors caused an abnormal localization to ring-like structures. In addition to the reduced phosphorylation of the mutagenized recombinant AoHex1[S151A] (Ser151 to alanine substitution) by PKC in vitro, the overexpression of Aohex1[S151A] as dsred2 fusion against the wild-type background also showed reduction of the oligomeric forms of the endogenous AoHex1 and its perturbed localization to ring-like structures in vivo. In conclusion, the present study implicates the relevance of PKC-dependent phosphorylation of the Woronin body protein, AoHex1, for its multimerization and proper localization. PMID:17441786

  11. Phosphorylation of inhibitor-2 and activation of MgATP-dependent protein phosphatase by rat skeletal muscle glycogen synthase kinase

    SciTech Connect

    Hegazy, M.G.; Reimann, E.M.; Thysseril, T.J.; Schlender, K.K.

    1986-05-01

    Rat skeletal muscle contains a glycogen synthase kinase (GSK-M) which is not stimulated by Ca/sup 2 +/ or cAMP. This kinase has an apparent Mr of 62,000 and uses ATP but not GTP as a phosphoryl donor. GSK-M phosphorylated glycogen synthase at sites 2 and 3. It phosphorylated ATP-citrate lyase and activated MgATP-dependent phosphatase in the presence of ATP but not GTP. As expected, the kinase also phosphorylated phosphatase inhibitor 2 (I-2). Phosphatase incorporation reached approximately 0.3 mol/mol of I-2. Phosphopeptide maps were obtained by digesting /sup 32/P-labeled I-2 with trypsin and separating the peptides by reversed phase HPLC. Two partially separated /sup 32/P-labeled peaks were obtained when I-2 was phosphorylated with either GSK-M or glycogen synthase kinase 3 (GSK-3) and these peptides were different from those obtained when I-2 was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase (CSU) or casein kinase II (CK-II). When I-2 was phosphorylated with GSK-M or GSK-3 and cleaved by CNBr, a single radioactive peak was obtained. Phosphoamino acid analysis showed that I-2 was phosphorylated by GSK-M or GSK-3 predominately in Thr whereas CSU and CK-II phosphorylated I-2 exclusively in Ser. These results indicate that GSK-M is similar to GSK-3 and to ATP-citrate lyase kinase. However, it appears to differ in Mr from ATP-citrate lyase kinase and it differs from GSK-3 in that it phosphorylates glycogen synthase at site 2 and it does not use GTP as a phosphoryl donor.

  12. A mutation of the fission yeast EB1 overcomes negative regulation by phosphorylation and stabilizes microtubules

    SciTech Connect

    Iimori, Makoto; Ozaki, Kanako; Chikashige, Yuji; Habu, Toshiyuki; Hiraoka, Yasushi; Maki, Takahisa; Hayashi, Ikuko; Obuse, Chikashi; Matsumoto, Tomohiro

    2012-02-01

    Mal3 is a fission yeast homolog of EB1, a plus-end tracking protein (+ TIP). We have generated a mutation (89R) replacing glutamine with arginine in the calponin homology (CH) domain of Mal3. Analysis of the 89R mutant in vitro has revealed that the mutation confers a higher affinity to microtubules and enhances the intrinsic activity to promote the microtubule-assembly. The mutant Mal3 is no longer a + TIP, but binds strongly the microtubule lattice. Live cell imaging has revealed that while the wild type Mal3 proteins dissociate from the tip of the growing microtubules before the onset of shrinkage, the mutant Mal3 proteins persist on microtubules and reduces a rate of shrinkage after a longer pausing period. Consequently, the mutant Mal3 proteins cause abnormal elongation of microtubules composing the spindle and aster. Mal3 is phosphorylated at a cluster of serine/threonine residues in the linker connecting the CH and EB1-like C-terminal motif domains. The phosphorylation occurs in a microtubule-dependent manner and reduces the affinity of Mal3 to microtubules. We propose that because the 89R mutation is resistant to the effect of phosphorylation, it can associate persistently with microtubules and confers a stronger stability of microtubules likely by reinforcing the cylindrical structure. -- Highlights: Black-Right-Pointing-Pointer We characterize a mutation (mal3-89R) in fission yeast homolog of EB1. Black-Right-Pointing-Pointer The mutation enhances the activity to assemble microtubules. Black-Right-Pointing-Pointer Mal3 is phosphorylated in a microtubule-dependent manner. Black-Right-Pointing-Pointer The phosphorylation negatively regulates the Mal3 activity.

  13. Correlation between persistent forms of zeaxanthin-dependent energy dissipation and thylakoid protein phosphorylation.

    PubMed

    Ebbert, V; Demmig-Adams, B; Adams, W W; Mueh, K E; Staehelin, L A

    2001-01-01

    High light stress induced not only a sustained form of xanthophyll cycle-dependent energy dissipation but also sustained thylakoid protein phosphorylation. The effect of protein phosphatase inhibitors (fluoride and molybdate ions) on recovery from a 1-h exposure to a high PFD was examined in leaf discs of Parthenocissus quinquefolia (Virginia creeper). Inhibition of protein dephosphorylation induced zeaxanthin retention and sustained energy dissipation (NPQ) upon return to low PFD for recovery, but had no significant effects on pigment and Chl fluorescence characteristics under high light exposure. In addition, whole plants of Monstera deliciosa and spinach grown at low to moderate PFDs were transferred to high PFDs, and thylakoid protein phosphorylation pattern (assessed with anti-phosphothreonine antibody) as well as pigment and Chl fluorescence characteristics were examined over several days. A correlation was obtained between dark-sustained D1/D2 phosphorylation and dark-sustained zeaxanthin retention and maintenance of PS II in a state primed for energy dissipation in both species. The degree of these dark-sustained phenomena was more pronounced in M. deliciosa compared with spinach. Moreover, M. deliciosa but not spinach plants showed unusual phosphorylation patterns of Lhcb proteins with pronounced dark-sustained Lhcb phosphorylation even under low PFD growth conditions. Subsequent to the transfer to a high PFD, dark-sustained Lhcb protein phosphorylation was further enhanced. Thus, phosphorylation patterns of D1/D2 and Lhcb proteins differed from each other as well as among plant species. The results presented here suggest an association between dark-sustained D1/D2 phosphorylation and sustained retention of zeaxanthin and energy dissipation (NPQ) in light-stressed, and particularly 'photoinhibited', leaves. Functional implications of these observations are discussed. PMID:16228317

  14. Induced expression of nucleolin phosphorylation-deficient mutant confers dominant-negative effect on cell proliferation.

    PubMed

    Xiao, Shu; Caglar, Elif; Maldonado, Priscilla; Das, Dibash; Nadeem, Zaineb; Chi, Angela; Trinité, Benjamin; Li, Xin; Saxena, Anjana

    2014-01-01

    Nucleolin (NCL) is a major nucleolar phosphoprotein that has pleiotropic effects on cell proliferation and is elevated in a variety of tumors. NCL is highly phosphorylated at the N-terminus by two major kinases: interphase casein kinase 2 (CK2) and mitotic cyclin-dependent kinase 1 (CDK1). Earlier we demonstrated that a NCL-mutant that is partly defective in undergoing phosphorylation by CK2 inhibits chromosomal replication through its interactions with Replication Protein A, mimicking the cellular response to DNA damage. We further delineated that the N-terminus of NCL associates with Hdm2, the most common E3 ubiquitin ligase of p53. We reported that NCL antagonizes Hdm2 to stabilize p53 and stimulates p53 transcriptional activity. Although NCL-phosphorylation by CK2 and ribosomal DNA transcription are closely coordinated during interphase, the role of NCL phosphorylation in regulating cell proliferation remains unexplored. We have therefore engineered unique human cells that specifically induce expression of NCL-wild type (WT) or a phosphorylation-deficient NCL-mutant, 6/S*A where all the six CK2 consensus serine sites residing in the N-terminus NCL were mutated to alanine. Here we show that this NCL-mutant is defective in undergoing phosphorylation by CK2. We also demonstrate that NCL-phosphorylation by CK2 is required through the S-phase progression in cell cycle and hence proliferation. Induced expression of NCL with mutated CK2 phosphorylation sites stabilizes p53, results in higher expression of Bcl2 (B-cell lymphoma 2) homology 3 (BH3)-only apoptotic markers and causes a dominant-negative effect on cell viability. Our unique cellular system thus provides the first evidential support to delineate phospho-specific functions of NCL on cell proliferation. PMID:25313645

  15. Phosphorylation of myosin regulatory light chain controls myosin head conformation in cardiac muscle.

    PubMed

    Kampourakis, Thomas; Irving, Malcolm

    2015-08-01

    The effect of phosphorylation on the conformation of the regulatory light chain (cRLC) region of myosin in ventricular trabeculae from rat heart was determined by polarized fluorescence from thiophosphorylated cRLCs labelled with bifunctional sulforhodamine (BSR). Less than 5% of cRLCs were endogenously phosphorylated in this preparation, and similarly low values of basal cRLC phosphorylation were measured in fresh intact ventricle from both rat and mouse hearts. BSR-labelled cRLCs were thiophosphorylated by a recombinant fragment of human cardiac myosin light chain kinase, which was shown to phosphorylate cRLCs specifically at serine 15 in a calcium- and calmodulin-dependent manner, both in vitro and in situ. The BSR-cRLCs were exchanged into demembranated trabeculae, and polarized fluorescence intensities measured for each BSR-cRLC in relaxation, active isometric contraction and rigor were combined with RLC crystal structures to calculate the orientation distribution of the C-lobe of the cRLC in each state. Only two of the four C-lobe orientation populations seen during relaxation and active isometric contraction in the unphosphorylated state were present after cRLC phosphorylation. Thus cRLC phosphorylation alters the equilibrium between defined conformations of the cRLC regions of the myosin heads, rather than simply disordering the heads as assumed previously. cRLC phosphorylation also changes the orientation of the cRLC C-lobe in rigor conditions, showing that the orientation of this part of the myosin head is determined by its interaction with the thick filament even when the head is strongly bound to actin. These results suggest that cRLC phosphorylation controls the contractility of the heart by modulating the interaction of the cRLC region of the myosin heads with the thick filament backbone. PMID:26057075

  16. Induced Expression of Nucleolin Phosphorylation-Deficient Mutant Confers Dominant-Negative Effect on Cell Proliferation

    PubMed Central

    Xiao, Shu; Caglar, Elif; Maldonado, Priscilla; Das, Dibash; Nadeem, Zaineb; Chi, Angela; Trinité, Benjamin; Li, Xin; Saxena, Anjana

    2014-01-01

    Nucleolin (NCL) is a major nucleolar phosphoprotein that has pleiotropic effects on cell proliferation and is elevated in a variety of tumors. NCL is highly phosphorylated at the N-terminus by two major kinases: interphase casein kinase 2 (CK2) and mitotic cyclin-dependent kinase 1 (CDK1). Earlier we demonstrated that a NCL-mutant that is partly defective in undergoing phosphorylation by CK2 inhibits chromosomal replication through its interactions with Replication Protein A, mimicking the cellular response to DNA damage. We further delineated that the N-terminus of NCL associates with Hdm2, the most common E3 ubiquitin ligase of p53. We reported that NCL antagonizes Hdm2 to stabilize p53 and stimulates p53 transcriptional activity. Although NCL-phosphorylation by CK2 and ribosomal DNA transcription are closely coordinated during interphase, the role of NCL phosphorylation in regulating cell proliferation remains unexplored. We have therefore engineered unique human cells that specifically induce expression of NCL-wild type (WT) or a phosphorylation-deficient NCL-mutant, 6/S*A where all the six CK2 consensus serine sites residing in the N-terminus NCL were mutated to alanine. Here we show that this NCL-mutant is defective in undergoing phosphorylation by CK2. We also demonstrate that NCL-phosphorylation by CK2 is required through the S-phase progression in cell cycle and hence proliferation. Induced expression of NCL with mutated CK2 phosphorylation sites stabilizes p53, results in higher expression of Bcl2 (B-cell lymphoma 2) homology 3 (BH3)-only apoptotic markers and causes a dominant-negative effect on cell viability. Our unique cellular system thus provides the first evidential support to delineate phospho-specific functions of NCL on cell proliferation. PMID:25313645

  17. Phosphorylation of myosin regulatory light chain controls myosin head conformation in cardiac muscle

    PubMed Central

    Kampourakis, Thomas; Irving, Malcolm

    2015-01-01

    The effect of phosphorylation on the conformation of the regulatory light chain (cRLC) region of myosin in ventricular trabeculae from rat heart was determined by polarized fluorescence from thiophosphorylated cRLCs labelled with bifunctional sulforhodamine (BSR). Less than 5% of cRLCs were endogenously phosphorylated in this preparation, and similarly low values of basal cRLC phosphorylation were measured in fresh intact ventricle from both rat and mouse hearts. BSR-labelled cRLCs were thiophosphorylated by a recombinant fragment of human cardiac myosin light chain kinase, which was shown to phosphorylate cRLCs specifically at serine 15 in a calcium- and calmodulin-dependent manner, both in vitro and in situ. The BSR-cRLCs were exchanged into demembranated trabeculae, and polarized fluorescence intensities measured for each BSR-cRLC in relaxation, active isometric contraction and rigor were combined with RLC crystal structures to calculate the orientation distribution of the C-lobe of the cRLC in each state. Only two of the four C-lobe orientation populations seen during relaxation and active isometric contraction in the unphosphorylated state were present after cRLC phosphorylation. Thus cRLC phosphorylation alters the equilibrium between defined conformations of the cRLC regions of the myosin heads, rather than simply disordering the heads as assumed previously. cRLC phosphorylation also changes the orientation of the cRLC C-lobe in rigor conditions, showing that the orientation of this part of the myosin head is determined by its interaction with the thick filament even when the head is strongly bound to actin. These results suggest that cRLC phosphorylation controls the contractility of the heart by modulating the interaction of the cRLC region of the myosin heads with the thick filament backbone. PMID:26057075

  18. Phosphorylation of Measles Virus Nucleoprotein Affects Viral Growth by Changing Gene Expression and Genomic RNA Stability

    PubMed Central

    Sugai, Akihiro; Sato, Hiroki; Yoneda, Misako

    2013-01-01

    The measles virus (MV) nucleoprotein associates with the viral RNA genome to form the N-RNA complex, providing a template for viral RNA synthesis. In our previous study, major phosphorylation sites of the nucleoprotein were identified as S479 and S510. However, the functions of these phosphorylation sites have not been clarified. In this study, we rescued recombinant MVs (rMVs) whose phosphorylation sites in the nucleoprotein were substituted (rMV-S479A, rMV-S510A, and rMV-S479A/S510A) by reverse genetics and used them in subsequent analyses. In a one-step growth experiment, rMVs showed rapid growth kinetics compared with wild-type MV, although the peak titer of the wild-type MV was the same as or slightly higher than those of the rMVs. Time course analysis of nucleoprotein accumulation also revealed that viral gene expression of rMV was enhanced during the early phase of infection. These findings suggest that nucleoprotein phosphorylation has an important role in controlling viral growth rate through the regulation of viral gene expression. Conversely, multistep growth curves revealed that nucleoprotein-phosphorylation intensity inversely correlated with viral titer at the plateau phase. Additionally, the phosphorylation intensity of the wild-type nucleoprotein in infected cells was significantly reduced through nucleoprotein-phosphoprotein binding. Excessive nucleoprotein-phosphorylation resulted in lower stability against RNase and faster turnover of viral genomic RNA. These results suggest that nucleoprotein-phosphorylation is also involved in viral genomic RNA stability. PMID:23966404

  19. Regulation of GABA-modulin phosphorylation and GABA receptor binding by excitatory amino acids

    SciTech Connect

    Vaccarino, F.; Guidotti, A.

    1987-05-01

    Primary cultures of cerebellar granule cells phosphorylate numerous proteins including GABA-modulin (GM), which is a putative allosteric modulator of GABA receptors. Cell depolarization and treatment with dicarboxylic excitatory amino acids, which activate PI turnover, Ca/sup 2 +/ influx and guanylate cyclase in granule cells increase the phosphorylation of specific proteins. To determine GM phosphorylation by endogenous protein kinases in living granule cell cultures, GM was isolated by immunoprecipitation and reverse-phase HPLC. High K/sup +/, veratridine, glutamate and NMDA treatment stimulated GM phosphorylation over 2-fold. This increase was abolished by the absence of extracellular Ca/sup 2 +/ and was antagonized by Mg/sup 2 +/ ions and by AVP. The excitatory amino acid action was mimicked by phorbol esters but not by forskolin or by cGMP, and thus may be mediated by an activation of protein kinase C (PKC). Moreover, excitatory amino acids increase /sup 3/H-labelled phorbol ester binding sites in granule cell membrane. The same cultures, treated with glutamate or kainate, showed a 50-fold greater efficacy of muscimol for the stimulation of benzodiazepine (BZ) binding. These data-suggest that excitatory amino acid stimulation of neurons triggers PKC translocation and the activated enzyme phosphorylates GM. The extent of GM phosphorylation may regulate the coupling between GABA and BZ binding sites.

  20. Nrf2 reduces levels of phosphorylated tau protein by inducing autophagy adaptor protein NDP52

    NASA Astrophysics Data System (ADS)

    Jo, Chulman; Gundemir, Soner; Pritchard, Susanne; Jin, Youngnam N.; Rahman, Irfan; Johnson, Gail V. W.

    2014-03-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a pivotal transcription factor in the defence against oxidative stress. Here we provide evidence that activation of the Nrf2 pathway reduces the levels of phosphorylated tau by induction of an autophagy adaptor protein NDP52 (also known as CALCOCO2) in neurons. The expression of NDP52, which we show has three antioxidant response elements (AREs) in its promoter region, is strongly induced by Nrf2, and its overexpression facilitates clearance of phosphorylated tau in the presence of an autophagy stimulator. In Nrf2-knockout mice, phosphorylated and sarkosyl-insoluble tau accumulates in the brains concurrent with decreased levels of NDP52. Moreover, NDP52 associates with phosphorylated tau from brain cortical samples of Alzheimer disease cases, and the amount of phosphorylated tau in sarkosyl-insoluble fractions is inversely proportional to that of NDP52. These results suggest that NDP52 plays a key role in autophagy-mediated degradation of phosphorylated tau in vivo.

  1. Histidine phosphorylation relieves copper inhibition in the mammalian potassium channel KCa3.1.

    PubMed

    Srivastava, Shekhar; Panda, Saswati; Li, Zhai; Fuhs, Stephen R; Hunter, Tony; Thiele, Dennis J; Hubbard, Stevan R; Skolnik, Edward Y

    2016-01-01

    KCa2.1, KCa2.2, KCa2.3 and KCa3.1 constitute a family of mammalian small- to intermediate-conductance potassium channels that are activated by calcium-calmodulin. KCa3.1 is unique among these four channels in that activation requires, in addition to calcium, phosphorylation of a single histidine residue (His358) in the cytoplasmic region, by nucleoside diphosphate kinase-B (NDPK-B). The mechanism by which KCa3.1 is activated by histidine phosphorylation is unknown. Histidine phosphorylation is well characterized in prokaryotes but poorly understood in eukaryotes. Here, we demonstrate that phosphorylation of His358 activates KCa3.1 by antagonizing copper-mediated inhibition of the channel. Furthermore, we show that activated CD4(+) T cells deficient in intracellular copper exhibit increased KCa3.1 histidine phosphorylation and channel activity, leading to increased calcium flux and cytokine production. These findings reveal a novel regulatory mechanism for a mammalian potassium channel and for T-cell activation, and highlight a unique feature of histidine versus serine/threonine and tyrosine as a regulatory phosphorylation site. PMID:27542194

  2. The Functions of Serine 687 Phosphorylation of Human DNA Polymerase η in UV Damage Tolerance.

    PubMed

    Dai, Xiaoxia; You, Changjun; Wang, Yinsheng

    2016-06-01

    DNA polymerase η (polη) is a Y-family translesion synthesis polymerase that plays a key role in the cellular tolerance toward UV irradiation-induced DNA damage. Here, we identified, for the first time, the phosphorylation of serine 687 (Ser(687)), which is located in the highly conserved nuclear localization signal (NLS) region of human polη and is mediated by cyclin-dependent kinase 2 (CDK2). We also showed that this phosphorylation is stimulated in human cells upon UV light exposure and results in diminished interaction of polη with proliferating cell nuclear antigen (PCNA). Furthermore, we demonstrated that the phosphorylation of Ser(687) in polη confers cellular protection from UV irradiation and increases the efficiency in replication across a site-specifically incorporated cyclobutane pyrimidine dimer in human cells. Based on these results, we proposed a mechanistic model where Ser(687) phosphorylation functions in the reverse polymerase switching step of translesion synthesis: The phosphorylation brings negative charges to the NLS of polη, which facilitates its departure from PCNA, thereby resetting the replication fork for highly accurate and processive DNA replication. Thus, our study, together with previous findings, supported that the posttranslational modifications of NLS of polη played a dual role in polymerase switching, where Lys(682) deubiquitination promotes the recruitment of polη to PCNA immediately prior to lesion bypass and Ser(687) phosphorylation stimulates its departure from the replication fork immediately after lesion bypass. PMID:26988343

  3. A cdc2-like kinase phosphorylates histone H1 in the amitotic macronucleus of Tetrahymena.

    PubMed Central

    Roth, S Y; Collini, M P; Draetta, G; Beach, D; Allis, C D

    1991-01-01

    Genetic and biochemical studies have shown that cdc2 protein kinase plays a pivotal role in a highly conserved mechanism controlling the entry of cells into mitosis. It is generally believed that one function of cdc2 kinase is to phosphorylate histone H1 which in turn promotes mitotic chromosome condensation. However, direct evidence linking H1 phosphorylation to mitotic chromatin condensation is limited and the exact cellular function(s) of H1 phosphorylation remains unclear. In this study, we show that mammalian cdc2 kinase phosphorylates H1 from the amitotic macronucleus of Tetrahymena with remarkable fidelity. Furthermore, we demonstrate that macronuclei from Tetrahymena contain a growth-associated H1 kinase activity which closely resembles cdc2 kinase from other eukaryotes. Using polyclonal antibodies raised against yeast p34cdc2, we have detected a 36 kd immunoactive polypeptide in macronuclei which binds to Suc1 (p13)-coated beads and closely follows H1 kinase activity. Since macronuclei divide without mitotic chromosome condensation, these data demonstrate that H1 phosphorylation by cdc2 kinase may be necessary, but is not sufficient to promote mitotic chromatin condensation. The fact that an activity which strongly resembles mammalian cdc2 kinase is active during cell growth in a nucleus which does not undergo mitosis and chromosome condensation suggests that other factors are needed for a true mitotic division to occur. These data also reinforce the notion that H1 phosphorylation has important functions outside mitosis both in Tetrahymena and in mammalian cells. Images PMID:2065655

  4. Identification and analysis of phosphorylation status of proteins in dormant terminal buds of poplar

    PubMed Central

    2011-01-01

    Background Although there has been considerable progress made towards understanding the molecular mechanisms of bud dormancy, the roles of protein phosphorylation in the process of dormancy regulation in woody plants remain unclear. Results We used mass spectrometry combined with TiO2 phosphopeptide-enrichment strategies to investigate the phosphoproteome of dormant terminal buds (DTBs) in poplar (Populus simonii × P. nigra). There were 161 unique phosphorylated sites in 161 phosphopeptides from 151 proteins; 141 proteins have orthologs in Arabidopsis, and 10 proteins are unique to poplar. Only 34 sites in proteins in poplar did not match well with the equivalent phosphorylation sites of their orthologs in Arabidopsis, indicating that regulatory mechanisms are well conserved between poplar and Arabidopsis. Further functional classifications showed that most of these phosphoproteins were involved in binding and catalytic activity. Extraction of the phosphorylation motif using Motif-X indicated that proline-directed kinases are a major kinase group involved in protein phosphorylation in dormant poplar tissues. Conclusions This study provides evidence about the significance of protein phosphorylation during dormancy, and will be useful for similar studies on other woody plants. PMID:22074553

  5. HIPK2 restricts SIRT1 activity upon severe DNA damage by a phosphorylation-controlled mechanism.

    PubMed

    Conrad, E; Polonio-Vallon, T; Meister, M; Matt, S; Bitomsky, N; Herbel, C; Liebl, M; Greiner, V; Kriznik, B; Schumacher, S; Krieghoff-Henning, E; Hofmann, T G

    2016-01-01

    Upon severe DNA damage a cellular signalling network initiates a cell death response through activating tumour suppressor p53 in association with promyelocytic leukaemia (PML) nuclear bodies. The deacetylase Sirtuin 1 (SIRT1) suppresses cell death after DNA damage by antagonizing p53 acetylation. To facilitate efficient p53 acetylation, SIRT1 function needs to be restricted. How SIRT1 activity is regulated under these conditions remains largely unclear. Here we provide evidence that SIRT1 activity is limited upon severe DNA damage through phosphorylation by the DNA damage-responsive kinase HIPK2. We found that DNA damage provokes interaction of SIRT1 and HIPK2, which phosphorylates SIRT1 at Serine 682 upon lethal damage. Furthermore, upon DNA damage SIRT1 and HIPK2 colocalize at PML nuclear bodies, and PML depletion abrogates DNA damage-induced SIRT1 Ser682 phosphorylation. We show that Ser682 phosphorylation inhibits SIRT1 activity and impacts on p53 acetylation, apoptotic p53 target gene expression and cell death. Mechanistically, we found that DNA damage-induced SIRT1 Ser682 phosphorylation provokes disruption of the complex between SIRT1 and its activator AROS. Our findings indicate that phosphorylation-dependent restriction of SIRT1 activity by HIPK2 shapes the p53 response. PMID:26113041

  6. HIPK2 restricts SIRT1 activity upon severe DNA damage by a phosphorylation-controlled mechanism

    PubMed Central

    Conrad, E; Polonio-Vallon, T; Meister, M; Matt, S; Bitomsky, N; Herbel, C; Liebl, M; Greiner, V; Kriznik, B; Schumacher, S; Krieghoff-Henning, E; Hofmann, T G

    2016-01-01

    Upon severe DNA damage a cellular signalling network initiates a cell death response through activating tumour suppressor p53 in association with promyelocytic leukaemia (PML) nuclear bodies. The deacetylase Sirtuin 1 (SIRT1) suppresses cell death after DNA damage by antagonizing p53 acetylation. To facilitate efficient p53 acetylation, SIRT1 function needs to be restricted. How SIRT1 activity is regulated under these conditions remains largely unclear. Here we provide evidence that SIRT1 activity is limited upon severe DNA damage through phosphorylation by the DNA damage-responsive kinase HIPK2. We found that DNA damage provokes interaction of SIRT1 and HIPK2, which phosphorylates SIRT1 at Serine 682 upon lethal damage. Furthermore, upon DNA damage SIRT1 and HIPK2 colocalize at PML nuclear bodies, and PML depletion abrogates DNA damage-induced SIRT1 Ser682 phosphorylation. We show that Ser682 phosphorylation inhibits SIRT1 activity and impacts on p53 acetylation, apoptotic p53 target gene expression and cell death. Mechanistically, we found that DNA damage-induced SIRT1 Ser682 phosphorylation provokes disruption of the complex between SIRT1 and its activator AROS. Our findings indicate that phosphorylation-dependent restriction of SIRT1 activity by HIPK2 shapes the p53 response. PMID:26113041

  7. Pyrimidine nucleoside phosphorylation in developing seeds and germinating seedlings of wheat

    SciTech Connect

    Rowe, M.L.

    1988-01-01

    Uridine- and thymidine-phosphorylating enzymes were measured in developing and germinating seeds of Triticum aestivum v. Arthur and T. aestivum v. Lemhi. Because crude extracts were to be used in the developmental study, characteristics of unpurified nucleoside phosphotransferase (NPTase) were examined. In the developmental study with two varieties of wheat, NPTase activity was found to be very low in all of the true seed tissues during seed maturation. Uridine-phosphorylating activity was due to primarily to uridine kinase. Thymidine phosphorylation was very low in all tissues throughout seed maturation, with a brief appearance by thymidine kinase in the developing embryo. In germinating seeds, uridine-phosphorylating activity was present from earliest stages of germination but showed a decrease in activity followed by a recovery after 48 hours inbibition. Experiments using ({alpha}-{sup 32}P)ATP indicated that uridine kinase was present during early germination but had disappeared by 96 hours. Uridine phosphorylation at later stages of germination was accomplished by NTPase. Thymidine phosphorylation did not begin until after 36 hours of germination and was the result of NPTase activity.

  8. Vinculin phosphorylation differentially regulates mechanotransduction at cell–cell and cell–matrix adhesions

    PubMed Central

    Bays, Jennifer L.; Peng, Xiao; Tolbert, Catlin E.; Guilluy, Christophe; Angell, Ashley E.; Pan, Yuan; Superfine, Richard; Burridge, Keith

    2014-01-01

    Cells experience mechanical forces throughout their lifetimes. Vinculin is critical for transmitting these forces, yet how it achieves its distinct functions at cell–cell and cell–matrix adhesions remains unanswered. Here, we show vinculin is phosphorylated at Y822 in cell–cell, but not cell–matrix, adhesions. Phosphorylation at Y822 was elevated when forces were applied to E-cadherin and was required for vinculin to integrate into the cadherin complex. The mutation Y822F ablated these activities and prevented cells from stiffening in response to forces on E-cadherin. In contrast, Y822 phosphorylation was not required for vinculin functions in cell–matrix adhesions, including integrin-induced cell stiffening. Finally, forces applied to E-cadherin activated Abelson (Abl) tyrosine kinase to phosphorylate vinculin; Abl inhibition mimicked the loss of vinculin phosphorylation. These data reveal an unexpected regulatory mechanism in which vinculin Y822 phosphorylation determines whether cadherins transmit force and provides a paradigm for how a shared component of adhesions can produce biologically distinct functions. PMID:24751539

  9. Threonine phosphorylation prevents promoter DNA binding of the Group B Streptococcus response regulator CovR.

    PubMed

    Lin, Wan-Jung; Walthers, Don; Connelly, James E; Burnside, Kellie; Jewell, Kelsea A; Kenney, Linda J; Rajagopal, Lakshmi

    2009-03-01

    All living organisms communicate with the external environment for their survival and existence. In prokaryotes, communication is achieved by two-component systems (TCS) comprising histidine kinases and response regulators. In eukaryotes, signalling is accomplished by serine/threonine and tyrosine kinases. Although TCS and serine/threonine kinases coexist in prokaryotes, direct cross-talk between these families was first described in Group B Streptococcus (GBS). A serine/threonine kinase (Stk1) and a TCS (CovR/CovS) co-regulate toxin expression in GBS. Typically, promoter binding of regulators like CovR is controlled by phosphorylation of the conserved active site aspartate (D53). In this study, we show that Stk1 phosphorylates CovR at threonine 65. The functional consequence of threonine phosphorylation of CovR in GBS was evaluated using phosphomimetic and silencing substitutions. GBS encoding the phosphomimetic T65E allele are deficient for CovR regulation unlike strains encoding the non-phosphorylated T65A allele. Further, compared with wild-type or T65A CovR, the T65E CovR is unable to bind promoter DNA and is decreased for phosphorylation at D53, similar to Stk1-phosphorylated CovR. Collectively, we provide evidence for a novel mechanism of response regulator control that enables GBS (and possibly other prokaryotes) to fine-tune gene expression for environmental adaptation. PMID:19170889

  10. Analysis of Phosphorylation of the Receptor-Like Protein Kinase HAESA during Arabidopsis Floral Abscission

    PubMed Central

    Taylor, Isaiah; Wang, Ying; Seitz, Kati; Baer, John; Bennewitz, Stefan; Mooney, Brian P.; Walker, John C.

    2016-01-01

    Receptor-like protein kinases (RLKs) are the largest family of plant transmembrane signaling proteins. Here we present functional analysis of HAESA, an RLK that regulates floral organ abscission in Arabidopsis. Through in vitro and in vivo analysis of HAE phosphorylation, we provide evidence that a conserved phosphorylation site on a region of the HAE protein kinase domain known as the activation segment positively regulates HAE activity. Additional analysis has identified another putative activation segment phosphorylation site common to multiple RLKs that potentially modulates HAE activity. Comparative analysis suggests that phosphorylation of this second activation segment residue is an RLK specific adaptation that may regulate protein kinase activity and substrate specificity. A growing number of RLKs have been shown to exhibit biologically relevant dual specificity toward serine/threonine and tyrosine residues, but the mechanisms underlying dual specificity of RLKs are not well understood. We show that a phospho-mimetic mutant of both HAE activation segment residues exhibits enhanced tyrosine auto-phosphorylation in vitro, indicating phosphorylation of this residue may contribute to dual specificity of HAE. These results add to an emerging framework for understanding the mechanisms and evolution of regulation of RLK activity and substrate specificity. PMID:26784444

  11. Phosphorylation and SCF-mediated degradation regulate CREB-H transcription of metabolic targets

    PubMed Central

    Barbosa, Sónia; Carreira, Suzanne; Bailey, Daniel; Abaitua, Fernando; O'Hare, Peter

    2015-01-01

    CREB‑H, an endoplasmic reticulum–anchored transcription factor, plays a key role in regulating secretion and in metabolic and inflammatory pathways, but how its activity is modulated remains unclear. We examined processing of the nuclear active form and identified a motif around S87–S90 with homology to DSG-type phosphodegrons. We show that this region is subject to multiple phosphorylations, which regulate CREB-H stability by targeting it to the SCFFbw1a E3 ubiquitin ligase. Data from phosphatase treatment, use of phosophospecific antibody, and substitution of serine residues demonstrate phosphorylation of candidate serines in the region, with the core S87/S90 motif representing a critical determinant promoting proteasome-mediated degradation. Candidate kinases CKII and GSK-3b phosphorylate CREB-H in vitro with specificities for different serines. Prior phosphorylation with GSK-3 at one or more of the adjacent serines substantially increases S87/S90-dependent phosphorylation by CKII. In vivo expression of a dominant-negative Cul1 enhances steady-state levels of CREB‑H, an effect augmented by Fbw1a. CREB-H directly interacts with Fbw1a in a phosphorylation-dependent manner. Finally, mutations within the phosphodegron, when incorporated into the full-length protein, result in increased levels of constitutively cleaved nuclear protein and increased transcription and secretion of a key endogenous target gene, apolipoprotein A IV. PMID:26108621

  12. Phosphorylation-Dependent Regulation of G-Protein Cycle during Nodule Formation in Soybean[OPEN

    PubMed Central

    2015-01-01

    Signaling pathways mediated by heterotrimeric G-protein complexes comprising Gα, Gβ, and Gγ subunits and their regulatory RGS (Regulator of G-protein Signaling) protein are conserved in all eukaryotes. We have shown that the specific Gβ and Gγ proteins of a soybean (Glycine max) heterotrimeric G-protein complex are involved in regulation of nodulation. We now demonstrate the role of Nod factor receptor 1 (NFR1)-mediated phosphorylation in regulation of the G-protein cycle during nodulation in soybean. We also show that during nodulation, the G-protein cycle is regulated by the activity of RGS proteins. Lower or higher expression of RGS proteins results in fewer or more nodules, respectively. NFR1 interacts with RGS proteins and phosphorylates them. Analysis of phosphorylated RGS protein identifies specific amino acids that, when phosphorylated, result in significantly higher GTPase accelerating activity. These data point to phosphorylation-based regulation of G-protein signaling during nodule development. We propose that active NFR1 receptors phosphorylate and activate RGS proteins, which help maintain the Gα proteins in their inactive, trimeric conformation, resulting in successful nodule development. Alternatively, RGS proteins might also have a direct role in regulating nodulation because overexpression of their phospho-mimic version leads to partial restoration of nodule formation in nod49 mutants. PMID:26498905

  13. How Phosphorylation and ATPase Activity Regulate Anion Flux though the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).

    PubMed

    Zwick, Matthias; Esposito, Cinzia; Hellstern, Manuel; Seelig, Anna

    2016-07-01

    The cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7), mutations of which cause cystic fibrosis, belongs to the ATP-binding cassette (ABC) transporter family and works as a channel for small anions, such as chloride and bicarbonate. Anion channel activity is known to depend on phosphorylation by cAMP-dependent protein kinase A (PKA) and CFTR-ATPase activity. Whereas anion channel activity has been extensively investigated, phosphorylation and CFTR-ATPase activity are still poorly understood. Here, we show that the two processes can be measured in a label-free and non-invasive manner in real time in live cells, stably transfected with CFTR. This study reveals three key findings. (i) The major contribution (≥90%) to the total CFTR-related ATP hydrolysis rate is due to phosphorylation by PKA and the minor contribution (≤10%) to CFTR-ATPase activity. (ii) The mutant CFTR-E1371S that is still conductive, but defective in ATP hydrolysis, is not phosphorylated, suggesting that phosphorylation requires a functional nucleotide binding domain and occurs in the post-hydrolysis transition state. (iii) CFTR-ATPase activity is inversely related to CFTR anion flux. The present data are consistent with a model in which CFTR is in a closed conformation with two ATPs bound. The open conformation is induced by ATP hydrolysis and corresponds to the post-hydrolysis transition state that is stabilized by phosphorylation and binding of chloride channel potentiators. PMID:27226582

  14. Tousled-like kinases phosphorylate Asf1 to promote histone supply during DNA replication

    NASA Astrophysics Data System (ADS)

    Klimovskaia, Ilnaz M.; Young, Clifford; Strømme, Caroline B.; Menard, Patrice; Jasencakova, Zuzana; Mejlvang, Jakob; Ask, Katrine; Ploug, Michael; Nielsen, Michael L.; Jensen, Ole N.; Groth, Anja

    2014-03-01

    During DNA replication, nucleosomes are rapidly assembled on newly synthesized DNA to restore chromatin organization. Asf1, a key histone H3-H4 chaperone required for this process, is phosphorylated by Tousled-like kinases (TLKs). Here, we identify TLK phosphorylation sites by mass spectrometry and dissect how phosphorylation has an impact on human Asf1 function. The divergent C-terminal tail of Asf1a is phosphorylated at several sites, and this is required for timely progression through S phase. Consistent with this, biochemical analysis of wild-type and phospho-mimetic Asf1a shows that phosphorylation enhances binding to histones and the downstream chaperones CAF-1 and HIRA. Moreover, we find that TLK phosphorylation of Asf1a is induced in cells experiencing deficiency of new histones and that TLK interaction with Asf1a involves its histone-binding pocket. We thus propose that TLK signalling promotes histone supply in S phase by targeting histone-free Asf1 and stimulating its ability to shuttle histones to sites of chromatin assembly.

  15. Thr-161 phosphorylation of monomeric Cdc2. Regulation by protein phosphatase 2C in Xenopus oocytes.

    PubMed

    De Smedt, Veronique; Poulhe, Robert; Cayla, Xavier; Dessauge, Frederic; Karaiskou, Anthi; Jessus, Catherine; Ozon, Rene

    2002-08-01

    Fully grown Xenopus oocyte is arrested at prophase I of meiosis. Re-entry into meiosis depends on the activation of MPF (M-phase promoting factor or cyclin B.Cdc2 complex), triggered by progesterone. The prophase-arrested oocyte contains a store of Cdc2. Most of the protein is present as a monomer whereas a minor fraction, called pre-MPF, is found to be associated with cyclin B. Activation of Cdc2 depends on two key events: cyclin binding and an activating phosphorylation on Thr-161 residue located in the T-loop. To get new insights into the regulation of Thr-161 phosphorylation of Cdc2, monomeric Cdc2 was isolated from prophase oocytes. Based on its activation upon cyclin addition and detection by an antibody directed specifically against Cdc2 phosphorylated on Thr-161, we show for the first time that the prophase oocyte contains a significant amount of monomeric Cdc2 phosphorylated on Thr-161. PP2C, a Mg2+-dependent phosphatase, negatively controls Thr-161 phosphorylation of Cdc2. The unexpected presence of a population of free Cdc2 already phosphorylated on Thr-161 could contribute to the generation of the Cdc2 kinase activity threshold required to initiate MPF amplification. PMID:12036957

  16. Histidine phosphorylation relieves copper inhibition in the mammalian potassium channel KCa3.1

    PubMed Central

    Srivastava, Shekhar; Panda, Saswati; Li, Zhai; Fuhs, Stephen R; Hunter, Tony; Thiele, Dennis J; Hubbard, Stevan R; Skolnik, Edward Y

    2016-01-01

    KCa2.1, KCa2.2, KCa2.3 and KCa3.1 constitute a family of mammalian small- to intermediate-conductance potassium channels that are activated by calcium-calmodulin. KCa3.1 is unique among these four channels in that activation requires, in addition to calcium, phosphorylation of a single histidine residue (His358) in the cytoplasmic region, by nucleoside diphosphate kinase-B (NDPK-B). The mechanism by which KCa3.1 is activated by histidine phosphorylation is unknown. Histidine phosphorylation is well characterized in prokaryotes but poorly understood in eukaryotes. Here, we demonstrate that phosphorylation of His358 activates KCa3.1 by antagonizing copper-mediated inhibition of the channel. Furthermore, we show that activated CD4+ T cells deficient in intracellular copper exhibit increased KCa3.1 histidine phosphorylation and channel activity, leading to increased calcium flux and cytokine production. These findings reveal a novel regulatory mechanism for a mammalian potassium channel and for T-cell activation, and highlight a unique feature of histidine versus serine/threonine and tyrosine as a regulatory phosphorylation site. DOI: http://dx.doi.org/10.7554/eLife.16093.001 PMID:27542194

  17. Protein kinase CK2 interacts with Chk2 and phosphorylates Mre11 on serine 649

    SciTech Connect

    Kim, Seong-Tae . E-mail: stkim@med.skku.ac.kr

    2005-05-27

    The Mre11-Rad50-Nbs1 protein complex has been known to be involved in a variety of DNA metabolic events that involve DNA double-strand breaks (DSBs). The phosphorylation of Mre11 is increased in response to ionizing radiation, which suggests that phosphorylation of Mre11 may be an important regulatory mechanism of this complex. Mre11-phosphorylating kinase activities were observed in Chk2 immunoprecipitates and HeLa nuclear extracts. Through the tandem affinity tagging system and conventional chromatography, this kinase was purified and identified as protein kinase CK2. CK2 phosphorylates Mre11 in vitro. In vitro kinase assay with a series of truncated Mre11 proteins as substrates for CK2 and site-directed mutagenesis showed that serine 649 of Mre11 is mainly phosphorylated by CK2 in vitro. In vivo labeling and phosphopeptide mapping analysis revealed that this phosphorylation occurs in vivo. These data implicate CK2 as a potential upstream regulator of Mre11 function.

  18. Brain-derived neurotrophic factor rapidly increases NMDA receptor channel activity through Fyn-mediated phosphorylation.

    PubMed

    Xu, Fei; Plummer, Mark R; Len, Guo-Wei; Nakazawa, Takanobu; Yamamoto, Tadashi; Black, Ira B; Wu, Kuo

    2006-11-22

    Brain-derived neurotrophic factor (BDNF) is a potent modulator of hippocampal synaptic plasticity. Previously, we found that one of the targets of BDNF modulation is NR2B-containing NMDA receptors. Furthermore, exposure to the trophin rapidly increases NMDA receptor activity and enhances tyrosine phosphorylation of NR2B in cortical and hippocampal postsynaptic densities (PSDs), potentially linking receptor phosphorylation to synaptic plasticity. To define the specific NR2B residue(s) regulated by BDNF, we focused on tyrosine 1472, phosphorylation of which increases after LTP. BDNF rapidly increased phosphorylation in cortical PSDs. The tyrosine kinase Fyn is critical since BDNF-dependent phosphorylation was abolished in Fyn knockout mice. Single-channel patch clamp recordings showed that Fyn is required for the increase in NMDA receptor activity elicited by BDNF. Collectively, our results suggest that BDNF enhances phosphorylation of NR2B tyrosine 1472 through activation of Fyn, leading to alteration of NMDA receptor activity and increased synaptic transmission. PMID:17045972

  19. Phosphorylation in vitro of human fibrinogen with casein kinase TS and characterization of phosphorylated sites

    SciTech Connect

    Heldin, P.

    1987-09-01

    Human fibrinogen was phosphorylated by casein kinase TS. The (/sup 32/P)phosphate incorporated varied between 0.5 and 1 mol of phosphate per mole of fibrinogen. The phosphate was localized to Ser523 and Ser590 and serine and threonine residues between amino acids 259 and 268 in the A alpha-chain. In addition, Thr416 and Ser420 were phosphorylated in the gamma'-chain, which is a variant of the gamma-chain, constituting 7-10% of the gamma-chain population. The functional significance of casein kinase TS-induced phosphorylation of fibrinogen remains unknown; however, a slight but consistent increase of the turbidity in a gelation assay was observed for phosphorylated compared to unphosphorylated fibrinogen.

  20. Skeletal muscle PLIN3 and PLIN5 are serine phosphorylated at rest and following lipolysis during adrenergic or contractile stimulation.

    PubMed

    Macpherson, Rebecca E K; Vandenboom, Rene; Roy, Brian D; Peters, Sandra J

    2013-09-01

    In adipose tissue, access of adipose triglyceride and hormone-sensitive lipases (ATGL and HSL) to the lipid droplet depends on PLIN1 phosphorylation, however, PLIN1 is not expressed in skeletal muscle and the phosphorylation of the expressed PLINs has yet to be investigated. Further, direct interactions between skeletal muscle PLINs and HSL are unknown. We investigated the isolated and combined effects of epinephrine and contraction on PLIN-to-lipase interactions as well as phosphorylation. Isolated rat solei were assigned to one of four 30 min in vitro conditions (25°C): (1) rest; (2) intermittent tetanic stimulation (60 Hz for 150 msec; train rate 20/min); (3) 5 nmol/L epinephrine; (4) intermittent tetanic stimulation and 5 nmol/L epinephrine. Immunoprecipitation of serine phosphorylated proteins followed by Western blotting for PLIN2, PLIN3, PLIN5, revealed that only PLIN2 is not phosphorylated under any of the experimental conditions. This is the first study to show that in whole rat skeletal muscle PLIN3 and PLIN5 are serine phosphorylated. The degree of serine phosphorylation remained unchanged following adrenergic and/or contractile stimulation. Oil red O staining of muscle sections for lipid content shows a significant decrease following each condition, confirming lipolysis occurred (P < 0.05). PLIN2, 3, and 5 all interact with HSL and ATGL, but these interactions were unchanged following treatments. Our results show that in skeletal muscle, PLIN2 is not serine phosphorylated at rest or with lipolytic stimulation and that while PLIN3, PLIN5 are serine phosphorylated at rest, the degree of phosphorylation does not change with lipolytic stimulation. PMID:24303154

  1. Cyclin-dependent kinase 5 phosphorylation of familial prion protein mutants exacerbates conversion into amyloid structure.

    PubMed

    Rouget, Raphaël; Sharma, Gyanesh; LeBlanc, Andréa C

    2015-02-27

    Familial prion protein (PrP) mutants undergo conversion from soluble and protease-sensitive to insoluble and partially protease-resistant proteins. Cyclin-dependent kinase 5 (Cdk5) phosphorylation of wild type PrP (pPrP) at serine 43 induces a conversion of PrP into aggregates and fibrils. Here, we investigated whether familial PrP mutants are predisposed to Cdk5 phosphorylation and whether phosphorylation of familial PrP mutants increases conversion. PrP mutants representing three major familial PrP diseases and different PrP structural domains were studied. We developed a novel in vitro kinase reaction coupled with Thioflavin T binding to amyloid structure assay to monitor phosphorylation-dependent amyloid conversion. Although non-phosphorylated full-length wild type or PrP mutants did not convert into amyloid, Cdk5 phosphorylation rapidly converted these into Thioflavin T-positive structures following first order kinetics. Dephosphorylation partially reversed conversion. Phosphorylation-dependent conversion of PrP from α-helical structures into β-sheet structures was confirmed by circular dichroism. Relative to wild type pPrP, most PrP mutants showed increased rate constants of conversion. In contrast, non-phosphorylated truncated PrP Y145X (where X represents a stop codon) and Q160X mutants converted spontaneously into Thioflavin T-positive fibrils after a lag phase of over 20 h, indicating nucleation-dependent polymerization. Phosphorylation reduced the lag phase by over 50% and thus accelerated the formation of the nucleating event. Consistently, phosphorylated Y145X and phosphorylated Q160X exacerbated conversion in a homologous seeding reaction, whereas WT pPrP could not seed WT PrP. These results demonstrate an influence of both the N terminus and the C terminus of PrP on conversion. We conclude that post-translational modifications of the flexible N terminus of PrP can cause or exacerbate PrP mutant conversion. PMID:25572400

  2. Study of phosphorylation events for cancer diagnoses and treatment.

    PubMed

    López Villar, Elena; Madero, Luis; A López-Pascual, Juan; C Cho, William

    2015-12-01

    The activation of signaling cascades in response to extracellular and intracellular stimuli to control cell growth, proliferation and survival, is orchestrated by protein kinases via phosphorylation. A critical issue is the study of the mechanisms of cancer cells for the development of more effective drugs. With the application of the new proteomic technologies, together with the advancement in the sequencing of the human proteome, patients will therefore be benefited by the discovery of novel therapeutic and/or diagnostic protein targets. Furthermore, the advances in proteomic approaches and the Human Proteome Organization (HUPO) have opened a new door which is helpful in the identification of patients at risk and towards improving current therapies. Modification of the signaling-networks via mutations or abnormal protein expression underlies the cause or consequence of many diseases including cancer. Resulting data is used to reveal connections between genes proteins and compounds and the related molecular pathways for underlining disease states. As a delegate of HUPO, for human proteome on children assays and studies, we, at Hospital Universitario Niño Jesús, are seeking to support the human proteome in this context. Clinical goals have to be clearly established and proteomics experts have to set up the appropriate proteomic strategy, which coupled to bioinformatics will make it possible to achieve new therapies for patients with poor prognosis. We envision to combine our up-coming data to the HUPO organization in order to support international efforts to advance the cure of cancer disease. PMID:26055493

  3. Rapamycin enhances eIF4E phosphorylation by activating MAP kinase-interacting kinase 2a (Mnk2a).

    PubMed

    Stead, Rebecca L; Proud, Christopher G

    2013-08-19

    Eukaryotic initiation factor eIF4E and its phosphorylation play key roles in cell transformation and tumorigenesis. eIF4E is phosphorylated by the Mnks (MAP (mitogen-activated protein) kinase-interacting kinases). Rapamycin increases eIF4E phosphorylation in cancer cells, potentially limiting their anti-cancer effects. Here we show that the rapamycin-induced increase in eIF4E phosphorylation reflects increased activity of Mnk2 but not Mnk1. This activation requires a novel phosphorylation site in Mnk2a, Ser437. Our findings have potentially important implications for the use of rapamycin and its analogues in cancer therapy, suggesting that inhibitors of mTOR and Mnk (or Mnk2) may be more efficacious than rapalogs alone. PMID:23831578

  4. Claudin-18 inhibits cell proliferation and motility mediated by inhibition of phosphorylation of PDK1 and Akt in human lung adenocarcinoma A549 cells.

    PubMed

    Shimobaba, Shun; Taga, Saeko; Akizuki, Risa; Hichino, Asami; Endo, Satoshi; Matsunaga, Toshiyuki; Watanabe, Ryo; Yamaguchi, Masahiko; Yamazaki, Yasuhiro; Sugatani, Junko; Ikari, Akira

    2016-06-01

    Abnormal expression of claudin subtypes has been reported in various cancers. However, the pathological role of each claudin has not been clarified in detail. Claudin-18 was absent in human non-small cell and small cell lung cancers, although it is expressed in normal lung tissues. Here, we examined the effect of claudin-18 expression on the expression of junctional proteins, cell proliferation, and cell motility using human lung adenocarcinoma A549 cells. Real-time PCR and western blotting showed that exogenous expression of claudin-18 had no effect on the expression of junctional proteins including claudin-1, zonula occludens-1 (ZO-1), occludin, and E-cadherin. Claudin-18 was mainly distributed in cell-cell contact areas concomitant with ZO-1. Cell proliferation was significantly decreased at 48 and 72h after seeding of claudin 18-expressing cells. Claudin-18 suppressed cell motility, whereas it increased cell death in anoikis. Claudin-18 decreased phosphorylated (p)-3-phosphoinositide-dependent protein kinase-1 (PDK1) and p-Akt levels without affecting p-epidermal growth factor receptor and p-phosphatidylinositol-3 kinase (PI3K) levels. Furthermore, claudin-18 was bound with PDK1 and suppressed the nuclear localization of PDK1. We suggest that claudin-18 suppresses the abnormal proliferation and motility of lung epithelial cells mediated by inhibition of the PI3K/PDK1/Akt signaling pathway. PMID:26919807

  5. Protein phosphorylation in isolated human adipocytes - Adrenergic control of the phosphorylation of hormone-sensitive lipase

    SciTech Connect

    Smiley, R.M. Columbia Univ College of Physicians and Surgeons, New York, NY ); Paul, S.; Browning, M.D.; Leibel, R.L.; Hirsch, J. )

    1990-01-01

    The effect of adrenergic agents on protein phosphorylation in human adipocytes was examined. Freshly isolated human fat cells were incubated with {sup 32}PO{sub 4} in order to label intracellular ATP, then treated with a variety of adrenergic and other pharmacologic agents. Treatment with the {beta}-adrenergic agonist isoproterenol led to a significant increase in phosphate content of at least five protein bands (M{sub r} 52, 53, 63, 67, 84 kDa). The increase in phosphorylation was partially inhibited by the {alpha}-2 agonist clonidine. Epinephrine, a combined {alpha} and {beta} agonist, was less effective at increasing phosphate content of the proteins than was isoproterenol. Neither insulin nor the {alpha}-1 agonist phenylephrine had any discernible effect on the pattern of protein phosphorylation. The 84 kDa phosphorylated peptide band appears to contain hormone-sensitive lipase, a key enzyme in the lipolytic pathway which is activated by phosphorylation. These results are somewhat different than previously reported results for rat adipocytes, and represent the first report of overall pattern and adrenergic modulation of protein phosphorylation in human adipocytes.

  6. G Protein-Coupled Receptor Kinase 2 (GRK2) and 5 (GRK5) Exhibit Selective Phosphorylation of the Neurotensin Receptor in Vitro

    PubMed Central

    2015-01-01

    G protein-coupled receptor kinases (GRKs) play an important role in the desensitization of G protein-mediated signaling of G protein-coupled receptors (GPCRs). The level of interest in mapping their phosphorylation sites has increased because recent studies suggest that the differential pattern of receptor phosphorylation has distinct biological consequences. In vitro phosphorylation experiments using well-controlled systems are useful for deciphering the complexity of these physiological reactions and understanding the targeted event. Here, we report on the phosphorylation of the class A GPCR neurotensin receptor 1 (NTSR1) by GRKs under defined experimental conditions afforded by nanodisc technology. Phosphorylation of NTSR1 by GRK2 was agonist-dependent, whereas phosphorylation by GRK5 occurred in an activation-independent manner. In addition, the negatively charged lipids in the immediate vicinity of NTSR1 directly affect phosphorylation by GRKs. Identification of phosphorylation sites in agonist-activated NTSR1 revealed that GRK2 and GRK5 target different residues located on the intracellular receptor elements. GRK2 phosphorylates only the C-terminal Ser residues, whereas GRK5 phosphorylates Ser and Thr residues located in intracellular loop 3 and the C-terminus. Interestingly, phosphorylation assays using a series of NTSR1 mutants show that GRK2 does not require acidic residues upstream of the phospho-acceptors for site-specific phosphorylation, in contrast to the β2-adrenergic and μ-opioid receptors. Differential phosphorylation of GPCRs by GRKs is thought to encode a particular signaling outcome, and our in vitro study revealed NTSR1 differential phosphorylation by GRK2 and GRK5. PMID:26120872

  7. Phosphorylation of the amino-terminus of the AGC kinase Gad8 prevents its interaction with TORC2.

    PubMed

    Du, Wei; Forte, Gabriella M; Smith, Duncan; Petersen, Janni

    2016-03-01

    Cell proliferation, metabolism, migration and survival are coordinated through the tight control of two target of rapamycin (TOR) kinase complexes: TORC1 and TORC2. Here, we show that a novel phosphorylation of fission yeast Gad8 (AGC kinase) on the evolutionarily conserved threonine 6 (Thr6) prevents the physical association between Gad8 and TORC2. Accordingly, this block to protein interactions by Gad8 Thr6 phosphorylation decreases TORC2-controlled activation of Gad8. Likewise, phosphorylation of Gad8 Thr6, possibly by PKC, prevents the association of Gad8 with TORC2 thereby increasing TORC2 activity, because it reduces Gad8-mediated feedback inhibition of TORC2. Consistently, the introduction of a Gad8 T6D mutant, that mimics phosphorylation, increased TORC2 activity. Increased PKC(Pck2) expression prevented Gad8-TORC2 binding and so reduced the TORC2-mediated phosphorylation of Gad8 serine 546 that activates Gad8. Interestingly, independent of the Ser546 phosphorylation status, Gad8 Thr6 phosphorylation is important for remodelling the actin cytoskeleton and survival upon potassium ion and heat stresses. In contrast, Ser546 phosphorylation is required for the control of G1 arrest, mating, cell length at division and vascular size. Finally, these findings reveal a novel mode of TORC2 activation that is essential for cell survival following stress. PMID:26935949

  8. Evolution of nuclear retinoic acid receptor alpha (RARα) phosphorylation sites. Serine gain provides fine-tuned regulation.

    PubMed

    Samarut, Eric; Amal, Ismail; Markov, Gabriel V; Stote, Roland; Dejaegere, Annick; Laudet, Vincent; Rochette-Egly, Cécile

    2011-07-01

    The human nuclear retinoic acid (RA) receptor alpha (hRARα) is a ligand-dependent transcriptional regulator, which is controlled by a phosphorylation cascade. The cascade starts with the RA-induced phosphorylation of a serine residue located in the ligand-binding domain, S(LBD), allowing the recruitment of the cdk7/cyclin H/MAT1 subcomplex of TFIIH through the docking of cyclin H. It ends by the subsequent phosphorylation by cdk7 of an other serine located in the N-terminal domain, S(NTD). Here, we show that this cascade relies on an increase in the flexibility of the domain involved in cyclin H binding, subsequently to the phosphorylation of S(LBD). Owing to the functional importance of RARα in several vertebrate species, we investigated whether the phosphorylation cascade was conserved in zebrafish (Danio rerio), which expresses two RARα genes: RARα-A and RARα-B. We found that in zebrafish RARαs, S(LBD) is absent, whereas S(NTD) is conserved and phosphorylated. Therefore, we analyzed the pattern of conservation of the phosphorylation sites and traced back their evolution. We found that S(LBD) is most often absent outside mammalian RARα and appears late during vertebrate evolution. In contrast, S(NTD) is conserved, indicating that the phosphorylation of this functional site has been under ancient high selection constraint. This suggests that, during evolution, different regulatory circuits control RARα activity. PMID:21297158

  9. Phosphorylation of the amino-terminus of the AGC kinase Gad8 prevents its interaction with TORC2

    PubMed Central

    Du, Wei; Forte, Gabriella M.; Smith, Duncan; Petersen, Janni

    2016-01-01

    Cell proliferation, metabolism, migration and survival are coordinated through the tight control of two target of rapamycin (TOR) kinase complexes: TORC1 and TORC2. Here, we show that a novel phosphorylation of fission yeast Gad8 (AGC kinase) on the evolutionarily conserved threonine 6 (Thr6) prevents the physical association between Gad8 and TORC2. Accordingly, this block to protein interactions by Gad8 Thr6 phosphorylation decreases TORC2-controlled activation of Gad8. Likewise, phosphorylation of Gad8 Thr6, possibly by PKC, prevents the association of Gad8 with TORC2 thereby increasing TORC2 activity, because it reduces Gad8-mediated feedback inhibition of TORC2. Consistently, the introduction of a Gad8 T6D mutant, that mimics phosphorylation, increased TORC2 activity. Increased PKCPck2 expression prevented Gad8–TORC2 binding and so reduced the TORC2-mediated phosphorylation of Gad8 serine 546 that activates Gad8. Interestingly, independent of the Ser546 phosphorylation status, Gad8 Thr6 phosphorylation is important for remodelling the actin cytoskeleton and survival upon potassium ion and heat stresses. In contrast, Ser546 phosphorylation is required for the control of G1 arrest, mating, cell length at division and vascular size. Finally, these findings reveal a novel mode of TORC2 activation that is essential for cell survival following stress. PMID:26935949

  10. Phosphorylation of the Light-Harvesting Complex II Isoform Lhcb2 Is Central to State Transitions1[OPEN

    PubMed Central

    Cariti, Federica; Fucile, Geoffrey; Goldschmidt-Clermont, Michel

    2015-01-01

    Light-harvesting complex II (LHCII) is a crucial component of the photosynthetic machinery, with central roles in light capture and acclimation to changing light. The association of an LHCII trimer with PSI in the PSI-LHCII supercomplex is strictly dependent on LHCII phosphorylation mediated by the kinase STATE TRANSITION7, and is directly related to the light acclimation process called state transitions. In Arabidopsis (Arabidopsis thaliana), the LHCII trimers contain isoforms that belong to three classes: Lhcb1, Lhcb2, and Lhcb3. Only Lhcb1 and Lhcb2 can be phosphorylated in the N-terminal region. Here, we present an improved Phos-tag-based method to determine the absolute extent of phosphorylation of Lhcb1 and Lhcb2. Both classes show very similar phosphorylation kinetics during state transition. Nevertheless, only Lhcb2 is extensively phosphorylated (>98%) in PSI-LHCII, whereas phosphorylated Lhcb1 is largely excluded from this supercomplex. Both isoforms are phosphorylated to different extents in other photosystem supercomplexes and in different domains of the thylakoid membranes. The data imply that, despite their high sequence similarity, differential phosphorylation of Lhcb1 and Lhcb2 plays contrasting roles in light acclimation of photosynthesis. PMID:26438789

  11. The Natively Disordered Loop of Bcl-2 Undergoes Phosphorylation-Dependent Conformational Change and Interacts with Pin1

    PubMed Central

    Kang, CongBao; Bharatham, Nagakumar; Chia, Joel; Mu, Yuguang; Baek, Kwanghee; Yoon, Ho Sup

    2012-01-01

    Bcl-2 plays a central role in the regulation of apoptosis. Structural studies of Bcl-2 revealed the presence of a flexible and natively disordered loop that bridges the Bcl-2 homology motifs, BH3 and BH4. This loop is phosphorylated on multiple sites in response to a variety of external stimuli, including the microtubule-targeting drugs, paclitaxel and colchicine. Currently, the underlying molecular mechanism of Bcl-2 phosphorylation and its biological significance remain elusive. In this study, we investigated the molecular characteristics of this anti-apoptotic protein. To this end, we generated synthetic peptides derived from the Bcl-2 loop, and multiple Bcl-2 loop truncation mutants that include the phosphorylation sites. Our results demonstrate that S87 in the flexible loop of Bcl-2 is the primary phosphorylation site for JNK and ERK2, suggesting some sequence or structural specificity for the phosphorylation by these kinases. Our NMR studies and molecular dynamics simulation studies support indicate that phosphorylation of S87 induces a conformational change in the peptide. Finally, we show that the phosphorylated peptides of the Bcl-2 loop can bind Pin1, further substantiating the phosphorylation-mediated conformation change of Bcl-2. PMID:23272207

  12. Partial purification of a spinach thylakoid protein kinase that can phosphorylate light-harvesting chlorophyll a/b proteins

    SciTech Connect

    Clark, R.D.; Hind, G.; Bennett, J.

    1985-01-01

    Protein phosphorylation in plant tissues is particularly marked in chloroplasts, protein kinase activity being associated with the outer envelope, the soluble stromal fraction, and the thylakoid membrane. Furthermore, thylakoid-bound activity probably includes several distinct kinases, as suggested by studies of divalent cation specificity and thermal lability carried out with intact thylakoids and by subfractionation of solubilized membranes. Illumination of thylakoids, particularly with red light, promotes the rapid and extensive phosphorylation of the light-harvesting chlorophyll a/b complex (LHCII) on a threonine residue near the amino terminus of the protein. This phosphorylation is thought to be involved in regulating the distribution of absorbed quanta between photosystems II and I and is modulated by the redox state of the thylakoid plastoquinone pool. Neither of the thylakoid kinases reported to date was capable of phosphorylating purified LHCII in vitro or of incorporating phosphate into threonyl residues of exogenous substrates, that some LHCII phosphorylation was catalyzed by a preliminary fraction led workers to suggest that at least one other kinase remained to be isolated. Here, the authors report the solubilization and partial purification of a protein kinase from spinach thylakoids that is capable of phosphorylating LHCII in vitro, and they show that the specific site of phosphorylation is very nearly the same as, if not identical with, the site phosphorylated in organello.

  13. The declined phosphorylation of Hsp27 in rat cardiac muscle after simulated microgravity induced by hindlimb unloading

    NASA Astrophysics Data System (ADS)

    Yuan, Ming; Jiang, Shizhong; Li, Zhili; Yuan, Min; Dong, Weijun

    Many studies have shown that simulated microgravity induced by hindlimb unloading can decrease the contractility of rat cardiac muscle however the mechanisms responsible for which remain unclear Actin polymerization which can be regulated by Hsp27 has important role in the transmission of stress force during the contraction of cardiac muscle In this study western blot analysis was used to detect the expression of Hsp27 and phosphorylated Hsp27 FAK and phosphorylated FAK P38 MAPK and phosphorylated P38 MAPK in rat cardiac muscle after 14d hindlimb unloading The results showed that the phosphorylation levels of both Hsp27 and P38 MAPK were declined significantly which may decrease actin polymerization and inhibit the transmission of stress force during the contraction of rat cardiac muscle after hindlimb unloading However the phosphorylation level of FAK was not declined significantly in cardiac muscle The results suggested that the declined phosphorylation level of Hsp27 which may be ascribable to the decline of contractility of rat cardiac muscle after 14d hindlimb unloading may be induced by the declined phosphorylation level of P38 MAPK but not phosphorylation level of FAK

  14. FUS is phosphorylated by DNA-PK and accumulates in the cytoplasm after DNA damage.

    PubMed

    Deng, Qiudong; Holler, Christopher J; Taylor, Georgia; Hudson, Kathryn F; Watkins, William; Gearing, Marla; Ito, Daisuke; Murray, Melissa E; Dickson, Dennis W; Seyfried, Nicholas T; Kukar, Thomas

    2014-06-01

    Abnormal cytoplasmic accumulation of Fused in Sarcoma (FUS) in neurons defines subtypes of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). FUS is a member of the FET protein family that includes Ewing's sarcoma (EWS) and TATA-binding protein-associated factor 2N (TAF15). FET proteins are predominantly localized to the nucleus, where they bind RNA and DNA to modulate transcription, mRNA splicing, and DNA repair. In ALS cases with FUS inclusions (ALS-FUS), mutations in the FUS gene cause disease, whereas FTLD cases with FUS inclusions (FTLD-FUS) do not harbor FUS mutations. Notably, in FTLD-FUS, all FET proteins accumulate with their nuclear import receptor Transportin 1 (TRN1), in contrast ALS-FUS inclusions are exclusively positive for FUS. In the present study, we show that induction of DNA damage replicates several pathologic hallmarks of FTLD-FUS in immortalized human cells and primary human neurons and astrocytes. Treatment with the antibiotic calicheamicin γ1, which causes DNA double-strand breaks, leads to the cytoplasmic accumulation of FUS, TAF15, EWS, and TRN1. Moreover, cytoplasmic translocation of FUS is mediated by phosphorylation of its N terminus by the DNA-dependent protein kinase. Finally, we observed elevated levels of phospho-H2AX in FTLD-FUS brains, indicating that DNA damage occurs in patients. Together, our data reveal a novel regulatory mechanism for FUS localization in cells and suggest that DNA damage may contribute to the accumulation of FET proteins observed in human FTLD-FUS cases, but not in ALS-FUS. PMID:24899704

  15. The importance of intrinsic disorder for protein phosphorylation.

    PubMed

    Iakoucheva, Lilia M; Radivojac, Predrag; Brown, Celeste J; O'Connor, Timothy R; Sikes, Jason G; Obradovic, Zoran; Dunker, A Keith

    2004-01-01

    Reversible protein phosphorylation provides a major regulatory mechanism in eukaryotic cells. Due to the high variability of amino acid residues flanking a relatively limited number of experimentally identified phosphorylation sites, reliable prediction of such sites still remains an important issue. Here we report the development of a new web-based tool for the prediction of protein phosphorylation sites, DISPHOS (DISorder-enhanced PHOSphorylation predictor, http://www.ist.temple. edu/DISPHOS). We observed that amino acid compositions, sequence complexity, hydrophobicity, charge and other sequence attributes of regions adjacent to phosphorylation sites are very similar to those of intrinsically disordered protein regions. Thus, DISPHOS uses position-specific amino acid frequencies and disorder information to improve the discrimination between phosphorylation and non-phosphorylation sites. Based on the estimates of phosphorylation rates in various protein categories, the outputs of DISPHOS are adjusted in order to reduce the total number of misclassified residues. When tested on an equal number of phosphorylated and non-phosphorylated residues, the accuracy of DISPHOS reaches 76% for serine, 81% for threonine and 83% for tyrosine. The significant enrichment in disorder-promoting residues surrounding phosphorylation sites together with the results obtained by applying DISPHOS to various protein functional classes and proteomes, provide strong support for the hypothesis that protein phosphorylation predominantly occurs within intrinsically disordered protein regions. PMID:14960716

  16. Syntheses and insulin-like activity of phosphorylated galactose derivatives.

    PubMed

    Caro, H N; Martín-Lomas, M; Bernabé, M

    1993-02-24

    The syntheses of the poly-phosphorylated galactosides 6, 8, 10, 13, 16, and 20, isolated as sodium salts, have been performed. The non-phosphorylated disaccharide 17 and trisaccharide 21 have been prepared via glycosylation of the 2-(trimethylsilyl)ethyl galactosides 3 and 2, respectively, and subsequent complete deprotection. Preliminary insulin-like activity of the phosphorylated derivatives is reported. PMID:8458006

  17. Plk4-dependent phosphorylation of STIL is required for centriole duplication.

    PubMed

    Kratz, Anne-Sophie; Bärenz, Felix; Richter, Kai T; Hoffmann, Ingrid

    2015-01-01

    Duplication of centrioles, namely the formation of a procentriole next to the parental centriole, is regulated by the polo-like kinase Plk4. Only a few other proteins, including STIL (SCL/TAL1 interrupting locus, SIL) and Sas-6, are required for the early step of centriole biogenesis. Following Plk4 activation, STIL and Sas-6 accumulate at the cartwheel structure at the initial stage of the centriole assembly process. Here, we show that STIL interacts with Plk4 in vivo. A STIL fragment harboring both the coiled-coil domain and the STAN motif shows the strongest binding affinity to Plk4. Furthermore, we find that STIL is phosphorylated by Plk4. We identified Plk4-specific phosphorylation sites within the C-terminal domain of STIL and show that phosphorylation of STIL by Plk4 is required to trigger centriole duplication. PMID:25701666

  18. Biochemical abnormalities in Pearson syndrome.

    PubMed

    Crippa, Beatrice Letizia; Leon, Eyby; Calhoun, Amy; Lowichik, Amy; Pasquali, Marzia; Longo, Nicola

    2015-03-01

    Pearson marrow-pancreas syndrome is a multisystem mitochondrial disorder characterized by bone marrow failure and pancreatic insufficiency. Children who survive the severe bone marrow dysfunction in childhood develop Kearns-Sayre syndrome later in life. Here we report on four new cases with this condition and define their biochemical abnormalities. Three out of four patients presented with failure to thrive, with most of them having normal development and head size. All patients had evidence of bone marrow involvement that spontaneously improved in three out of four patients. Unique findings in our patients were acute pancreatitis (one out of four), renal Fanconi syndrome (present in all patients, but symptomatic only in one), and an unusual organic aciduria with 3-hydroxyisobutyric aciduria in one patient. Biochemical analysis indicated low levels of plasma citrulline and arginine, despite low-normal ammonia levels. Regression analysis indicated a significant correlation between each intermediate of the urea cycle and the next, except between ornithine and citrulline. This suggested that the reaction catalyzed by ornithine transcarbamylase (that converts ornithine to citrulline) might not be very efficient in patients with Pearson syndrome. In view of low-normal ammonia levels, we hypothesize that ammonia and carbamylphosphate could be diverted from the urea cycle to the synthesis of nucleotides in patients with Pearson syndrome and possibly other mitochondrial disorders. PMID:25691415

  19. The XXXXY Sex Chromosome Abnormality

    PubMed Central

    Barr, M. L.; Carr, D. H.; Pozsonyi, J.; Wilson, R. A.; Dunn, H. G.; Jacobson, T. S.; Miller, J. R.; Chown, B.

    1962-01-01

    The most common sex chromosome complex in sex chromatin-positive males with Klinefelter's syndrome is XXY. When the complex is XXYY or XXXY, the clinical findings do not seem to differ materially from those seen in XXY subjects, although more patients with these intersexual chromosome complements need to be studied to establish possible phenotypical expressions of the chromosomal variants. Two male children with an XXXXY sex chromosome abnormality are described. The data obtained from the study of these cases and five others described in the literature suggest that the XXXXY patient is likely to have congenital defects not usually seen in the common form of the Klinefelter syndrome. These include a triad of (1) skeletal anomalies (including radioulnar synostosis), (2) hypogenitalism (hypoplasia of penis and scrotum, incomplete descent of testes and defective prepubertal development of seminiferous tubules), and (3) greater risk of severe mental deficiency. That the conclusions are based on data from a small number of patients is emphasized, together with the need for a cytogenetic survey of a large control or unselected population. ImagesFig. 1Fig. 2Fig. 3Fig. 4Fig. 5Fig. 6Fig. 7Fig. 8Fig. 9Fig. 10 PMID:13969480

  20. Abnormal Mitochondrial Dynamics and Neurodegenerative Diseases

    PubMed Central

    Su, Bo; Wang, Xinglong; Zheng, Ling; Perry, George; Smith, Mark A.; Zhu, Xiongwei

    2009-01-01

    Mitochondrial dysfunction is a prominent feature of various neurodegenerative diseases. A deeper understanding of the remarkably dynamic nature of mitochondria, characterized by a delicate balance of fission and fusion, has helped to fertilize a recent wave of new studies demonstrating abnormal mitochondrial dynamics in neurodegenerative diseases. This review highlights mitochondrial dysfunction and abnormal mitochondrial dynamics in Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis, and Huntington disease and discusses how these abnormal mitochondrial dynamics may contribute to mitochondrial and neuronal dysfunction. We propose that abnormal mitochondrial dynamics represents a key common pathway that mediates or amplifies mitochondrial dysfunction and neuronal dysfunction during the course of neurodegeneration. PMID:19799998

  1. Chromosomal abnormalities in child psychiatric patients.

    PubMed

    Hong, K E; Kim, J H; Moon, S Y; Oh, S K

    1999-08-01

    To determine the frequency of chromosomal abnormalities in a child psychiatric population, and to evaluate possible associations between types of abnormalities and patient's clinical characteristics, cytogenetic examination was performed on 604 patients. Demographic data, reasons for karyotyping, clinical signs, and other patient characteristics were assessed and correlated with the results from karyotyping. Chromosomal abnormalities were found in 69 patients (11.3%); these were structural in 49 cases and numerical in 20. Inversion of chromosome nine was found in 15 subjects, trisomy of chromosome 21 in 11, and fragile X in five patients. When karyotyping was performed because of intellectual impairment or multiple developmental delay, significantly more abnormalities were found than average; when performed because autistic disorder was suspected, the number of abnormalities was significantly fewer. There were no differences in clinical variables between structural and numerical abnormalities, nor among nine types of chromosomal abnormalities, except that numerical abnormalities and polymorphism were found at a later age, and that walking was more delayed and IQ was lower in patients with Down syndrome. Clinicians should be aware of the possible presence of chromosomal abnormalities in child psychiatric populations; the close collaboration with geneticists and the use of more defined guidelines for cytogenetic investigation are important. PMID:10485616

  2. Radiologic atlas of pulmonary abnormalities in children

    SciTech Connect

    Singleton, E.B.; Wagner, M.L.; Dutton, R.V.

    1988-01-01

    This book is an atlas about thoracic abnormalities in infants and children. The authors include computed tomographic, digital subtraction angiographic, ultrasonographic, and a few magnetic resonance (MR) images. They recognize and discuss how changes in the medical treatment of premature infants and the management of infection and pediatric tumors have altered some of the appearances and considerations in these diseases. Oriented toward all aspects of pulmonary abnormalities, the book starts with radiographic techniques and then discusses the normal chest, the newborn, infections, tumors, and pulmonary vascular diseases. There is comprehensive treatment of mediastinal abnormalities and a discussion of airway abnormalities.

  3. Phosphorylation of Kraft fibers with phosphate esters.

    PubMed

    Shi, Ying; Belosinschi, Dan; Brouillette, François; Belfkira, Ahmed; Chabot, Bruno

    2014-06-15

    Phosphate esters, derived from two different long-chain aliphatic alcohols, were used as phosphorylating reagents for Kraft pulp fibers. High phosphorus contents and almost non-degraded fibers were obtained by following this pathway. The phosphorylation efficiency was influenced by the alkyl chain length of PEs since the phosphorus content in modified fibers was higher for the shorter chain reagent. Due to the heterogeneous reaction environment, the amount of grafted phosphorus was found to be almost three times higher at the surface than in the bulk of the fibers. Analyses also indicated that the phosphorus was bonded to fibers as a phosphate-like structure. Furthermore, the situation seemed to be different for the fiber surface where significant amounts of phosphorus were present in more complex structures like pyrophosphate or even oligo-phosphate. PMID:24721058

  4. Functions of the major tyrosine phosphorylation site of the PDGF receptor beta subunit.

    PubMed Central

    Kazlauskas, A; Durden, D L; Cooper, J A

    1991-01-01

    Two tyrosine phosphorylation sites in the human platelet-derived growth factor receptor (PDGFR) beta subunit have been mapped previously to tyrosine (Y)751, in the kinase insert, and Y857, in the kinase domain. Y857 is the major site of tyrosine phosphorylation in PDGF-stimulated cells. To evaluate the importance of these phosphorylations, we have characterized the wild-type (WT) and mutant human PDGF receptor beta subunits in dog kidney epithelial cells. Replacement of either Y751 or Y857 with phenylalanine (F) reduced PDGF-stimulated DNA synthesis to approximately 50% of the WT level. A mutant receptor with both tyrosines mutated was unable to initiate DNA synthesis, as was a kinase-inactive mutant receptor. Transmodulation of the epidermal growth factor receptor required Y857 but not Y751. We also tested the effects of phosphorylation site mutations on PDGF-stimulated receptor kinase activity. PDGF-induced tyrosine phosphorylation of two cellular proteins, phospholipase C gamma 1 (PLC gamma 1) and the GTPase activating protein of Ras (GAP), was assayed in epithelial cells expressing each of the mutant receptors. Tyrosine phosphorylation of GAP and PLC gamma 1 was reduced markedly by the F857 mutation but not significantly by the F751 mutation. Reduced kinase activity of F857 receptors was also evident in vitro. Immunoprecipitated WT receptors showed a two- to fourfold increase in specific kinase activity if immunoprecipitated from PDGF-stimulated cells. The F751 receptors showed a similar increase in activity, but F857 receptors did not. Our data suggest that phosphorylation of Y857 may be important for stimulation of kinase activity of the receptors and for downstream actions such as epidermal growth factor receptor transmodulation and mitogenesis. Images PMID:1653029

  5. Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and trimeric complex formation

    PubMed Central

    Montagnoli, Alessia; Fiore, Francesca; Eytan, Esther; Carrano, Andrea C.; Draetta, Giulio F.; Hershko, Avram; Pagano, Michele

    1999-01-01

    The cellular abundance of the cyclin-dependent kinase (Cdk) inhibitor p27 is regulated by the ubiquitin–proteasome system. Activation of p27 degradation is seen in proliferating cells and in many types of aggressive human carcinomas. p27 can be phosphorylated on threonine 187 by Cdks, and cyclin E/Cdk2 overexpression can stimulate the degradation of wild-type p27, but not of a threonine 187-to-alanine p27 mutant [p27(T187A)]. However, whether threonine 187 phosphorylation stimulates p27 degradation through the ubiquitin–proteasome system or an alternative pathway is still not known. Here, we demonstrate that p27 ubiquitination (as assayed in vivo and in an in vitro reconstituted system) is cell-cycle regulated and that Cdk activity is required for the in vitro ubiquitination of p27. Furthermore, ubiquitination of wild-type p27, but not of p27(T187A), can occur in G1-enriched extracts only upon addition of cyclin E/Cdk2 or cyclin A/Cdk2. Using a phosphothreonine 187 site-specific antibody for p27, we show that threonine 187 phosphorylation of p27 is also cell-cycle dependent, being present in proliferating cells but undetectable in G1 cells. Finally, we show that in addition to threonine 187 phosphorylation, efficient p27 ubiquitination requires formation of a trimeric complex with the cyclin and Cdk subunits. In fact, cyclin B/Cdk1 which can phosphorylate p27 efficiently, but cannot form a stable complex with it, is unable to stimulate p27 ubiquitination by G1 extracts. Furthermore, another p27 mutant [p27(CK−)] that can be phosphorylated by cyclin E/Cdk2 but cannot bind this kinase complex, is refractory to ubiquitination. Thus throughout the cell cycle, both phosphorylation and trimeric complex formation act as signals for the ubiquitination of a Cdk inhibitor. PMID:10323868

  6. Phosphorylation of TCF proteins by homeodomain-interacting protein kinase 2.

    PubMed

    Hikasa, Hiroki; Sokol, Sergei Y

    2011-04-01

    Wnt pathways play essential roles in cell proliferation, morphogenesis, and cell fate specification during embryonic development. According to the consensus view, the Wnt pathway prevents the degradation of the key signaling component β-catenin by the protein complex containing the negative regulators Axin and glycogen synthase kinase 3 (GSK3). Stabilized β-catenin associates with TCF proteins and enters the nucleus to promote target gene expression. This study examines the involvement of HIPK2 (homeodomain-interacting protein kinase 2) in the regulation of different TCF proteins in Xenopus embryos in vivo. We show that the TCF family members LEF1, TCF4, and TCF3 are phosphorylated in embryonic ectoderm after Wnt8 stimulation and HIPK2 overexpression. We also find that TCF3 phosphorylation is triggered by canonical Wnt ligands, LRP6, and dominant negative mutants for Axin and GSK3, indicating that this process shares the same upstream regulators with β-catenin stabilization. HIPK2-dependent phosphorylation caused the dissociation of LEF1, TCF4, and TCF3 from a target promoter in vivo. This result provides a mechanistic explanation for the context-dependent function of HIPK2 in Wnt signaling; HIPK2 up-regulates transcription by phosphorylating TCF3, a transcriptional repressor, but inhibits transcription by phosphorylating LEF1, a transcriptional activator. Finally, we show that upon HIPK2-mediated phosphorylation, TCF3 is replaced with positively acting TCF1 at a target promoter. These observations emphasize a critical role for Wnt/HIPK2-dependent TCF phosphorylation and suggest that TCF switching is an important mechanism of Wnt target gene activation in vertebrate embryos. PMID:21285352

  7. Global Detection of Protein Kinase D-dependent Phosphorylation Events in Nocodazole-treated Human Cells*

    PubMed Central

    Franz-Wachtel, Mirita; Eisler, Stephan A.; Krug, Karsten; Wahl, Silke; Carpy, Alejandro; Nordheim, Alfred; Pfizenmaier, Klaus; Hausser, Angelika; Macek, Boris

    2012-01-01

    Protein kinase D (PKD) is a cytosolic serine/threonine kinase implicated in regulation of several cellular processes such as response to oxidative stress, directed cell migration, invasion, differentiation, and fission of the vesicles at the trans-Golgi network. Its variety of functions must be mediated by numerous substrates; however, only a couple of PKD substrates have been identified so far. Here we perform stable isotope labeling of amino acids in cell culture-based quantitative phosphoproteomic analysis to detect phosphorylation events dependent on PKD1 activity in human cells. We compare relative phosphorylation levels between constitutively active and kinase dead PKD1 strains of HEK293 cells, both treated with nocodazole, a microtubule-depolymerizing reagent that disrupts the Golgi complex and activates PKD1. We identify 124 phosphorylation sites that are significantly down-regulated upon decrease of PKD1 activity and show that the PKD target motif is significantly enriched among down-regulated phosphorylation events, pointing to the presence of direct PKD1 substrates. We further perform PKD1 target motif analysis, showing that a proline residue at position +1 relative to the phosphorylation site serves as an inhibitory cue for PKD1 activity. Among PKD1-dependent phosphorylation events, we detect predominantly proteins with localization at Golgi membranes and function in protein sorting, among them several sorting nexins and members of the insulin-like growth factor 2 receptor pathway. This study presents the first global detection of PKD1-dependent phosphorylation events and provides a wealth of information for functional follow-up of PKD1 activity upon disruption of the Golgi network in human cells. PMID:22496350

  8. Phosphorylation and Activation of RhoA by ERK in Response to Epidermal Growth Factor Stimulation.

    PubMed

    Tong, Junfeng; Li, Laiji; Ballermann, Barbara; Wang, Zhixiang

    2016-01-01

    The small GTPase RhoA has been implicated in various cellular activities, including the formation of stress fibers, cell motility, and cytokinesis. In addition to the canonical GTPase cycle, recent findings have suggested that phosphorylation further contributes to the tight regulation of Rho GTPases. Indeed, RhoA is phosphorylated on serine 188 (188S) by a number of protein kinases. We have recently reported that Rac1 is phosphorylated on threonine 108 (108T) by extracellular signal-regulated kinases (ERK) in response to epidermal growth factor (EGF) stimulation. Here, we provide evidence that RhoA is phosphorylated by ERK on 88S and 100T in response to EGF stimulation. We show that ERK interacts with RhoA and that this interaction is dependent on the ERK docking site (D-site) at the C-terminus of RhoA. EGF stimulation enhanced the activation of the endogenous RhoA. The phosphomimetic mutant, GFP-RhoA S88E/T100E, when transiently expressed in COS-7 cells, displayed higher GTP-binding than wild type RhoA. Moreover, the expression of GFP-RhoA S88E/T100E increased actin stress fiber formation in COS-7 cells, which is consistent with its higher activity. In contrast to Rac1, phosphorylation of RhoA by ERK does not target RhoA to the nucleus. Finally, we show that regardless of the phosphorylation status of RhoA and Rac1, substitution of the RhoA PBR with the Rac1 PBR targets RhoA to the nucleus and substitution of Rac1 PBR with RhoA PBR significantly reduces the nuclear localization of Rac1. In conclusion, ERK phosphorylates RhoA on 88S and 100T in response to EGF, which upregulates RhoA activity. PMID:26816343

  9. Phosphorylation and Activation of RhoA by ERK in Response to Epidermal Growth Factor Stimulation

    PubMed Central

    Tong, Junfeng; Li, Laiji; Ballermann, Barbara; Wang, Zhixiang

    2016-01-01

    The small GTPase RhoA has been implicated in various cellular activities, including the formation of stress fibers, cell motility, and cytokinesis. In addition to the canonical GTPase cycle, recent findings have suggested that phosphorylation further contributes to the tight regulation of Rho GTPases. Indeed, RhoA is phosphorylated on serine 188 (188S) by a number of protein kinases. We have recently reported that Rac1 is phosphorylated on threonine 108 (108T) by extracellular signal-regulated kinases (ERK) in response to epidermal growth factor (EGF) stimulation. Here, we provide evidence that RhoA is phosphorylated by ERK on 88S and 100T in response to EGF stimulation. We show that ERK interacts with RhoA and that this interaction is dependent on the ERK docking site (D-site) at the C-terminus of RhoA. EGF stimulation enhanced the activation of the endogenous RhoA. The phosphomimetic mutant, GFP-RhoA S88E/T100E, when transiently expressed in COS-7 cells, displayed higher GTP-binding than wild type RhoA. Moreover, the expression of GFP-RhoA S88E/T100E increased actin stress fiber formation in COS-7 cells, which is consistent with its higher activity. In contrast to Rac1, phosphorylation of RhoA by ERK does not target RhoA to the nucleus. Finally, we show that regardless of the phosphorylation status of RhoA and Rac1, substitution of the RhoA PBR with the Rac1 PBR targets RhoA to the nucleus and substitution of Rac1 PBR with RhoA PBR significantly reduces the nuclear localization of Rac1. In conclusion, ERK phosphorylates RhoA on 88S and 100T in response to EGF, which upregulates RhoA activity. PMID:26816343

  10. Solid polymer electrolyte from phosphorylated chitosan

    SciTech Connect

    Fauzi, Iqbal Arcana, I Made

    2014-03-24

    Recently, the need of secondary battery application continues to increase. The secondary battery which using a liquid electrolyte was indicated had some weakness. A solid polymer electrolyte is an alternative electrolytes membrane which developed in order to replace the liquid electrolyte type. In the present study, the effect of phosphorylation on to polymer electrolyte membrane which synthesized from chitosan and lithium perchlorate salts was investigated. The effect of the component’s composition respectively on the properties of polymer electrolyte, was carried out by analyzed of it’s characterization such as functional groups, ion conductivity, and thermal properties. The mechanical properties i.e tensile resistance and the morphology structure of membrane surface were determined. The phosphorylation processing of polymer electrolyte membrane of chitosan and lithium perchlorate was conducted by immersing with phosphoric acid for 2 hours, and then irradiated on a microwave for 60 seconds. The degree of deacetylation of chitosan derived from shrimp shells was obtained around 75.4%. Relative molecular mass of chitosan was obtained by viscometry method is 796,792 g/mol. The ionic conductivity of chitosan membrane was increase from 6.33 × 10{sup −6} S/cm up to 6.01 × 10{sup −4} S/cm after adding by 15 % solution of lithium perchlorate. After phosphorylation, the ionic conductivity of phosphorylated lithium chitosan membrane was observed 1.37 × 10{sup −3} S/cm, while the tensile resistance of 40.2 MPa with a better thermal resistance. On the strength of electrolyte membrane properties, this polymer electrolyte membrane was suggested had one potential used for polymer electrolyte in field of lithium battery applications.

  11. IER5 generates a novel hypo-phosphorylated active form of HSF1 and contributes to tumorigenesis.

    PubMed

    Asano, Yoshinori; Kawase, Tatsuya; Okabe, Atsushi; Tsutsumi, Shuichi; Ichikawa, Hitoshi; Tatebe, Satoko; Kitabayashi, Issay; Tashiro, Fumio; Namiki, Hideo; Kondo, Tadashi; Semba, Kentaro; Aburatani, Hiroyuki; Taya, Yoichi; Nakagama, Hitoshi; Ohki, Rieko

    2016-01-01

    The transcription factors HSF1 and p53 both modulate the stress response, thereby protecting and facilitating the recovery of stressed cells, but both have the potential to promote tumor development. Here we show that a p53 target gene, IER5, encodes an activator of HSF1. IER5 forms a ternary complex with HSF1 and the phosphatase PP2A, and promotes the dephosphorylation of HSF1 at numbers of serine and threonine residues, generating a novel, hypo-phosphorylated active form of HSF1. IER5 is also transcriptionally upregulated in various cancers, although this upregulation is not always p53-dependent. The IER5 locus is associated with a so-called super enhancer, frequently associated with hyperactivated oncogenes in cancer cell lines. Enhanced expression of IER5 induces abnormal HSF1 activation in cancer cells and contributes to the proliferation of these cells under stressed conditions. These results reveal the existence of a novel IER5-mediated cancer regulation pathway that is responsible for the activation of HSF1 observed in various cancers. PMID:26754925

  12. Phosphorylation of threonine 3 on histone H3 by haspin kinase is required for meiosis I in mouse oocytes

    PubMed Central

    Nguyen, Alexandra L.; Gentilello, Amanda S.; Balboula, Ahmed Z.; Shrivastava, Vibha; Ohring, Jacob; Schindler, Karen

    2014-01-01

    ABSTRACT Meiosis I (MI), the division that generates haploids, is prone to errors that lead to aneuploidy in females. Haspin is a kinase that phosphorylates histone H3 on threonine 3, thereby recruiting Aurora kinase B (AURKB) and the chromosomal passenger complex (CPC) to kinetochores to regulate mitosis. Haspin and AURKC, an AURKB homolog, are enriched in germ cells, yet their significance in regulating MI is not fully understood. Using inhibitors and overexpression approaches, we show a role for haspin during MI in mouse oocytes. Haspin-perturbed oocytes display abnormalities in chromosome morphology and alignment, improper kinetochore–microtubule attachments at metaphase I and aneuploidy at metaphase II. Unlike in mitosis, kinetochore localization remained intact, whereas the distribution of the CPC along chromosomes was absent. The meiotic defects following haspin inhibition were similar to those observed in oocytes where AURKC was inhibited, suggesting that the correction of microtubule attachments during MI requires AURKC along chromosome arms rather than at kinetochores. Our data implicate haspin as a regulator of the CPC and chromosome segregation during MI, while highlighting important differences in how chromosome segregation is regulated between MI and mitosis. PMID:25315835

  13. IER5 generates a novel hypo-phosphorylated active form of HSF1 and contributes to tumorigenesis

    PubMed Central

    Asano, Yoshinori; Kawase, Tatsuya; Okabe, Atsushi; Tsutsumi, Shuichi; Ichikawa, Hitoshi; Tatebe, Satoko; Kitabayashi, Issay; Tashiro, Fumio; Namiki, Hideo; Kondo, Tadashi; Semba, Kentaro; Aburatani, Hiroyuki; Taya, Yoichi; Nakagama, Hitoshi; Ohki, Rieko

    2016-01-01

    The transcription factors HSF1 and p53 both modulate the stress response, thereby protecting and facilitating the recovery of stressed cells, but both have the potential to promote tumor development. Here we show that a p53 target gene, IER5, encodes an activator of HSF1. IER5 forms a ternary complex with HSF1 and the phosphatase PP2A, and promotes the dephosphorylation of HSF1 at numbers of serine and threonine residues, generating a novel, hypo-phosphorylated active form of HSF1. IER5 is also transcriptionally upregulated in various cancers, although this upregulation is not always p53-dependent. The IER5 locus is associated with a so-called super enhancer, frequently associated with hyperactivated oncogenes in cancer cell lines. Enhanced expression of IER5 induces abnormal HSF1 activation in cancer cells and contributes to the proliferation of these cells under stressed conditions. These results reveal the existence of a novel IER5-mediated cancer regulation pathway that is responsible for the activation of HSF1 observed in various cancers. PMID:26754925

  14. Geniposide ameliorates learning memory deficits, reduces tau phosphorylation and decreases apoptosis via GSK3β pathway in streptozotocin-induced alzheimer rat model.

    PubMed

    Gao, Chong; Liu, Yueze; Jiang, Yuanhong; Ding, Jianming; Li, Lin

    2014-04-01

    Intracerebral-ventricular (ICV) injection of streptozotocin (STZ) induces an insulin-resistant brain state that may underlie the neural pathogenesis of sporadic Alzheimer disease (AD). Our previous work showed that prior ICV treatment of glucagon-like peptide-1 (GLP-1) could prevent STZ-induced learning memory impairment and tau hyperphosphorylation in the rat brain. The Chinese herbal medicine geniposide is known to relieve symptoms of type 2 diabetes. Because geniposide is thought to act as a GLP-1 receptor agonist, we investigated the potential therapeutic effect of geniposide on STZ-induced AD model in rats. Our result showed that a single injection of geniposide (50 μM, 10 μL) to the lateral ventricle prevented STZ-induced spatial learning deficit by about 40% and reduced tau phosphorylation by about 30% with Morris water maze test and quantitative immunohistochemical analysis, respectively. It has been known that tau protein can be phosphorylated by glycogen synthase kinase-3 (GSK3) and STZ can increase the activity of GSK3β. Our result with Western blot analysis showed that central administration of geniposide resulted in an elevated expression of GSK3β(pS-9) but suppressed GSK3β(pY-216) indicating that geniposide reduced STZ-induced GSK3β hyperactivity. In addition, ultrastructure analysis showed that geniposide averted STZ-induced neural pathology, including paired helical filament (PHF)-like structures, accumulation of vesicles in synaptic terminal, abnormalities of endoplasmic reticulum (ER) and early stage of apoptosis. In summary, our study suggests that the water soluble and orally active monomer of Chinese herbal medicine geniposide may serve as a novel therapeutic agent for the treatment of sporadic AD. PMID:24329968

  15. Mechanisms and consequences of paternally transmitted chromosomal abnormalities

    SciTech Connect

    Marchetti, F; Wyrobek, A J

    2005-04-05

    Paternally transmitted chromosomal damage has been associated with pregnancy loss, developmental and morphological defects, infant mortality, infertility, and genetic diseases in the offspring including cancer. There is epidemiological evidence linking paternal exposure to occupational or environmental agents with an increased risk of abnormal reproductive outcomes. There is also a large body of literature on germ cell mutagenesis in rodents showing that treatment of male germ cells with mutagens has dramatic consequences on reproduction producing effects such as those observed in human epidemiological studies. However, we know very little about the etiology, transmission and early embryonic consequences of paternally-derived chromosomal abnormalities. The available evidence suggests that: (1) there are distinct patterns of germ cell-stage differences in the sensitivity of induction of transmissible genetic damage with male postmeiotic cells being the most sensitive; (2) cytogenetic abnormalities at first metaphase after fertilization are critical intermediates between paternal exposure and abnormal reproductive outcomes; and, (3) there are maternally susceptibility factors that may have profound effects on the amount of sperm DNA damage that is converted into chromosomal aberrations in the zygote and directly affect the risk for abnormal reproductive outcomes.

  16. 42 CFR 37.53 - Notification of abnormal roentgenographic findings.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... suggesting, enlarged heart, tuberculosis, lung cancer, or any other significant abnormal findings other than... to the miner by MSHA in accordance with section 203 of the act (see 30 CFR part 90). Positive... files and the most recent examination was interpreted to show enlarged heart, tuberculosis,...

  17. Regulation of peroxisome dynamics by phosphorylation.

    PubMed

    Oeljeklaus, Silke; Schummer, Andreas; Mastalski, Thomas; Platta, Harald W; Warscheid, Bettina

    2016-05-01

    Peroxisomes are highly dynamic organelles that can rapidly change in size, abundance, and protein content in response to alterations in nutritional and other environmental conditions. These dynamic changes in peroxisome features, referred to as peroxisome dynamics, rely on the coordinated action of several processes of peroxisome biogenesis. Revealing the regulatory mechanisms of peroxisome dynamics is an emerging theme in cell biology. These mechanisms are inevitably linked to and synchronized with the biogenesis and degradation of peroxisomes. To date, the key players and basic principles of virtually all steps in the peroxisomal life cycle are known, but regulatory mechanisms remained largely elusive. A number of recent studies put the spotlight on reversible protein phosphorylation for the control of peroxisome dynamics and highlighted peroxisomes as hubs for cellular signal integration and regulation. Here, we will present and discuss the results of several studies performed using yeast and mammalian cells that convey a sense of the impact protein phosphorylation may have on the modulation of peroxisome dynamics by regulating peroxisomal matrix and membrane protein import, proliferation, inheritance, and degradation. We further put forward the idea to make use of current data on phosphorylation sites of peroxisomal and peroxisome-associated proteins reported in advanced large-scale phosphoproteomic studies. PMID:26775584

  18. Phosphorylation Stoichiometries of Human Eukaryotic Initiation Factors

    PubMed Central

    Andaya, Armann; Villa, Nancy; Jia, Weitao; Fraser, Christopher S.; Leary, Julie A.

    2014-01-01

    Eukaryotic translation initiation factors are the principal molecular effectors regulating the process converting nucleic acid to functional protein. Commonly referred to as eIFs (eukaryotic initiation factors), this suite of proteins is comprised of at least 25 individual subunits that function in a coordinated, regulated, manner during mRNA translation. Multiple facets of eIF regulation have yet to be elucidated; however, many of the necessary protein factors are phosphorylated. Herein, we have isolated, identified and quantified phosphosites from eIF2, eIF3, and eIF4G generated from log phase grown HeLa cell lysates. Our investigation is the first study to globally quantify eIF phosphosites and illustrates differences in abundance of phosphorylation between the residues of each factor. Thus, identification of those phosphosites that exhibit either high or low levels of phosphorylation under log phase growing conditions may aid researchers to concentrate their investigative efforts to specific phosphosites that potentially harbor important regulatory mechanisms germane to mRNA translation. PMID:24979134

  19. Phosphorylation-dephosphorylation of yeast pyruvate dehydrogenase

    SciTech Connect

    Uhlinger, D.J.; Reed, L.J.

    1986-05-01

    Pyruvate dehydrogenase complex (PDC) was purified to homogeneity from baker's yeast (Saccharomyces cerevisiae). No pyruvate dehydrogenase (PDH) kinase activity was detected at any stage of the purification. However, the purified PDC was phosphorylated and inactivated by purified PDH kinase from bovine kidney mitochondria, Mg/sup 2 +/, and (..gamma..-/sup 32/P)ATP. The protein-bound radioactivity was localized in the PDH ..cap alpha.. subunit. The phosphorylated, inactivated PDC was dephosphorylated and reactivated with purified bovine PDH phosphatase, Mg/sup 2 +/, and Ca/sup 2 +/. From a tryptic digest of phosphorylated yeast PDC a radioactive peptide was isolated by anion and reverse phase HPLC. The sequence of this tetradecapeptide is Tyr-Gly-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Thr-Thr-Tyr-Arg. This sequence is very similar to the sequence of a tryptic phosphopeptide derived from the ..cap alpha.. subunit of bovine kidney and heart PDH: Tyr-His-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Val-Ser-Tyr-Arg.

  20. Phosphorylation network rewiring by gene duplication

    PubMed Central

    Freschi, Luca; Courcelles, Mathieu; Thibault, Pierre; Michnick, Stephen W; Landry, Christian R

    2011-01-01

    Elucidating how complex regulatory networks have assembled during evolution requires a detailed understanding of the evolutionary dynamics that follow gene duplication events, including changes in post-translational modifications. We compared the phosphorylation profiles of paralogous proteins in the budding yeast Saccharomyces cerevisiae to that of a species that diverged from the budding yeast before the duplication of those genes. We found that 100 million years of post-duplication divergence are sufficient for the majority of phosphorylation sites to be lost or gained in one paralog or the other, with a strong bias toward losses. However, some losses may be partly compensated for by the evolution of other phosphosites, as paralogous proteins tend to preserve similar numbers of phosphosites over time. We also found that up to 50% of kinase–substrate relationships may have been rewired during this period. Our results suggest that after gene duplication, proteins tend to subfunctionalize at the level of post-translational regulation and that even when phosphosites are preserved, there is a turnover of the kinases that phosphorylate them. PMID:21734643

  1. Phosphorylation of proteins in Clostridium thermohydrosulfuricum

    SciTech Connect

    Londesborough, J.

    1986-02-01

    Cell extracts of the thermophile Clostridium thermohydrosulfuricum catalyzed the phosphorylation by (..gamma..-/sup 32/P)ATP of several endogenous proteins with M/sub r/s between 13,000 and 100,000. Serine and tyrosine were the main acceptors. Distinct substrate proteins were found in the soluble (e.g., proteins p66, p63, and p53 of M/sub r/s 66,000, 63,000, and 53,000, respectively) and particulate (p76 and p30) fractions, both of which contained protein kinase and phosphatase activity. The soluble fraction suppressed the phosphorylation of particulate proteins and contained a protein kinase inhibitor. Phosphorylation of p53 was promoted by 10..mu..M fructose 1,6-bisphosphate or glucose 1,6-bisphosphate and suppressed by hexose monophosphates, whereas p30 and p13 were suppressed by 5 ..mu..M brain (but not spinach) calmodulin. Polyamines, including the odd polyamines characteristic of thermophiles, modulated the labeling of most of the phosphoproteins. Apart from p66, all the proteins labeled in vitro were also rapidly labeled in intact cells by /sub 32/P/sub i/. Several proteins strongly labeled in vivo were labeled slowly or not at all in vitro.

  2. Using Reduced Interference Distribution to Analyze Abnormal Cardiac Signal

    NASA Astrophysics Data System (ADS)

    Mousa, Allam; Saleem, Rashid

    2011-05-01

    Due to the non-stationary, multicomponent nature of biomedical signals, the use of time-frequency analysis can be inevitable for these signals. The choice and selection of the proper Time-Frequency Distribution (TFD) that can reveal the exact multicomponent structure of biological signals is vital in many applications, including the diagnosis of medical abnormalities. In this paper, the instantaneous frequency techniques using two distribution functions are applied for analysis of biological signals. These distributions are the Wigner-Ville Distribution and the Bessel Distribution. The simulation performed on normaland abnormal cardiac signals show that the Bessel Distribution can clearly detect the QRS complexes. However, Wigner-Ville Distribution was able to detect the QRS complexes in the normal signa, but fails to detect these complexes in the abnormal cardiac signal.

  3. Detection of Abnormal Events via Optical Flow Feature Analysis

    PubMed Central

    Wang, Tian; Snoussi, Hichem

    2015-01-01

    In this paper, a novel algorithm is proposed to detect abnormal events in video streams. The algorithm is based on the histogram of the optical flow orientation descriptor and the classification method. The details of the histogram of the optical flow orientation descriptor are illustrated for describing movement information of the global video frame or foreground frame. By combining one-class support vector machine and kernel principal component analysis methods, the abnormal events in the current frame can be detected after a learning period characterizing normal behaviors. The difference abnormal detection results are analyzed and explained. The proposed detection method is tested on benchmark datasets, then the experimental results show the effectiveness of the algorithm. PMID:25811227

  4. Altered protein phosphorylation in sciatic nerve from rats with streptozocin-induced diabetes

    SciTech Connect

    Schrama, L.H.; Berti-Mattera, L.N.; Eichberg, J.

    1987-11-01

    The effect of experimental diabetes on the phosphorylation of proteins in the rat sciatic nerve was studied. Nerves from animals made diabetic with streptozocin were incubated in vitro with (/sup 32/P)orthophosphate and divided in