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Sample records for 14-3-3 proteins regulate

  1. Up-regulated 14-3-3β and 14-3-3ζ proteins in prefrontal cortex of subjects with schizophrenia: effect of psychotropic treatment.

    PubMed

    Rivero, Guadalupe; Gabilondo, Ane M; García-Sevilla, Jesús A; La Harpe, Romano; Morentín, Benito; Meana, J Javier

    2015-02-01

    14-3-3 is a family of conserved regulatory proteins that bind to a multitude of functionally diverse signalling proteins. Various genetic studies and gene expression and proteomic analyses have involved 14-3-3 proteins in schizophrenia (SZ). On the other hand, studies about the status of these proteins in major depressive disorder (MD) are still missing. Immunoreactivity values of cytosolic 14-3-3β and 14-3-3ζ proteins were evaluated by Western blot in prefrontal cortex (PFC) of subjects with schizophrenia (SZ; n=22), subjects with major depressive disorder (MD; n=21) and age-, gender- and postmortem delay-matched control subjects (n=52). The modulation of 14-3-3β and 14-3-3ζ proteins by psychotropic medication was also assessed. The analysis of both proteins in SZ subjects with respect to matched control subjects showed increased 14-3-3β (Δ=33±10%, p<0.05) and 14-3-3ζ (Δ=29±6%, p<0.05) immunoreactivity in antipsychotic-free but not in antipsychotic-treated SZ subjects. Immunoreactivity values of 14-3-3β and 14-3-3ζ were not altered in MD subjects. These results show the specific up-regulation of 14-3-3β and 14-3-3ζ proteins in PFC of SZ subjects and suggest a possible down-regulation of both proteins by antipsychotic treatment. PMID:25549848

  2. Involvement of 14-3-3 Proteins in Regulating Tumor Progression of Hepatocellular Carcinoma.

    PubMed

    Wu, Yi-Ju; Jan, Yee-Jee; Ko, Bor-Sheng; Liang, Shu-Man; Liou, Jun-Yang

    2015-01-01

    There are seven mammalian isoforms of the 14-3-3 protein, which regulate multiple cellular functions via interactions with phosphorylated partners. Increased expression of 14-3-3 proteins contributes to tumor progression of various malignancies. Several isoforms of 14-3-3 are overexpressed and associate with higher metastatic risks and poorer survival rates of hepatocellular carcinoma (HCC). 14-3-3β and 14-3-3ζ regulate HCC cell proliferation, tumor growth and chemosensitivity via modulating mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK) and p38 signal pathways. Moreover, 14-3-3ε suppresses E-cadherin and induces focal adhesion kinase (FAK) expression, thereby enhancing epithelial-mesenchymal transition (EMT) and HCC cell migration. 14-3-3ζ forms complexes with αB-crystallin, which induces EMT and is the cause of sorafenib resistance in HCC. Finally, a recent study has indicated that 14-3-3σ induces heat shock protein 70 (HSP70) expression, which increases HCC cell migration. These results suggest that selective 14-3-3 isoforms contribute to cell proliferation, EMT and cell migration of HCC by regulating distinct targets and signal pathways. Targeting 14-3-3 proteins together with specific downstream effectors therefore has potential to be therapeutic and prognostic factors of HCC. In this article, we will overview 14-3-3's regulation of its downstream factors and contributions to HCC EMT, cell migration and proliferation. PMID:26083935

  3. 14-3-3 proteins regulate Tctp-Rheb interaction for organ growth in Drosophila.

    PubMed

    Le, Thao Phuong; Vuong, Linh Thuong; Kim, Ah-Ram; Hsu, Ya-Chieh; Choi, Kwang-Wook

    2016-01-01

    14-3-3 family proteins regulate multiple signalling pathways. Understanding biological functions of 14-3-3 proteins has been limited by the functional redundancy of conserved isotypes. Here we provide evidence that 14-3-3 proteins regulate two interacting components of Tor signalling in Drosophila, translationally controlled tumour protein (Tctp) and Rheb GTPase. Single knockdown of 14-3-3ɛ or 14-3-3ζ isoform does not show obvious defects in organ development but causes synergistic genetic interaction with Tctp and Rheb to impair tissue growth. 14-3-3 proteins physically interact with Tctp and Rheb. Knockdown of both 14-3-3 isoforms abolishes the binding between Tctp and Rheb, disrupting organ development. Depletion of 14-3-3s also reduces the level of phosphorylated S6 kinase, phosphorylated Thor/4E-BP and cyclin E (CycE). Growth defects from knockdown of 14-3-3 and Tctp are suppressed by CycE overexpression. This study suggests a novel mechanism of Tor regulation mediated by 14-3-3 interaction with Tctp and Rheb. PMID:27151460

  4. 14-3-3 proteins regulate Tctp–Rheb interaction for organ growth in Drosophila

    PubMed Central

    Le, Thao Phuong; Vuong, Linh Thuong; Kim, Ah-Ram; Hsu, Ya-Chieh; Choi, Kwang-Wook

    2016-01-01

    14-3-3 family proteins regulate multiple signalling pathways. Understanding biological functions of 14-3-3 proteins has been limited by the functional redundancy of conserved isotypes. Here we provide evidence that 14-3-3 proteins regulate two interacting components of Tor signalling in Drosophila, translationally controlled tumour protein (Tctp) and Rheb GTPase. Single knockdown of 14-3-3ɛ or 14-3-3ζ isoform does not show obvious defects in organ development but causes synergistic genetic interaction with Tctp and Rheb to impair tissue growth. 14-3-3 proteins physically interact with Tctp and Rheb. Knockdown of both 14-3-3 isoforms abolishes the binding between Tctp and Rheb, disrupting organ development. Depletion of 14-3-3s also reduces the level of phosphorylated S6 kinase, phosphorylated Thor/4E-BP and cyclin E (CycE). Growth defects from knockdown of 14-3-3 and Tctp are suppressed by CycE overexpression. This study suggests a novel mechanism of Tor regulation mediated by 14-3-3 interaction with Tctp and Rheb. PMID:27151460

  5. Regulation of starch accumulation by granule-associated plant 14-3-3 proteins.

    PubMed

    Sehnke, P C; Chung, H J; Wu, K; Ferl, R J

    2001-01-16

    In higher plants the production of starch is orchestrated by chloroplast-localized biosynthetic enzymes, namely starch synthases, ADP-glucose pyrophosphorylase, and starch branching and debranching enzymes. Diurnal regulation of these enzymes, as well as starch-degrading enzymes, influences both the levels and composition of starch, and is dependent in some instances upon phosphorylation-linked regulation. The phosphoserine/threonine-binding 14-3-3 proteins participate in environmentally responsive phosphorylation-related regulatory functions in plants, and as such are potentially involved in starch regulation. We report here that reduction of the epsilon subgroup of Arabidopsis 14-3-3 proteins by antisense technology resulted in a 2- to 4-fold increase in leaf starch accumulation. Dark-governed starch breakdown was unaffected in these "antisense plants," indicating an unaltered starch-degradation pathway and suggesting a role for 14-3-3 proteins in regulation of starch synthesis. Absorption spectra and gelatinization properties indicate that the starch from the antisense plants has an altered branched glucan composition. Biochemical characterization of protease-treated starch granules from both Arabidopsis leaves and maize endosperm showed that 14-3-3 proteins are internal intrinsic granule proteins. These data suggest a direct role for 14-3-3 proteins in starch accumulation. The starch synthase III family is a possible target for 14-3-3 protein regulation because, uniquely among plastid-localized starch metabolic enzymes, all members of the family contain the conserved 14-3-3 protein phosphoserine/threonine-binding consensus motif. This possibility is strengthened by immunocapture using antibodies to DU1, a maize starch synthase III family member, and direct interaction with biotinylated 14-3-3 protein, both of which demonstrated an association between 14-3-3 proteins and DU1 or DU1-like proteins. PMID:11149942

  6. Dual binding of 14-3-3 protein regulates Arabidopsis nitrate reductase activity.

    PubMed

    Chi, Jen-Chih; Roeper, Juliane; Schwarz, Guenter; Fischer-Schrader, Katrin

    2015-03-01

    14-3-3 proteins represent a family of ubiquitous eukaryotic proteins involved in numerous signal transduction processes and metabolic pathways. One important 14-3-3 target in higher plants is nitrate reductase (NR), whose activity is regulated by different physiological conditions. Intra-molecular electron transfer in NR is inhibited following 14-3-3 binding to a conserved phospho-serine motif located in hinge 1, a surface exposed loop between the catalytic molybdenum and central heme domain. Here we describe a novel 14-3-3 binding site within the NR N-terminus, an acidic motif conserved in NRs of higher plants, which significantly contributes to 14-3-3-mediated inhibition of NR. Deletion or mutation of the N-terminal acidic motif resulted in a significant loss of 14-3-3 mediated inhibition of Ser534 phosphorylated NR-Mo-heme (residues 1-625), a previously established model of NR regulation. Co-sedimentation and crosslinking studies with NR peptides comprising each of the two binding motifs demonstrated direct binding of either peptide to 14-3-3. Surface plasmon resonance spectroscopy disclosed high-affinity binding of 14-3-3ω to the well-known phospho-hinge site and low-affinity binding to the N-terminal acidic motif. A binding groove-deficient 14-3-3ω variant retained interaction to the acidic motif, but lost binding to the phospho-hinge motif. To our knowledge, NR is the first enzyme that harbors two independent 14-3-3 binding sites with different affinities, which both need to be occupied by 14-3-3ω to confer full inhibition of NR activity under physiological conditions. PMID:25578809

  7. Regulation of the Regulators: Post-Translational Modifications, Subcellular, and Spatiotemporal Distribution of Plant 14-3-3 Proteins

    PubMed Central

    Wilson, Rashaun S.; Swatek, Kirby N.; Thelen, Jay J.

    2016-01-01

    14-3-3 proteins bind to and modulate the activity of phosphorylated proteins that regulate a variety of metabolic processes in eukaryotes. Multiple 14-3-3 isoforms are expressed in most organisms and display redundancy in both sequence and function. Plants contain the largest number of 14-3-3 isoforms. For example, Arabidopsis thaliana contains thirteen 14-3-3 genes, each of which is expressed. Interest in the plant 14-3-3 field has swelled over the past decade, largely due to the vast number of possibilities for 14-3-3 metabolic regulation. As the field progresses, it is essential to understand these proteins' activities at both the spatiotemporal and subcellular levels. This review summarizes current knowledge of 14-3-3 proteins in plants, including 14-3-3 interactions, regulatory functions, isoform specificity, and post-translational modifications. We begin with a historical overview and structural analysis of 14-3-3 proteins, which describes the basic principles of 14-3-3 function, and then discuss interactions and regulatory effects of plant 14-3-3 proteins in specific tissues and subcellular compartments. We conclude with a summary of 14-3-3 phosphorylation and current knowledge of the functional effects of this modification in plants. PMID:27242818

  8. 14-3-3 Proteins regulate Akt Thr308 phosphorylation in intestinal epithelial cells.

    PubMed

    Gómez-Suárez, M; Gutiérrez-Martínez, I Z; Hernández-Trejo, J A; Hernández-Ruiz, M; Suárez-Pérez, D; Candelario, A; Kamekura, R; Medina-Contreras, O; Schnoor, M; Ortiz-Navarrete, V; Villegas-Sepúlveda, N; Parkos, C; Nusrat, A; Nava, P

    2016-06-01

    Akt activation has been associated with proliferation, differentiation, survival and death of epithelial cells. Phosphorylation of Thr308 of Akt by phosphoinositide-dependent kinase 1 (PDK1) is critical for optimal stimulation of its kinase activity. However, the mechanism(s) regulating this process remain elusive. Here, we report that 14-3-3 proteins control Akt Thr308 phosphorylation during intestinal inflammation. Mechanistically, we found that IFNγ and TNFα treatment induce degradation of the PDK1 inhibitor, 14-3-3η, in intestinal epithelial cells. This mechanism requires association of 14-3-3ζ with raptor in a process that triggers autophagy and leads to 14-3-3η degradation. Notably, inhibition of 14-3-3 function by the chemical inhibitor BV02 induces uncontrolled Akt activation, nuclear Akt accumulation and ultimately intestinal epithelial cell death. Our results suggest that 14-3-3 proteins control Akt activation and regulate its biological functions, thereby providing a new mechanistic link between cell survival and apoptosis of intestinal epithelial cells during inflammation. PMID:26846144

  9. 14-3-3 proteins: Macro-regulators with great potential for improving abiotic stress tolerance in plants.

    PubMed

    Liu, Qing; Zhang, Shaohong; Liu, Bin

    2016-08-12

    14-3-3 proteins (14-3-3s) are highly conserved regulatory proteins that are uniquely eukaryotic, and deeply involved in protein-protein interactions that mediate diverse signaling pathways. In plants, 14-3-3s have been validated to regulate many biological processes, such as metabolism, light and hormone signaling, cell-cycle control and protein trafficking. Recent years we have also witnessed an increasing number of reports describing the functions of 14-3-3s in plant stress responses through interactions with key proteins in both biotic and abiotic stresses. In this review, we highlight the advances that have been made in investigating the roles of 14-3-3s in plant abiotic stress tolerance. These advances provide a framework for our understanding of how signals are integrated to perceive and respond to the abiotic stresses in plants. PMID:27233603

  10. A 14-3-3 Family Protein from Wild Soybean (Glycine Soja) Regulates ABA Sensitivity in Arabidopsis

    PubMed Central

    Sun, Xiaoli; Sun, Mingzhe; Jia, Bowei; Chen, Chao; Qin, Zhiwei; Yang, Kejun; Shen, Yang; Meiping, Zhang; Mingyang, Cong; Zhu, Yanming

    2015-01-01

    It is widely accepted that the 14-3-3 family proteins are key regulators of multiple stress signal transduction cascades. By conducting genome-wide analysis, researchers have identified the soybean 14-3-3 family proteins; however, until now, there is still no direct genetic evidence showing the involvement of soybean 14-3-3s in ABA responses. Hence, in this study, based on the latest Glycine max genome on Phytozome v10.3, we initially analyzed the evolutionary relationship, genome organization, gene structure and duplication, and three-dimensional structure of soybean 14-3-3 family proteins systematically. Our results suggested that soybean 14-3-3 family was highly evolutionary conserved and possessed segmental duplication in evolution. Then, based on our previous functional characterization of a Glycine soja 14-3-3 protein GsGF14o in drought stress responses, we further investigated the expression characteristics of GsGF14o in detail, and demonstrated its positive roles in ABA sensitivity. Quantitative real-time PCR analyses in Glycine soja seedlings and GUS activity assays in PGsGF14O:GUS transgenic Arabidopsis showed that GsGF14o expression was moderately and rapidly induced by ABA treatment. As expected, GsGF14o overexpression in Arabidopsis augmented the ABA inhibition of seed germination and seedling growth, promoted the ABA induced stomata closure, and up-regulated the expression levels of ABA induced genes. Moreover, through yeast two hybrid analyses, we further demonstrated that GsGF14o physically interacted with the AREB/ABF transcription factors in yeast cells. Taken together, results presented in this study strongly suggested that GsGF14o played an important role in regulation of ABA sensitivity in Arabidopsis. PMID:26717241

  11. Neuroprotective Function of 14-3-3 Proteins in Neurodegeneration

    PubMed Central

    Shimada, Tadayuki; Fournier, Alyson E.; Yamagata, Kanato

    2013-01-01

    14-3-3 proteins are abundantly expressed adaptor proteins that interact with a vast number of binding partners to regulate their cellular localization and function. They regulate substrate function in a number of ways including protection from dephosphorylation, regulation of enzyme activity, formation of ternary complexes and sequestration. The diversity of 14-3-3 interacting partners thus enables 14-3-3 proteins to impact a wide variety of cellular and physiological processes. 14-3-3 proteins are broadly expressed in the brain, and clinical and experimental studies have implicated 14-3-3 proteins in neurodegenerative disease. A recurring theme is that 14-3-3 proteins play important roles in pathogenesis through regulating the subcellular localization of target proteins. Here, we review the evidence that 14-3-3 proteins regulate aspects of neurodegenerative disease with a focus on their protective roles against neurodegeneration. PMID:24364034

  12. Binding and Transcriptional Regulation by 14-3-3 (Bmh) Proteins Requires Residues Outside of the Canonical Motif

    PubMed Central

    Parua, Pabitra K.

    2014-01-01

    Evolutionarily conserved 14-3-3 proteins have important functions as dimers in numerous cellular signaling processes, including regulation of transcription. Yeast 14-3-3 proteins, known as Bmh, inhibit a post-DNA binding step in transcription activation by Adr1, a glucose-regulated transcription factor, by binding to its regulatory domain, residues 226 to 240. The domain was originally defined by regulatory mutations, ADR1c alleles that alter activator-dependent gene expression. Here, we report that ADR1c alleles and other mutations in the regulatory domain impair Bmh binding and abolish Bmh-dependent regulation both directly and indirectly. The indirect effect is caused by mutations that inhibit phosphorylation of Ser230 and thus inhibit Bmh binding, which requires phosphorylated Ser230. However, several mutations inhibit Bmh binding without inhibiting phosphorylation and thus define residues that provide important interaction sites between Adr1 and Bmh. Our proposed model of the Adr1 regulatory domain bound to Bmh suggests that residues Ser238 and Tyr239 could provide cross-dimer contacts to stabilize the complex and that this might explain the failure of a dimerization-deficient Bmh mutant to bind Adr1 and to inhibit its activity. A bioinformatics analysis of Bmh-interacting proteins suggests that residues outside the canonical 14-3-3 motif might be a general property of Bmh target proteins and might help explain the ability of 14-3-3 to distinguish target and nontarget proteins. Bmh binding to the Adr1 regulatory domain, and its failure to bind when mutations are present, explains at a molecular level the transcriptional phenotype of ADR1c mutants. PMID:24142105

  13. Protein kinase B (AKT) regulates SYK activity and shuttling through 14-3-3 and importin 7.

    PubMed

    Mohammad, Dara K; Nore, Beston F; Gustafsson, Manuela O; Mohamed, Abdalla J; Smith, C I Edvard

    2016-09-01

    The Protein kinase B (AKT) regulates a plethora of intracellular signaling proteins to fine-tune signaling of multiple pathways. Here, we found that following B-cell receptor (BCR)-induced tyrosine phosphorylation of the cytoplasmic tyrosine kinase SYK and the adaptor BLNK, the AKT/PKB enzyme strongly induced BLNK (>100-fold) and SYK (>100-fold) serine/threonine phosphorylation (pS/pT). Increased phosphorylation promoted 14-3-3 binding to BLNK (37-fold) and SYK (2.5-fold) in a pS/pT-concentration dependent manner. We also demonstrated that the AKT inhibitor MK2206 reduced pS/pT of both BLNK (3-fold) and SYK (2.5-fold). Notably, the AKT phosphatase, PHLPP2 maintained the activating phosphorylation of BLNK at Y84 and increased protein stability (8.5-fold). In addition, 14-3-3 was required for the regulation SYK's interaction with BLNK and attenuated SYK binding to Importin 7 (5-fold), thereby perturbing shuttling to the nucleus. Moreover, 14-3-3 proteins also sustained tyrosine phosphorylation of SYK and BLNK. Furthermore, substitution of S295 or S297 for alanine abrogated SYK's binding to Importin 7. SYK with S295A or S297A replacements showed intense pY525/526 phosphorylation, and BLNK pY84 phosphorylation correlated with the SYK pY525/526 phosphorylation level. Conversely, the corresponding mutations to aspartic acid in SYK reduced pY525/526 phosphorylation. Collectively, these and previous results suggest that AKT and 14-3-3 proteins down-regulate the activity of several BCR-associated components, including BTK, BLNK and SYK and also inhibit SYK's interaction with Importin 7. PMID:27381982

  14. Protein Modifications Regulate the Role of 14-3-3γ Adaptor Protein in cAMP-induced Steroidogenesis in MA-10 Leydig Cells*

    PubMed Central

    Aghazadeh, Yasaman; Ye, Xiaoying; Blonder, Josip; Papadopoulos, Vassilios

    2014-01-01

    The 14-3-3 protein family comprises adaptors and scaffolds that regulate intracellular signaling pathways. The 14-3-3γ isoform is a negative regulator of steroidogenesis that is hormonally induced and transiently functions at the initiation of steroidogenesis by delaying maximal steroidogenesis in MA-10 mouse tumor Leydig cells. Treatment of MA-10 cells with the cAMP analog 8-bromo-cAMP (8-Br-cAMP), which stimulates steroidogenesis, triggers the interaction of 14-3-3γ with the steroidogenic acute regulatory protein (STAR) in the cytosol, limiting STAR activity to basal levels. Over time, this interaction ceases, allowing for a 2-fold induction in STAR activity and maximal increase in the rate of steroid formation. The 14-3-3γ/STAR pattern of interaction was found to be opposite that of the 14-3-3γ homodimerization pattern. Phosphorylation and acetylation of 14-3-3γ showed similar patterns to homodimerization and STAR binding, respectively. 14-3-3γ Ser58 phosphorylation and 14-3-3γ Lys49 acetylation were blocked using trans-activator of HIV transcription factor 1 peptides coupled to 14-3-3γ sequences containing Ser58 or Lys49. Blocking either one of these modifications further induced 8-Br-cAMP-induced steroidogenesis while reducing lipid storage, suggesting that the stored cholesterol is used for steroid formation. Taken together, these results indicate that Ser58 phosphorylation and Lys49 acetylation of 14-3-3γ occur in a coordinated time-dependent manner to regulate 14-3-3γ homodimerization. 14-3-3γ Ser58 phosphorylation is required for STAR interactions under control conditions, and 14-3-3γ Lys49 acetylation is important for the cAMP-dependent induction of these interactions. PMID:25086053

  15. 14-3-3 Proteins Bind to the Brassinosteroid Receptor Kinase, BRI1 and are Positive Regulators of Brassinosteroid Signaling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Multiple members of the 14-3-3 protein family have been found in all eukaryotes, the biological functions of which are to interact physically with specific client proteins and thereby effect a change in the client. Thus, 14-3-3s are involved in many processes. The plant brassinosteroid (BR) recepto...

  16. The 14-3-3 protein PAR-5 regulates the asymmetric localization of the LET-99 spindle positioning protein.

    PubMed

    Wu, Jui-Ching; Espiritu, Eugenel B; Rose, Lesilee S

    2016-04-15

    PAR proteins play important roles in establishing cytoplasmic polarity as well as regulating spindle positioning during asymmetric division. However, the molecular mechanisms by which the PAR proteins generate asymmetry in different cell types are still being elucidated. Previous studies in Caenorhabditis elegans revealed that PAR-3 and PAR-1 regulate the asymmetric localization of LET-99, which in turn controls spindle positioning by affecting the distribution of the conserved force generating complex. In wild-type embryos, LET-99 is localized in a lateral cortical band pattern, via inhibition at the anterior by PAR-3 and at the posterior by PAR-1. In this report, we show that the 14-3-3 protein PAR-5 is also required for cortical LET-99 asymmetry. PAR-5 associated with LET-99 in pull-down assays, and two PAR-5 binding sites were identified in LET-99 using the yeast two-hybrid assay. Mutation of these sites abolished binding in yeast and altered LET-99 localization in vivo: LET-99 was present at the highest levels at the posterior pole of the embryo instead of a band in par-5 embryos. Together the results indicate that PAR-5 acts in a mechanism with PAR-1 to regulate LET-99 cortical localization. PMID:26921457

  17. Identification of 14-3-3 proteins as a target of ATL31 ubiquitin ligase, a regulator of the C/N response in Arabidopsis.

    PubMed

    Sato, Takeo; Maekawa, Shugo; Yasuda, Shigetaka; Domeki, Yukie; Sueyoshi, Kuni; Fujiwara, Masayuki; Fukao, Yoichiro; Goto, Derek B; Yamaguchi, Junji

    2011-10-01

    The balance between carbon (C) and nitrogen (N) availability is an important determinant for various phases of plant growth; however, the detailed mechanisms regulating the C/N response are not well understood. We previously described two related ubiquitin ligases, ATL31 and ATL6, that function in the C/N response in Arabidopsis thaliana. Here, we used FLAG tag affinity purification and MS analysis to identify proteins targeted by ATL31, and thus likely to be involved in regulating the phase transition checkpoint based on C/N status. This analysis revealed that 14-3-3 proteins were associated with ATL31, and one of these, 14-3-3χ, was selected for detailed characterization. The interaction between ATL31 and 14-3-3χ was confirmed by yeast two-hybrid and co-immunoprecipitation analyses. In vitro assays showed that ubiquitination of 14-3-3χ is catalyzed by ATL31. Degradation of 14-3-3χin vivo was shown to be correlated with ATL31 activity, and to occur in a proteasome-dependent manner. Furthermore, 14-3-3 protein accumulation was induced by a shift to high-C/N stress conditions in Arabidopsis seedlings, and this regulated response required both ATL31 and ATL6. It was also shown that over-expression of 14-3-3χ leads to hypersensitivity of Arabidopsis seedlings to C/N stress conditions. These results indicate that ATL31 targets and ubiquitinates 14-3-3 proteins for degradation via the ubiquitin-proteasome system during the response to cellular C/N status. PMID:21668537

  18. 14-3-3 Proteins in Guard Cell Signaling

    PubMed Central

    Cotelle, Valérie; Leonhardt, Nathalie

    2016-01-01

    Guard cells are specialized cells located at the leaf surface delimiting pores which control gas exchanges between the plant and the atmosphere. To optimize the CO2 uptake necessary for photosynthesis while minimizing water loss, guard cells integrate environmental signals to adjust stomatal aperture. The size of the stomatal pore is regulated by movements of the guard cells driven by variations in their volume and turgor. As guard cells perceive and transduce a wide array of environmental cues, they provide an ideal system to elucidate early events of plant signaling. Reversible protein phosphorylation events are known to play a crucial role in the regulation of stomatal movements. However, in some cases, phosphorylation alone is not sufficient to achieve complete protein regulation, but is necessary to mediate the binding of interactors that modulate protein function. Among the phosphopeptide-binding proteins, the 14-3-3 proteins are the best characterized in plants. The 14-3-3s are found as multiple isoforms in eukaryotes and have been shown to be involved in the regulation of stomatal movements. In this review, we describe the current knowledge about 14-3-3 roles in the regulation of their binding partners in guard cells: receptors, ion pumps, channels, protein kinases, and some of their substrates. Regulation of these targets by 14-3-3 proteins is discussed and related to their function in guard cells during stomatal movements in response to abiotic or biotic stresses. PMID:26858725

  19. 14-3-3 family members act coordinately to regulate mitotic progression.

    PubMed

    Dalal, Sorab N; Yaffe, Michael B; DeCaprio, James A

    2004-05-01

    The mitosis promoting phosphatase, cdc25C, is a target of both the DNA replication and DNA damage checkpoint pathways. These pathways regulate cdc25C function, in part, by promoting the association of cdc25C with 14-3-3 proteins, which results in the retention of cdc25C in the cytoplasm. To determine which 14-3-3 proteins were required to regulate cdc25C function, we tested the ability of various 14-3-3 family members to form a complex with and negatively regulate cdc25C in human cells. Two 14-3-3 family members, 14-3-3epsilon and 14-3-3gamma specifically formed a complex with cdc25C but not with the 14-3-3 binding defective cdc25C mutant, S216A. In addition, 14-3-3epsilon and 14-3-3gamma inhibited the ability of cdc25C, but not the S216A mutant, to induce premature chromatin condensation (PCC) in U-2OS cells. These results suggested that the reduction in PCC by 14-3-3epsilon and 14-3-3gamma was due to inhibition of cdc25C function. In contrast, 14-3-3sigma was unable to form a complex with cdc25C, but was able to inhibit the ability of both wild type cdc25C and S216A to induce PCC. This suggests that 14-3-3sigma regulates entry into mitosis independently of cdc25C and 14-3-3epsilon and 14-3-3gamma. Thus, specific members of the 14-3-3 family of proteins may act coordinately to maintain the DNA replication checkpoint by regulating the activity of different cell cycle proteins. PMID:15107609

  20. The role of the 14-3-3 protein family in health, disease, and drug development.

    PubMed

    Aghazadeh, Yasaman; Papadopoulos, Vassilios

    2016-02-01

    14-3-3 proteins regulate intracellular signaling pathways, such as signal transduction, protein trafficking, cell cycle, and apoptosis. In addition to the ubiquitous roles of 14-3-3 isoforms, unique tissue-specific functions are also described for each isoform. Owing to their role in regulating cell cycle, protein trafficking, and steroidogenesis, 14-3-3 proteins are prevalent in human diseases, such as cancer, neurodegeneration, and reproductive disorders, and, therefore, serve as valuable drug targets. In this review, we summarize the role of 14-3-3 proteins in normal and disease states, with a focus on 14-3-3γ and ɛ. We also discuss drug compounds targeting 14-3-3 proteins and their potential therapeutic uses. PMID:26456530

  1. Determining novel functions of Arabidopsis 14-3-3 proteins in central metabolic processes

    PubMed Central

    2011-01-01

    Background 14-3-3 proteins are considered master regulators of many signal transduction cascades in eukaryotes. In plants, 14-3-3 proteins have major roles as regulators of nitrogen and carbon metabolism, conclusions based on the studies of a few specific 14-3-3 targets. Results In this study, extensive novel roles of 14-3-3 proteins in plant metabolism were determined through combining the parallel analyses of metabolites and enzyme activities in 14-3-3 overexpression and knockout plants with studies of protein-protein interactions. Decreases in the levels of sugars and nitrogen-containing-compounds and in the activities of known 14-3-3-interacting-enzymes were observed in 14-3-3 overexpression plants. Plants overexpressing 14-3-3 proteins also contained decreased levels of malate and citrate, which are intermediate compounds of the tricarboxylic acid (TCA) cycle. These modifications were related to the reduced activities of isocitrate dehydrogenase and malate dehydrogenase, which are key enzymes of TCA cycle. In addition, we demonstrated that 14-3-3 proteins interacted with one isocitrate dehydrogenase and two malate dehydrogenases. There were also changes in the levels of aromatic compounds and the activities of shikimate dehydrogenase, which participates in the biosynthesis of aromatic compounds. Conclusion Taken together, our findings indicate that 14-3-3 proteins play roles as crucial tuners of multiple primary metabolic processes including TCA cycle and the shikimate pathway. PMID:22104211

  2. Molecular evolution of the 14-3-3 protein family.

    PubMed

    Wang, W; Shakes, D C

    1996-10-01

    Members of the highly conserved and ubiquitous 14-3-3 protein family modulate a wide variety of cellular processes. To determine the evolutionary relationships among specific 14-3-3 proteins in different plant, animal, and fungal species and to initiate a predictive analysis of isoform-specific differences in light of the latest functional and structural studies of 14-3-3, multiple alignments were constructed from forty-six 14-3-3 sequences retrieved from the GenBank and SwissProt databases and a newly identified second 14-3-3 gene from Caenorhabditis elegans. The alignment revealed five highly conserved sequence blocks. Blocks 2-5 correlate well with the alpha helices 3, 5, 7, and 9 which form the proposed internal binding domain in the three-dimensional structure model of the functioning dimer. Amino acid differences within the functional and structural domains of plant and animal 14-3-3 proteins were identified which may account for functional diversity amongst isoforms. Protein phylogenic trees were constructed using both the maximum parsimony and neighbor joining methods of the PHYLIP(3.5c) package; 14-3-3 proteins from Entamoeba histolytica, an amitochondrial protozoa, were employed as an outgroup in our analysis. Epsilon isoforms from the animal lineage form a distinct grouping in both trees, which suggests an early divergence from the other animal isoforms. Epsilons were found to be more similar to yeast and plant isoforms than other animal isoforms at numerous amino acid positions, and thus epsilon may have retained functional characteristics of the ancestral protein. The known invertebrate proteins group with the nonepsilon mammalian isoforms. Most of the current 14-3-3 isoform diversity probably arose through independent duplication events after the divergence of the major eukaryotic kingdoms. Divergence of the seven mammalian isoforms beta, zeta, gamma, eta, epsilon, tau, and sigma (stratifin/HME1) occurred before the divergence of mammalian and perhaps

  3. Anchoring of both PKA-RIIα and 14-3-3θ regulates retinoic acid induced 16 mediated phosphorylation of heat shock protein 70

    PubMed Central

    Tang, Hai-Lin; Zhu, Shi-Ying; Zhao, Lan-Juan; Ren, Hao; Zhao, Ping; Qi, Zhong-Tian; Wang, Wen

    2015-01-01

    Our previous study reported that retinoic acid induced 16 (RAI16) could enhance tumorigenesis in hepatocellular carcinoma (HCC). However, the cellular functions of RAI16 are still unclear. In this study, by immunoprecipitation and tandem (MS/MS) mass spectrometry analysis, we identified that RAI16 interacted with the type II regulatory subunit of PKA (PKA-RIIα), acting as a novel protein kinase A anchoring protein (AKAP). In addition, RAI16 also interacted with heat shock protein 70 (HSP70) and 14-3-3θ. Further studies indicated that RAI16 mediated PKA phosphorylation of HSP70 at serine 486, resulting in anti-apoptosis events. RAI16 was also phosphorylated by the anchored PKA at serine 325, which promoted the recruitment of 14-3-3θ, which, in turn, inhibited RAI16 mediated PKA phosphorylation of HSP70. These findings offer mechanism insight into RAI16 mediated anti-apoptosis signaling in HCC. PMID:25900241

  4. The Hsp70 homolog Ssb and the 14-3-3 protein Bmh1 jointly regulate transcription of glucose repressed genes in Saccharomyces cerevisiae.

    PubMed

    Hübscher, Volker; Mudholkar, Kaivalya; Chiabudini, Marco; Fitzke, Edith; Wölfle, Tina; Pfeifer, Dietmar; Drepper, Friedel; Warscheid, Bettina; Rospert, Sabine

    2016-07-01

    Chaperones of the Hsp70 family interact with a multitude of newly synthesized polypeptides and prevent their aggregation. Saccharomyces cerevisiae cells lacking the Hsp70 homolog Ssb suffer from pleiotropic defects, among others a defect in glucose-repression. The highly conserved heterotrimeric kinase SNF1/AMPK (AMP-activated protein kinase) is required for the release from glucose-repression in yeast and is a key regulator of energy balance also in mammalian cells. When glucose is available the phosphatase Glc7 keeps SNF1 in its inactive, dephosphorylated state. Dephosphorylation depends on Reg1, which mediates targeting of Glc7 to its substrate SNF1. Here we show that the defect in glucose-repression in the absence of Ssb is due to the ability of the chaperone to bridge between the SNF1 and Glc7 complexes. Ssb performs this post-translational function in concert with the 14-3-3 protein Bmh, to which Ssb binds via its very C-terminus. Raising the intracellular concentration of Ssb or Bmh enabled Glc7 to dephosphorylate SNF1 even in the absence of Reg1. By that Ssb and Bmh efficiently suppressed transcriptional deregulation of Δreg1 cells. The findings reveal that Ssb and Bmh comprise a new chaperone module, which is involved in the fine tuning of a phosphorylation-dependent switch between respiration and fermentation. PMID:27001512

  5. The Hsp70 homolog Ssb and the 14-3-3 protein Bmh1 jointly regulate transcription of glucose repressed genes in Saccharomyces cerevisiae

    PubMed Central

    Hübscher, Volker; Mudholkar, Kaivalya; Chiabudini, Marco; Fitzke, Edith; Wölfle, Tina; Pfeifer, Dietmar; Drepper, Friedel; Warscheid, Bettina; Rospert, Sabine

    2016-01-01

    Chaperones of the Hsp70 family interact with a multitude of newly synthesized polypeptides and prevent their aggregation. Saccharomyces cerevisiae cells lacking the Hsp70 homolog Ssb suffer from pleiotropic defects, among others a defect in glucose-repression. The highly conserved heterotrimeric kinase SNF1/AMPK (AMP-activated protein kinase) is required for the release from glucose-repression in yeast and is a key regulator of energy balance also in mammalian cells. When glucose is available the phosphatase Glc7 keeps SNF1 in its inactive, dephosphorylated state. Dephosphorylation depends on Reg1, which mediates targeting of Glc7 to its substrate SNF1. Here we show that the defect in glucose-repression in the absence of Ssb is due to the ability of the chaperone to bridge between the SNF1 and Glc7 complexes. Ssb performs this post-translational function in concert with the 14-3-3 protein Bmh, to which Ssb binds via its very C-terminus. Raising the intracellular concentration of Ssb or Bmh enabled Glc7 to dephosphorylate SNF1 even in the absence of Reg1. By that Ssb and Bmh efficiently suppressed transcriptional deregulation of Δreg1 cells. The findings reveal that Ssb and Bmh comprise a new chaperone module, which is involved in the fine tuning of a phosphorylation-dependent switch between respiration and fermentation. PMID:27001512

  6. A fusicoccin binding protein belongs to the family of 14-3-3 brain protein homologs.

    PubMed Central

    Korthout, H A; de Boer, A H

    1994-01-01

    The fusicoccin binding protein (FCBP) is a highly conserved plasma membrane protein present in all higher plants tested thus far. It exhibits high- and low-affinity binding for the fungal toxin fusicoccin (FC). We purified the active FCBP from a fraction highly enriched in plasma membrane by selective precipitation and anion exchange chromatography. After SDS-PAGE, the two FCBP subunits of 30 and 31 kD were detected as major bands. Amino acid sequence analysis of the 31-kD polypeptide displayed a high degree of identity with so-called 14-3-3 proteins, a class of mammalian brain proteins initially described as regulators of neurotransmitter synthesis and protein kinase C inhibitors. Thereafter, we affinity purified the 30- and 31-kD FCBP subunits, using biotinylated FC in combination with a monomeric avidin column. Immunodecoration of these 30- and 31-kD FCBP subunits with polyclonal antibodies raised against a 14-3-3 homolog from yeast confirmed the identity of the FCBP as a 14-3-3 homolog. Similar to all 14-3-3 protein homologs, the FCBP seems to exist as a dimer in native form. Thus far, the FCBP is the only 14-3-3 homolog with a receptor-like function. The conserved structure of the 14-3-3 protein family is a further indication that the FCBP plays an important role in the physiology of higher plants. PMID:7827499

  7. Differential neuroprotective effects of 14-3-3 proteins in models of Parkinson's disease.

    PubMed

    Yacoubian, T A; Slone, S R; Harrington, A J; Hamamichi, S; Schieltz, J M; Caldwell, K A; Caldwell, G A; Standaert, D G

    2010-01-01

    14-3-3 proteins are important negative regulators of cell death pathways. Recent studies have revealed alterations in 14-3-3s in Parkinson's disease (PD) and the ability of 14-3-3s to interact with alpha-synuclein (α-syn), a protein central to PD pathophysiology. In a transgenic α-syn mouse model, we found reduced expression of 14-3-3θ, ε, and γ. These same isoforms prevent α-syn inclusion formation in an H4 neuroglioma cell model. Using dopaminergic cell lines stably overexpressing each 14-3-3 isoform, we found that overexpression of 14-3-3θ, ε, or γ led to resistance to both rotenone and 1-methyl-4-phenylpyridinium (MPP(+)), while other isoforms were not protective against both toxins. Inhibition of a single protective isoform, 14-3-3θ, by shRNA did not increase vulnerability to neurotoxic injury, but toxicity was enhanced by broad-based inhibition of 14-3-3 action with the peptide inhibitor difopein. Using a transgenic C. elegans model of PD, we confirmed the ability of both human 14-3-3θ and a C. elegans 14-3-3 homolog (ftt-2) to protect dopaminergic neurons from α-syn toxicity. Collectively, these data show a strong neuroprotective effect of enhanced 14-3-3 expression - particularly of the 14-3-3θ, ε, and γ isoforms - in multiple cellular and animal models of PD, and point to the potential value of these proteins in the development of neuroprotective therapies for human PD. PMID:21152247

  8. Hyperglycemia decreases expression of 14-3-3 proteins in an animal model of stroke.

    PubMed

    Jeon, Seong-Jun; Sung, Jin-Hee; Koh, Phil-Ok

    2016-07-28

    Diabetes is a severe metabolic disorder and a major risk factor for stroke. Stroke severity is worse in patients with diabetes compared to the non-diabetic population. The 14-3-3 proteins are a family of conserved acidic proteins that are ubiquitously expressed in cells and tissues. These proteins are involved in many cellular processes including metabolic pathways, signal transduction, protein trafficking, protein synthesis, and cell cycle control. This study investigated 14-3-3 proteins expression in the cerebral cortex of animals with diabetes, cerebral ischemic injury and a combination of both diabetes and cerebral ischemic injury. Diabetes was induced by intraperitoneal injection of streptozotocin (40mg/kg) in adult male rats. After 4 weeks of treatment, middle cerebral artery occlusion (MCAO) was performed for the induction of focal cerebral ischemia and cerebral cortex tissue was collected 24h after MCAO. We confirmed that diabetes increases infarct volume following MCAO compared to non-diabetic animals. In diabetic animals with MCAO injury, reduction of 14-3-3 β/α, 14-3-3 ζ/δ, 14-3-3 γ, and 14-3-3 ε isoforms was detected. The expression of these proteins was significantly decreased in diabetic animals with MCAO injury compared to diabetic-only and MCAO-only animals. Moreover, Western blot analysis ascertained the decreased expression of 14-3-3 family proteins in diabetic animals with MCAO injury, including β/α, ζ/δ, γ, ε, τ, and η isoforms. These results show the changes of 14-3-3 proteins expression in streptozotocin-induced diabetic animals with MCAO injury. Thus, these findings suggest that decreases in 14-3-3 proteins might be involved in the regulation of 14-3-3 proteins under the presence of diabetes following MCAO. PMID:27177727

  9. Identification of 14-3-3zeta associated protein networks in oral cancer.

    PubMed

    Matta, Ajay; Masui, Olena; Siu, K W Michael; Ralhan, Ranju

    2016-04-01

    Advancements in genomics, proteomics, and bioinformatics have improved our understanding of gene/protein networks involved in intra- and intercellular communication and tumor-host interactions. Using proteomics integrated with bioinformatics, previously we reported overexpression of 14-3-3ζ in premalignant oral lesions and oral squamous cell carcinoma tissues in comparison with normal oral epithelium. 14-3-3ζ emerged as a novel molecular target for therapeutics and a potential prognostic marker in oral squamous cell carcinoma patients. However, the role of 14-3-3ζ in development and progression of oral cancer is not known yet. This study aimed to identify the 14-3-3ζ associated protein networks in oral cancer cell lines using IP-MS/MS and bioinformatics. A total of 287 binding partners of 14-3-3ζ were identified in metastatic (MDA1986) and nonmetastatic (SCC4) oral cancer cell lines including other 14-3-3 isoforms (2%), proteins involved in apoptosis (2%), cytoskeleton (9%), metabolism (16%), and maintenance of redox potential (2%). Our bioinformatics analysis revealed involvement of 14-3-3ζ in protein networks regulating cell cycle, proliferation, apoptosis, cellular trafficking, and endocytosis in oral cancer. In conclusion, our data revealed several novel protein interaction networks involving 14-3-3ζ in oral cancer progression and metastasis. PMID:26857332

  10. Akirin interacts with Bap60 and 14-3-3 proteins to regulate the expression of antimicrobial peptides in the kuruma shrimp (Marsupenaeus japonicus).

    PubMed

    Liu, Ning; Wang, Xian-Wei; Sun, Jie-Jie; Wang, Lei; Zhang, Hong-Wei; Zhao, Xiao-Fan; Wang, Jin-Xing

    2016-02-01

    Akirin is a recently discovered nuclear factor that plays important roles in innate immune responses. Akirin is a positive regulator of the NF-κB factor of the Drosophila immune deficiency (IMD) pathway, which shares extensive similarities with the mammalian tumor necrosis factor receptor (TNFR) signaling pathway. However, some studies found that the NF-κB transcriptional targets were also strongly repressed in akirin2 knockout mice following TLR, IL-1β and TNFα treatment. Therefore, the function of Akirin in the immune response requires further clarification. In this study, an Akirin homolog in the kuruma shrimp (Marsupenaeus japonicus) was identified. It was mainly expressed in hemocytes, heart and intestines. The expression of Akirin was upregulated by challenge with the Gram-negative bacterium Vibrio anguillarum, but was not significantly influenced by challenge with the Gram-positive bacterium Staphylococcus aureus. Knockdown of Akirin suppressed the expression of several IMD-Relish target effectors (antimicrobial peptides, AMPs). The limited regulating spectrum of Akirin might be associated with Bap60, a component of the Brahma (SWI/SNF) ATP-dependent chromatin-remodeling complex. In addition, Akirin also interacts with 14-3-3, which inhibited the expression of Akirin-target AMPs. The results suggested that Akirin is involved in the IMD-Relish pathway by interacting with Relish. The interaction of Akirin with Bap60 positively regulated the Akirin-Relish function, and its interaction with 14-3-3 negatively regulated the Akirin-Relish function. PMID:26493016

  11. Proteomic profiling of tandem affinity purified 14-3-3 protein complexes in Arabidopsis thaliana

    PubMed Central

    Chang, Ing-Feng; Curran, Amy; Woolsey, Rebekah; Quilici, David; Cushman, John; Mittler, Ron; Harmon, Alice; Harper, Jeffrey

    2014-01-01

    In eukaryotes, 14-3-3 dimers regulate hundreds of functionally diverse proteins (clients), typically in phosphorylation-dependent interactions. To uncover new clients, a 14-3-3 omega (At1g78300) from Arabidopsis was engineered with a “tandem affinity purification” (TAP) tag and expressed in transgenic plants. Purified complexes were analyzed by tandem mass spectrometry. Results indicate that 14-3-3 omega can dimerize with at least 10 of the 12 14-3-3 isoforms expressed in Arabidopsis. The identification here of 121 putative clients provides support for in vivo 14-3-3 interactions with a diverse array of proteins, including those involved in: (1) Ion transport, such as a K+ channel (GORK), a Cl− channel (CLCg), Ca2+ channels belonging to the glutamate receptor family (GLRs 1.2, 2.1, 2.9, 3.4, 3.7); (2) hormone signaling, such as ACC synthase (isoforms ACS-6, 7 and 8 involved in ethylene synthesis) and the brassinolide receptors BRI1 and BAK1; (3) transcription, such as 7 WRKY family transcription factors; (4) metabolism, such as phosphoenol pyruvate (PEP) carboxylase; and (5) lipid signaling, such as phospholipase D (β, and γ). More than 80% (101) of these putative clients represent previously unidentified 14-3-3 interactors. These results raise the number of putative 14-3-3 clients identified in plants to over 300. PMID:19452453

  12. Phosphoregulatory protein 14-3-3 facilitates SAC1 transport from the endoplasmic reticulum

    PubMed Central

    Bajaj Pahuja, Kanika; Wang, Jinzhi; Blagoveshchenskaya, Anastasia; Lim, Lillian; Madhusudhan, M. S.; Mayinger, Peter; Schekman, Randy

    2015-01-01

    Most secretory cargo proteins in eukaryotes are synthesized in the endoplasmic reticulum and actively exported in membrane-bound vesicles that are formed by the cytosolic coat protein complex II (COPII). COPII proteins are assisted by a variety of cargo-specific adaptor proteins required for the concentration and export of secretory proteins from the endoplasmic reticulum (ER). Adaptor proteins are key regulators of cargo export, and defects in their function may result in disease phenotypes in mammals. Here we report the role of 14-3-3 proteins as a cytosolic adaptor in mediating SAC1 transport in COPII-coated vesicles. Sac1 is a phosphatidyl inositol-4 phosphate (PI4P) lipid phosphatase that undergoes serum dependent translocation between the endoplasmic reticulum and Golgi complex and controls cellular PI4P lipid levels. We developed a cell-free COPII vesicle budding reaction to examine SAC1 exit from the ER that requires COPII and at least one additional cytosolic factor, the 14-3-3 protein. Recombinant 14-3-3 protein stimulates the packaging of SAC1 into COPII vesicles and the sorting subunit of COPII, Sec24, interacts with 14-3-3. We identified a minimal sorting motif of SAC1 that is important for 14-3-3 binding and which controls SAC1 export from the ER. This LS motif is part of a 7-aa stretch, RLSNTSP, which is similar to the consensus 14-3-3 binding sequence. Homology models, based on the SAC1 structure from yeast, predict this region to be in the exposed exterior of the protein. Our data suggest a model in which the 14-3-3 protein mediates SAC1 traffic from the ER through direct interaction with a sorting signal and COPII. PMID:26056309

  13. Regulation of the Yeast Hxt6 Hexose Transporter by the Rod1 α-Arrestin, the Snf1 Protein Kinase, and the Bmh2 14-3-3 Protein.

    PubMed

    Llopis-Torregrosa, Vicent; Ferri-Blázquez, Alba; Adam-Artigues, Anna; Deffontaines, Emilie; van Heusden, G Paul H; Yenush, Lynne

    2016-07-15

    Cell viability requires adaptation to changing environmental conditions. Ubiquitin-mediated endocytosis plays a crucial role in this process, because it provides a mechanism to remove transport proteins from the membrane. Arrestin-related trafficking proteins are important regulators of the endocytic pathway in yeast, facilitating selective ubiquitylation of target proteins by the E3 ubiquitin ligase, Rsp5. Specifically, Rod1 (Art4) has been reported to regulate the endocytosis of both the Hxt1, Hxt3, and Hxt6 glucose transporters and the Jen1 lactate transporter. Also, the AMP kinase homologue, Snf1, and 14-3-3 proteins have been shown to regulate Jen1 via Rod1. Here, we further characterized the role of Rod1, Snf1, and 14-3-3 in the signal transduction route involved in the endocytic regulation of the Hxt6 high affinity glucose transporter by showing that Snf1 interacts specifically with Rod1 and Rog3 (Art7), that the interaction between the Bmh2 and several arrestin-related trafficking proteins may be modulated by carbon source, and that both the 14-3-3 protein Bmh2 and the Snf1 regulatory domain interact with the arrestin-like domain containing the N-terminal half of Rod1 (amino acids 1-395). Finally, using both co-immunoprecipitation and bimolecular fluorescence complementation, we demonstrated the interaction of Rod1 with Hxt6 and showed that the localization of the Rod1-Hxt6 complex at the plasma membrane is affected by carbon source and is reduced upon overexpression of SNF1 and BMH2. PMID:27261460

  14. 14-3-3ζ Interacts with Stat3 and Regulates Its Constitutive Activation in Multiple Myeloma Cells

    PubMed Central

    Li, Wenliang; Xiong, Qian; Yang, Mingkun; Zheng, Peng; Li, Chongyang; Pei, Jianfeng; Ge, Feng

    2012-01-01

    The 14-3-3 proteins are a family of regulatory signaling molecules that interact with other proteins in a phosphorylation-dependent manner and function as adapter or scaffold proteins in signal transduction pathways. One family member, 14-3-3ζ, is believed to function in cell signaling, cycle control, and apoptotic death. A systematic proteomic analysis done in our laboratory has identified signal transducers and activators of transcription 3 (Stat3) as a novel 14-3-3ζ interacting protein. Following our initial finding, in this study, we provide evidence that 14-3-3ζ interacts physically with Stat3. We further demonstrate that phosphorylation of Stat3 at Ser727 is vital for 14-3-3ζ interaction and mutation of Ser727 to Alanine abolished 14-3-3ζ/Stat3 association. Inhibition of 14-3-3ζ protein expression in U266 cells inhibited Stat3 Ser727 phosphorylation and nuclear translocation, and decreased both Stat3 DNA binding and transcriptional activity. Moreover, 14-3-3ζ is involved in the regulation of protein kinase C (PKC) activity and 14-3-3ζ binding to Stat3 protects Ser727 dephosphorylation from protein phosphatase 2A (PP2A). Taken together, our findings support the model that multiple signaling events impinge on Stat3 and that 14-3-3ζ serves as an essential coordinator for different pathways to regulate Stat3 activation and function in MM cells. PMID:22279540

  15. 14-3-3σ regulates keratinocyte proliferation and differentiation by modulating Yap1 cellular localization

    PubMed Central

    Sambandam, Sumitha A.T.; Kasetti, Ramesh Babu; Xue, Lei; Dean, Douglas C.; Lu, Qingxian; Li, Qiutang

    2015-01-01

    The homozygous repeated epilation (Er/Er) mouse mutant of the gene encoding 14-3-3σ displays an epidermal phenotype characterized by hyperproliferative keratinocytes and undifferentiated epidermis. Heterozygous Er/+ mice develop spontaneous skin tumors and are highly sensitive to tumor-promoting DMBA/TPA induction. The molecular mechanisms underlying 14-3-3σ regulation of epidermal proliferation, differentiation, and tumor formation have not been well elucidated. In the present study, we found that Er/Er keratinocytes failed to sequester Yap1 in the cytoplasm, leading to its nuclear localization during epidermal development in vivo and under differentiation-inducing culture conditions in vitro. In addition, enhanced Yap1 nuclear localization was also evident in DMBA/TPA-induced tumors from Er/+ skin. Furthermore, shRNA knockdown of Yap1 expression in Er/Er keratinocytes inhibited their proliferation, suggesting that YAP1 functions as a downstream effector of 14-3-3σ controlling epidermal proliferation. We then demonstrated that keratinocytes express all seven 14-3-3 protein isoforms, some of which form heterodimers with 14-3-3σ, either full-length WT or the mutant form found in Er/Er mice. However Er 14-3-3σ does not interact with Yap1, as demonstrated by co-immunoprecipitation. We conclude that Er 14-3-3σ disrupts the interaction between 14-3-3 and Yap1, thus fails to block Yap1 nuclear transcriptional function, causing continued progenitor expansion and inhibition of differentiation in Er/Er epidermis. PMID:25668240

  16. Alternations of 14-3-3 θ and β protein levels in brain during experimental sepsis.

    PubMed

    Memos, Nikolaos; Kataki, Agapi; Chatziganni, Emmy; Nikolopoulou, Marilena; Skoulakis, Euthimios; Consoulas, Christos; Zografos, George; Konstadoulakis, Manousos

    2011-09-01

    The 14-3-3 family members play a crucial role in the determination of cell fate, exerting their antiapoptotic activity through directly interfering with the critical function of the mitochondrial core proapoptotic machinery. Dimerization of 14-3-3 is vital for the interaction with many of its client proteins and is regulated by phosphorylation. In a previous study, we observed time-dependent neuronal apoptosis during sepsis. Therefore, in the present study, we sought to evaluate the expression of 14-3-3 θ and β isoforms in septic brain and their association with apoptosis. Sepsis was induced by a CLP model in Wistar rats that were sacrificed at predefined time points. Flow cytometric analysis showed a sepsis-induced, time-dependent alteration of 14-3-3 θ and β isoforms in both Neun(+) and GFAP(+) cells. 14-3-3 θ was linearly correlated with apoptosis, and stratified analysis for alive and apoptotic neuronal cells demonstrated a gradual down-regulation of θ isoform in alive neurons and astrocytes. The phospho-P38 (pP38) MAP kinase levels were altered in a time-dependent manner during sepsis, presenting a peak at 6 hr post-CLP. A significant correlation between the two isoforms of 14-3-3 was observed in septic rats, with the θ isoform predominant at all time points. The hippocampus, Purkinje cells, and glia-like cells showed intense immunohistochemical reactivity for 14-3-3 θ isoform, whereas the choroid plexus showed constantly increased β isoform expression. Our results showed that sepsis alters the expression of both 14-3-3 θ and β isoforms in a time-, cell-, and topography-dependent manner. PMID:21618583

  17. 14-3-3 Proteins Buffer Intracellular Calcium Sensing Receptors to Constrain Signaling

    PubMed Central

    Grant, Michael P.; Cavanaugh, Alice; Breitwieser, Gerda E.

    2015-01-01

    Calcium sensing receptors (CaSR) interact with 14-3-3 binding proteins at a carboxyl terminal arginine-rich motif. Mutations identified in patients with familial hypocalciuric hypercalcemia, autosomal dominant hypocalcemia, pancreatitis or idiopathic epilepsy support the functional importance of this motif. We combined total internal reflection fluorescence microscopy and biochemical approaches to determine the mechanism of 14-3-3 protein regulation of CaSR signaling. Loss of 14-3-3 binding caused increased basal CaSR signaling and plasma membrane levels, and a significantly larger signaling-evoked increase in plasma membrane receptors. Block of core glycosylation with tunicamycin demonstrated that changes in plasma membrane CaSR levels were due to differences in exocytic rate. Western blotting to quantify time-dependent changes in maturation of expressed wt CaSR and a 14-3-3 protein binding-defective mutant demonstrated that signaling increases synthesis to maintain constant levels of the immaturely and maturely glycosylated forms. CaSR thus operates by a feed-forward mechanism, whereby signaling not only induces anterograde trafficking of nascent receptors but also increases biosynthesis to maintain steady state levels of net cellular CaSR. Overall, these studies suggest that 14-3-3 binding at the carboxyl terminus provides an important buffering mechanism to increase the intracellular pool of CaSR available for signaling-evoked trafficking, but attenuates trafficking to control the dynamic range of responses to extracellular calcium. PMID:26317416

  18. 14-3-3 proteins restrain the Exo1 nuclease to prevent overresection.

    PubMed

    Chen, Xiaoqing; Kim, In-Kwon; Honaker, Yuchi; Paudyal, Sharad C; Koh, Won Kyun; Sparks, Melanie; Li, Shan; Piwnica-Worms, Helen; Ellenberger, Tom; You, Zhongsheng

    2015-05-01

    The DNA end resection process dictates the cellular response to DNA double strand break damage and is essential for genome maintenance. Although insufficient DNA resection hinders homology-directed repair and ATR (ataxia telangiectasia and Rad3 related)-dependent checkpoint activation, overresection produces excessive single-stranded DNA that could lead to genomic instability. However, the mechanisms controlling DNA end resection are poorly understood. Here we show that the major resection nuclease Exo1 is regulated both positively and negatively by protein-protein interactions to ensure a proper level of DNA resection. We have shown previously that the sliding DNA clamp proliferating cell nuclear antigen (PCNA) associates with the C-terminal domain of Exo1 and promotes Exo1 damage association and DNA resection. In this report, we show that 14-3-3 proteins interact with a central region of Exo1 and negatively regulate Exo1 damage recruitment and subsequent resection. 14-3-3s limit Exo1 damage association, at least in part, by suppressing its association with PCNA. Disruption of the Exo1 interaction with 14-3-3 proteins results in elevated sensitivity of cells to DNA damage. Unlike Exo1, the Dna2 resection pathway is apparently not regulated by PCNA and 14-3-3s. Our results provide critical insights into the mechanism and regulation of the DNA end resection process and may have implications for cancer treatment. PMID:25833945

  19. A novel sphingosine-dependent protein kinase (SDK1) specifically phosphorylates certain isoforms of 14-3-3 protein.

    PubMed

    Megidish, T; Cooper, J; Zhang, L; Fu, H; Hakomori, S

    1998-08-21

    Protein kinases activated by sphingosine or N,N'-dimethylsphingosine, but not by other lipids, have been detected and are termed sphingosine-dependent protein kinases (SDKs). These SDKs were previously shown to phosphorylate endogenous 14-3-3 proteins (Megidish, T., White, T., Takio, K., Titani, K., Igarashi, Y., and Hakomori, S. (1995) Biochem. Biophys. Res. Commun. 216, 739-747). We have now partially purified one SDK, termed SDK1, from cytosol of mouse Balb/c 3T3(A31) fibroblasts. SDK1 is a serine kinase with molecular mass 50-60 kDa that is strongly activated by N, N'-dimethylsphingosine and sphingosine, but not by ceramide, sphingosine 1-phosphate, or other sphingo-, phospho-, or glycerolipids tested. Its activity is inhibited by the protein kinase C activator phosphatidylserine. Activity of SDK1 is clearly distinct from other types of serine kinases tested, including casein kinase II, the alpha and zeta isoforms of protein kinase C, extracellular signal-regulated mitogene-activated protein kinase 1 (Erk-1), Erk-2, and Raf-1. SDK1 specifically phosphorylates certain isoforms of 14-3-3 (eta, beta, zeta) but not others (sigma, tau). The phosphorylation site was identified as Ser* in the sequence Arg-Arg-Ser-Ser*-Trp-Arg in 14-3-3 beta. The sigma and tau isoforms of 14-3-3 lack serine at this position, potentially explaining their lack of phosphorylation by SDK1. Interestingly, the phosphorylation site is located on the dimer interface of 14-3-3. Phosphorylation of this site by SDK1 was studied in 14-3-3 mutants. Mutation of a lysine residue, located 9 amino acids N-terminal to the phosphorylation site, abolished 14-3-3 phosphorylation. Furthermore, co-immunoprecipitation experiments demonstrate an association between an SDK and 14-3-3 in situ. Exogenous N, N'-dimethylsphingosine stimulates 14-3-3 phosphorylation in Balb/c 3T3 fibroblasts, suggesting that SDK1 may phosphorylate 14-3-3 in situ. These data support a biological role of SDK1 activation and consequent

  20. Tomato 14-3-3 protein 7 (TFT7) positively regulates immunity-associated programmed cell death by enhancing accumulation and signaling ability of MAPKKKalpha

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Programmed cell death (PCD) is triggered when Pto, a serine-threonine protein kinase recognizes either the AvrPto or AvrPtoB effector from Pseudomonas syringae pv. tomato. This PCD requires MAPKKKalpha as a positive regulator in tomato and Nicotiana benthamiana. To examine how PCD-eliciting activi...

  1. Phosphorylation-dependent inhibition of Cdc42 GEF Gef1 by 14-3-3 protein Rad24 spatially regulates Cdc42 GTPase activity and oscillatory dynamics during cell morphogenesis

    PubMed Central

    Das, Maitreyi; Nuñez, Illyce; Rodriguez, Marbelys; Wiley, David J.; Rodriguez, Juan; Sarkeshik, Ali; Yates, John R.; Buchwald, Peter; Verde, Fulvia

    2015-01-01

    Active Cdc42 GTPase, a key regulator of cell polarity, displays oscillatory dynamics that are anticorrelated at the two cell tips in fission yeast. Anticorrelation suggests competition for active Cdc42 or for its effectors. Here we show how 14-3-3 protein Rad24 associates with Cdc42 guanine exchange factor (GEF) Gef1, limiting Gef1 availability to promote Cdc42 activation. Phosphorylation of Gef1 by conserved NDR kinase Orb6 promotes Gef1 binding to Rad24. Loss of Rad24–Gef1 interaction increases Gef1 protein localization and Cdc42 activation at the cell tips and reduces the anticorrelation of active Cdc42 oscillations. Increased Cdc42 activation promotes precocious bipolar growth activation, bypassing the normal requirement for an intact microtubule cytoskeleton and for microtubule-dependent polarity landmark Tea4-PP1. Further, increased Cdc42 activation by Gef1 widens cell diameter and alters tip curvature, countering the effects of Cdc42 GTPase-activating protein Rga4. The respective levels of Gef1 and Rga4 proteins at the membrane define dynamically the growing area at each cell tip. Our findings show how the 14-3-3 protein Rad24 modulates the availability of Cdc42 GEF Gef1, a homologue of mammalian Cdc42 GEF DNMBP/TUBA, to spatially control Cdc42 GTPase activity and promote cell polarization and cell shape emergence. PMID:26246599

  2. Quantitative proteomic dissection of a native 14-3-3ε interacting protein complex associated with hepatocellular carcinoma.

    PubMed

    Bai, Chen; Tang, Siwei; Bai, Chen; Chen, Xian

    2014-04-01

    The 14-3-3 proteins regulate diverse biological processes that are implicated in cancer development, and seven 14-3-3 isoforms were identified with isoform-specific roles in different human tumors. In our previous work, we dissected the interactome of 14-3-3ε formed during the DNA damage response in a hepatocellular carcinoma (HCC) cell using an AACT/SILAC-based quantitative proteomic approach. In this study, we used a similar proteomic approach to profile/identify the 14-3-3ε interactome formed in native HCC cells. Functional categorization and data-dependent network analysis of the native HCC-specific 14-3-3ε interactome revealed that 14-3-3ε is involved in the regulation of multiple biological processes (BPs)/pathways, including cell cycle control, apoptosis, signal transduction, transport, cell adhesion, carbohydrate metabolism, and nucleic acid metabolism. Biological validation further supports that 14-3-3ε, via association with multiple BP/pathway-specific proteins, coordinates the regulation of proliferation, survival, and metastasis of HCC. The findings in this study, together with those of our previous study, provide an extensive profile of the 14-3-3ε interaction network in HCC cells, which should be valuable for understanding the pathology of HCC and HCC therapy. PMID:24363202

  3. Destabilisation of dimeric 14-3-3 proteins as a novel approach to anti-cancer therapeutics.

    PubMed

    Woodcock, Joanna M; Coolen, Carl; Goodwin, Katy L; Baek, Dong Jae; Bittman, Robert; Samuel, Michael S; Pitson, Stuart M; Lopez, Angel F

    2015-06-10

    14-3-3 proteins play a pivotal role in controlling cell proliferation and survival, two commonly dysregulated hallmarks of cancers. 14-3-3 protein expression is enhanced in many human cancers and correlates with more aggressive tumors and poor prognosis, suggesting a role for 14-3-3 proteins in tumorigenesis and/or progression. We showed previously that the dimeric state of 14-3-3 proteins is regulated by the lipid sphingosine, a physiological inducer of apoptosis. As the functions of 14-3-3 proteins are dependent on their dimeric state, this sphingosine-mediated 14-3-3 regulation provides a possible means to target dimeric 14-3-3 for therapeutic effect. However, sphingosine mimics are needed that are not susceptible to sphingolipid metabolism. We show here the identification and optimization of sphingosine mimetics that render dimeric 14-3-3 susceptible to phosphorylation at a site buried in the dimer interface and induce mitochondrial-mediated apoptosis. Two such compounds, RB-011 and RB-012, disrupt 14-3-3 dimers at low micromolar concentrations and induce rapid down-regulation of Raf-MAPK and PI3K-Akt signaling in Jurkat cells. Importantly, both RB-011 and RB-012 induce apoptosis of human A549 lung cancer cells and RB-012, through disruption of MAPK signaling, reduces xenograft growth in mice. Thus, these compounds provide proof-of-principle for this novel 14-3-3-targeting approach for anti-cancer drug discovery. PMID:25971334

  4. Inhibition of the Arabidopsis Salt Overly Sensitive Pathway by 14-3-3 Proteins[C][W

    PubMed Central

    Zhou, Huapeng; Lin, Huixin; Chen, She; Becker, Katia; Yang, Yongqing; Zhao, Jinfeng; Kudla, Jörg; Schumaker, Karen S.; Guo, Yan

    2014-01-01

    The Salt Overly Sensitive (SOS) pathway regulates intracellular sodium ion (Na+) homeostasis and salt tolerance in plants. Until recently, little was known about the mechanisms that inhibit the SOS pathway when plants are grown in the absence of salt stress. In this study, we report that the Arabidopsis thaliana 14-3-3 proteins λ and κ interact with SOS2 and repress its kinase activity. Growth in the presence of salt decreases the interaction between SOS2 and the 14-3-3 proteins, leading to kinase activation in planta. 14-3-3 λ interacts with the SOS2 junction domain, which is important for its kinase activity. A phosphorylation site (Ser-294) is identified within this domain by mass spectrometry. Mutation of Ser-294 to Ala or Asp does not affect SOS2 kinase activity in the absence of the 14-3-3 proteins. However, in the presence of 14-3-3 proteins, the inhibition of SOS2 activity is decreased by the Ser-to-Ala mutation and enhanced by the Ser-to-Asp exchange. These results identify 14-3-3 λ and κ as important regulators of salt tolerance. The inhibition of SOS2 mediated by the binding of 14-3-3 proteins represents a novel mechanism that confers basal repression of the SOS pathway in the absence of salt stress. PMID:24659330

  5. Differential interaction and aggregation of 3-repeat and 4-repeat tau isoforms with 14-3-3{zeta} protein

    SciTech Connect

    Sadik, Golam; Tanaka, Toshihisa; Kato, Kiyoko; Yanagi, Kentaro; Kudo, Takashi; Takeda, Masatoshi

    2009-05-22

    Tau isoforms, 3-repeat (3R) and 4-repeat tau (4R), are differentially involved in neuronal development and in several tauopathies. 14-3-3 protein binds to tau and 14-3-3/tau association has been found both in the development and in tauopathies. To understand the role of 14-3-3 in the differential regulation of tau isoforms, we have performed studies on the interaction and aggregation of 3R-tau and 4R-tau, either phosphorylated or unphosphorylated, with 14-3-3{zeta}. We show by surface plasmon resonance studies that the interaction between unphosphorylated 3R-tau and 14-3-3{zeta} is {approx}3-folds higher than that between unphosphorylated 4R-tau and 14-3-3{zeta}. Phosphorylation of tau by protein kinase A (PKA) increases the affinity of both 3R- and 4R-tau for 14-3-3{zeta} to a similar level. An in vitro aggregation assay employing both transmission electron microscopy and fluorescence spectroscopy revealed the aggregation of unphosphorylated 4R-tau to be significantly higher than that of unphosphorylated 3R-tau following the induction of 14-3-3{zeta}. The filaments formed from 3R- and 4R-tau were almost similar in morphology. In contrast, the aggregation of both 3R- and 4R-tau was reduced to a similar low level after phosphorylation with PKA. Taken together, these results suggest that 14-3-3{zeta} exhibits a similar role for tau isoforms after PKA-phosphorylation, but a differential role for unphosphorylated tau. The significant aggregation of 4R-tau by 14-3-3{zeta} suggests that 14-3-3 may act as an inducer in the generation of 4R-tau-predominant neurofibrillary tangles in tauopathies.

  6. 14-3-3 proteins interact with the insulin-like growth factor receptor but not the insulin receptor.

    PubMed Central

    Furlanetto, R W; Dey, B R; Lopaczynski, W; Nissley, S P

    1997-01-01

    We have used a yeast two-hybrid system to identify proteins which bind to the cytosolic portion of the type 1 insulin-like growth factor (IGF) receptor (IGFIR) but not the insulin receptor (IR). This analysis identified 14-3-3beta and zeta proteins. 14-3-3beta also binds to the IGFIR but not the IR in vitro and 14-3-3-IGFIR complexes are present in insect cells overexpressing the IGFIR cytoplasmic domain. 14-3-3 proteins are substrates of the IGFIR in the yeast system and in vitro. The interaction of 14-3-3 with the IGFIR requires receptor-kinase activity and maps to the C-terminus of the receptor, but does not depend on tyrosine residues in this or the juxtamembrane regions. Instead, the binding maps to serine residue 1283 and requires phosphorylation of this residue. 14-3-3 proteins are phosphoserine-binding proteins which have been shown to interact directly with components of the mitogenic and apoptotic signalling pathways, suggesting that they participate in growth regulation. Our findings suggest that 14-3-3 proteins may play a role in IGFIR signal transduction and may contribute to the differences in IGF and IR signalling capabilities. PMID:9581554

  7. Identification of a redox-modulatory interaction between selenoprotein W and 14-3-3 protein.

    PubMed

    Jeon, Yeong Ha; Ko, Kwan Young; Lee, Jea Hwang; Park, Ki Jun; Jang, Jun Ki; Kim, Ick Young

    2016-01-01

    Selenoprotein W (SelW) contains a selenocysteine (Sec, U) in a conserved CXXU motif corresponding to the CXXC redox motif of thioredoxin, suggesting a putative redox function of SelW. We have previously reported that the binding of 14-3-3 protein to its target proteins, including CDC25B, Rictor and TAZ, is inhibited by the interaction of 14-3-3 protein with SelW. However, the binding mechanism is unclear. In this study, we sought to determine the binding site of SelW to understand the regulatory mechanism of the interaction between SelW and 14-3-3 and its biological effects. Phosphorylated Ser(pS) or Thr(pT) residues in RSXpSXP or RXXXp(S/T)XP motifs are well-known common 14-3-3-binding sites, but Thr41, Ser59, and T69 of SelW, which are computationally predicted to serve are phosphorylation sites, were neither phosphorylation sites nor sites involved in the interaction. A mutant SelW in which Sec13 is changed to Ser (U13S) was unable to interact with 14-3-3 protein and thus did not inhibit the interaction of 14-3-3 to other target proteins. However, other Cys mutants of SelW(C10S, C33S and C37S) normally interacted with 14-3-3 protein. The interaction of SelW to 14-3-3 protein was enhanced by diamide or H2O2 and decreased by dithiothreitol (DTT). Taken together, these findings demonstrate that the Sec of SelW is involved in its interaction with 14-3-3 protein and that this interaction is increased under oxidative stress conditions. Thus, SelW may have a regulatory function in redox cell signaling by interacting with 14-3-3 protein. PMID:26474786

  8. 14-3-3ε and ζ Regulate Neurogenesis and Differentiation of Neuronal Progenitor Cells in the Developing Brain

    PubMed Central

    Wachi, Tomoka; Hunt, Robert F.; Baraban, Scott C.; Taya, Shinichiro; Ramshaw, Hayley; Kaibuchi, Kozo; Schwarz, Quenten P.; Lopez, Angel F.

    2014-01-01

    During brain development, neural progenitor cells proliferate and differentiate into neural precursors. These neural precursors migrate along the radial glial processes and localize at their final destination in the cortex. Numerous reports have revealed that 14-3-3 proteins are involved in many neuronal activities, although their functions in neurogenesis remain unclear. Here, using 14-3-3ε/ζ double knock-out mice, we found that 14-3-3 proteins are important for proliferation and differentiation of neural progenitor cells in the cortex, resulting in neuronal migration defects and seizures. 14-3-3 deficiency resulted in the increase of δ-catenin and the decrease of β-catenin and αN-catenin. 14-3-3 proteins regulated neuronal differentiation into neurons via direct interactions with phosphorylated δ-catenin to promote F-actin formation through a catenin/Rho GTPase/Limk1/cofilin signaling pathway. Conversely, neuronal migration defects seen in the double knock-out mice were restored by phosphomimic Ndel1 mutants, but not δ-catenin. Our findings provide new evidence that 14-3-3 proteins play important roles in neurogenesis and neuronal migration via the regulation of distinct signaling cascades. PMID:25186760

  9. 14-3-3ε and ζ regulate neurogenesis and differentiation of neuronal progenitor cells in the developing brain.

    PubMed

    Toyo-oka, Kazuhito; Wachi, Tomoka; Hunt, Robert F; Baraban, Scott C; Taya, Shinichiro; Ramshaw, Hayley; Kaibuchi, Kozo; Schwarz, Quenten P; Lopez, Angel F; Wynshaw-Boris, Anthony

    2014-09-01

    During brain development, neural progenitor cells proliferate and differentiate into neural precursors. These neural precursors migrate along the radial glial processes and localize at their final destination in the cortex. Numerous reports have revealed that 14-3-3 proteins are involved in many neuronal activities, although their functions in neurogenesis remain unclear. Here, using 14-3-3ε/ζ double knock-out mice, we found that 14-3-3 proteins are important for proliferation and differentiation of neural progenitor cells in the cortex, resulting in neuronal migration defects and seizures. 14-3-3 deficiency resulted in the increase of δ-catenin and the decrease of β-catenin and αN-catenin. 14-3-3 proteins regulated neuronal differentiation into neurons via direct interactions with phosphorylated δ-catenin to promote F-actin formation through a catenin/Rho GTPase/Limk1/cofilin signaling pathway. Conversely, neuronal migration defects seen in the double knock-out mice were restored by phosphomimic Ndel1 mutants, but not δ-catenin. Our findings provide new evidence that 14-3-3 proteins play important roles in neurogenesis and neuronal migration via the regulation of distinct signaling cascades. PMID:25186760

  10. Isoform-specific cleavage of 14-3-3 proteins in apoptotic JURL-MK1 cells.

    PubMed

    Kuzelová, Katerina; Grebenová, Dana; Pluskalová, Michaela; Kavan, Daniel; Halada, Petr; Hrkal, Zbynek

    2009-03-01

    The proteins of 14-3-3 family are substantially involved in the regulation of many biological processes including the apoptosis. We studied the changes in the expression of five 14-3-3 isoforms (beta, gamma, epsilon, tau, and zeta) during the apoptosis of JURL-MK1 and K562 cells. The expression level of all these proteins markedly decreased in relation with the apoptosis progression and all isoforms underwent truncation, which probably corresponds to the removal of several C-terminal amino acids. The observed 14-3-3 modifications were partially blocked by caspase-3 inhibition. In addition to caspases, a non-caspase protease is likely to contribute to 14-3-3's cleavage in an isoform-specific manner. While 14-3-3 gamma seems to be cleaved mainly by caspase-3, the alternative mechanism is essentially involved in the case of 14-3-3 tau, and a combined effect was observed for the isoforms epsilon, beta, and zeta. We suggest that the processing of 14-3-3 proteins could form an integral part of the programmed cell death or at least of some apoptotic pathways. PMID:19173300

  11. Phosphodiesterase 3A binds to 14-3-3 proteins in response to PMA-induced phosphorylation of Ser428

    PubMed Central

    Pozuelo Rubio, Mercedes; Campbell, David G.; Morrice, Nicholas A.; Mackintosh, Carol

    2005-01-01

    PDE3A (phosphodiesterase 3A) was identified as a phosphoprotein that co-immunoprecipitates with endogenous 14-3-3 proteins from HeLa cell extracts, and binds directly to 14-3-3 proteins in a phosphorylation-dependent manner. Among cellular stimuli tested, PMA promoted maximal binding of PDE3A to 14-3-3 proteins. While p42/p44 MAPK (mitogen-activated protein kinase), SAPK2 (stress-activated protein kinase 2)/p38 and PKC (protein kinase C) were all activated by PMA in HeLa cells, the PMA-induced binding of PDE3A to 14-3-3 proteins was inhibited by the non-specific PKC inhibitors Ro 318220 and H-7, but not by PD 184352, which inhibits MAPK activation, nor by SB 203580 and BIRB0796, which inhibit SAPK2 activation. Binding of PDE3A to 14-3-3 proteins was also blocked by the DNA replication inhibitors aphidicolin and mimosine, but the PDE3A–14-3-3 interaction was not cell-cycle-regulated. PDE3A isolated from cells was able to bind to 14-3-3 proteins after in vitro phosphorylation with PKC isoforms. Using MS/MS of IMAC (immobilized metal ion affinity chromatography)-enriched tryptic phosphopeptides and phosphospecific antibodies, at least five sites on PDE3A were found to be phosphorylated in vivo, of which Ser428 was selectively phosphorylated in response to PMA and dephosphorylated in cells treated with aphidicolin and mimosine. Phosphorylation of Ser428 therefore correlated with 14-3-3 binding to PDE3A. Ser312 of PDE3A was phosphorylated in an H-89-sensitive response to forskolin, indicative of phosphorylation by PKA (cAMP-dependent protein kinase), but phosphorylation at this site did not stimulate 14-3-3 binding. Thus 14-3-3 proteins can discriminate between sites in a region of multisite phosphorylation on PDE3A. An additional observation was that the cytoskeletal cross-linker protein plectin-1 coimmunoprecipitated with PDE3A independently of 14-3-3 binding. PMID:16153182

  12. Phosphorylation and Interaction with the 14-3-3 Protein of the Plasma Membrane H+-ATPase are Involved in the Regulation of Magnesium-Mediated Increases in Aluminum-Induced Citrate Exudation in Broad Bean (Vicia faba. L).

    PubMed

    Chen, Qi; Kan, Qi; Wang, Ping; Yu, Wenqian; Yu, Yuzhen; Zhao, Yan; Yu, Yongxiong; Li, Kunzhi; Chen, Limei

    2015-06-01

    Several studies have shown that external application of micromolar magnesium (Mg) can increase the resistance of legumes to aluminum (Al) stress by enhancing Al-induced citrate exudation. However, the exact mechanism underlying this regulation remains unknown. In this study, the physiological and molecular mechanisms by which Mg enhances Al-induced citrate exudation to alleviate Al toxicity were investigated in broad bean. Micromolar concentrations of Mg that alleviated Al toxicity paralleled the stimulation of Al-induced citrate exudation and increased the activity of the plasma membrane (PM) H(+)-ATPase. Northern blot analysis shows that a putative MATE-like gene (multidrug and toxic compound extrusion) was induced after treatment with Al for 4, 8 and 12 h, whereas the mRNA abundance of the MATE-like gene showed no significant difference between Al plus Mg and Al-only treatments during the entire treatment period. Real-time reverse transcription-PCR (RT-PCR) and Western blot analyses suggest that the transcription and translation of the PM H(+)-ATPase were induced by Al but not by Mg. In contrast, immunoprecipitation suggests that Mg enhanced the phosphorylation levels of VHA2 and its interaction with the vf14-3-3b protein under Al stress. Taken together, our results suggest that micromolar concentrations of Mg can alleviate the Al rhizotoxicity by increasing PM H(+)-ATPase activity and Al-induced citrate exudation in YD roots. This enhancement is likely to be attributable to Al-induced increases in the expression of the MATE-like gene and vha2 and Mg-induced changes in the phosphorylation levels of VHA2, thus changing its interaction with the vf14-3-3b protein. PMID:25745032

  13. 14-3-3β protein expression in eosinophilic meningitis caused by Angiostrongylus cantonensis infection

    PubMed Central

    2014-01-01

    Background Angiostrongylus cantonensis is a parasite endemic in the Southeast Asian and Pacific regions. Humans are incidentally infected either by eating uncooked intermediate hosts or by consuming vegetables containing the living third-stage larvae. The 14-3-3β protein is a cerebrospinal fluid (CSF) marker of neuronal damage during the development of Creutzfeldt-Jakob disease. In addition, increased 14-3-3β protein is also found in CSF from patients with a variety of neurological disorders. The goal of this study is to determine the roles of serum/CSF14-3-3β protein in patients with eosinophilic meningitis. Methods In a cohort study among nine Thai laborers with eosinophilic meningitis due to eating raw snails (Pomacea canaliculata), we examined the CSF weekly while patients were still hospitalized and followed up the serum for 6 months. The levels of 14-3-3β protein in CSF were analyzed by western blot and an in-house 14-3-3β enzyme-linked immunosorbent assay (ELISA) measurement was established and tested in an animal model of eosinophilic meningitis. Results The elevated 14-3-3β level was detected in the CSF from eight out of nine (81%) patients After 2 weeks of treatment, all patients showed a declined level or cleared of 14-3-3β protein in the CSF. By developing an in-house ELISA for measurement of 14-3-3β protein, it was found that the serum 14-3-3β level was significantly increased in patients during initial visit. . This finding was consistent to the animal experiment result in which there was severe blood brain barrier damage three weeks after infection and increased 14-3-3β protein expression in the CSF and serum by western blot and in house ELISA. After treatment, the serum 14-3-3β level in meningitis patients was rapidly returned to normal threshold. There was a correlation between initial CSF 14-3-3β level with severity of headache (r = 0.692, p = 0.039), CSF pleocytosis (r = 0.807, p = 0.009) and eosinophilia (r = 0

  14. 14-3-3sigma is a cruciform DNA binding protein and associates in vivo with origins of DNA replication.

    PubMed

    Alvarez, David; Novac, Olivia; Callejo, Mario; Ruiz, Marcia T; Price, Gerald B; Zannis-Hadjopoulos, Maria

    2002-01-01

    A human cruciform binding protein (CBP) was previously shown to bind to cruciform DNA in a structure-specific manner and be a member of the 14-3-3 protein family. CBP had been found to contain the 14-3-3 isoforms beta, gamma, epsilon, and zeta. Here, we show by Western blot analysis that the CBP-cruciform DNA complex eluted from band-shift polyacrylamide gels also contains the 14-3-3sigma isoform, which is present in HeLa cell nuclear extracts. An antibody specific for the 14-3-3sigma isoform was able to interfere with the formation of the CBP-cruciform DNA complex. The effect of the same anti-14-3-3sigma antibody in the in vitro replication of p186, a plasmid containing the minimal replication origin of the monkey origin ors8, was also analyzed. Pre-incubation of total HeLa cell extracts with this antibody decreased p186 in vitro replication to approximately 30% of control levels, while non-specific antibodies had no effect. 14-3-3sigma was found to associate in vivo with the monkey origins of DNA replication ors8 and ors12 in a cell cycle-dependent manner, as assayed by a chromatin immunoprecipitation (ChIP) assay that involved formaldehyde cross-linking, followed by immunoprecipitation with anti-14-3-3sigma antibody and quantitative PCR. The association of 14-3-3sigma with the replication origins was maximal at the G(1)/S phase. The results indicate that 14-3-3sigma is an origin binding protein involved in the regulation of DNA replication via cruciform DNA binding. PMID:12244572

  15. The cell cycle regulator 14-3-3σ opposes and reverses cancer metabolic reprogramming

    PubMed Central

    Phan, Liem; Chou, Ping-Chieh; Velazquez-Torres, Guermarie; Samudio, Ismael; Parreno, Kenneth; Huang, Yaling; Tseng, Chieh; Vu, Thuy; Gully, Chris; Su, Chun-Hui; Wang, Edward; Chen, Jian; Choi, Hyun-Ho; Fuentes-Mattei, Enrique; Shin, Ji-Hyun; Shiang, Christine; Grabiner, Brian; Blonska, Marzenna; Skerl, Stephen; Shao, Yiping; Cody, Dianna; Delacerda, Jorge; Kingsley, Charles; Webb, Douglas; Carlock, Colin; Zhou, Zhongguo; Hsieh, Yun-Chih; Lee, Jaehyuk; Elliott, Andrew; Ramirez, Marc; Bankson, Jim; Hazle, John; Wang, Yongxing; Li, Lei; Weng, Shaofan; Rizk, Nibal; Wen, Yu Ye; Lin, Xin; Wang, Hua; Wang, Huamin; Zhang, Aijun; Xia, Xuefeng; Wu, Yun; Habra, Mouhammed; Yang, Wei; Pusztai, Lajos; Yeung, Sai-Ching; Lee, Mong-Hong

    2015-01-01

    Summary Extensive reprogramming of cellular energy metabolism is a hallmark of cancer. Despite its importance, the molecular mechanism controlling this tumour metabolic shift remains not fully understood. Here we show that 14-3-3σ regulates cancer metabolic reprogramming and protects cells from tumourigenic transformation. 14-3-3σ opposes tumour-promoting metabolic programs by enhancing c-Myc poly-ubiquitination and subsequent degradation. 14-3-3σ demonstrates the suppressive impact on cancer glycolysis, glutaminolysis, mitochondrial biogenesis and other major metabolic processes of tumours. Importantly, 14-3-3σ expression levels predict overall and recurrence-free survival rates, tumour glucose uptake and metabolic gene expression in breast cancer patients. Thus, these results highlight that 14-3-3σ is an important regulator of tumour metabolism, and loss of 14-3-3σ expression is critical for cancer metabolic reprogramming. We anticipate that pharmacologically elevating the function of 14-3-3σ in tumours could be a promising direction for targeted anti-cancer metabolism therapy development in future. PMID:26179207

  16. Phosphoproteomic analysis identifies the tumor suppressor PDCD4 as a RSK substrate negatively regulated by 14-3-3

    PubMed Central

    Galan, Jacob A.; Geraghty, Kathryn M.; Lavoie, Geneviève; Kanshin, Evgeny; Tcherkezian, Joseph; Calabrese, Viviane; Jeschke, Grace R.; Turk, Benjamin E.; Ballif, Bryan A.; Blenis, John; Thibault, Pierre; Roux, Philippe P.

    2014-01-01

    The Ras/MAPK signaling cascade regulates various biological functions, including cell growth and proliferation. As such, this pathway is frequently deregulated in several types of cancer, including most cases of melanoma. RSK (p90 ribosomal S6 kinase) is a MAPK-activated protein kinase required for melanoma growth and proliferation, but relatively little is known about its exact function and the nature of its substrates. Herein, we used a quantitative phosphoproteomics approach to define the signaling networks regulated by RSK in melanoma. To more accurately predict direct phosphorylation substrates, we defined the RSK consensus phosphorylation motif and found significant overlap with the binding consensus of 14-3-3 proteins. We thus characterized the phospho-dependent 14-3-3 interactome in melanoma cells and found that a large proportion of 14-3-3 binding proteins are also potential RSK substrates. Our results show that RSK phosphorylates the tumor suppressor PDCD4 (programmed cell death protein 4) on two serine residues (Ser76 and Ser457) that regulate its subcellular localization and interaction with 14-3-3 proteins. We found that 14-3-3 binding promotes PDCD4 degradation, suggesting an important role for RSK in the inactivation of PDCD4 in melanoma. In addition to this tumor suppressor, our results suggest the involvement of RSK in a vast array of unexplored biological functions with relevance in oncogenesis. PMID:25002506

  17. Interaction of a 14-3-3 protein with the plant microtubule-associated protein EDE1

    PubMed Central

    Pignocchi, Cristina; Doonan, John H.

    2011-01-01

    Background and Aims The cell cycle-regulated protein ENDOSPERM DEFECTIVE 1 (EDE1) is a novel plant microtubule-associated protein essential for plant cell division and for microtubule organization in endosperm. EDE1 is only present on microtubules at mitosis and its expression is highly cell cycle regulated both at the protein and the transcript levels. Methods To search for EDE1-interacting proteins, a yeast two-hybrid screen was used in which EDE1 was fused with GAL4 DNA binding domain and expressed in a yeast strain that was then mated with a strain carrying a cDNA library fused to the GAL4 transactivation domain. Candidate interacting proteins were identified and confirmed in vitro. Key Results 14-3-3 upsilon was isolated several times from the library screen. In in vitro tests, it also interacted with EDE1: 14-3-3 upsilon most strongly associates with EDE1 in its free form, but also weakly when EDE1 is bound to microtubules. This study shows that EDE1 is a cyclin-dependent kinase substrate but that phosphorylation is not required for interaction with 14-3-3 upsilon. Conclusions The results suggest that 14-3-3 proteins may play a role in cytoskeletal organization of plant cells. The potential role of this interaction in the dynamics of EDE1 during the cell cycle is discussed. PMID:21558460

  18. Identification of 14-3-3 Proteins Phosphopeptide-Binding Specificity Using an Affinity-Based Computational Approach

    PubMed Central

    Li, Zhao; Tang, Jijun; Guo, Fei

    2016-01-01

    The 14-3-3 proteins are a highly conserved family of homodimeric and heterodimeric molecules, expressed in all eukaryotic cells. In human cells, this family consists of seven distinct but highly homologous 14-3-3 isoforms. 14-3-3σ is the only isoform directly linked to cancer in epithelial cells, which is regulated by major tumor suppressor genes. For each 14-3-3 isoform, we have 1,000 peptide motifs with experimental binding affinity values. In this paper, we present a novel method for identifying peptide motifs binding to 14-3-3σ isoform. First, we propose a sampling criteria to build a predictor for each new peptide sequence. Then, we select nine physicochemical properties of amino acids to describe each peptide motif. We also use auto-cross covariance to extract correlative properties of amino acids in any two positions. Finally, we consider elastic net to predict affinity values of peptide motifs, based on ridge regression and least absolute shrinkage and selection operator (LASSO). Our method tests on the 1,000 known peptide motifs binding to seven 14-3-3 isoforms. On the 14-3-3σ isoform, our method has overall pearson-product-moment correlation coefficient (PCC) and root mean squared error (RMSE) values of 0.84 and 252.31 for N–terminal sublibrary, and 0.77 and 269.13 for C–terminal sublibrary. We predict affinity values of 16,000 peptide sequences and relative binding ability across six permutated positions similar with experimental values. We identify phosphopeptides that preferentially bind to 14-3-3σ over other isoforms. Several positions on peptide motifs are in the same amino acid category with experimental substrate specificity of phosphopeptides binding to 14-3-3σ. Our method is fast and reliable and is a general computational method that can be used in peptide-protein binding identification in proteomics research. PMID:26828594

  19. Identification of 14-3-3 Proteins Phosphopeptide-Binding Specificity Using an Affinity-Based Computational Approach.

    PubMed

    Li, Zhao; Tang, Jijun; Guo, Fei

    2016-01-01

    The 14-3-3 proteins are a highly conserved family of homodimeric and heterodimeric molecules, expressed in all eukaryotic cells. In human cells, this family consists of seven distinct but highly homologous 14-3-3 isoforms. 14-3-3σ is the only isoform directly linked to cancer in epithelial cells, which is regulated by major tumor suppressor genes. For each 14-3-3 isoform, we have 1,000 peptide motifs with experimental binding affinity values. In this paper, we present a novel method for identifying peptide motifs binding to 14-3-3σ isoform. First, we propose a sampling criteria to build a predictor for each new peptide sequence. Then, we select nine physicochemical properties of amino acids to describe each peptide motif. We also use auto-cross covariance to extract correlative properties of amino acids in any two positions. Finally, we consider elastic net to predict affinity values of peptide motifs, based on ridge regression and least absolute shrinkage and selection operator (LASSO). Our method tests on the 1,000 known peptide motifs binding to seven 14-3-3 isoforms. On the 14-3-3σ isoform, our method has overall pearson-product-moment correlation coefficient (PCC) and root mean squared error (RMSE) values of 0.84 and 252.31 for N-terminal sublibrary, and 0.77 and 269.13 for C-terminal sublibrary. We predict affinity values of 16,000 peptide sequences and relative binding ability across six permutated positions similar with experimental values. We identify phosphopeptides that preferentially bind to 14-3-3σ over other isoforms. Several positions on peptide motifs are in the same amino acid category with experimental substrate specificity of phosphopeptides binding to 14-3-3σ. Our method is fast and reliable and is a general computational method that can be used in peptide-protein binding identification in proteomics research. PMID:26828594

  20. 14-3-3 protein targets misfolded chaperone-associated proteins to aggresomes

    PubMed Central

    Xu, Zhe; Graham, Kourtney; Foote, Molly; Liang, Fengshan; Rizkallah, Raed; Hurt, Myra; Wang, Yanchang; Wu, Yuying; Zhou, Yi

    2013-01-01

    Summary The aggresome is a key cytoplasmic organelle for sequestration and clearance of toxic protein aggregates. Although loading misfolded proteins cargos to dynein motors has been recognized as an important step in the aggresome formation process, the molecular machinery that mediates the association of cargos with the dynein motor is poorly understood. Here, we report a new aggresome-targeting pathway that involves isoforms of 14-3-3, a family of conserved regulatory proteins. 14-3-3 interacts with both the dynein-intermediate chain (DIC) and an Hsp70 co-chaperone Bcl-2-associated athanogene 3 (BAG3), thereby recruiting chaperone-associated protein cargos to dynein motors for their transport to aggresomes. This molecular cascade entails functional dimerization of 14-3-3, which we show to be crucial for the formation of aggresomes in both yeast and mammalian cells. These results suggest that 14-3-3 functions as a molecular adaptor to promote aggresomal targeting of misfolded protein aggregates and may link such complexes to inclusion bodies observed in various neurodegenerative diseases. PMID:23843611

  1. Molecular tweezers modulate 14-3-3 protein-protein interactions.

    PubMed

    Bier, David; Rose, Rolf; Bravo-Rodriguez, Kenny; Bartel, Maria; Ramirez-Anguita, Juan Manuel; Dutt, Som; Wilch, Constanze; Klärner, Frank-Gerrit; Sanchez-Garcia, Elsa; Schrader, Thomas; Ottmann, Christian

    2013-03-01

    Supramolecular chemistry has recently emerged as a promising way to modulate protein functions, but devising molecules that will interact with a protein in the desired manner is difficult as many competing interactions exist in a biological environment (with solvents, salts or different sites for the target biomolecule). We now show that lysine-specific molecular tweezers bind to a 14-3-3 adapter protein and modulate its interaction with partner proteins. The tweezers inhibit binding between the 14-3-3 protein and two partner proteins--a phosphorylated (C-Raf) protein and an unphosphorylated one (ExoS)--in a concentration-dependent manner. Protein crystallography shows that this effect arises from the binding of the tweezers to a single surface-exposed lysine (Lys214) of the 14-3-3 protein in the proximity of its central channel, which normally binds the partner proteins. A combination of structural analysis and computer simulations provides rules for the tweezers' binding preferences, thus allowing us to predict their influence on this type of protein-protein interactions. PMID:23422566

  2. Molecular tweezers modulate 14-3-3 protein-protein interactions

    NASA Astrophysics Data System (ADS)

    Bier, David; Rose, Rolf; Bravo-Rodriguez, Kenny; Bartel, Maria; Ramirez-Anguita, Juan Manuel; Dutt, Som; Wilch, Constanze; Klärner, Frank-Gerrit; Sanchez-Garcia, Elsa; Schrader, Thomas; Ottmann, Christian

    2013-03-01

    Supramolecular chemistry has recently emerged as a promising way to modulate protein functions, but devising molecules that will interact with a protein in the desired manner is difficult as many competing interactions exist in a biological environment (with solvents, salts or different sites for the target biomolecule). We now show that lysine-specific molecular tweezers bind to a 14-3-3 adapter protein and modulate its interaction with partner proteins. The tweezers inhibit binding between the 14-3-3 protein and two partner proteins—a phosphorylated (C-Raf) protein and an unphosphorylated one (ExoS)—in a concentration-dependent manner. Protein crystallography shows that this effect arises from the binding of the tweezers to a single surface-exposed lysine (Lys214) of the 14-3-3 protein in the proximity of its central channel, which normally binds the partner proteins. A combination of structural analysis and computer simulations provides rules for the tweezers' binding preferences, thus allowing us to predict their influence on this type of protein-protein interactions.

  3. 14-3-3zeta is indispensable for aggregate formation of polyglutamine-expanded huntingtin protein.

    PubMed

    Omi, Kazuya; Hachiya, Naomi S; Tanaka, Mayumi; Tokunaga, Katsushi; Kaneko, Kiyotoshi

    2008-01-24

    Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder caused by polyglutamine (polyQ) expansions in the huntingtin (Htt) protein. A hallmark of HD is the presence of aggregates-predominantly composed of NH(2)-terminal fragments of polyQ-expanded Htt-in the nucleus and cytoplasm of affected neurons. We previously proposed that 14-3-3zeta might act as a sweeper of misfolded proteins by facilitating the formation of aggregates possibly for neuroprotection; these aggregates are referred to as inclusion bodies. However, evidence available in this regard is indirect and circumstantial. In this study, analysis of the aggregation-prone protein Htt encoded by HD gene exon 1 containing polyglutamine expansions (Htt86Q) revealed that 17 residues in the NH(2)-terminal of this protein are indispensable for its aggregate formation. Immunoprecipitation assays revealed that 14-3-3beta, gamma, eta, and zeta interact with Htt86Q transfected in N2a cells. Interestingly, the small interfering ribonucleic acid (siRNA) suppression of 14-3-3zeta exclusively abolished Htt86Q aggregate formation, whereas 14-3-3beta or eta siRNA suppression did not. This indicates that 14-3-3zeta participates in aggregate formation under nonnative conditions. Our data support a novel role for 14-3-3zeta in the aggregate formation of nonnative, aggregation-prone proteins. PMID:18078716

  4. Diagnosing Sporadic Creutzfeldt-Jakob Disease: Accuracy of CSF 14-3-3 Protein Test of the Spinal Fluid

    MedlinePlus

    ... JAKOB DISEASE: ACCURACY OF THE 14-3-3 PROTEIN TEST OF THE SPINAL FLUID This information sheet ... help you understand how the 14-3-3 protein test helps in diagnosing sporadic Creutzfeldt-Jakob disease ( ...

  5. Proteomic screen in the simple metazoan Hydra identifies 14-3-3 binding proteins implicated in cellular metabolism, cytoskeletal organisation and Ca2+ signalling

    PubMed Central

    Pauly, Barbara; Lasi, Margherita; MacKintosh, Carol; Morrice, Nick; Imhof, Axel; Regula, Jörg; Rudd, Stephen; David, Charles N; Böttger, Angelika

    2007-01-01

    Background 14-3-3 proteins have been implicated in many signalling mechanisms due to their interaction with Ser/Thr phosphorylated target proteins. They are evolutionarily well conserved in eukaryotic organisms from single celled protozoans and unicellular algae to plants and humans. A diverse array of target proteins has been found in higher plants and in human cell lines including proteins involved in cellular metabolism, apoptosis, cytoskeletal organisation, secretion and Ca2+ signalling. Results We found that the simple metazoan Hydra has four 14-3-3 isoforms. In order to investigate whether the diversity of 14-3-3 target proteins is also conserved over the whole animal kingdom we isolated 14-3-3 binding proteins from Hydra vulgaris using a 14-3-3-affinity column. We identified 23 proteins that covered most of the above-mentioned groups. We also isolated several novel 14-3-3 binding proteins and the Hydra specific secreted fascin-domain-containing protein PPOD. In addition, we demonstrated that one of the 14-3-3 isoforms, 14-3-3 HyA, interacts with one Hydra-Bcl-2 like protein in vitro. Conclusion Our results indicate that 14-3-3 proteins have been ubiquitous signalling components since the start of metazoan evolution. We also discuss the possibility that they are involved in the regulation of cell numbers in response to food supply in Hydra. PMID:17651497

  6. Modulation of GluK2a subunit-containing kainate receptors by 14-3-3 proteins.

    PubMed

    Sun, Changcheng; Qiao, Haifa; Zhou, Qin; Wang, Yan; Wu, Yuying; Zhou, Yi; Li, Yong

    2013-08-23

    Kainate receptors (KARs) are one of the ionotropic glutamate receptors that mediate excitatory postsynaptic currents (EPSCs) with characteristically slow kinetics. Although mechanisms for the slow kinetics of KAR-EPSCs are not totally understood, recent evidence has implicated a regulatory role of KAR-associated proteins. Here, we report that decay kinetics of GluK2a-containing receptors is modulated by closely associated 14-3-3 proteins. 14-3-3 binding requires PKC-dependent phosphorylation of serine residues localized in the carboxyl tail of the GluK2a subunit. In transfected cells, 14-3-3 binding to GluK2a slows desensitization kinetics of both homomeric GluK2a and heteromeric GluK2a/GluK5 receptors. Moreover, KAR-EPSCs at mossy fiber-CA3 synapses decay significantly faster in the 14-3-3 functional knock-out mice. Collectively, these results demonstrate that 14-3-3 proteins are an important regulator of GluK2a-containing KARs and may contribute to the slow decay kinetics of native KAR-EPSCs. PMID:23861400

  7. Phosphorylation of Arabidopsis Ubiquitin Ligase ATL31 Is Critical for Plant Carbon/Nitrogen Nutrient Balance Response and Controls the Stability of 14-3-3 Proteins*

    PubMed Central

    Yasuda, Shigetaka; Sato, Takeo; Maekawa, Shugo; Aoyama, Shoki; Fukao, Yoichiro; Yamaguchi, Junji

    2014-01-01

    Ubiquitin ligase plays a fundamental role in regulating multiple cellular events in eukaryotes by fine-tuning the stability and activity of specific target proteins. We have previously shown that ubiquitin ligase ATL31 regulates plant growth in response to nutrient balance between carbon and nitrogen (C/N) in Arabidopsis. Subsequent study demonstrated that ATL31 targets 14-3-3 proteins for ubiquitination and modulates the protein abundance in response to C/N-nutrient status. However, the underlying mechanism for the targeting of ATL31 to 14-3-3 proteins remains unclear. Here, we show that ATL31 interacts with 14-3-3 proteins in a phosphorylation-dependent manner. We identified Thr209, Ser247, Ser270, and Ser303 as putative 14-3-3 binding sites on ATL31 by motif analysis. Mutation of these Ser/Thr residues to Ala in ATL31 inhibited the interaction with 14-3-3 proteins, as demonstrated by yeast two-hybrid and co-immunoprecipitation analyses. Additionally, we identified in vivo phosphorylation of Thr209 and Ser247 on ATL31 by MS analysis. A peptide competition assay showed that the application of synthetic phospho-Thr209 peptide, but not the corresponding unphosphorylated peptide, suppresses the interaction between ATL31 and 14-3-3 proteins. Moreover, Arabidopsis plants overexpressing mutated ATL31, which could not bind to 14-3-3 proteins, showed accumulation of 14-3-3 proteins and growth arrest in disrupted C/N-nutrient conditions similar to wild-type plants, although overexpression of intact ATL31 resulted in repression of 14-3-3 accumulation and tolerance to the conditions. Together, these results demonstrate that the physiological role of phosphorylation at 14-3-3 binding sites on ATL31 is to modulate the binding ability and stability of 14-3-3 proteins to control plant C/N-nutrient response. PMID:24722992

  8. Interaction of 14-3-3 proteins with the Estrogen Receptor Alpha F domain provides a drug target interface

    PubMed Central

    De Vries-van Leeuwen, Ingrid J.; da Costa Pereira, Daniel; Flach, Koen D.; Piersma, Sander R.; Haase, Christian; Bier, David; Yalcin, Zeliha; Michalides, Rob; Feenstra, K. Anton; Jiménez, Connie R.; de Greef, Tom F. A.; Brunsveld, Luc; Ottmann, Christian; Zwart, Wilbert; de Boer, Albertus H.

    2013-01-01

    Estrogen receptor alpha (ERα) is involved in numerous physiological and pathological processes, including breast cancer. Breast cancer therapy is therefore currently directed at inhibiting the transcriptional potency of ERα, either by blocking estrogen production through aromatase inhibitors or antiestrogens that compete for hormone binding. Due to resistance, new treatment modalities are needed and as ERα dimerization is essential for its activity, interference with receptor dimerization offers a new opportunity to exploit in drug design. Here we describe a unique mechanism of how ERα dimerization is negatively controlled by interaction with 14-3-3 proteins at the extreme C terminus of the receptor. Moreover, the small-molecule fusicoccin (FC) stabilizes this ERα/14-3-3 interaction. Cocrystallization of the trimeric ERα/14-3-3/FC complex provides the structural basis for this stabilization and shows the importance of phosphorylation of the penultimate Threonine (ERα-T594) for high-affinity interaction. We confirm that T594 is a distinct ERα phosphorylation site in the breast cancer cell line MCF-7 using a phospho-T594–specific antibody and by mass spectrometry. In line with its ERα/14-3-3 interaction stabilizing effect, fusicoccin reduces the estradiol-stimulated ERα dimerization, inhibits ERα/chromatin interactions and downstream gene expression, resulting in decreased cell proliferation. Herewith, a unique functional phosphosite and an alternative regulation mechanism of ERα are provided, together with a small molecule that selectively targets this ERα/14-3-3 interface. PMID:23676274

  9. Polycations Globally Enhance Binding of 14-3-3 omega to Target Proteins in Spinach Leaves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The binding of 14-3-3' to phosphorylated NR (pNR) is stimulated by cations such as Mg2+ or spermine, and decreased by 5'-AMP. In order to determine whether binding to other cellular proteins is affected similarly, Far-Western overlays of extracts prepared from light- or dark-treated spinach (Spinac...

  10. Revealing the binding modes and the unbinding of 14-3-3σ proteins and inhibitors by computational methods

    PubMed Central

    Hu, Guodong; Cao, Zanxia; Xu, Shicai; Wang, Wei; Wang, Jihua

    2015-01-01

    The 14-3-3σ proteins are a family of ubiquitous conserved eukaryotic regulatory molecules involved in the regulation of mitogenic signal transduction, apoptotic cell death, and cell cycle control. A lot of small-molecule inhibitors have been identified for 14-3-3 protein-protein interactions (PPIs). In this work, we carried out molecular dynamics (MD) simulations combined with molecular mechanics generalized Born surface area (MM-GBSA) method to study the binding mechanism between a 14-3-3σ protein and its eight inhibitors. The ranking order of our calculated binding free energies is in agreement with the experimental results. We found that the binding free energies are mainly from interactions between the phosphate group of the inhibitors and the hydrophilic residues. To improve the binding free energy of Rx group, we designed the inhibitor R9 with group R9 = 4-hydroxypheny. However, we also found that the binding free energy of inhibitor R9 is smaller than that of inhibitor R1. By further using the steer molecular dynamics (SMD) simulations, we identified a new hydrogen bond between the inhibitor R8 and residue Arg64 in the pulling paths. The information obtained from this study may be valuable for future rational design of novel inhibitors, and provide better structural understanding of inhibitor binding to 14-3-3σ proteins. PMID:26568041

  11. Molecular Dynamics Simulations and Structural Analysis of Giardia duodenalis 14-3-3 Protein-Protein Interactions.

    PubMed

    Cau, Ylenia; Fiorillo, Annarita; Mori, Mattia; Ilari, Andrea; Botta, Maurizo; Lalle, Marco

    2015-12-28

    Giardiasis is a gastrointestinal diarrheal illness caused by the protozoan parasite Giardia duodenalis, which affects annually over 200 million people worldwide. The limited antigiardial drug arsenal and the emergence of clinical cases refractory to standard treatments dictate the need for new chemotherapeutics. The 14-3-3 family of regulatory proteins, extensively involved in protein-protein interactions (PPIs) with pSer/pThr clients, represents a highly promising target. Despite homology with human counterparts, the single 14-3-3 of G. duodenalis (g14-3-3) is characterized by a constitutive phosphorylation in a region critical for target binding, thus affecting the function and the conformation of g14-3-3/clients interaction. However, to approach the design of specific small molecule modulators of g14-3-3 PPIs, structural elucidations are required. Here, we present a detailed computational and crystallographic study exploring the implications of g14-3-3 phosphorylation on protein structure and target binding. Self-Guided Langevin Dynamics and classical molecular dynamics simulations show that phosphorylation affects locally and globally g14-3-3 conformation, inducing a structural rearrangement more suitable for target binding. Profitable features for g14-3-3/clients interaction were highlighted using a hydrophobicity-based descriptor to characterize g14-3-3 client peptides. Finally, the X-ray structure of g14-3-3 in complex with a mode-1 prototype phosphopeptide was solved and combined with structure-based simulations to identify molecular features relevant for clients binding to g14-3-3. The data presented herein provide a further and structural understanding of g14-3-3 features and set the basis for drug design studies. PMID:26551337

  12. Discovery and structural characterization of a small molecule 14-3-3 protein-protein interaction inhibitor

    SciTech Connect

    Zhao, Jing; Du, Yuhong; Horton, John R.; Upadhyay, Anup K.; Lou, Bin; Bai, Yan; Zhang, Xing; Du, Lupei; Li, Minyong; Wang, Binghe; Zhang, Lixin; Barbieri, Joseph T.; Khuri, Fadlo R.; Cheng, Xiaodong; Fu, Haian

    2013-02-14

    The 14-3-3 family of phosphoserine/threonine-recognition proteins engage multiple nodes in signaling networks that control diverse physiological and pathophysiological functions and have emerged as promising therapeutic targets for such diseases as cancer and neurodegenerative disorders. Thus, small molecule modulators of 14-3-3 are much needed agents for chemical biology investigations and therapeutic development. To analyze 14-3-3 function and modulate its activity, we conducted a chemical screen and identified 4-[(2Z)-2-[4-formyl-6-methyl-5-oxo-3-(phosphonatooxymethyl)pyridin-2-ylidene]hydrazinyl]benzoate as a 14-3-3 inhibitor, which we termed FOBISIN (FOurteen-three-three BInding Small molecule INhibitor) 101. FOBISIN101 effectively blocked the binding of 14-3-3 with Raf-1 and proline-rich AKT substrate, 40 kD{sub a} and neutralized the ability of 14-3-3 to activate exoenzyme S ADP-ribosyltransferase. To provide a mechanistic basis for 14-3-3 inhibition, the crystal structure of 14-3-3{zeta} in complex with FOBISIN101 was solved. Unexpectedly, the double bond linking the pyridoxal-phosphate and benzoate moieties was reduced by X-rays to create a covalent linkage of the pyridoxal-phosphate moiety to lysine 120 in the binding groove of 14-3-3, leading to persistent 14-3-3 inactivation. We suggest that FOBISIN101-like molecules could be developed as an entirely unique class of 14-3-3 inhibitors, which may serve as radiation-triggered therapeutic agents for the treatment of 14-3-3-mediated diseases, such as cancer.

  13. Discovery and structural characterization of a small molecule 14-3-3 protein-protein interaction inhibitor.

    PubMed

    Zhao, Jing; Du, Yuhong; Horton, John R; Upadhyay, Anup K; Lou, Bin; Bai, Yan; Zhang, Xing; Du, Lupei; Li, Minyong; Wang, Binghe; Zhang, Lixin; Barbieri, Joseph T; Khuri, Fadlo R; Cheng, Xiaodong; Fu, Haian

    2011-09-27

    The 14-3-3 family of phosphoserine/threonine-recognition proteins engage multiple nodes in signaling networks that control diverse physiological and pathophysiological functions and have emerged as promising therapeutic targets for such diseases as cancer and neurodegenerative disorders. Thus, small molecule modulators of 14-3-3 are much needed agents for chemical biology investigations and therapeutic development. To analyze 14-3-3 function and modulate its activity, we conducted a chemical screen and identified 4-[(2Z)-2-[4-formyl-6-methyl-5-oxo-3-(phosphonatooxymethyl)pyridin-2-ylidene]hydrazinyl]benzoate as a 14-3-3 inhibitor, which we termed FOBISIN (FOurteen-three-three BInding Small molecule INhibitor) 101. FOBISIN101 effectively blocked the binding of 14-3-3 with Raf-1 and proline-rich AKT substrate, 40 kD(a) and neutralized the ability of 14-3-3 to activate exoenzyme S ADP-ribosyltransferase. To provide a mechanistic basis for 14-3-3 inhibition, the crystal structure of 14-3-3ζ in complex with FOBISIN101 was solved. Unexpectedly, the double bond linking the pyridoxal-phosphate and benzoate moieties was reduced by X-rays to create a covalent linkage of the pyridoxal-phosphate moiety to lysine 120 in the binding groove of 14-3-3, leading to persistent 14-3-3 inactivation. We suggest that FOBISIN101-like molecules could be developed as an entirely unique class of 14-3-3 inhibitors, which may serve as radiation-triggered therapeutic agents for the treatment of 14-3-3-mediated diseases, such as cancer. PMID:21908710

  14. Ablation of the 14-3-3gamma Protein Results in Neuronal Migration Delay and Morphological Defects in the Developing Cerebral Cortex.

    PubMed

    Wachi, Tomoka; Cornell, Brett; Marshall, Courtney; Zhukarev, Vladimir; Baas, Peter W; Toyo-Oka, Kazuhito

    2016-06-01

    14-3-3 proteins are ubiquitously-expressed and multifunctional proteins. There are seven isoforms in mammals with a high level of homology, suggesting potential functional redundancy. We previously found that two of seven isoforms, 14-3-3epsilon and 14-3-3zeta, are important for brain development, in particular, radial migration of pyramidal neurons in the developing cerebral cortex. In this work, we analyzed the function of another isoform, the protein 14-3-3gamma, with respect to neuronal migration in the developing cortex. We found that in utero 14-3-3gamma-deficiency resulted in delays in neuronal migration as well as morphological defects. Migrating neurons deficient in 14-3-3gamma displayed a thicker leading process stem, and the basal ends of neurons were not able to reach the boundary between the cortical plate and the marginal zone. Consistent with the results obtained from in utero electroporation, time-lapse live imaging of brain slices revealed that the ablation of the 14-3-3gamma proteins in pyramidal neurons slowed down their migration. In addition, the 14-3-3gamma deficient neurons showed morphological abnormalities, including increased multipolar neurons with a thicker leading processes stem during migration. These results indicate that the 14-3-3gamma proteins play an important role in radial migration by regulating the morphology of migrating neurons in the cerebral cortex. The findings underscore the pathological phenotypes of brain development associated with the disruption of different 14-3-3 proteins and will advance the preclinical data regarding disorders caused by neuronal migration defects. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 600-614, 2016. PMID:26297819

  15. Validation of 14-3-3 Protein as a Marker in Sporadic Creutzfeldt-Jakob Disease Diagnostic.

    PubMed

    Schmitz, Matthias; Ebert, Elisabeth; Stoeck, Katharina; Karch, André; Collins, Steven; Calero, Miguel; Sklaviadis, Theodor; Laplanche, Jean-Louis; Golanska, Ewa; Baldeiras, Ines; Satoh, Katsuya; Sanchez-Valle, Raquel; Ladogana, Anna; Skinningsrud, Anders; Hammarin, Anna-Lena; Mitrova, Eva; Llorens, Franc; Kim, Yong Sun; Green, Alison; Zerr, Inga

    2016-05-01

    At present, the testing of 14-3-3 protein in cerebrospinal fluid (CSF) is a standard biomarker test in suspected sporadic Creutzfeldt-Jakob disease (sCJD) diagnosis. Increasing 14-3-3 test referrals in CJD reference laboratories in the last years have led to an urgent need to improve established 14-3-3 test methods. The main result of our study was the validation of a commercially available 14-3-3 ELISA next to the commonly used Western blot method as a high-throughput screening test. Hereby, 14-3-3 protein expression was quantitatively analyzed in CSF of 231 sCJD and 2035 control patients. We obtained excellent sensitivity/specificity values of 88 and 96% that are comparable to the established Western blot method. Since standard protocols and preanalytical sample handling have become more important in routine diagnostic, we investigated in a further step the reproducibility and stability of 14-3-3 as a biomarker for human prion diseases. Ring trial data from 2009 to 2013 revealed an increase of Fleiss' kappa from 0.51 to 0.68 indicating an improving reliability of 14-3-3 protein detection. The stability of 14-3-3 protein under short-term and long-term storage conditions at various temperatures and after repeated freezing/thawing cycles was confirmed. Contamination of CSF samples with blood appears likely to be an important factor at a concentration of more than 2500 erythrocytes/μL. Hemolysis of erythrocytes with significant release of 14-3-3 protein started after 2 days at room temperature. We first define clear standards for the sample handling, short- and long-term storage of CSF samples as well as the handling of blood- contaminated samples which may result in artificially elevated CSF levels of 14-3-3. PMID:25947081

  16. Site-specific regulatory interaction between spinach leaf sucrose-phosphate synthase and 14-3-3 proteins

    NASA Technical Reports Server (NTRS)

    Toroser, D.; Athwal, G. S.; Huber, S. C.; Davies, E. (Principal Investigator)

    1998-01-01

    We report an Mg2+-dependent interaction between spinach leaf sucrose-phosphate synthase (SPS) and endogenous 14-3-3 proteins, as evidenced by co-elution during gel filtration and co-immunoprecipitation. The content of 14-3-3s associated with an SPS immunoprecipitate was inversely related to activity, and was specifically reduced when tissue was pretreated with 5-aminoimidazole-4-carboxamide riboside, suggesting metabolite control in vivo. A synthetic phosphopeptide based on Ser-229 was shown by surface plasmon resonance to bind a recombinant plant 14-3-3, and addition of the phosphorylated SPS-229 peptide was found to stimulate the SPS activity of an SPS:14-3-3 complex. Taken together, the results suggest a regulatory interaction of 14-3-3 proteins with Ser-229 of SPS.

  17. Small-Molecule Stabilization of the 14-3-3/Gab2 Protein-Protein Interaction (PPI) Interface.

    PubMed

    Bier, David; Bartel, Maria; Sies, Katharina; Halbach, Sebastian; Higuchi, Yusuke; Haranosono, Yu; Brummer, Tilman; Kato, Nobuo; Ottmann, Christian

    2016-04-19

    Small-molecule modulation of protein-protein interactions (PPIs) is one of the most promising new areas in drug discovery. In the vast majority of cases only inhibition or disruption of PPIs is realized, whereas the complementary strategy of targeted stabilization of PPIs is clearly under-represented. Here, we report the example of a semi-synthetic natural product derivative-ISIR-005-that stabilizes the cancer-relevant interaction of the adaptor protein 14-3-3 and Gab2. The crystal structure of ISIR-005 in complex with 14-3-3 and the binding motif of Gab2 comprising two phosphorylation sites (Gab2pS210pT391) showed how the stabilizing molecule binds to the rim-of-the-interface of the protein complex. Only in the direct vicinity of 14-3-3/Gab2pT391 site is a pre-formed pocket occupied by ISIR-005; binding of the Gab2pS210 motif to 14-3-3 does not create an interface pocket suitable for the molecule. Accordingly, ISIR-005 only stabilizes the binding of the Gab2pT391 but not the Gab2pS210 site. This study represents structural and biochemical proof of the druggability of the 14-3-3/Gab2 PPI interface with important implications for the development of PPI stabilizers. PMID:26644359

  18. Transcription variants of the prostate-specific PrLZ gene and their interaction with 14-3-3 proteins

    SciTech Connect

    Wang, Ruoxiang; He, Hui; Sun, Xiaojuan; Xu, Jianchun; Marshall, Fray F.; Zhau, Haiyen; Chung, Leland W.K.; Fu, Haian; He, Dalin

    2009-11-20

    We have reported isolation and characterization of the prostate-specific and androgen-regulated PrLZ gene abnormally expressed in prostate cancer. PrLZ is a potential biomarker for prostate cancer and a candidate oncogene promoting cell proliferation and survival in prostate cancer cells. A full delineation of the PrLZ gene and its gene products may provide clues to the mechanisms regulating its expression and function. In this report, we identified three additional exons in the PrLZ gene and recognized five transcript variants from alternative splicing that could be detected by RT-PCR and Western blotting. Structural comparison demonstrated that the PrLZ proteins are highly conserved among species. PrLZ contains multiple potential sites for interaction with other proteins. We used mammalian two-hybrid assays to demonstrate that PrLZ isoforms interact with 14-3-3 proteins, and multiple sites in the PrLZ may be involved in the interaction. Alternative splicing may contribute to abnormally enhanced PrLZ levels in prostate cancer, and interaction with 14-3-3 proteins may be a mechanism by which PrLZ promotes cell proliferation and survival during prostate cancer development and progression. This information is a valuable addition to the investigation of the oncogenic properties of the PrLZ gene.

  19. Functions of Saccharomyces cerevisiae 14-3-3 proteins in response to DNA damage and to DNA replication stress.

    PubMed Central

    Lottersberger, Francisca; Rubert, Fabio; Baldo, Veronica; Lucchini, Giovanna; Longhese, Maria Pia

    2003-01-01

    Two members of the 14-3-3 protein family, involved in key biological processes in different eukaryotes, are encoded by the functionally redundant Saccharomyces cerevisiae BMH1 and BMH2 genes. We produced and characterized 12 independent bmh1 mutant alleles, whose presence in the cell as the sole 14-3-3 source causes hypersensitivity to genotoxic agents, indicating that Bmh proteins are required for proper response to DNA damage. In particular, the bmh1-103 and bmh1-266 mutant alleles cause defects in G1/S and G2/M DNA damage checkpoints, whereas only the G2/M checkpoint is altered by the bmh1-169 and bmh1-221 alleles. Impaired checkpoint responses correlate with the inability to maintain phosphorylated forms of Rad53 and/or Chk1, suggesting that Bmh proteins might regulate phosphorylation/dephosphorylation of these checkpoint kinases. Moreover, several bmh1 bmh2Delta mutants are defective in resuming DNA replication after transient deoxynucleotide depletion, and all display synthetic effects when also carrying mutations affecting the polalpha-primase and RPA DNA replication complexes, suggesting a role for Bmh proteins in DNA replication stress response. Finally, the bmh1-169 bmh2Delta and bmh1-170 bmh2Delta mutants show increased rates of spontaneous gross chromosomal rearrangements, indicating that Bmh proteins are required to suppress genome instability. PMID:14704161

  20. Association of 14-3-3 Proteins to β1-Adrenergic Receptors Modulates Kv11.1 K+ Channel Activity in Recombinant Systems

    PubMed Central

    Tutor, Antonio S.; Delpón, Eva; Caballero, Ricardo; Gómez, Ricardo; Núñez, Lucía; Vaquero, Miguel; Tamargo, Juan; Penela, Petronila

    2006-01-01

    We identify a new mechanism for the β1-adrenergic receptor (β1AR)-mediated regulation of human ether-a-go-go–related gene (HERG) potassium channel (Kv11.1). We find that the previously reported modulatory interaction between Kv11.1 channels and 14-3-3ε proteins is competed by wild type β1AR by means of a novel interaction between this receptor and 14-3-3ε. The association between β1AR and 14-3-3ε is increased by agonist stimulation in both transfected cells and heart tissue and requires cAMP-dependent protein kinase (PKA) activity. The β1AR/14-3-3ε association is direct, since it can be recapitulated using purified 14-3-3ε and β1AR fusion proteins and is abolished in cells expressing β1AR phosphorylation–deficient mutants. Biochemical and electrophysiological studies of the effects of isoproterenol on Kv11.1 currents recorded using the whole-cell patch clamp demonstrated that β1AR phosphorylation–deficient mutants do not recruit 14-3-3ε away from Kv11.1 and display a markedly altered agonist-mediated modulation of Kv11.1 currents compared with wild-type β1AR, increasing instead of inhibiting current amplitudes. Interestingly, such differential modulation is not observed in the presence of 14-3-3 inhibitors. Our results suggest that the dynamic association of 14-3-3 proteins to both β1AR and Kv11.1 channels is involved in the adrenergic modulation of this critical regulator of cardiac repolarization and refractoriness. PMID:16914520

  1. The alternative role of 14-3-3 zeta as a sweeper of misfolded proteins in disease conditions.

    PubMed

    Kaneko, Kiyotoshi; Hachiya, Naomi S

    2006-01-01

    Here, we propose a novel hypothesis that 14-3-3 zeta might act as a sweeper of misfolded proteins by facilitating the formation of aggregates, which are referred to as inclusion bodies. Studies on the localization of the 14-3-3 proteins in different types of inclusion bodies in the brain including neurofibrillary tangle in Alzheimer's disease, pick bodies in Pick's disease, Lewy body-like hyaline inclusions in sporadic amyotrophic lateral sclerosis, prion/florid plaques in sporadic/variant Creutzfeldt-Jakob disease, nuclear inclusions in spinocerebellar ataxia-1, and possibly Lewy bodies in Parkinson's disease suggest a close association of these diseases with 14-3-3 zeta. The highly conserved hydrophobic surface of the amphipathic groove in 14-3-3 zeta represents a general mechanism with diverse cellular proteins, and it may also allow for the molecular recognition of misfolded proteins by hydrophobic interaction in disease conditions. When the abnormal processing of misfolded proteins overwhelms the quality control systems of the cell, it is likely that 14-3-3 zeta is recruited to form deposits of protein aggregates with nonnative, misfolded proteins in order to protect the cell against toxicity. Hence, 14-3-3 zeta may be considered as an auxiliary therapeutic tool in the protein aggregation disorders. PMID:16516399

  2. Proteomic analysis of media from lung cancer cells reveals role of 14-3-3 proteins in cachexia

    PubMed Central

    McLean, Julie B.; Moylan, Jennifer S.; Horrell, Erin M. W.; Andrade, Francisco H.

    2015-01-01

    Aims: At the time of diagnosis, 60% of lung cancer patients present with cachexia, a severe wasting syndrome that increases morbidity and mortality. Tumors secrete multiple factors that contribute to cachectic muscle wasting, and not all of these factors have been identified. We used Orbitrap electrospray ionization mass spectrometry to identify novel cachexia-inducing candidates in media conditioned with Lewis lung carcinoma cells (LCM). Results: One-hundred and 58 proteins were confirmed in three biological replicates. Thirty-three were identified as secreted proteins, including 14-3-3 proteins, which are highly conserved adaptor proteins known to have over 200 binding partners. We confirmed the presence of extracellular 14-3-3 proteins in LCM via western blot and discovered that LCM contained less 14-3-3 content than media conditioned with C2C12 myotubes. Using a neutralizing antibody, we depleted extracellular 14-3-3 proteins in myotube culture medium, which resulted in diminished myosin content. We identified the proposed receptor for 14-3-3 proteins, CD13, in differentiated C2C12 myotubes and found that inhibiting CD13 via Bestatin also resulted in diminished myosin content. Conclusions: Our novel findings show that extracellular 14-3-3 proteins may act as previously unidentified myokines and may signal via CD13 to help maintain muscle mass. PMID:25972815

  3. Verteporfin inhibits YAP function through up-regulating 14-3-3σ sequestering YAP in the cytoplasm

    PubMed Central

    Wang, Chao; Zhu, Xiaoyong; Feng, Weiwei; Yu, Yinhua; Jeong, Kangjin; Guo, Wei; Lu, Yiling; Mills, Gordon B

    2016-01-01

    Yes-associated protein (YAP), the central mediator of Hippo pathway, not only regulates a diversity of cellular processes during development but also plays a pivotal role in tumorigenesis. YAP is overexpressed in many types of human cancers with its expression level being associated with patient outcomes. Thus, inhibiting YAP function could provide a novel therapeutic approach. Verteporfin, a photosensitizer, which has been used in photodynamic therapy (PDT), was recently identified as an inhibitor of the interaction of YAP with TEAD, which, in turn, blocks transcriptional activation of targets downstream of YAP. However, the mechanism by which Verteporfin inhibits YAP activity remains to be elucidated. We demonstrate that overexpression of YAP stimulates cell proliferation whereas knocking down YAP or treating cells with Verteporfin inhibited cell proliferation, even in the presence of growth factors. Protoporphyrin IX, another photosensitizer, did not have similar activity demonstrating specificity to Verteporfin. Verteporfin induced sequestration of YAP in cytoplasm through increasing levels of 14-3-3σ, a YAP chaperon protein that retains YAP in cytoplasm and targets it for degradation in the proteosome. Interestingly, while knockdown of YAP had no effect on the ability of Verteporfin to induce 14-3-3σ, p53 is required for this effect of Verteporfin. This provides potential approaches to select patients likely to benefit from Verteporfin. PMID:27073720

  4. Regulation of Molecular Chaperone Gene Transcription Involves the Serine Phosphorylation, 14-3-3ɛ Binding, and Cytoplasmic Sequestration of Heat Shock Factor 1

    PubMed Central

    Wang, XiaoZhe; Grammatikakis, Nicholas; Siganou, Aliki; Calderwood, Stuart K.

    2003-01-01

    Heat shock factor 1 (HSF1) regulates the transcription of molecular chaperone hsp genes. However, the cellular control mechanisms that regulate HSF1 activity are not well understood. In this study, we have demonstrated for the first time that human HSF1 binds to the essential cell signaling protein 14-3-3ɛ. Binding of HSF1 to 14-3-3ɛ occurs in cells in which extracellular signal regulated kinase (ERK) is activated and blockade of the ERK pathway by treatment with the specific ERK pathway inhibitor PD98059 in vivo strongly suppresses the binding. We previously showed that ERK1 phosphorylates HSF1 on serine 307 and leads to secondary phosphorylation by glycogen synthase kinase 3 (GSK3) on serine 303 within the regulatory domain and that these phosphorylation events repress HSF1. We show here that HSF1 binding to 14-3-3ɛ requires HSF1 phosphorylation on serines 303 and 307. Furthermore, the serine phosphorylation-dependent binding of HSF1 to 14-3-3ɛ results in the transcriptional repression of HSF1 and its sequestration in the cytoplasm. Leptomycin B, a specific inhibitor of nuclear export receptor CRM1, was found to reverse the cytoplasmic sequestration of HSF1 mediated by 14-3-3ɛ, suggesting that CRM1/14-3-3ɛ directed nuclear export plays a major role in repression of HSF1 by the ERK/GSK3/14-3-3ɛ pathway. Our experiments indicate a novel pathway for HSF1 regulation and suggest a mechanism for suppression of its activity during cellular proliferation. PMID:12917326

  5. Phosphorylation of Thr-948 at the C terminus of the plasma membrane H(+)-ATPase creates a binding site for the regulatory 14-3-3 protein.

    PubMed Central

    Svennelid, F; Olsson, A; Piotrowski, M; Rosenquist, M; Ottman, C; Larsson, C; Oecking, C; Sommarin, M

    1999-01-01

    The plant plasma membrane H(+)-ATPase is activated by the binding of 14-3-3 protein to the C-terminal region of the enzyme, thus forming an H(+)-ATPase-14-3-3 complex that can be stabilized by the fungal toxin fusicoccin. A novel 14-3-3 binding motif, QQXYpT(948)V, at the C terminus of the H(+)-ATPase is identified and characterized, and the protein kinase activity in the plasma membrane fraction that phosphorylates this threonine residue in the H(+)-ATPase is identified. A synthetic peptide that corresponds to the C-terminal 16 amino acids of the H(+)-ATPase and that is phosphorylated on Thr-948 prevents the in vitro activation of the H(+)-ATPase that is obtained in the presence of recombinant 14-3-3 and fusicoccin. Furthermore, binding of 14-3-3 to the H(+)-ATPase in the absence of fusicoccin is absolutely dependent on the phosphorylation of Thr-948, whereas binding of 14-3-3 in the presence of fusicoccin occurs independently of phosphorylation but still involves the C-terminal motif YTV. Finally, by complementing yeast that lacks its endogenous H(+)-ATPase with wild-type and mutant forms of the Nicotiana plumbaginifolia H(+)-ATPase isoform PMA2, we provide physiological evidence for the importance of the phosphothreonine motif in 14-3-3 binding and, hence, in the activation of the H(+)-ATPase in vivo. Indeed, replacing Thr-948 in the plant H(+)-ATPase with alanine is lethal because this mutant fails to functionally replace the yeast H(+)-ATPase. Considering the importance of the motif QQXYpTV for 14-3-3 binding and yeast growth, this motif should be of vital importance for regulating H(+)-ATPase activity in the plant and thus for plant growth. PMID:10590165

  6. CSF Tau proteins reduce misdiagnosis of sporadic Creutzfeldt-Jakob disease suspected cases with inconclusive 14-3-3 result.

    PubMed

    Leitão, M J; Baldeiras, I; Almeida, M R; Ribeiro, M H; Santos, A C; Ribeiro, M; Tomás, J; Rocha, S; Santana, I; Oliveira, C R

    2016-09-01

    Cerebrospinal fluid (CSF) 14-3-3 protein supports sporadic Creutzfeldt-Jakob (sCJD) diagnosis, but often leads to weak-positive results and lacks standardization. In this study, we explored the added diagnostic value of Total Tau (t-Tau) and phosphorylated Tau (p-Tau) in sCJD diagnosis, particularly in the cases with inconclusive 14-3-3 result. 95 definite sCJD and 287 patients without prion disease (non-CJD) were included in this study. CSF samples were collected in routine clinical diagnosis and analysed for 14-3-3 detection by Western blot (WB). CSF t-Tau and p-Tau were quantified by commercial ELISA kits and PRNP and APOE genotyping assessed by PCR-RFLP. In a regression analysis of the whole cohort, 14-3-3 protein revealed an overall accuracy of 82 % (sensitivity = 96.7 %; specificity = 75.6 %) for sCJD. Regarding 14-3-3 clear positive results, we observed no added value either of t-Tau alone or p-Tau/t-Tau ratio in the model. On the other hand, considering 14-3-3 weak-positive cases, t-Tau protein increased the overall accuracy of 14-3-3 alone from 91 to 94 % and specificity from 74 to 93 % (p < 0.05), with no sensitivity improvement. However, inclusion of p-Tau/t-Tau ratio did not significantly improve the first model (p = 0.0595). Globally, t-Tau protein allowed a further discrimination of 65 % within 14-3-3 inconclusive results. Furthermore, PRNP MV genotype showed a trend to decrease 14-3-3 sensitivity (p = 0.051), but such effect was not seen on t-Tau protein. In light of these results, we suggest that t-Tau protein assay is of significant importance as a second marker in identifying 14-3-3 false-positive results among sCJD probable cases. PMID:27357003

  7. Analysis of the cruciform binding activity of recombinant 14-3-3zeta-MBP fusion protein, its heterodimerization profile with endogenous 14-3-3 isoforms, and effect on mammalian DNA replication in vitro.

    PubMed

    Alvarez, David; Callejo, Mario; Shoucri, Rami; Boyer, Lee; Price, Gerald B; Zannis-Hadjopoulos, Maria

    2003-06-17

    The human cruciform binding protein (CBP), a member of the 14-3-3 protein family, has been recently identified as an origin of DNA replication binding protein and involved in DNA replication. Here, pure recombinant 14-3-3zeta tagged with maltose binding protein (r14-3-3zeta-MBP) at its N-terminus was tested for binding to cruciform DNA either in the absence or presence of F(TH), a CBP-enriched fraction, by electromobility shift assay (EMSA), followed by Western blot analysis of the electroeluted CBP-cruciform DNA complex. The r14-3-3zeta-MBP was found to have cruciform binding activity only after preincubation with F(TH). Anti-MBP antibody immunoprecipitation of F(TH) preincubated with r14-3-3zeta-MBP, followed by Western blot analysis with antibodies specific to the beta, gamma, epsilon, zeta, and sigma 14-3-3 isoforms showed that r14-3-3zeta-MBP heterodimerized with the endogenous beta, epsilon, and zeta isoforms present in the F(TH) but not with the gamma or sigma isoforms. Immunoprecipitation of endogenous 14-3-3zeta from nuclear extracts (NE) of HeLa cells that were either serum-starved (s-s) or blocked at the G(1)/S or G(2)/M phases of the cell cycle revealed that at G(1)/S and G(2)/M, the zeta isoform heterodimerized only with the beta and epsilon isoforms, while in s-s extracts, the 14-3-3zeta/epsilon heterodimer was never detected, and the 14-3-3zeta/beta heterodimer was seldom detected. Furthermore, addition of r14-3-3zeta-MBP to HeLa cell extracts used in a mammalian in vitro replication system increased the replication level of p186, a plasmid bearing the minimal 186-bp origin of the monkey origin of DNA replication ors8, by approximately 3.5-fold. The data suggest that specific dimeric combinations of the 14-3-3 isoforms have CBP activity and that upregulation of this activity leads to an increase in DNA replication. PMID:12795617

  8. 14-3-3 isoforms bind directly exon B of the 5′-UTR of human surfactant protein A2 mRNA

    PubMed Central

    Noutsios, Georgios T.; Ghattas, Paul; Bennett, Stephanie

    2015-01-01

    Human surfactant protein (SP) A (SP-A), an innate immunity molecule, is encoded by two genes, SFTPA1 and SFTPA2. The 5′-untranslated splice variant of SP-A2 (ABD), but not SP-A1 (AD), contains exon B (eB). eB is an enhancer for transcription and translation and contains cis-regulatory elements. Specific trans-acting factors, including 14-3-3, bind eB. The 14-3-3 protein family contains seven isoforms that have been found by mass spectrometry in eB electromobility shift assays (Noutsios et al. Am J Physiol Lung Cell Mol Physiol 304: L722–L735, 2013). We used four different approaches to investigate whether 14-3-3 isoforms bind directly to eB. 1) eB RNA pulldown assays showed that 14-3-3 isoforms specifically bind eB. 2) RNA electromobility shift assay complexes were formed using purified 14-3-3 isoforms β, γ, ε, η, σ, and τ, but not isoform ζ, with wild-type eB RNA. 3 and 4) RNA affinity chromatography assays and surface plasmon resonance analysis showed that 14-3-3 isoforms β, γ, ε, η, σ, and τ, but not isoform ζ, specifically and directly bind eB. Inhibition of 14-3-3 isoforms γ, ε, η, and τ/θ with shRNAs in NCI-H441 cells resulted in downregulation of SP-A2 levels but did not affect SP-A1 levels. However, inhibition of 14-3-3 isoform σ was correlated with lower levels of SP-A1 and SP-A2. Inhibition of 14-3-3 isoform ζ/δ, which does not bind eB, had no effect on expression levels of SP-A1 and SP-A2. In conclusion, the 14-3-3 protein family affects differential regulation of SP-A1 and SP-A2 by binding directly to SP-A2 5′-UTR mRNA. PMID:26001776

  9. Klotho Regulates 14-3-3ζ Monomerization and Binding to the ASK1 Signaling Complex in Response to Oxidative Stress

    PubMed Central

    Brobey, Reynolds K.; Dheghani, Mehdi; Foster, Philip P.; Kuro-o, Makoto; Rosenblatt, Kevin P

    2015-01-01

    The reactive oxygen species (ROS)-sensitive apoptosis signal-regulating kinase 1 (ASK1) signaling complex is a key regulator of p38 MAPK activity, a major modulator of stress-associated with aging disorders. We recently reported that the ratio of free ASK1 to the complex-bound ASK1 is significantly decreased in Klotho-responsive manner and that Klotho-deficient tissues have elevated levels of free ASK1 which coincides with increased oxidative stress. Here, we tested the hypothesis that: 1) covalent interactions exist among three identified proteins constituting the ASK1 signaling complex; 2) in normal unstressed cells the ASK1, 14-3-3ζ and thioredoxin (Trx) proteins simultaneously engage in a tripartite complex formation; 3) Klotho’s stabilizing effect on the complex relied solely on 14-3-3ζ expression and its apparent phosphorylation and dimerization changes. To verify the hypothesis, we performed 14-3-3ζ siRNA knock-down experiments in conjunction with cell-based assays to measure ASK1-client protein interactions in the presence and absence of Klotho, and with or without an oxidant such as rotenone. Our results show that Klotho activity induces posttranslational modifications in the complex targeting 14-3-3ζ monomer/dimer changes to effectively protect against ASK1 oxidation and dissociation. This is the first observation implicating all three proteins constituting the ASK1 signaling complex in close proximity. PMID:26517365

  10. The Saccharomyces cerevisiae 14-3-3 proteins are required for the G1/S transition, actin cytoskeleton organization and cell wall integrity.

    PubMed

    Lottersberger, Francisca; Panza, Andrea; Lucchini, Giovanna; Piatti, Simonetta; Longhese, Maria Pia

    2006-06-01

    14-3-3 proteins are highly conserved polypeptides that participate in many biological processes by binding phosphorylated target proteins. The Saccharomyces cerevisiae BMH1 and BMH2 genes, whose concomitant deletion is lethal, encode two functionally redundant 14-3-3 isoforms. To gain insights into the essential function(s) shared by these proteins, we searched for high-dosage suppressors of the growth defects of temperature-sensitive bmh mutants. Both the protein kinase C1 (Pkc1) and its upstream regulators Wsc2 and Mid2 were found to act as high dosage suppressors of bmh mutants' temperature sensitivity, indicating a functional interaction between 14-3-3 and Pkc1. Consistent with a role of 14-3-3 proteins in Pkc1-dependent cellular processes, shift to the restrictive temperature of bmh mutants severely impaired initiation of DNA replication, polarization of the actin cytoskeleton, and budding, as well as cell wall integrity. Because Pkc1 acts in concert with the Swi4-Swi6 (SBF) transcriptional activator to control all these processes, the defective G(1)/S transition of bmh mutants might be linked to impaired SBF activity. Indeed, the levels of the G(1) cyclin CLN2 transcripts, which are positively regulated by SBF, were dramatically reduced in bmh mutants. Remarkably, budding and DNA replication defects of bmh mutants were suppressed by CLN2 expression from an SBF-independent promoter, suggesting that 14-3-3 proteins might contribute to regulating the late G(1) transcriptional program. PMID:16648583

  11. Identification of a host 14-3-3 Protein that Interacts with Xanthomonas effector AvrRxv

    Technology Transfer Automated Retrieval System (TEKTRAN)

    AvrRxv is a member of a family of pathogen effectors from both plant and mammalian pathogens. Using a yeast two hybrid screen, we identified a 14-3-3 protein from tomato that interacts with AvrRxv called AvrRxv Interactor 1 (ARI1). The interaction was confirmed in vitro with affinity chromatograph...

  12. 14-3-3γ Regulates Lipopolysaccharide-Induced Inflammatory Responses and Lactation in Dairy Cow Mammary Epithelial Cells by Inhibiting NF-κB and MAPKs and Up-Regulating mTOR Signaling

    PubMed Central

    Liu, Lixin; Lin, Ye; Liu, Lili; Bian, Yanjie; Zhang, Li; Gao, Xuejun; Li, Qingzhang

    2015-01-01

    As a protective factor for lipopolysaccharide (LPS)-induced injury, 14-3-3γ has been the subject of recent research. Nevertheless, whether 14-3-3γ can regulate lactation in dairy cow mammary epithelial cells (DCMECs) induced by LPS remains unknown. Here, the anti-inflammatory effect and lactation regulating ability of 14-3-3γ in LPS-induced DCMECs are investigated for the first time, and the molecular mechanisms responsible for their effects are explored. The results of qRT-PCR showed that 14-3-3γ overexpression significantly inhibited the mRNA expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS). Enzyme-linked immunosorbent assay (ELISA) analysis revealed that 14-3-3γ overexpression also suppressed the production of TNF-α and IL-6 in cell culture supernatants. Meanwhile, CASY-TT Analyser System showed that 14-3-3γ overexpression clearly increased the viability and proliferation of cells. The results of kit methods and western blot analysis showed that 14-3-3γ overexpression promoted the secretion of triglycerides and lactose and the synthesis of β-casein. Furthermore, the expression of genes relevant to nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPKs) and lactation-associated proteins were assessed by western blot, and the results suggested that 14-3-3γ overexpression inactivated the NF-κB and MAPK signaling pathways by down-regulating extracellular signal regulated protein kinase (ERK), p38 mitogen-activated protein kinase (p38MAPK) and inhibitor of NF-κB (IκB) phosphorylation levels, as well as by inhibiting NF-κB translocation. Meanwhile, 14-3-3γ overexpression enhanced the expression levels of β-casein, mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase 1 (S6K1), serine/threonine protein kinase Akt 1 (AKT1), sterol regulatory element binding protein 1 (SREBP1) and peroxisome proliferator-activated receptor gamma (PPAR

  13. Proteomics Profiling Reveals Carbohydrate Metabolic Enzymes and 14-3-3 Proteins Play Important Roles for Starch Accumulation during Cassava Root Tuberization.

    PubMed

    Wang, Xuchu; Chang, Lili; Tong, Zheng; Wang, Dongyang; Yin, Qi; Wang, Dan; Jin, Xiang; Yang, Qian; Wang, Liming; Sun, Yong; Huang, Qixing; Guo, Anping; Peng, Ming

    2016-01-01

    Cassava is one of the most important root crops as a reliable source of food and carbohydrates. Carbohydrate metabolism and starch accumulation in cassava storage root is a cascade process that includes large amounts of proteins and cofactors. Here, comparative proteomics were conducted in cassava root at nine developmental stages. A total of 154 identified proteins were found to be differentially expressed during starch accumulation and root tuberization. Many enzymes involved in starch and sucrose metabolism were significantly up-regulated, and functional classification of the differentially expressed proteins demonstrated that the majority were binding-related enzymes. Many proteins were took part in carbohydrate metabolism to produce energy. Among them, three 14-3-3 isoforms were induced to be clearly phosphorylated during storage root enlargement. Overexpression of a cassava 14-3-3 gene in Arabidopsis thaliana confirmed that the older leaves of these transgenic plants contained higher sugar and starch contents than the wild-type leaves. The 14-3-3 proteins and their binding enzymes may play important roles in carbohydrate metabolism and starch accumulation during cassava root tuberization. These results not only deepened our understanding of the tuberous root proteome, but also uncovered new insights into carbohydrate metabolism and starch accumulation during cassava root enlargement. PMID:26791570

  14. Proteomics Profiling Reveals Carbohydrate Metabolic Enzymes and 14-3-3 Proteins Play Important Roles for Starch Accumulation during Cassava Root Tuberization

    PubMed Central

    Wang, Xuchu; Chang, Lili; Tong, Zheng; Wang, Dongyang; Yin, Qi; Wang, Dan; Jin, Xiang; Yang, Qian; Wang, Liming; Sun, Yong; Huang, Qixing; Guo, Anping; Peng, Ming

    2016-01-01

    Cassava is one of the most important root crops as a reliable source of food and carbohydrates. Carbohydrate metabolism and starch accumulation in cassava storage root is a cascade process that includes large amounts of proteins and cofactors. Here, comparative proteomics were conducted in cassava root at nine developmental stages. A total of 154 identified proteins were found to be differentially expressed during starch accumulation and root tuberization. Many enzymes involved in starch and sucrose metabolism were significantly up-regulated, and functional classification of the differentially expressed proteins demonstrated that the majority were binding-related enzymes. Many proteins were took part in carbohydrate metabolism to produce energy. Among them, three 14-3-3 isoforms were induced to be clearly phosphorylated during storage root enlargement. Overexpression of a cassava 14-3-3 gene in Arabidopsis thaliana confirmed that the older leaves of these transgenic plants contained higher sugar and starch contents than the wild-type leaves. The 14-3-3 proteins and their binding enzymes may play important roles in carbohydrate metabolism and starch accumulation during cassava root tuberization. These results not only deepened our understanding of the tuberous root proteome, but also uncovered new insights into carbohydrate metabolism and starch accumulation during cassava root enlargement. PMID:26791570

  15. Proteomic analysis of human norepinephrine transporter complexes reveals associations with protein phosphatase 2A anchoring subunit and 14-3-3 proteins

    SciTech Connect

    Sung, Uhna; Jennings, Jennifer L.; Link, Andrew J.; Blakely, Randy D.; E-mail: andy.blakely@vanderbilt.edu

    2005-08-05

    The norepinephrine transporter (NET) terminates noradrenergic signals by clearing released NE at synapses. NET regulation by receptors and intracellular signaling pathways is supported by a growing list of associated proteins including syntaxin1A, protein phosphatase 2A (PP2A) catalytic subunit (PP2A-C), PICK1, and Hic-5. In the present study, we sought evidence for additional partnerships by mass spectrometry-based analysis of proteins co-immunoprecipitated with human NET (hNET) stably expressed in a mouse noradrenergic neuroblastoma cell line. Our initial proteomic analyses reveal multiple peptides derived from hNET, peptides arising from the mouse PP2A anchoring subunit (PP2A-Ar) and peptides derived from 14-3-3 proteins. We verified physical association of NET with PP2A-Ar via co-immunoprecipitation studies using mouse vas deferens extracts and with 14-3-3 via a fusion pull-down approach, implicating specifically the hNET NH{sub 2}-terminus for interactions. The transporter complexes described likely support mechanisms regulating transporter activity, localization, and trafficking.

  16. ISOFORM-SPECIFIC BINDING OF 14-3-3 PROTEINS TO NITRATE REDUCTASE AND THE BRASSINOSTEROID INSENSITIVE 1 RECEPTOR KINASE SIGNALING COMPLEX

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The 14-3-3 proteins are known to bind many different soluble protein clients, but less is known about binding to integral membrane proteins, and in both cases the issue of isoform specificity remains largely unexplored. Using an array of anti-14-3-3 antibodies and 2-dimensional electrophoresis (2-DE...

  17. 14-3-3-Pred: improved methods to predict 14-3-3-binding phosphopeptides

    PubMed Central

    Madeira, Fábio; Tinti, Michele; Murugesan, Gavuthami; Berrett, Emily; Stafford, Margaret; Toth, Rachel; Cole, Christian; MacKintosh, Carol; Barton, Geoffrey J.

    2015-01-01

    Motivation: The 14-3-3 family of phosphoprotein-binding proteins regulates many cellular processes by docking onto pairs of phosphorylated Ser and Thr residues in a constellation of intracellular targets. Therefore, there is a pressing need to develop new prediction methods that use an updated set of 14-3-3-binding motifs for the identification of new 14-3-3 targets and to prioritize the downstream analysis of >2000 potential interactors identified in high-throughput experiments. Results: Here, a comprehensive set of 14-3-3-binding targets from the literature was used to develop 14-3-3-binding phosphosite predictors. Position-specific scoring matrix, support vector machines (SVM) and artificial neural network (ANN) classification methods were trained to discriminate experimentally determined 14-3-3-binding motifs from non-binding phosphopeptides. ANN, position-specific scoring matrix and SVM methods showed best performance for a motif window spanning from −6 to +4 around the binding phosphosite, achieving Matthews correlation coefficient of up to 0.60. Blind prediction showed that all three methods outperform two popular 14-3-3-binding site predictors, Scansite and ELM. The new methods were used for prediction of 14-3-3-binding phosphosites in the human proteome. Experimental analysis of high-scoring predictions in the FAM122A and FAM122B proteins confirms the predictions and suggests the new 14-3-3-predictors will be generally useful. Availability and implementation: A standalone prediction web server is available at http://www.compbio.dundee.ac.uk/1433pred. Human candidate 14-3-3-binding phosphosites were integrated in ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome database. Contact: cmackintosh@dundee.ac.uk or gjbarton@dundee.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25735772

  18. The EFF-1A Cytoplasmic Domain Influences Hypodermal Cell Fusions in C. elegans But Is Not Dependent on 14-3-3 Proteins

    PubMed Central

    Shinn-Thomas, Jessica H.; del Campo, Jacob J.; Wang, Jianjun; Mohler, William A.

    2016-01-01

    Background Regulatory and biophysical mechanisms of cell-cell fusion are largely unknown despite the fundamental requirement for fused cells in eukaryotic development. Only two cellular fusogens that are not of clear recent viral origin have been identified to date, both in nematodes. One of these, EFF-1, is necessary for most cell fusions in Caenorhabditis elegans. Unregulated EFF-1 expression causes lethality due to ectopic fusion between cells not developmentally programmed to fuse, highlighting the necessity of tight fusogen regulation for proper development. Identifying factors that regulate EFF-1 and its paralog AFF-1 could lead to discovery of molecular mechanisms that control cell fusion upstream of the action of a membrane fusogen. Bioinformatic analysis of the EFF-1A isoform’s predicted cytoplasmic domain (endodomain) previously revealed two motifs that have high probabilities of interacting with 14-3-3 proteins when phosphorylated. Mutation of predicted phosphorylation sites within these motifs caused measurable loss of eff-1 gene function in cell fusion in vivo. Moreover, a human 14-3-3 isoform bound to EFF-1::GFP in vitro. We hypothesized that the two 14-3-3 proteins in C. elegans, PAR-5 and FTT-2, may regulate either localization or fusion-inducing activity of EFF-1. Methodology/Principal Findings Timing of fusion events was slightly but significantly delayed in animals unable to produce full-length EFF-1A. Yet, mutagenesis and live imaging showed that phosphoserines in putative 14-3-3 binding sites are not essential for EFF-1::GFP accumulation at the membrane contact between fusion partner cells. Moreover, although the EFF-1A endodomain was required for normal rates of eff-1-dependent epidermal cell fusions, reduced levels of FTT-2 and PAR-5 did not visibly affect the function of wild-type EFF-1 in the hypodermis. Conclusions/Significance Deletion of the EFF-1A endodomain noticeably affects the timing of hypodermal cell fusions in vivo. However

  19. A Glycine soja 14-3-3 protein GsGF14o participates in stomatal and root hair development and drought tolerance in Arabidopsis thaliana.

    PubMed

    Sun, Xiaoli; Luo, Xiao; Sun, Mingzhe; Chen, Chao; Ding, Xiaodong; Wang, Xuedong; Yang, Shanshan; Yu, Qingyue; Jia, Bowei; Ji, Wei; Cai, Hua; Zhu, Yanming

    2014-01-01

    It is well established that 14-3-3 proteins are key regulators of multiple stress signal transduction cascades. However, the biological functions of soybean 14-3-3 proteins, especially in plant drought response, are not yet known. In this study, we characterized a Glycine soja 14-3-3 gene, GsGF14o, which is involved in plant development and drought response. GsGF14o expression was greatly induced by drought stress, as evidenced by the quantitative real-time PCR and β-glucuronidase (GUS) activity analysis. GsGF14o overexpression in Arabidopsis thaliana resulted in decreased drought tolerance during seed germination and seedling growth. Furthermore, silencing of AtGF14µ, the most homologous 14-3-3 gene of GsGF14o, led to enhanced drought tolerance at both the seed germination and seedling stage. Unexpectedly, GsGF14o transgenic lines showed reduced water loss and transpiration rates compared with wild-type plants, which was demonstrated to be the consequence of the decreased stomatal size. At the same time, the smaller stomata due to GsGF14o overexpression led to a relatively slow net photosynthesis rate, which led to a growth penalty under drought stress. We further demonstrated that GsGF14o overexpression caused deficits in root hair formation and development, and thereby reduced the water intake capacity of the transgenic root system. In addition, GsGF14o overexpression down-regulated the transcript levels of drought-responsive marker genes. Finally, we also investigated the tissue-specific accumulation of GsGF14o by using a GUS activity assay. Collectively, the results presented here confirm that GsGF14o plays a dual role in drought stress responses through its involvement in the regulation of stomatal size and root hair development. PMID:24272249

  20. Arsenite Stress Down-regulates Phosphorylation and 14-3-3 Binding of Leucine-rich Repeat Kinase 2 (LRRK2), Promoting Self-association and Cellular Redistribution*

    PubMed Central

    Mamais, Adamantios; Chia, Ruth; Beilina, Alexandra; Hauser, David N.; Hall, Christine; Lewis, Patrick A.; Cookson, Mark R.; Bandopadhyay, Rina

    2014-01-01

    Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are a common genetic cause of Parkinson disease, but the mechanisms whereby LRRK2 is regulated are unknown. Phosphorylation of LRRK2 at Ser910/Ser935 mediates interaction with 14-3-3. Pharmacological inhibition of its kinase activity abolishes Ser910/Ser935 phosphorylation and 14-3-3 binding, and this effect is also mimicked by pathogenic mutations. However, physiological situations where dephosphorylation occurs have not been defined. Here, we show that arsenite or H2O2-induced stresses promote loss of Ser910/Ser935 phosphorylation, which is reversed by phosphatase inhibition. Arsenite-induced dephosphorylation is accompanied by loss of 14-3-3 binding and is observed in wild type, G2019S, and kinase-dead D2017A LRRK2. Arsenite stress stimulates LRRK2 self-association and association with protein phosphatase 1α, decreases kinase activity and GTP binding in vitro, and induces translocation of LRRK2 to centrosomes. Our data indicate that signaling events induced by arsenite and oxidative stress may regulate LRRK2 function. PMID:24942733

  1. 14-3-3σ Gene Loss Leads to Activation of the Epithelial to Mesenchymal Transition Due to the Stabilization of c-Jun Protein.

    PubMed

    Raychaudhuri, Kumarkrishna; Chaudhary, Neelam; Gurjar, Mansa; D'Souza, Roseline; Limzerwala, Jazeel; Maddika, Subbareddy; Dalal, Sorab N

    2016-07-29

    Loss of 14-3-3σ has been observed in multiple tumor types; however, the mechanisms by which 14-3-3σ loss leads to tumor progression are not understood. The experiments in this report demonstrate that loss of 14-3-3σ leads to a decrease in the expression of epithelial markers and an increase in the expression of mesenchymal markers, which is indicative of an induction of the epithelial to mesenchymal transition (EMT). The EMT was accompanied by an increase in migration and invasion in the 14-3-3σ(-/-) cells. 14-3-3σ(-/-) cells show increased stabilization of c-Jun, resulting in an increase in the expression of the EMT transcription factor slug. 14-3-3σ induces the ubiquitination and degradation of c-Jun in an FBW7-dependent manner. c-Jun ubiquitination is dependent on the presence of an intact nuclear export pathway as c-Jun is stabilized and localized to the nucleus in the presence of a nuclear export inhibitor. Furthermore, the absence of 14-3-3σ leads to the nuclear accumulation and stabilization of c-Jun, suggesting that 14-3-3σ regulates the subcellular localization of c-Jun. Our results have identified a novel mechanism by which 14-3-3σ maintains the epithelial phenotype by inhibiting EMT and suggest that this property of 14-3-3σ might contribute to its function as a tumor suppressor gene. PMID:27261462

  2. Phosphate differentially regulates 14-3-3 family members and GRF9 plays a role in Pi-starvation induced responses.

    PubMed

    Cao, Aiqin; Jain, Ajay; Baldwin, James C; Raghothama, Kashchandra G

    2007-10-01

    The 14-3-3s are phosphoserine-binding proteins that act as key regulators of many metabolic pathways. Several biotic and abiotic stresses have been shown to modulate the expression of 14-3-3 genes. In Arabidopsis thaliana, 15 genes are known to code for 14-3-3 isoforms belonging to epsilon and non-epsilon groups. Since phosphorus is one of the essential macronutrients for plants, we examined its role in the regulation of the expression of 14-3-3 isoforms belonging to epsilon (GRF9, GRF10, GRF11, GRF13) and non-epsilon (GRF1, GRF3, GRF6, GRF8) groups. The effect of Pi deprivation was differential on the members of non-epsilon group ranging from a significant reduction in the transcripts of GRF3 to non-perceptible changes in the transcripts of other members. Suppressive effect of Pi-deficiency was more pronounced on some of the members of epsilon group with transcripts levels of GRF9 and GRF13 barely detectable. A concurrent increase in the transcript levels of GRF9 with an increase in the Pi concentration suggested a correlation between gene expression and Pi availability. However, neither Pi deficiency at low temperature nor Fe and K deficiency failed to suppress GRF9 expression. In planta role of GRF9 was elucidated by the analysis of the loss-of-function mutant under Pi-replete condition. The analyses revealed exaggerated Pi-starvation responses in the form of starch accumulation in the leaves and modulated root system architecture (RSA). An inverse relationship between the abundance of GRF9 transcripts and accumulation of starch in transgenic lines over-expressing this gene provided further evidence towards the role of GRF9 in modulation of metabolic pathways during Pi-starvation responses. PMID:17598127

  3. Controllability of protein-protein interaction phosphorylation-based networks: Participation of the hub 14-3-3 protein family.

    PubMed

    Uhart, Marina; Flores, Gabriel; Bustos, Diego M

    2016-01-01

    Posttranslational regulation of protein function is an ubiquitous mechanism in eukaryotic cells. Here, we analyzed biological properties of nodes and edges of a human protein-protein interaction phosphorylation-based network, especially of those nodes critical for the network controllability. We found that the minimal number of critical nodes needed to control the whole network is 29%, which is considerably lower compared to other real networks. These critical nodes are more regulated by posttranslational modifications and contain more binding domains to these modifications than other kinds of nodes in the network, suggesting an intra-group fast regulation. Also, when we analyzed the edges characteristics that connect critical and non-critical nodes, we found that the former are enriched in domain-to-eukaryotic linear motif interactions, whereas the later are enriched in domain-domain interactions. Our findings suggest a possible structure for protein-protein interaction networks with a densely interconnected and self-regulated central core, composed of critical nodes with a high participation in the controllability of the full network, and less regulated peripheral nodes. Our study offers a deeper understanding of complex network control and bridges the controllability theorems for complex networks and biological protein-protein interaction phosphorylation-based networked systems. PMID:27195976

  4. Controllability of protein-protein interaction phosphorylation-based networks: Participation of the hub 14-3-3 protein family

    PubMed Central

    Uhart, Marina; Flores, Gabriel; Bustos, Diego M.

    2016-01-01

    Posttranslational regulation of protein function is an ubiquitous mechanism in eukaryotic cells. Here, we analyzed biological properties of nodes and edges of a human protein-protein interaction phosphorylation-based network, especially of those nodes critical for the network controllability. We found that the minimal number of critical nodes needed to control the whole network is 29%, which is considerably lower compared to other real networks. These critical nodes are more regulated by posttranslational modifications and contain more binding domains to these modifications than other kinds of nodes in the network, suggesting an intra-group fast regulation. Also, when we analyzed the edges characteristics that connect critical and non-critical nodes, we found that the former are enriched in domain-to-eukaryotic linear motif interactions, whereas the later are enriched in domain-domain interactions. Our findings suggest a possible structure for protein-protein interaction networks with a densely interconnected and self-regulated central core, composed of critical nodes with a high participation in the controllability of the full network, and less regulated peripheral nodes. Our study offers a deeper understanding of complex network control and bridges the controllability theorems for complex networks and biological protein-protein interaction phosphorylation-based networked systems. PMID:27195976

  5. Spinach 14-3-3 protein interacts with the plasma membrane H(+)-ATPase and nitrate reductase in response to excess nitrate stress.

    PubMed

    Xu, Huini; Zhao, Xiuling; Guo, Chuanlong; Chen, Limei; Li, Kunzhi

    2016-09-01

    To investigate the function of 14-3-3 protein in response to excess nitrate stress, a 14-3-3 protein, designated as So14-3-3, was isolated from spinach. Phylogenetic analysis demonstrated that So14-3-3 belongs to non-ε group of 14-3-3 superfamily. Real time-quantitative RT-PCR and western blot analysis showed that So14-3-3 was induced by excess nitrate stress in spinach roots and leaves. After nitrate treatment, the phosphorylated H(+)-ATPase and nitrate reductase (NR) increased and decreased respectively. Co-Immunoprecipitation (Co-IP) suggested that the interaction of So14-3-3 with the phosphorylated H(+)-ATPase enhanced, but reduced with phosphorylated NR in spinach roots after nitrate treatment. Besides, 5 proteins interacted with So14-3-3 were found by Co-IP and LC-MS/MS analysis. So14-3-3 overexpressing transgenic tobacco plants showed enhanced tolerance to nitrate treatment at the germination and young seedlings stage. The transgenic plants showed longer root length, lower malondialdehyde (MDA), H2O2, protein carbonyl contents, relatively higher soluble sugar and protein contents, than the WT plants after nitrate treatment. The phosphorylation levels of H(+)-ATPase in transgenic plants were higher than the WT plants after nitrate treatment, whereas NR were lower. Additionally, in transgenic plants, the interaction of So14-3-3 with phosphorylated H(+)-ATPase and NR increased and decreased more than the WT plants under nitrate stress, leading to higher H(+)-ATPase and NR activities in transgenic plants. These data suggested that So14-3-3 might be involved in nitrate stress response by interacting with H(+)-ATPase and NR. PMID:27161584

  6. Down-regulation of 14-3-3β exerts anti-cancer effects through inducing ER stress in human glioma U87 cells: Involvement of CHOP–Wnt pathway

    SciTech Connect

    Cao, Lei; Lei, Hui; Chang, Ming-Ze; Liu, Zhi-Qin; Bie, Xiao-Hua

    2015-07-10

    We previously identified 14-3-3β as a tumor-specific isoform of 14-3-3 protein in astrocytoma, but its functional role in glioma cells and underlying mechanisms are poorly understood. In the present study, we investigated the effects of 14-3-3β inhibition in human glioma U87 cells using specific targeted small interfering RNA (siRNA). The results showed that 14-3-3β is highly expressed in U87 cells but not in normal astrocyte SVGp12 cells. Knockdown of 14-3-3β by Si-14-3-3β transfection significantly decreased the cell viability but increased the LDH release in a time-dependent fashion in U87 cells, and these effects were accompanied with G0/G1 cell cycle arrest and apoptosis. In addition, 14-3-3β knockdown induced ER stress in U87 cells, as evidenced by ER calcium release, increased expression of XBP1S mRNA and induction of ER related pro-apoptotic factors. Down-regulation of 14-3-3β significantly decreased the nuclear localization of β-catenin and inhibited Topflash activity, which was shown to be reversely correlated with CHOP. Furthermore, Si-CHOP and sFRP were used to inhibit CHOP and Wnt, respectively. The results showed that the anti-cancer effects of 14-3-3β knockdown in U87 cells were mediated by increased expression of CHOP and followed inhibition of Wnt/β-catenin pathway. In summary, the remarkable efficiency of 14-3-3β knockdown to induce apoptotic cell death in U87 cells may find therapeutic application for the treatment of glioma patients. - Highlights: • Knockdown of 14-3-3β leads to cytotoxicity in human glioma U87 cells. • Knockdown of 14-3-3β induces cell cycle arrest and apoptosis in U87 cells. • Knockdown of 14-3-3β results in ER stress in U87 cells. • Knockdown of 14-3-3β inhibits Wnt/β-catenin pathway via CHOP activation.

  7. Multiple elements regulate nuclear/cytoplasmic shuttling of FOXO1: characterization of phosphorylation- and 14-3-3-dependent and -independent mechanisms.

    PubMed Central

    Zhao, Xiangshan; Gan, Lixia; Pan, Haiyun; Kan, Donghui; Majeski, Michael; Adam, Stephen A; Unterman, Terry G

    2004-01-01

    FOXO1, a Forkhead transcription factor, is an important target of insulin and growth factor action. Phosphorylation of Thr-24, Ser-256 and Ser-319 promotes nuclear exclusion of FOXO1, yet the mechanisms regulating nuclear/cytoplasmic shuttling of FOXO1 are poorly understood. Previous studies have identified an NLS (nuclear localization signal) in the C-terminal basic region of the DBD (DNA-binding domain), and a leucine-rich, leptomycin-B sensitive NES (nuclear export signal) located further downstream. Here, we find that other elements in the DBD also contribute to nuclear localization, and that multiple mechanisms contribute to nuclear exclusion of FOXO1. Phosphorylation of Ser-319 and a cluster of nearby residues (Ser-322, Ser-325 and Ser-329) functions co-operatively with the nearby NES to promote nuclear exclusion. The N-terminal region of FOXO1 (amino acids 1-149) also is sufficient to promote nuclear exclusion, and does so through multiple mechanisms. Amino acids 1-50 are sufficient to promote nuclear exclusion of green fluorescent protein fusion proteins, and the phosphorylation of Thr-24 is required for this effect. A leucine-rich, leptomycin B-sensitive export signal is also present nearby. Phosphorylated FOXO1 binds 14-3-3 proteins, and co-precipitation studies with tagged proteins indicate that 14-3-3 binding involves co-operative interactions with both Thr-24 and Ser-256. Ser-256 is located in the C-terminal region of the DBD, where 14-3-3 proteins may interfere both with DNA-binding and with nuclear-localization functions. Together, these studies demonstrate that multiple elements contribute to nuclear/cytoplasmic shuttling of FOXO1, and that phosphorylation and 14-3-3 binding regulate the cellular distribution and function of FOXO1 through multiple mechanisms. The presence of these redundant mechanisms supports the concept that the regulation of FOXO1 function plays a critical role in insulin and growth factor action. PMID:14664696

  8. Role for the PP2A/B56delta phosphatase in regulating 14-3-3 release from Cdc25 to control mitosis

    PubMed Central

    Margolis, Seth S.; Perry, Jennifer A.; Forester, Craig M.; Nutt, Leta K.; Guo, Yanxiang; Jardim, Melanie J.; Thomenius, Michael J.; Freel, Christopher D.; Darbandi, Rashid; Ahn, Jung-Hyuck; Arroyo, Jason D.; Wang, Xiao-Fan; Shenolikar, Shirish; Nairn, Angus C.; Dunphy, William G.; Hahn, William C.; Virshup, David M.; Kornbluth, Sally

    2009-01-01

    Summary DNA-responsive checkpoints prevent cell cycle progression following DNA damage or replication inhibition. The mitotic activator Cdc25 is suppressed by checkpoints through inhibitory phosphorylation at Ser287 (Xenopus numbering) and docking of 14-3-3. S287 phosphorylation is a major locus of G2/M checkpoint control, though several checkpoint-independent kinases can phosphorylate this site. We reported previously that mitotic entry requires 14-3-3 removal and S287 dephosphorylation. We show here that DNA-responsive checkpoints activate PP2A/B56δ phosphatase complexes to dephosphorylate Cdc25 at a site (T138) whose phosphorylation is required for 14-3-3 release. However, phosphorylation of T138 is not sufficient for 14-3-3 release from Cdc25. Rather, our data suggest that creation of a 14-3-3 “sink”, consisting of phosphorylated 14-3-3-binding intermediate filament proteins, coupled with reduced Cdc25-14-3-3 affinity, contribute to Cdc25 activation. These observations identify PP2A/B56δ as a central checkpoint effector, and suggest a mechanism for controlling 14-3-3 interactions to promote mitosis. PMID:17110335

  9. Regulation of Aldo-keto-reductase family 1 B10 by 14-3-3ε and their prognostic impact of hepatocellular carcinoma

    PubMed Central

    Lu, Yi-Jhu; Liang, Shu-Man; Liu, Chia-Chia; Chen, Shyh-Chang; Wang, John; Shyue, Song-Kun; Liou, Jun-Yang

    2015-01-01

    14-3-3ε is overexpressed in hepatocellular carcinoma (HCC) and its expression significantly associates with a poor prognostic outcome. To uncover how 14-3-3ε contributes to the tumor progression of HCC, we investigated the potential downstream targets regulated by 14-3-3ε. We found that 14-3-3ε increases expression and nuclear translocation of β-catenin and that 14-3-3ε-induced cell proliferation is attenuated by β-catenin silencing in HCC cells. Moreover, 14-3-3ε induces aldo-keto reductase family 1 member B10 (AKR1B10) expression through the activation of β-catenin signaling. Knockdown of AKR1B10 by siRNAs abolished 14-3-3ε-induced in vitro cell proliferation, anchorage-independent growth as well as in vivo tumor growth. Furthermore, AKR1B10 silencing increased retinoic acid (RA) levels in the serum of tumor-bearing mice and RA treatment attenuated 14-3-3ε-induced HCC cell proliferation. We further examined 14-3-3ε and AKR1B10 expression and clinicopathological characteristics of HCC tumors. Although the expression of AKR1B10 was significantly correlated with 14-3-3ε, an increase of AKR1B10 expression in 14-3-3ε positive patients paradoxically had better overall survival and disease-free survival rates as well as lower metastatic incidence than those without an AKR1B10 increase. Finally, we found a loss of AKR1B10 expression in cells exhibiting a high capacity of invasiveness. Silencing of AKR1B10 resulted in inducing snail and vimentin expression in HCC cells. These results indicate that AKR1B10 may play a dual role during HCC tumor progression. Our results also indicate that 14-3-3ε regulates AKR1B10 expression by activating β-catenin signaling. A combination of 14-3-3ε with AKR1B10 is a potential therapeutic target and novel prognostic biomarker of HCC. PMID:26516929

  10. Involvement of 14-3-3 protein GRF9 in root growth and response under polyethylene glycol-induced water stress.

    PubMed

    He, Yuchi; Wu, Jingjing; Lv, Bing; Li, Jia; Gao, Zhiping; Xu, Weifeng; Baluška, František; Shi, Weiming; Shaw, Pang Chui; Zhang, Jianhua

    2015-04-01

    Plant 14-3-3 proteins are phosphoserine-binding proteins that regulate a wide array of targets via direct protein-protein interactions. In this study, the role of a 14-3-3 protein, GRF9, in plant response to water stress was investigated. Arabidopsis wild-type, GRF9-deficient mutant (grf9), and GRF9-overexpressing (OE) plants were treated with polyethylene glycol (PEG) to induce mild water stress. OE plant showed better whole-plant growth and root growth than the wild type under normal or water stress conditions while the grf9 mutant showed worse growth. In OE plants, GRF9 favours the allocation of shoot carbon to roots. In addition, GRF9 enhanced proton extrusion, mainly in the root elongation zone and root hair zone, and maintained root growth under mild water stress. Grafting among the wild type, OE, and grf9 plants showed that when OE plants were used as the scion and GRF9 was overexpressed in the shoot, it enhanced sucrose transport into the root, and when OE plants were used as rootstock and GRF9 was overexpressed in the root, it caused more release of protons into the root surface under water stress. Taken together, the results suggest that under PEG-induced water stress, GRF9 is involved in allocating more carbon from the shoot to the root and enhancing proton secretion in the root growing zone, and this process is important for root response to mild water stress. PMID:25873671

  11. 14-3-3ζ up-regulates hypoxia-inducible factor-1α in hepatocellular carcinoma via activation of PI3K/Akt/NF-кB signal transduction pathway

    PubMed Central

    Tang, Yufu; Lv, Pengfei; Sun, Zhongyi; Han, Lei; Luo, Bichao; Zhou, Wenping

    2015-01-01

    14-3-3ζ protein, a member of 14-3-3 family, plays important roles in multiple cellular processes. Our previous study showed that 14-3-3ζ could bind to regulate the expression of hypoxia-inducible factor-1α (HIF-1α), which is induced by hypoxia and a crucial factor for induction of tumor metastasis. Moreover, we also have confirmed the response of 14-3-3ζ to hypoxia in our unpublished data as well. Thus, in the present study, we attempted to reveal that whether the regulation effect of 14-3-3ζ on HIF-1α functioned in a similar pattern as hypoxia. Stable regulation of 14-3-3ζ in human HCC cell line SMMC-772 and HCC-LM3 was achieved. The regulation of 14-3-3ζ on HIF-1α mRNA transcription was evaluated by luciferase activity assay and quantitative real-time PCR (qPCR). The effect of 14-3-3ζ on the production of HIF-1α and pathways determining HIF-1α’s response to hypoxia was assessed using western blotting assay. Our results showed that regulation of 14-3-3ζ expression influenced the activity of HIF-1α, phosphatidyl inositol 3-kinase (PI3K), Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), and nuclear factor kappa B (NF-кB). Blocking of these pathways using indicated inhibitors revealed that 14-3-3ζ enhanced the production of HIF-1α via the activation of PI3K/Akt/NF-кB pathway, which was identical to hypoxia induced HIF-1α expression. For the first time, our study described the key role of 14-3-3ζ in the HIF-1α production in HCC cells. And the molecule exerted its function on HIF-1α both by directly binding to it and via PI3K/Akt/NF-кB signal transduction pathway. PMID:26884855

  12. Wig-1 regulates cell cycle arrest and cell death through the p53 targets FAS and 14-3-3σ

    PubMed Central

    Bersani, C; Xu, L-D; Vilborg, A; Lui, W-O; Wiman, K G

    2014-01-01

    Wig-1, also known as ZMAT3, is a p53 target gene that encodes an RNA-binding zinc-finger protein involved in the regulation of mRNA stability through binding to AU-rich elements (AREs). We have used microarray analysis to identify novel Wig-1 target mRNAs. We identified 2447 transcripts with >fourfold differential expression between Wig-1 and control small interfering (si)RNA-treated HCT116 cells. Several p53 target genes were among the deregulated transcripts. We found that Wig-1 regulates FAS and 14-3-3σ mRNA independently of p53. We show that Wig-1 binds to FAS mRNA 3′-UTR and decreases its stability through an ARE in the 3′-UTR. Depletion of Wig-1 was associated with increased cell death and reduced cell cycle arrest upon DNA damage. Our results suggest a role of Wig-1 as a survival factor that directs the p53 stress response toward cell cycle arrest rather than apoptosis through the regulation of FAS and 14-3-3σ mRNA levels. PMID:24469038

  13. Phospho-specific recognition by 14-3-3 proteins and antibodies monitored by a high throughput label-free optical biosensor.

    PubMed

    Wu, Meng; Coblitz, Brian; Shikano, Sojin; Long, Shunyou; Spieker, Matt; Frutos, Anthony G; Mukhopadhyay, Sunil; Li, Min

    2006-10-16

    Label-free detection of molecular interactions has considerable potential in facilitating assay development. When combined with high throughput capability, it may be applied to small molecule screens for drug candidates. Phosphorylation is a key posttranslational process that confers diverse regulation in biological systems involving specific protein-protein interactions recognizing the phosphorylated motifs. Using a resonant waveguide grating biosensor, the Epic mark System, we have developed a generic assay to quantitatively measure phospho-specific interactions between a trafficking signal-phosphorylated SWTY peptide and 14-3-3 proteins or anti-phosphopeptide antibodies. Compared with a solution-based fluorescence anisotropy assay, our results support that the high throughput resonant waveguide grating biosensor system has favorable technical profiles in detecting protein-protein interactions that recognize phosphorylated motifs. Hence it provides a new generic HTS platform for phospho-detection. PMID:17011553

  14. Involvement of 14-3-3 protein GRF9 in root growth and response under polyethylene glycol-induced water stress

    PubMed Central

    He, Yuchi; Wu, Jingjing; Lv, Bing; Li, Jia; Gao, Zhiping; Xu, Weifeng; Baluška, František; Shi, Weiming; Shaw, Pang Chui; Zhang, Jianhua

    2015-01-01

    Plant 14-3-3 proteins are phosphoserine-binding proteins that regulate a wide array of targets via direct protein–protein interactions. In this study, the role of a 14-3-3 protein, GRF9, in plant response to water stress was investigated. Arabidopsis wild-type, GRF9-deficient mutant (grf9), and GRF9-overexpressing (OE) plants were treated with polyethylene glycol (PEG) to induce mild water stress. OE plant showed better whole-plant growth and root growth than the wild type under normal or water stress conditions while the grf9 mutant showed worse growth. In OE plants, GRF9 favours the allocation of shoot carbon to roots. In addition, GRF9 enhanced proton extrusion, mainly in the root elongation zone and root hair zone, and maintained root growth under mild water stress. Grafting among the wild type, OE, and grf9 plants showed that when OE plants were used as the scion and GRF9 was overexpressed in the shoot, it enhanced sucrose transport into the root, and when OE plants were used as rootstock and GRF9 was overexpressed in the root, it caused more release of protons into the root surface under water stress. Taken together, the results suggest that under PEG-induced water stress, GRF9 is involved in allocating more carbon from the shoot to the root and enhancing proton secretion in the root growing zone, and this process is important for root response to mild water stress. PMID:25873671

  15. A Negative Regulatory Mechanism Involving 14-3-3ζ Limits Signaling Downstream of ROCK to Regulate Tissue Stiffness in Epidermal Homeostasis.

    PubMed

    Kular, Jasreen; Scheer, Kaitlin G; Pyne, Natasha T; Allam, Amr H; Pollard, Anthony N; Magenau, Astrid; Wright, Rebecca L; Kolesnikoff, Natasha; Moretti, Paul A; Wullkopf, Lena; Stomski, Frank C; Cowin, Allison J; Woodcock, Joanna M; Grimbaldeston, Michele A; Pitson, Stuart M; Timpson, Paul; Ramshaw, Hayley S; Lopez, Angel F; Samuel, Michael S

    2015-12-21

    ROCK signaling causes epidermal hyper-proliferation by increasing ECM production, elevating dermal stiffness, and enhancing Fak-mediated mechano-transduction signaling. Elevated dermal stiffness in turn causes ROCK activation, establishing mechano-reciprocity, a positive feedback loop that can promote tumors. We have identified a negative feedback mechanism that limits excessive ROCK signaling during wound healing and is lost in squamous cell carcinomas (SCCs). Signal flux through ROCK was selectively tuned down by increased levels of 14-3-3ζ, which interacted with Mypt1, a ROCK signaling antagonist. In 14-3-3ζ(-/-) mice, unrestrained ROCK signaling at wound margins elevated ECM production and reduced ECM remodeling, increasing dermal stiffness and causing rapid wound healing. Conversely, 14-3-3ζ deficiency enhanced cutaneous SCC size. Significantly, inhibiting 14-3-3ζ with a novel pharmacological agent accelerated wound healing 2-fold. Patient samples of chronic non-healing wounds overexpressed 14-3-3ζ, while cutaneous SCCs had reduced 14-3-3ζ. These results reveal a novel 14-3-3ζ-dependent mechanism that negatively regulates mechano-reciprocity, suggesting new therapeutic opportunities. PMID:26702834

  16. Overexpression of the 14-3-3gamma protein in embryonic mice results in neuronal migration delay in the developing cerebral cortex.

    PubMed

    Cornell, Brett; Wachi, Tomoka; Zhukarev, Vladimir; Toyo-Oka, Kazuhito

    2016-08-15

    The 14-3-3 protein family is a group of multifunctional proteins that are highly expressed in the brain; however, their functions in brain development are largely unknown. Williams Syndrome is a neurodevelopmental disorder caused by a deletion in the 7q11.23 chromosome locus, including the gene encoding 14-3-3gamma, resulting in developmental delay, intellectual disabilities and epilepsy. We have previously shown that knocking down the 14-3-3gamma protein in utero in mice results in delays in neuronal migration of pyramidal neurons in the cortex. Importantly, there is a reciprocal duplication syndrome to Williams Syndrome where the 7q11.23 locus is duplicated, resulting in epilepsy and intellectual disabilities. Thus, the deletion or the duplication of the 7q11.23 chromosome locus results in epilepsy. Taken together with the fact that defects in neuronal migration are one of main causes for epilepsy, we analyzed if the overexpression of 14-3-3gamma causes neuronal migration defects. In this work, we found that the overexpression of 14-3-3gamma in utero in the developing mouse cortex results in delays in pyramidal neuron migration, similar to what was previously observed when 14-3-3gamma was knocked down. These results, in conjunction with our previous research, indicate that a balance of 14-3-3gamma expression is required during cortical development to prevent delays in neuronal migration. This work provides clear evidence as to the involvement of 14-3-3gamma in neurodevelopmental disorders and how a disruption in 14-3-3gamma expression may contribute to the neurodevelopmental disorders that manifest when the 7q11.23 locus is altered. PMID:27288018

  17. 14-3-3σ regulation of and interaction with YAP1 in acquired gemcitabine resistance via promoting ribonucleotide reductase expression

    PubMed Central

    Qin, Li; Dong, Zizheng; Zhang, Jian-Ting

    2016-01-01

    Gemcitabine is an important anticancer therapeutics approved for treatment of several human cancers including locally advanced or metastatic pancreatic ductal adenocarcinoma (PDAC). Its clinical effectiveness, however, is hindered by existence of intrinsic and development of acquired resistances. Previously, it was found that 14-3-3σ expression associates with poor clinical outcome of PDAC patients. It was also found that 14-3-3σ expression is up-regulated in gemcitabine resistant PDAC cells and contributes to the acquired gemcitabine resistance. In this study, we investigated the molecular mechanism of 14-3-3σ function in gemcitabine resistance and found that 14-3-3σ up-regulates YAP1 expression and then binds to YAP1 to inhibit gemcitabine-induced caspase 8 activation and apoptosis. 14-3-3σ association with YAP1 up-regulates the expression of ribonucleotide reductase M1 and M2, which may mediate 14-3-3σ/YAP1 function in the acquired gemcitabine resistance. These findings suggest a possible role of YAP1 signaling in gemcitabine resistance. PMID:26894857

  18. 14-3-3 proteins sequester a pool of soluble TRIM32 ubiquitin ligase to repress autoubiquitylation and cytoplasmic body formation.

    PubMed

    Ichimura, Tohru; Taoka, Masato; Shoji, Ikuo; Kato, Hiroki; Sato, Tomonobu; Hatakeyama, Shigetsugu; Isobe, Toshiaki; Hachiya, Naomi

    2013-05-01

    Deregulated expression of tripartite motif-containing protein 32 (TRIM32, an E3 ubiquitin-protein ligase) contributes to various diseases. Here we report, using quantitative proteomics and biochemistry, that 14-3-3 proteins bind to phosphorylated TRIM32 and prevent TRIM32 autoubiquitylation and the formation of TRIM32-containing cytoplasmic bodies, which are potential autoregulatory mechanisms that can reduce the concentration of soluble free TRIM32. The 14-3-3-TRIM32 interaction is dependent on protein-kinase-A-catalyzed phosphorylation of TRIM32 at Ser651. We found that the inhibitory effect of 14-3-3 is, in part, a consequence of disrupting the propensity of TRIM32 to undergo higher-order self-association without affecting its dimerization. Consequently, dimerized TRIM32 bound to 14-3-3 was sequestered in a distinct cytoplasmic pool away from the microtubule network, whereas a TRIM32 mutant that cannot bind 14-3-3 underwent multimerization and was unavailable to facilitate cell growth. Our results reveal a novel connection between ubiquitylation and phosphorylation pathways, which could modulate a variety of cell events by stimulating the formation of the 14-3-3-TRIM32 signaling complex. PMID:23444366

  19. 14-3-3ζ coordinates adipogenesis of visceral fat

    PubMed Central

    Lim, Gareth E.; Albrecht, Tobias; Piske, Micah; Sarai, Karnjit; Lee, Jason T. C; Ramshaw, Hayley S.; Sinha, Sunita; Guthridge, Mark A.; Acker-Palmer, Amparo; Lopez, Angel F.; Clee, Susanne M.; Nislow, Corey; Johnson, James D.

    2015-01-01

    The proteins that coordinate complex adipogenic transcriptional networks are poorly understood. 14-3-3ζ is a molecular adaptor protein that regulates insulin signalling and transcription factor networks. Here we report that 14-3-3ζ-knockout mice are strikingly lean from birth with specific reductions in visceral fat depots. Conversely, transgenic 14-3-3ζ overexpression potentiates obesity, without exacerbating metabolic complications. Only the 14-3-3ζ isoform is essential for adipogenesis based on isoform-specific RNAi. Mechanistic studies show that 14-3-3ζ depletion promotes autophagy-dependent degradation of C/EBP-δ, preventing induction of the master adipogenic factors, Pparγ and C/EBP-α. Transcriptomic data indicate that 14-3-3ζ acts upstream of hedgehog signalling-dependent upregulation of Cdkn1b/p27Kip1. Indeed, concomitant knockdown of p27Kip1 or Gli3 rescues the early block in adipogenesis induced by 14-3-3ζ knockdown in vitro. Adipocyte precursors in 14-3-3ζKO embryos also appear to have greater Gli3 and p27Kip1 abundance. Together, our in vivo and in vitro findings demonstrate that 14-3-3ζ is a critical upstream driver of adipogenesis. PMID:26220403

  20. Identification of a functional splice variant of 14-3-3E1 in rainbow trout

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The 14-3-3 proteins are a family of regulatory proteins involved in diverse cellular processes. The presence of 14-3-3 isoforms and the diversity of cellular processes regulated by 14-3-3 isoforms suggest functional specificity of the isoforms. In this study, we report the identification and charact...

  1. Genetic variations of 14-3-3E1 isoform in rainbow trout (Oncorhynchus mykiss)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The highly conserved family of 14-3-3 proteins functions in the regulation of a wide variety of cellular processes. The presence of 14-3-3 isoforms and the diversity of cellular processes regulated by 14-3-3 isoforms suggest functional specificity of the isoforms. Several studies have observed diffe...

  2. 14-3-3γ regulates cell viability and milk fat synthesis in lipopolysaccharide-induced dairy cow mammary epithelial cells

    PubMed Central

    LIU, LIXIN; ZHANG, LI; LIN, YE; BIAN, YANJIE; GAO, XUEJUN; QU, BO; LI, QINGZHANG

    2016-01-01

    Our previous study demonstrated that 14-3-3γ overexpression was able to inhibit the production of lipopolysaccharide (LPS)-induced cytokines in dairy cow mammary epithelial cells (DCMECs) by inhibiting the activation of nuclear factor-κB (NF-κB) signaling pathways. However, the association between 14-3-3γ overexpression and milk fat synthesis in LPS-induced DCMECs remains unclear. Therefore, the present study investigated the effect of 14-3-3γ on cell viability and milk fat synthesis in LPS-induced DCMECs. The results of the MTT assay and lactate dehydrogenase activity assay demonstrated that 14-3-3γ overexpression was able to attenuate LPS-induced cytotoxicity in DCMECs, and increase the viability of the cells. In addition, the results of reverse transcription-quantitative polymerase chain reaction suggested that mRNA expression levels of genes associated with milk fat synthesis, including sterol regulatory element binding protein (SREBP1), peroxisome proliferator-activated receptor-γ (PPARG), cluster of differentiation 36, acetyl-coA carboxylase (ACC), fatty acid synthase (FAS) and fatty acid binding protein-3, were significantly upregulated in cells overexpressing the 14-3-3γ protein. In addition, as compared with the LPS-treated group, the activities of FAS and ACC were significantly increased. Furthermore, western blotting demonstrated that 14-3-3γ overexpression enhanced the protein expression levels of phosphorylated SREBP1 and PPARG. These results suggested that high levels of 14-3-3γ protein were able to attenuate LPS-induced cell damage and promote milk fat synthesis in LPS-induced DCMECs by increasing the cell viability and upregulating the expression levels of transcription factors associated with milk fat synthesis. PMID:27073437

  3. 14-3-3σ confers cisplatin resistance in esophageal squamous cell carcinoma cells via regulating DNA repair molecules.

    PubMed

    Lai, Kenneth K Y; Chan, Kin Tak; Choi, Mei Yuk; Wang, Hector K; Fung, Eva Y M; Lam, Ho Yu; Tan, Winnie; Tung, Lai Nar; Tong, Daniel K H; Sun, Raymond W Y; Lee, Nikki P; Law, Simon

    2016-02-01

    Esophageal squamous cell carcinoma (ESCC) is the predominant type of esophageal cancer in Asia. Cisplatin is commonly used in chemoradiation for unresectable ESCC patients. However, the treatment efficacy is diminished in patients with established cisplatin resistance. To understand the mechanism leading to the development of cisplatin resistance in ESCC, we compared the proteomes from a cisplatin-resistant HKESC-2R cell line with its parental-sensitive counterpart HKESC-2 to identify key molecule involved in this process. Mass spectrometry analysis detected 14-3-3σ as the most abundant molecule expressed exclusively in HKESC-2R cells, while western blot result further validated it to be highly expressed in HKESC-2R cells when compared to HKESC-2 cells. Ectopic expression of 14-3-3σ increased cisplatin resistance in HKESC-2 cells, while its suppression sensitized SLMT-1 cells to cisplatin. Among the molecules involved in drug detoxification, drug transportation, and DNA repair, the examined DNA repair molecules HMGB1 and XPA were found to be highly expressed in HKESC-2R cells with high 14-3-3σ expression. Subsequent manipulation of 14-3-3σ by both overexpression and knockdown approaches concurrently altered the expression of HMGB1 and XPA. 14-3-3σ, HMGB1, and XPA were preferentially expressed in cisplatin-resistant SLMT-1 cells when compared to those more sensitive to cisplatin. In ESCC patients with poor response to cisplatin-based chemoradiation, their pre-treatment tumors expressed higher expression of HMGB1 than those with response to such treatment. In summary, our results demonstrate that 14-3-3σ induces cisplatin resistance in ESCC cells and that 14-3-3σ-mediated cisplatin resistance involves DNA repair molecules HMGB1 and XPA. Results from this study provide evidences for further work in researching the potential use of 14-3-3σ and DNA repair molecules HMGB1 and XPA as biomarkers and therapeutic targets for ESCC. PMID:26346170

  4. 14-3-3ɛ/ζ Affects the stability of δ-catenin and regulates δ-catenin-induced dendrogenesis.

    PubMed

    He, Yongfeng; Han, Jeong Ran; Chang, Ockyoung; Oh, Minsoo; James, Sarah E; Lu, Qun; Seo, Young-Woo; Kim, Hangun; Kim, Kwonseop

    2013-01-01

    Accumulated evidence suggests that aberrant regulation of δ-catenin leads to pathological consequences such as mental retardation and cognitive dysfunction. This study revealed that 14-3-3ɛ/ζ stabilizes δ-catenin, with different binding regions involved in the interaction. Furthermore, the specific inhibition of the interaction of 14-3-3 with δ-catenin reduced levels of δ-catenin and significantly impaired the capacity of δ-catenin to induce dendritic branching in both NIH3T3 fibroblasts and primary hippocampal neurons. However, the S1094A δ-catenin mutant, which cannot interact with 14-3-3ζ, still retained the capability of inducing dendrogenesis. Taken together, these results elucidate the underlying events that regulate the stability of δ-catenin and δ-catenin-induced dendrogenesis. PMID:23772369

  5. Properties of the monomeric form of human 14-3-3ζ protein and its interaction with tau and HspB6.

    PubMed

    Sluchanko, Nikolai N; Sudnitsyna, Maria V; Seit-Nebi, Alim S; Antson, Alfred A; Gusev, Nikolai B

    2011-11-15

    Dimers formed by seven isoforms of the human 14-3-3 protein participate in multiple cellular processes. The dimeric form has been extensively characterized; however, little is known about the structure and properties of the monomeric form of 14-3-3. The monomeric form is involved in the assembly of homo- and heterodimers, which could partially dissociate back into monomers in response to phosphorylation at Ser58. To obtain monomeric forms of human 14-3-3ζ, we produced four protein constructs with different combinations of mutated (M) or wild-type (W) segments E(5), (12)LAE(14), and (82)YREKIE(87). Under a wide range of expression conditions in Escherichia coli, the MMM and WMM mutants were insoluble, whereas WMW and MMW mutants were soluble, highly expressed, and purified to homogeneity. WMW and MMW mutants remained monomeric over a wide range of concentrations while retaining the α-helical structure characteristic of wild-type 14-3-3. However, WMW and MMW mutants were highly susceptible to proteolysis and had much lower thermal stabilities than the wild-type protein. Using WMW and MMW mutants, we show that the monomeric form interacts with the tau protein and with the HspB6 protein, in both cases forming complexes with a 1:1 stoichiometry, in contrast to the 2:1 and/or 2:2 complexes formed by wild-type 14-3-3. Significantly, this interaction requires phosphorylation of tau protein and HspB6. Because of minimal changes in structure, MMW and especially WMW mutant proteins are promising candidates for analyzing the effect of monomerization on the physiologically important properties of 14-3-3ζ. PMID:21978388

  6. Characterization of 14-3-3 isoforms expressed in the Echinococcus granulosus pathogenic larval stage.

    PubMed

    Teichmann, Aline; Vargas, Daiani M; Monteiro, Karina M; Meneghetti, Bruna V; Dutra, Cristine S; Paredes, Rodolfo; Galanti, Norbel; Zaha, Arnaldo; Ferreira, Henrique B

    2015-04-01

    The 14-3-3 protein family of eukaryotic regulators was studied in Echinococcus granulosus, the causative agent of cystic hydatid disease. These proteins mediate important cellular processes in eukaryotes and are expected to play important roles in parasite biology. Six isoforms of E. granulosus 14-3-3 genes and proteins (Eg14-3-3.1-6) were analyzed, and their phylogenetic relationships were established with bona fide 14-3-3 orthologous proteins from eukaryotic species. Eg14-3-3 isoforms with previous evidence of expression (Eg14-3-3.1-4) in E. granulosus pathogenic larval stage (metacestode) were cloned, and recombinant proteins were used for functional studies. These protein isoforms were detected in different components of E. granulosus metacestode, including interface components with the host. The roles that are played by Eg14-3-3 proteins in parasite biology were inferred from the repertoires of interacting proteins with each isoform, as assessed by gel overlay, cross-linking, and affinity chromatography assays. A total of 95 Eg14-3-3 protein ligands were identified by mass spectrometry. Eg14-3-3 isoforms have shared partners (44 proteins), indicating some overlapping functions; however, they also bind exclusive partners (51 proteins), suggesting Eg14-3-3 functional specialization. These ligand repertoires indicate the involvement of Eg14-3-3 proteins in multiple biochemical pathways in the E. granulosus metacestode and note some degree of isoform specialization. PMID:25748451

  7. Observation of interaction between bid and 14-3-3 proteins by FRET in living cell during TNF-a-induced apoptosis

    NASA Astrophysics Data System (ADS)

    Wang, Jinjun; Chen, Tongsheng; Xing, Da; Wang, Fang

    2005-01-01

    Caspase8 is activated and cleaves Bid into two fragments when cells are exposed to death-inducing molecules such as tumor necrosis factor-α (TNF-α). Then the C-terminal fragment relocates from cytosol to mitochondria and promotes the release of cytochrome c, in the final cellular apoptosis is induced. Despite recent progress in the study of Bid during apoptosis induction, it remains unclear how C-terminal fragment of Bid cleaved moves to mitochondria and then induces the release of cytochrome c and so on. The 14-3-3 proteins are known to sequester certain pro-apoptotic members of Bcl-2 family. In order to further study the biological action of Bid during apoptosis, especially under physiological condition of living cell, the plasmids pBid-CFP and pYFP-14-3-3 were constructed. By the transient transfection of pBid-CFP and pYFP-14-3-3, the dynamic process of interaction of Bid and 14-3-3 protein in individual living cell during the apoptosis was primarily investigated with FRET (fluorescent resonance energy transfer) technique by the use of fluorescence microscopy.

  8. Protein Kinase CK2 Interacts at the Neuromuscular Synapse with Rapsyn, Rac1, 14-3-3γ, and Dok-7 Proteins and Phosphorylates the Latter Two*

    PubMed Central

    Herrmann, Dustin; Straubinger, Marion; Hashemolhosseini, Said

    2015-01-01

    Previously, we demonstrated that the protein kinase CK2 associates with and phosphorylates the receptor tyrosine kinase MuSK (muscle specific receptor tyrosine kinase) at the neuromuscular junction (NMJ), thereby preventing fragmentation of the NMJs (Cheusova, T., Khan, M. A., Schubert, S. W., Gavin, A. C., Buchou, T., Jacob, G., Sticht, H., Allende, J., Boldyreff, B., Brenner, H. R., and Hashemolhosseini, S. (2006) Genes Dev. 20, 1800–1816). Here, we asked whether CK2 interacts with other proteins involved in processes at the NMJ, which would be consistent with the previous observation that CK2 appears enriched at the NMJ. We identified the following proteins to interact with protein kinase CK2: (a) the α and β subunits of the nicotinic acetylcholine receptors with weak interaction, (b) dishevelled (Dsh), and (c) another four proteins, Rapsyn, Rac1, 14-3-3γ, and Dok-7, with strong interaction. CK2 phosphorylated 14-3-3γ at serine residue 235 and Dok-7 at several serine residues but does not phosphorylate Rapsyn or Rac1. Furthermore, phosphomimetic Dok-7 mutants aggregated nicotinic acetylcholine receptors in C2C12 myotubes with significantly higher frequency than wild type Dok-7. Additionally, we mapped the interacting epitopes of all four binding partners to CK2 and thereby gained insights into the potential role of the CK2/Rapsyn interaction. PMID:26198629

  9. Rapid antidepressants stimulate the decoupling of GABAB receptors from GIRK/Kir3 channels through increased protein stability of 14-3-3η

    PubMed Central

    Workman, E R; Haddick, P C G; Bush, K; Dilly, G A; Niere, F; Zemelman, B V; Raab-Graham, K F

    2015-01-01

    A single injection of N-methyl-D-aspartate receptor (NMDAR) antagonists produces a rapid antidepressant response. Lasting changes in the synapse structure and composition underlie the effectiveness of these drugs. We recently discovered that rapid antidepressants cause a shift in the γ-aminobutyric acid receptor (GABABR) signaling pathway, such that GABABR activation shifts from opening inwardly rectifiying potassium channels (Kir/GIRK) to increasing resting dendritic calcium signal and mammalian Target of Rapamycin activity. However, little is known about the molecular and biochemical mechanisms that initiate this shift. Herein, we show that GABABR signaling to Kir3 (GIRK) channels decreases with NMDAR blockade. Blocking NMDAR signaling stabilizes the adaptor protein 14-3-3η, which decouples GABABR signaling from Kir3 and is required for the rapid antidepressant efficacy. Consistent with these results, we find that key proteins involved in GABABR signaling bidirectionally change in a depression model and with rapid antidepressants. In socially defeated rodents, a model for depression, GABABR and 14-3-3η levels decrease in the hippocampus. The NMDAR antagonists AP5 and Ro-25-6981, acting as rapid antidepressants, increase GABABR and 14-3-3η expression and decrease Kir3.2. Taken together, these data suggest that the shift in GABABR function requires a loss of GABABR-Kir3 channel activity mediated by 14-3-3η. Our findings support a central role for 14-3-3η in the efficacy of rapid antidepressants and define a critical molecular mechanism for activity-dependent alterations in GABABR signaling. PMID:25560757

  10. Influence of the Paracoccidioides brasiliensis 14-3-3 and gp43 proteins on the induction of apoptosis in A549 epithelial cells

    PubMed Central

    da Silva, Julhiany de Fátima; Vicentim, Juliana; de Oliveira, Haroldo Cesar; Marcos, Caroline Maria; Assato, Patricia Akemi; Andreotti, Patrícia Ferrari; da Silva, Juliana Leal Monteiro; Soares, Christiane Pienna; Benard, Gil; Almeida, Ana Marisa Fusco; Mendes-Giannini, Maria José Soares

    2015-01-01

    The fungal strain Paracoccidioides brasiliensis remains viable inside of epithelial cells and can induce apoptosis in this population. However, until now, the molecules that participate in this process remained unknown. Thus, this study evaluated the contribution of two P. brasiliensis molecules, the 14-3-3 and glycoprotein of 43 kDa proteins, which had been previously described as extracellular matrix adhesins and apoptosis inductors in human pneumocytes. Accordingly, epithelial cells were treated with these molecules for different periods of time and the expression of the apoptosis regulating-proteins Bak, Bax, Bcl-2, p53 and caspases were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling, flow cytometry and real-time polymerase chain reaction analysis. Our results demonstrated that treatment with these molecules induces apoptosis signalling in pulmonary epithelial cells, showing the same pattern of programmed cell-death as that observed during infection with P. brasiliensis. Thus, we could conclude that P. brasiliensis uses these molecules as virulence factors that participate not only in the fungal adhesion process to host cells, but also in other important cellular mechanisms such as apoptosis. PMID:26038961

  11. 14-3-3 Proteins SGF14c and SGF14l Play Critical Roles during Soybean Nodulation1[W][OA

    PubMed Central

    Radwan, Osman; Wu, Xia; Govindarajulu, Manjula; Libault, Marc; Neece, David J.; Oh, Man-Ho; Berg, R. Howard; Stacey, Gary; Taylor, Christopher G.; Huber, Steven C.; Clough, Steven J.

    2012-01-01

    The soybean (Glycine max) genome contains 18 members of the 14-3-3 protein family, but little is known about their association with specific phenotypes. Here, we report that the Glyma0529080 Soybean G-box Factor 14-3-3c (SGF14c) and Glyma08g12220 (SGF14l) genes, encoding 14-3-3 proteins, appear to play essential roles in soybean nodulation. Quantitative reverse transcription-polymerase chain reaction and western-immunoblot analyses showed that SGF14c mRNA and protein levels were specifically increased in abundance in nodulated soybean roots 10, 12, 16, and 20 d after inoculation with Bradyrhizobium japonicum. To investigate the role of SGF14c during soybean nodulation, RNA interference was employed to silence SGF14c expression in soybean roots using Agrobacterium rhizogenes-mediated root transformation. Due to the paleopolyploid nature of soybean, designing a specific RNA interference sequence that exclusively targeted SGF14c was not possible. Therefore, two highly similar paralogs (SGF14c and SGF14l) that have been shown to function as dimers were silenced. Transcriptomic and proteomic analyses showed that mRNA and protein levels were significantly reduced in the SGF14c/SGF14l-silenced roots, and these roots exhibited reduced numbers of mature nodules. In addition, SGF14c/SGF14l-silenced roots contained large numbers of arrested nodule primordia following B. japonicum inoculation. Transmission electron microscopy further revealed that the host cytoplasm and membranes, except the symbiosome membrane, were severely degraded in the failed nodules. Altogether, transcriptomic, proteomic, and cytological data suggest a critical role of one or both of these 14-3-3 proteins in early development stages of soybean nodules. PMID:23060368

  12. A rare case of rapidly progressive dementia with elevated RT-QuIC and negative 14-3-3 and tau proteins.

    PubMed

    Trikamji, Bhavesh; Hamlin, Clive; Baldwin, Kelly J

    2016-05-01

    Creutzfeldt-Jakob disease (CJD) is characterized by rapidly progressing dementia with death usually occurring within 6 months. There is no verified disease-specific pre-mortem diagnostic test besides brain biopsy. We describe a 66 y old previously high functioning male who presented with a 5 month history of rapidly progressive dementia. Neurological examination revealed a score of 19/30 on MOCA testing. An extensive workup into various causes of dementia including electroencephalography and imaging studies was unremarkable. The cerebrospinal fluid was sent to National Prion Disease Center and it revealed elevated RT-QuIC levels with negative 14-3-3 and T tau proteins. Based on literature review, our case is one of few living subjects with elevated RT-QuIC levels and negative 14-3-3 and tau proteins. PMID:27249661

  13. 14-3-3 and aggresome formation

    PubMed Central

    Jia, Baohui; Wu, Yuying; Zhou, Yi

    2014-01-01

    Protein misfolding and aggregation underlie the pathogenesis of many neurodegenerative diseases. In addition to chaperone-mediated refolding and proteasomal degradation, the aggresome-macroautophagy pathway has emerged as another defense mechanism for sequestration and clearance of toxic protein aggregates in cells. Previously, the 14-3-3 proteins were shown to be indispensable for the formation of aggresomes induced by mutant huntingtin proteins. In a recent study, we have determined that 14-3-3 functions as a molecular adaptor to recruit chaperone-associated misfolded proteins to dynein motors for transport to aggresomes. This molecular complex involves a dimeric binding of 14-3-3 to both the dynein-intermediate chain (DIC) and an Hsp70 co-chaperone Bcl-2-associated athanogene 3 (BAG3). As 14-3-3 has been implicated in various neurodegenerative diseases, our findings may provide mechanistic insights into its role in managing misfolded protein stress during the process of neurodegeneration. PMID:24549097

  14. Specificity of ε and Non-ε Isoforms of Arabidopsis 14-3-3 Proteins Towards the H+-ATPase and Other Targets

    PubMed Central

    Pallucca, Roberta; Visconti, Sabina; Camoni, Lorenzo; Cesareni, Giovanni; Melino, Sonia; Panni, Simona; Torreri, Paola; Aducci, Patrizia

    2014-01-01

    14-3-3 proteins are a family of ubiquitous dimeric proteins that modulate many cellular functions in all eukaryotes by interacting with target proteins. 14-3-3s exist as a number of isoforms that in Arabidopsis identifies two major groups named ε and non-ε. Although isoform specificity has been demonstrated in many systems, the molecular basis for the selection of specific sequence contexts has not been fully clarified. In this study we have investigated isoform specificity by measuring the ability of different Arabidopsis 14-3-3 isoforms to activate the H+-ATPase. We observed that GF14 isoforms of the non-ε group were more effective than ε group isoforms in the interaction with the H+-ATPase and in the stimulation of its activity. Kinetic and thermodynamic parameters of the binding of GF14ε and GF14ω isoforms, representative of ε and non-ε groups respectively, with the H+-ATPase, have been determined by Surface Plasmon Resonance analysis demonstrating that the higher affinity of GF14ω is mainly due to slower dissociation. The role of the C-terminal region and of a Gly residue located in the loop 8 and conserved in all non-ε isoforms has also been studied by deletion and site-specific mutagenesis. The C-terminal domains, despite their high divergence, play an auto-inhibitory role in both isoforms and they, in addition to a specific residue located in the loop 8, contribute to isoform specificity. To investigate the generality of these findings, we have used the SPOT-synthesis technology to array a number of phosphopeptides matching known or predicted 14-3-3 binding sites present in a number of clients. The results of this approach confirmed isoform specificity in the recognition of several target peptides, suggesting that the isoform specificity may have an impact on the modulation of a variety of additional protein activities, as suggested by probing of a phosphopeptide array with members of the two 14-3-3 groups. PMID:24603559

  15. Aqueous Extract from Hibiscus sabdariffa Linnaeus Ameliorate Diabetic Nephropathy via Regulating Oxidative Status and Akt/Bad/14-3-3γ in an Experimental Animal Model

    PubMed Central

    Wang, Shou-Chieh; Lee, Shiow-Fen; Wang, Chau-Jong; Lee, Chao-Hsin; Lee, Wen-Chin; Lee, Huei-Jane

    2011-01-01

    Several studies point out that oxidative stress maybe a major culprit in diabetic nephropathy. Aqueous extract of Hibiscus sabdariffa L. (HSE) has been demonstrated as having beneficial effects on anti-oxidation and lipid-lowering in experimental studies. This study aimed at investigating the effects of Hibiscus sabdariffa L. on diabetic nephropathy in streptozotocin induced type 1 diabetic rats. Our results show that HSE is capable of reducing lipid peroxidation, increasing catalase and glutathione activities significantly in diabetic kidney, and decreasing the plasma levels of triglyceride, low-density lipoprotein (LDL) and increasing high-density lipoprotein (HDL) value. In histological examination, HSE improves hyperglycemia-caused osmotic diuresis in renal proximal convoluted tubules (defined as hydropic change) in diabetic rats. The study also reveals that up-regulation of Akt/Bad/14-3-3γ and NF-κB-mediated transcription might be involved. In conclusion, our results show that HSE possesses the potential effects to ameliorate diabetic nephropathy via improving oxidative status and regulating Akt/Bad/14-3-3γ signaling. PMID:19965962

  16. Chloride intracellular channel protein CLIC4 (p64H1) binds directly to brain dynamin I in a complex containing actin, tubulin and 14-3-3 isoforms.

    PubMed Central

    Suginta, W; Karoulias, N; Aitken, A; Ashley, R H

    2001-01-01

    Mammalian chloride intracellular channel (CLIC) (p64-related) proteins are widely expressed, with an unusual dual localization as both soluble and integral membrane proteins. The molecular basis for their cellular localization and ion channel activity remains unclear. To help in addressing these problems, we identified novel rat brain CLIC4 (p64H1) binding partners by affinity chromatography, mass spectrometric analysis and microsequencing. Brain CLIC4 binds dynamin I, alpha-tubulin, beta-actin, creatine kinase and two 14-3-3 isoforms; the interactions are confirmed in vivo by immunoprecipitation. Gel overlay and reverse pull-down assays indicate that the binding of CLIC4 to dynamin I and 14-3-3zeta is direct. In HEK-293 cells, biochemical and immunofluorescence analyses show partial co-localization of recombinant CLIC4 with caveolin and with functional caveolae, which is consistent with a dynamin-associated role for CLIC4 in caveolar endocytosis. We speculate that brain CLIC4 might be involved in the dynamics of neuronal plasma membrane microdomains (micropatches) containing caveolin-like proteins and might also have other cellular roles related to membrane trafficking. Our results provide the basis for new hypotheses concerning novel ways in which CLIC proteins might be associated with cell membrane remodelling, the control of cell shape, and anion channel activity. PMID:11563969

  17. Positive 14-3-3 and tau proteins in a sporadic Creutzfeldt-Jakob disease case and a brief perspective of prion diseases in Colombia.

    PubMed

    Escandón-Vargas, Kevin; Zorrilla-Vaca, Andrés; Corral-Prado, Raúl Heli

    2016-01-01

    Prion diseases are rare neurodegenerative disorders occurring worldwide and affecting both humans and animals. Herein, we present the case of a patient diagnosed with definite sporadic Creutzfeldt-Jakob disease in Cali, Colombia. Besides neurological examination, 14-3-3 and tau proteins were valuable tools supporting the diagnosis. We also present a brief perspective of the prion diseases reported in Colombia to date. Although the incidence of prion diseases is unknown in Colombia, our literature review revealed that one case of scrapie in 1981 and 29 human sporadic cases of Creutzfeldt-Jakob disease have been documented and published in our country. PMID:27622622

  18. A calcium and free fatty acid-modulated protein kinase as putative effector of the fusicoccin 14-3-3 receptor.

    PubMed Central

    van der Hoeven, P C; Siderius, M; Korthout, H A; Drabkin, A V; de Boer, A H

    1996-01-01

    A protein kinase that is activated by calcium and cis-unsaturated fatty acids has been characterized from oat (Avena sativa L.) root plasma membranes. The kinase phosphorylates a synthetic peptide with a motif (-R-T-L-S-) that can be phosphorylated by both protein kinase C (PKC) and calcium-dependent protein kinase (CDPK)-type kinases. Calphostin C and chelerythrine, two PKC inhibitors, completely inhibited the kinase activity with values of inhibitor concentration for 50% inhibition of 0.7 and 30 microns, respectively. At low Ca2+ concentrations cis-unsaturated fatty acids (linolenic acid, linoleic acid, arachidonic acid, and oleic acid) stimulated the kinase activity almost 10-fold. The two inhibitors of the kinase, calphostin C and chelerythrin, strongly reduced the fusicoccin (FC)-induced H+ extrusion, and the activators of the kinase, the cis-unsaturated fatty acids, prevented [3H]FC binding to the FC 14-3-3 receptor. CDPK antibodies cross-reacted with a 43-kD band in the plasma membrane and in a purified FC receptor fraction. A polypeptide with the same apparent molecular mass was recognized by a synthetic peptide that has a sequence homologous to the annexin-like domain from barely 14-3-3. The possibility of the involvement of a kinase, with properties from both CDPK and PKC, and a phospholipase A2 in the FC Signal transduction pathway is discussed. PMID:8754686

  19. Exon B of human surfactant protein A2 mRNA, alone or within its surrounding sequences, interacts with 14-3-3; role of cis-elements and secondary structure

    PubMed Central

    Noutsios, Georgios T.; Silveyra, Patricia; Bhatti, Faizah

    2013-01-01

    Human surfactant protein A, an innate immunity molecule, is encoded by two genes: SFTPA1 (SP-A1) and SFTPA2 (SP-A2). The 5′ untranslated (5′UTR) splice variant of SP-A2 (ABD), but not of SP-A1 (AD), contains exon B (eB), which is an enhancer for transcription and translation. We investigated whether eB contains cis-regulatory elements that bind trans-acting factors in a sequence-specific manner as well as the role of the eB mRNA secondary structure. Binding of cytoplasmic NCI-H441 proteins to wild-type eB, eB mutant, AD, and ABD 5′UTR mRNAs were studied by RNA electromobility shift assays (REMSAs). The bound proteins were identified by mass spectroscopy and specific antibodies (Abs). We found that 1) proteins bind eB mRNA in a sequence-specific manner, with two cis-elements identified within eB to be important; 2) eB secondary structure is necessary for binding; 3) mass spectroscopy and specific Abs in REMSAs identified 14-3-3 proteins to bind (directly or indirectly) eB and the natural SP-A2 (ABD) splice variant but not the SP-A1 (AD) splice variant; 4) other ribosomal and cytoskeletal proteins, and translation factors, are also present in the eB mRNA-protein complex; 5) knockdown of 14-3-3 β/α isoform resulted in a downregulation of SP-A2 expression. In conclusion, proteins including the 14-3-3 family bind two cis-elements within eB of hSP-A2 mRNA in a sequence- and secondary structure-specific manner. Differential regulation of SP-A1 and SP-A2 is mediated by the 14-3-3 protein family as well as by a number of other proteins that bind UTRs with or without eB mRNA. PMID:23525782

  20. The Pseudomonas syringae Effector HopQ1 Promotes Bacterial Virulence and Interacts with Tomato 14-3-3 Proteins in a Phosphorylation-Dependent Manner1[C][W][OA

    PubMed Central

    Li, Wei; Yadeta, Koste A.; Elmore, James Mitch; Coaker, Gitta

    2013-01-01

    A key virulence strategy of bacterial pathogens is the delivery of multiple pathogen effector proteins into host cells during infection. The Hrp outer protein Q (HopQ1) effector from Pseudomonas syringae pv tomato (Pto) strain DC3000 is conserved across multiple bacterial plant pathogens. Here, we investigated the virulence function and host targets of HopQ1 in tomato (Solanum lycopersicum). Transgenic tomato lines expressing dexamethasone-inducible HopQ1 exhibited enhanced disease susceptibility to virulent Pto DC3000, the Pto ΔhrcC mutant, and decreased expression of a pathogen-associated molecular pattern-triggered marker gene after bacterial inoculation. HopQ1-interacting proteins were coimmunoprecipitated and identified by mass spectrometry. HopQ1 can associate with multiple tomato 14-3-3 proteins, including TFT1 and TFT5. HopQ1 is phosphorylated in tomato, and four phosphorylated peptides were identified by mass spectrometry. HopQ1 possesses a conserved mode I 14-3-3 binding motif whose serine-51 residue is phosphorylated in tomato and regulates its association with TFT1 and TFT5. Confocal microscopy and fractionation reveal that HopQ1 exhibits nucleocytoplasmic localization, while HopQ1 dephosphorylation mimics exhibit more pronounced nuclear localization. HopQ1 delivered from Pto DC3000 was found to promote bacterial virulence in the tomato genotype Rio Grande 76R. However, the HopQ1(S51A) mutant delivered from Pto DC3000 was unable to promote pathogen virulence. Taken together, our data demonstrate that HopQ1 enhances bacterial virulence and associates with tomato 14-3-3 proteins in a phosphorylation-dependent manner that influences HopQ1’s subcellular localization and virulence-promoting activities in planta. PMID:23417089

  1. Analysis of 14-3-3 Family Member Function in Xenopus Embryos by Microinjection of Antisense Morpholino Oligos

    NASA Astrophysics Data System (ADS)

    Lau, Jeffrey M. C.; Muslin, Anthony J.

    The 14-3-3 intracellular phosphoserine/threonine-binding proteins are adapter molecules that regulate signal transduction, cell cycle, nutrient sensing, apoptotic, and cytoskeletal pathways. There are seven 14-3-3 family members, encoded by separate genes, in vertebrate organisms. To evaluate the role of individual 14-3-3 proteins in vertebrate embryonic development, we utilized an antisense morpholino oligo microinjection technique in Xenopus laevis embryos. By use of this method, we showed that embryos lacking specific 14-3-3 proteins displayed unique phenotypic abnormalities. Specifically, embryos lacking 14-3-3 τ exhibited gastrulation and axial patterning defects, but embryos lacking 14-3-3 γ exhibited eye defects without other abnormalities, and embryos lacking 14-3-3 ζ appeared completely normal. These and other results demonstrate the power and specificity of the morpholino antisense oligo microinjection technique.

  2. 14-3-3 in Thoracic Aortic Aneurysms

    PubMed Central

    Chakravarti, Ritu; Gupta, Karishma; Swain, Mamuni; Willard, Belinda; Scholtz, Jaclyn; Svensson, Lars G.; Roselli, Eric E.; Pettersson, Gosta; Johnston, Douglas R.; Soltesz, Edward G.; Yamashita, Michifumi; Stuehr, Dennis; Daly, Thomas M.; Hoffman, Gary S.

    2015-01-01

    Objective Large vessel vasculitides (LVV) are a group of autoimmune diseases characterized by injury to and anatomic modifications of large vessels, including the aorta and its branch vessels. Disease etiology is unknown. This study was undertaken to identify antigen targets within affected vessel walls in aortic root, ascending aorta, and aortic arch surgical specimens from patients with LVV, including giant cell arteritis, Takayasu arteritis, and isolated focal aortitis. Methods Thoracic aortic aneurysm specimens and autologous blood were acquired from consenting patients who underwent aorta reconstruction procedures. Aorta proteins were extracted from both patients with LVV and age-, race-, and sex-matched disease controls with noninflammatory aneurysms. A total of 108 serum samples from patients with LVV, matched controls, and controls with antinuclear antibodies, different forms of vasculitis, or sepsis were tested. Results Evaluation of 108 serum samples and 22 aortic tissue specimens showed that 78% of patients with LVV produced antibodies to 14-3-3 proteins in the aortic wall (93.7% specificity), whereas controls were less likely to do so (6.7% produced antibodies). LVV patient sera contained autoantibody sufficient to immunoprecipitate 14-3-3 protein(s) from aortic lysates. Three of 7 isoforms of 14-3-3 were found to be up-regulated in aorta specimens from patients with LVV, and 2 isoforms (ε and ζ) were found to be antigenic in LVV. Conclusion This is the first study to use sterile, snap-frozen thoracic aorta biopsy specimens to identify autoantigens in LVV. Our findings indicate that 78% of patients with LVV have antibody reactivity to 14-3-3 protein(s). The precise role of these antibodies and 14-3-3 proteins in LVV pathogenesis deserves further study. PMID:25917817

  3. Identification and expression analysis of four 14-3-3 genes during fruit ripening in banana (Musa acuminata L. AAA group, cv. Brazilian).

    PubMed

    Li, Mei-Ying; Xu, Bi-Yu; Liu, Ju-Hua; Yang, Xiao-Liang; Zhang, Jian-Bin; Jia, Cai-Hong; Ren, Li-Cheng; Jin, Zhi-Qiang

    2012-02-01

    To investigate the regulation of 14-3-3 proteins in banana (Musa acuminata L. AAA group, cv. Brazilian) fruit postharvest ripening, four cDNAs encoding 14-3-3 proteins were isolated from banana and designated as Ma-14-3-3a, Ma-14-3-3c, Ma-14-3-3e, and Ma-14-3-3i, respectively. Amino acid sequence alignment showed that the four 14-3-3 proteins shared a highly conserved core structure and variable C-terminal as well as N-terminal regions with 14-3-3 proteins from other plant species. Phylogenetic analysis revealed that the four 14-3-3 genes belong to the non-ε groups. They were differentially and specifically expressed in various tissues. Real-time RT-PCR analysis indicated that these four genes function differentially during banana fruit postharvest ripening. Three genes, Ma-14-3-3a, Ma-14-3-3c, and Ma-14-3-3e, were significantly induced by exogenous ethylene treatment. However, gene function differed in naturally ripened fruits. Ethylene could induce Ma-14-3-3c expression during postharvest ripening, but expression patterns of Ma-14-3-3a and Ma-14-3-3e suggest that these two genes appear to be involved in regulating ethylene biosynthesis during fruit ripening. No obvious relationship emerged between Ma-14-3-3i expression in naturally ripened and 1-MCP (1-methylcyclopropene)-treated fruit groups during fruit ripening. These results indicate that the 14-3-3 proteins might be involved in various regulatory processes of banana fruit ripening. Further studies will mainly focus on revealing the detailed biological mechanisms of these four 14-3-3 genes in regulating banana fruit postharvest ripening. PMID:22009053

  4. Development of a dot blot assay with antibodies to recombinant “core” 14-3-3 protein: Evaluation of its usefulness in diagnosis of Creutzfeldt–Jakob disease

    PubMed Central

    Subramanian, Sarada; Mahadevan, Anita; Satishchandra, Parthasarathy; Shankar, Susarla Krishna

    2016-01-01

    Background and Purpose: Definitive diagnosis of Creutzfeldt–Jakob disease (CJD) requires demonstration of infective prion protein (PrPSc) in brain tissues by immunohistochemistry or immunoblot, making antemortem diagnosis of CJD difficult. The World Health Organization (WHO) recommends detection of 14-3-3 protein in cerebrospinal fluid (CSF) in cases of dementia, with clinical correlation, as a useful diagnostic marker for CJD, obviating the need for brain biopsy. This facility is currently available in only a few specialized centers in the West and no commercial kit is available for clinical diagnostic use in India. Hence the objective of this study was to develop an in-house sensitive assay for quantitation of 14-3-3 protein and to evaluate its diagnostic potential to detect 14-3-3 proteins in CSF as a biomarker in suspected cases of CJD. Materials and Methods: A minigene expressing the “core” 14-3-3 protein was synthesized by overlapping polymerase chain reaction (PCR) and the recombinant protein was produced by employing a bacterial expression system. Polyclonal antibodies raised in rabbit against the purified recombinant protein were used for developing a dot blot assay with avidin-biotin technology for signal amplification and quantitation of 14-3-3 protein in CSF. Results: The results in the present study suggest the diagnostic potential of the dot blot method with about 10-fold difference (P< 0.001) in the CSF levels of 14-3-3 protein between the CJD cases (N= 50) and disease controls (N= 70). The receiver operating characteristic (ROC) analysis of the results suggested an optimal cutoff value of 2 ng/mL. Conclusions: We have developed an indigenous, economical, and sensitive dot blot method for the quantitation of 14-3-3 protein in CSF. PMID:27293331

  5. The chaperone-like protein 14-3-3η interacts with human α-synuclein aggregation intermediates rerouting the amyloidogenic pathway and reducing α-synuclein cellular toxicity.

    PubMed

    Plotegher, Nicoletta; Kumar, Dhruv; Tessari, Isabella; Brucale, Marco; Munari, Francesca; Tosatto, Laura; Belluzzi, Elisa; Greggio, Elisa; Bisaglia, Marco; Capaldi, Stefano; Aioanei, Daniel; Mammi, Stefano; Monaco, Hugo L; Samo, Bruno; Bubacco, Luigi

    2014-11-01

    Familial and idiopathic Parkinson's disease (PD) is associated with the abnormal neuronal accumulation of α-synuclein (aS) leading to β-sheet-rich aggregates called Lewy Bodies (LBs). Moreover, single point mutation in aS gene and gene multiplication lead to autosomal dominant forms of PD. A connection between PD and the 14-3-3 chaperone-like proteins was recently proposed, based on the fact that some of the 14-3-3 isoforms can interact with genetic PD-associated proteins such as parkin, LRRK2 and aS and were found as components of LBs in human PD. In particular, a direct interaction between 14-3-3η and aS was reported when probed by co-immunoprecipitation from cell models, from parkinsonian brains and by surface plasmon resonance in vitro. However, the mechanisms through which 14-3-3η and aS interact in PD brains remain unclear. Herein, we show that while 14-3-3η is unable to bind monomeric aS, it interacts with aS oligomers which occur during the early stages of aS aggregation. This interaction diverts the aggregation process even when 14-3-3η is present in sub-stoichiometric amounts relative to aS. When aS level is overwhelmingly higher than that of 14-3-3η, the fibrillation process becomes a sequestration mechanism for 14-3-3η, undermining all processes governed by this protein. Using a panel of complementary techniques, we single out the stage of aggregation at which the aS/14-3-3η interaction occurs, characterize the products of the resulting processes, and show how the processes elucidated in vitro are relevant in cell models. Our findings constitute a first step in elucidating the molecular mechanism of aS/14-3-3η interaction and in understanding the critical aggregation step at which 14-3-3η has the potential to rescue aS-induced cellular toxicity. PMID:24895406

  6. Locomotor hyperactivity in 14-3-3ζ KO mice is associated with dopamine transporter dysfunction

    PubMed Central

    Ramshaw, H; Xu, X; Jaehne, E J; McCarthy, P; Greenberg, Z; Saleh, E; McClure, B; Woodcock, J; Kabbara, S; Wiszniak, S; Wang, Ting-Yi; Parish, C; van den Buuse, M; Baune, B T; Lopez, A; Schwarz, Q

    2013-01-01

    Dopamine (DA) neurotransmission requires a complex series of enzymatic reactions that are tightly linked to catecholamine exocytosis and receptor interactions on pre- and postsynaptic neurons. Regulation of dopaminergic signalling is primarily achieved through reuptake of extracellular DA by the DA transporter (DAT) on presynaptic neurons. Aberrant regulation of DA signalling, and in particular hyperactivation, has been proposed as a key insult in the presentation of schizophrenia and related neuropsychiatric disorders. We recently identified 14-3-3ζ as an essential component of neurodevelopment and a central risk factor in the schizophrenia protein interaction network. Our analysis of 14-3-3ζ-deficient mice now shows that baseline hyperactivity of knockout (KO) mice is rescued by the antipsychotic drug clozapine. 14-3-3ζ KO mice displayed enhanced locomotor hyperactivity induced by the DA releaser amphetamine. Consistent with 14-3-3ζ having a role in DA signalling, we found increased levels of DA in the striatum of 14-3-3ζ KO mice. Although 14-3-3ζ is proposed to modulate activity of the rate-limiting DA biosynthesis enzyme, tyrosine hydroxylase (TH), we were unable to identify any differences in total TH levels, TH localization or TH activation in 14-3-3ζ KO mice. Rather, our analysis identified significantly reduced levels of DAT in the absence of notable differences in RNA or protein levels of DA receptors D1–D5. Providing insight into the mechanisms by which 14-3-3ζ controls DAT stability, we found a physical association between 14-3-3ζ and DAT by co-immunoprecipitation. Taken together, our results identify a novel role for 14-3-3ζ in DA neurotransmission and provide support to the hyperdopaminergic basis of pathologies associated with schizophrenia and related disorders. PMID:24301645

  7. Ywhaz/14-3-3ζ Deletion Improves Glucose Tolerance Through a GLP-1-Dependent Mechanism.

    PubMed

    Lim, Gareth E; Piske, Micah; Lulo, James E; Ramshaw, Hayley S; Lopez, Angel F; Johnson, James D

    2016-07-01

    Multiple signaling pathways mediate the actions of metabolic hormones to control glucose homeostasis, but the proteins that coordinate such networks are poorly understood. We previously identified the molecular scaffold protein, 14-3-3ζ, as a critical regulator of in vitro β-cell survival and adipogenesis, but its metabolic roles in glucose homeostasis have not been studied in depth. Herein, we report that Ywhaz gene knockout mice (14-3-3ζKO) exhibited elevated fasting insulin levels while maintaining normal β-cell responsiveness to glucose when compared with wild-type littermate controls. In contrast with our observations after an ip glucose bolus, glucose tolerance was significantly improved in 14-3-3ζKO mice after an oral glucose gavage. This improvement in glucose tolerance was associated with significantly elevated fasting glucagon-like peptide-1 (GLP-1) levels. 14-3-3ζ knockdown in GLUTag L cells elevated GLP-1 synthesis and increased GLP-1 release. Systemic inhibition of the GLP-1 receptor attenuated the improvement in oral glucose tolerance that was seen in 14-3-3ζKO mice. When taken together these findings demonstrate novel roles of 14-3-3ζ in the regulation of glucose homeostasis and suggest that modulating 14-3-3ζ levels in intestinal L cells may have beneficial metabolic effects through GLP-1-dependent mechanisms. PMID:27167773

  8. Differential expression of 14-3-3 isoforms in human alcoholic brain

    PubMed Central

    MacKay, Rachel K.; Colson, Natalie J.; Dodd, Peter R.; Lewohl, Joanne M.

    2011-01-01

    Background Neuropathological damage due to chronic alcohol abuse often results in impairment of cognitive function. The damage is particularly marked in the frontal cortex. The 14-3-3 protein family consists of 7 proteins, β, γ, ε, ζ, η, θ and σ, encoded by 7 distinct genes. They are highly conserved molecular chaperones with roles in regulation of metabolism, signal transduction, cell-cycle control, protein trafficking, and apoptosis. They may also play an important role in neurodegeneration in chronic alcoholism. Methods We used Real-Time PCR to measure the expression of 14-3-3 mRNA transcripts in both the dorsolateral prefrontal cortex and motor cortex of human brains obtained at autopsy. Results We found significantly lower 14-3-3β, γ and θ expression in both cortical areas of alcoholics; but no difference in 14-3-3η expression, and higher expression of 14-3-3σ, in both areas. Levels of 14-3-3ζ and ε transcripts were significantly lower only in alcoholic motor cortex. Conclusions Altered 14-3-3 expression could contribute to synaptic dysfunction and altered neurotransmission in chronic alcohol misuse by human subjects. PMID:21332526

  9. 14-3-3ζ: A numbers game in adipocyte function?

    PubMed Central

    Lim, Gareth E.; Johnson, James D.

    2016-01-01

    ABSTRACT Molecular scaffolds are often viewed as passive signaling molecules that facilitate protein-protein interactions. However, new evidence gained from the use of loss-of-function or gain-of-function models is dispelling this notion. Our own recent discovery of 14-3-3ζ as an essential regulator of adipogenesis highlights the complex roles of this member of the 14-3-3 protein family. Depletion of the 14-3-3ζ isoform affected parallel pathways that drive adipocyte development, including pathways controlling the stability of key adipogenic transcription factors and cell cycle progression. Going beyond adipocyte differentiation, this study opens new avenues of research in the context of metabolism, as 14-3-3ζ binds to a variety of well-established metabolic proteins that harbor its canonical phosphorylation binding motifs. This suggests that 14-3-3ζ may contribute to key metabolic signaling pathways, such as those that facilitate glucose uptake and fatty acid metabolism. Herein, we discuss these novel areas of research, which will undoubtedly shed light onto novel roles of 14-3-3ζ, and perhaps its related family members, on glucose homeostasis. PMID:27386155

  10. Characterization of the Interactome of the Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 2 Reveals the Hyper Variable Region as a Binding Platform for Association with 14-3-3 Proteins.

    PubMed

    Xiao, Yihong; Wu, Weining; Gao, Jiming; Smith, Nikki; Burkard, Christine; Xia, Dong; Zhang, Minxia; Wang, Chengbao; Archibald, Alan; Digard, Paul; Zhou, En-Min; Hiscox, Julian A

    2016-05-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat to the swine industry worldwide and hence global food security, exacerbated by a newly emerged highly pathogenic (HP-PRRSV) strain from China. PRRSV nonstructural protein 2 (nsp2) is a multifunctional polypeptide with strain-dependent influences on pathogenicity. A number of discrete functional regions have been identified on the protein. Quantitative label free proteomics was used to identify cellular binding partners of nsp2 expressed by HP-PRRSV. This allowed the identification of potential cellular interacting partners and the discrimination of nonspecific interactions. The interactome data were further investigated and validated using biological replicates and also compared with nsp2 from a low pathogenic (LP) strain of PRRSV. Validation included both forward and reverse pulldowns and confocal microscopy. The data indicated that nsp2 interacted with a number of cellular proteins including 14-3-3, CD2AP, and other components of cellular aggresomes. The hyper-variable region of nsp2 protein was identified as a binding platform for association with 14-3-3 proteins. PMID:26709850

  11. Dynamic interaction between 14-3-3zeta and bax during TNF-α-induced apoptosis in living cells

    NASA Astrophysics Data System (ADS)

    Gao, Xuejuan; Xing, Da; Chen, Tongsheng

    2006-09-01

    Bax, a proapoptotic member of the Bcl-2 family, localizes largely in the cytoplasm but redistributes to mitochondria and undergoes oligomerization to induce the release of apoptogenic factors such as cytochrome c in response to apoptotic stimuli. Cytoplasmic protein 14-3-3zeta binds to Bax and, upon apoptotic stimulation, releases Bax by a caspase-independent mechanism. However, the direct interaction of the cytoplasmic 14-3-3zeta and Bax in living cells has not been observed. In present study, to monitor the dynamic interaction between 14-3-3zeta and Bax in living cells in real time during apoptosis induced by tumor necrosis factor (TNF-α), DsRed-14-3-3zeta plasmid is constructed. By cotransfecting DsRed- 14-3-3zeta and GFP-Bax plasmids into human lung adenocarcinoma cells (ASTC-a-1), we observe the dynamic interaction between Bax and 14-3-3zeta using fluorescence resonance energy transfer (FRET) technique on laser scanning confocal microscope. The results show that 14-3-3zeta remains in the cytoplasm but GFP-Bax translocates to mitochondria completely after TNF-α stimulation. These results reveal that 14-3-3zeta binds directly to Bax in healthy cells, and that 14-3-3zeta negatively regulates Bax translocation to mitochondria during TNF-α-induced apoptosis.

  12. Sphingosine induces apoptosis in hippocampal neurons and astrocytes by activating caspase-3/-9 via a mitochondrial pathway linked to SDK/14-3-3 protein/Bax/cytochrome c.

    PubMed

    Kanno, Takeshi; Nishizaki, Tomoyuki

    2011-09-01

    The present study examined sphingosine-induced apoptosis in cultured rat hippocampal neurons and astrocytes. Sphingosine induced apoptosis in a concentration (1-100 µM)-dependent manner, that is inhibited by the PKC-δ inhibitor rottlerin, and a similar effect was obtained with the sphingosine kinase inhibitors, to raise intracellular sphingosine concentrations. Sphingosine increased presence of sphingosine-dependent protein kinase (SDK), and the effect was suppressed by rottlerin. Sphingosine increased phosphorylated 14-3-3 protein, thereby transforming the protein from a dimeric structure into a monomeric structure. Sphingosine accumulated Bax in the mitochondria and stimulated cytochrome c release into the cytosol, and those effects were inhibited by rottlerin. Sphingosine disrupted mitochondrial membrane potentials, that was abolished by silencing the PKC-δ-targeted gene. Moreover, sphingosine activated caspase-9 and the effector caspase-3 in a PKC-δ-dependent manner. Taken together, the results of the present study indicate that sphingosine activates SDK, produced through proteolytic processing of an active form of PKC-δ, to phosphorylate 14-3-3 protein and transform into a monomeric structure, causing Bax dissociation from 14-3-3 protein and accumulation in the mitochondria, which perturbs mitochondrial membrane potentials allowing cytochrome c release into the cytosol, to activate caspase-9 and the effector caspase-3, responsible for apoptosis in hippocampal neurons and astrocytes. PMID:21660956

  13. 14-3-3σ is an independent prognostic biomarker for gastric cancer and is associated with apoptosis and proliferation in gastric cancer.

    PubMed

    Li, Yi-Liang; Liu, Lihua; Xiao, Yang; Zeng, Tao; Zeng, Chao

    2015-01-01

    14-3-3 proteins participate in various cellular processes, including apoptosis, proliferation and malignant transformation. 14-3-3σ, a member of the 14-3-3 protein family, is important in several types of cancer; however, little is known about the clinical significance and biological roles of 14-3-3σ in gastric cancer. The present study analyzed the expression pattern of 14-3-3σ in gastric cancer and investigated its correlation with the prognosis of gastric cancer patients. Furthermore, the association of 14-3-3σ with Ki-67, Bcl-2 and Bax was evaluated. 14-3-3σ was expressed at higher level in gastric cancer tissue compared with healthy gastric tissue, and 14-3-3σ expression was significantly correlated with tumor size and tumor node metastasis stage (P<0.05). To the best of our knowledge, the present study data are the first to suggest that 14-3-3σ expression has been significantly associated with poor prognosis in gastric cancer. Additionally, 14-3-3σ overexpression was positively correlated with Ki-67 and Bcl-2 expression levels. Thus, 14-3-3σ is a potential prognostic marker for gastric cancer patients, and may be involved in regulating the apoptosis and proliferation of gastric cancer cells. PMID:25435977

  14. Loss of the 14-3-3σ is essential for LASP1-mediated colorectal cancer progression via activating PI3K/AKT signaling pathway

    PubMed Central

    Shao, Ziyun; Cai, Yanjun; Xu, Lijun; Yao, Xueqing; Shi, Jiaolong; Zhang, Feifei; Luo, Yuhao; Zheng, Kehong; Liu, Jian; Deng, Fengliu; Li, Rui; Zhang, Lanzhi; Wang, Hui; Li, Mingyi; Ding, Yanqing; Zhao, Liang

    2016-01-01

    LIM and SH3 protein 1 (LASP1) can promote colorectal cancer (CRC) progression and metastasis, but the direct evidence that elucidates the molecular mechanism remains unclear. Here, our proteomic data showed that LASP1 interacted with 14-3-3σ and decreased the expression of 14-3-3σ in CRC. Deletion of 14-3-3σ was required for LASP1-mediated CRC cell aggressiveness. In vitro gain- and loss-of-function assays showed that 14-3-3σ suppressed the ability of cell migration and decreased the phosphorylation of AKT in CRC cells. We further observed clearly co-localization between AKT and 14-3-3σ in CRC cells. Treatment of PI3K inhibitor LY294002 markedly prevented phosphorylation of AKT and subsequently counteract aggressive phenotype mediated by siRNA of 14-3-3σ. Clinically, 14-3-3σ is frequently down-regulated in CRC tissues. Down-regulation of 14-3-3σ is associated with tumor progression and poor prognosis of patients with CRC. Multivariate analysis confirmed low expression of 14-3-3σ as an independent prognostic factor for CRC. A combination of low 14-3-3σ and high LASP1 expression shows a worse trend with overall survival of CRC patients. Our research paves the path to future investigation of the LASP1-14-3-3σ axis as a target for novel anticancer therapies of advanced CRC. PMID:27156963

  15. Characteristics of Korean patients with suspected Creutzfeldt-Jakob disease with 14-3-3 protein in cerebrospinal fluid: Preliminary study of the Korean Creutzfeldt-Jakob disease active surveillance program

    PubMed Central

    Lim, Jae-Sung; Kwon, Hyung-Min; Jang, Jae-Won; Ju, Young-Ran; Kim, SuYeon; Park, Young Ho; Park, So Young; Kim, SangYun

    2015-01-01

    Abstract Although Korea had a national surveillance system for Creutzfeldt-Jakob disease (CJD), it was mainly dependent on attending physician's reports. Thus, little prospective data about the epidemiology, characteristics, and final diagnoses of suspected patients were available. We have established a nationwide network for the active surveillance of patients with suspected CJD. When the requested cerebrospinal fluid (CSF) samples tested positive for 14-3-3 protein, we investigated the clinical characteristics of the corresponding patients and followed them until their final diagnoses were confirmed. A total of 218 samples were requested for CSF assays from May 2010 to August 2012, and 106 (48.6%) were positive for 14-3-3 protein. In 89 patients with complete clinical data, 38 (42.7%) were diagnosed with probable CJD and the estimated annual occurrence of CJD was 16.3 persons-per-year. The most common diagnoses of the remainder were central nervous system infection and any-cause encephalopathy. Non-CJD subjects showed worse initial consciousness levels than CJD patients. This preliminary study showed that the number of reported cases of CJD and the true positivity rates of CSF 14-3-3 protein assays were both low in Korea. An active surveillance system is urgently needed to provide the latest nationwide epidemiological data of CJD. PMID:25996401

  16. 14-3-3, an integrator of cell mechanics and cytokinesis.

    PubMed

    Robinson, Douglas N

    2010-11-01

    One of the goals of understanding cytokinesis is to uncover the molecular regulation of the cellular mechanical properties that drive cell shape change. Such regulatory pathways are likely to be used at multiple stages of a cell's life, but are highly featured during cell division. Recently, we demonstrated that 14-3-3 (encoded by a single gene in the social amoeba Dictyostelium discoideum) serves to integrate key cytoskeletal components-microtubules, Rac and myosin II-to control cell mechanics and cytokinesis. As 14-3-3 proteins are frequently altered in a variety of human tumors, we extend these observations to suggest possible additional roles for how 14-3-3 proteins may contribute to tumorigenesis. PMID:21686271

  17. A Characterization of the expression of 14-3-3 isoforms in psoriasis, basal cell carcinoma, atopic dermatitis and contact dermatitis

    PubMed Central

    Raaby, Line; Otkjær, Kristian; Salvskov-Iversen, Maria Luise; Johansen, Claus; Iversen, Lars

    2010-01-01

    14-3-3 is a highly conserved protein involved in a number of cellular processes including cell signalling, cell cycle regulation and gene transcription. Seven isoforms of the protein have been identified; β, γ, ε, ζ η σ and τ. The expression profile of the various isoforms in skin diseases is unknown. To investigate the expression of the seven 14-3-3 isoforms in involved and uninvolved skin from psoriasis, basal cell carcinoma (BCC), atopic dermatitis and nickel induced allergic contact dermatitis. Punch biopsies from involved and uninvolved skin were analyzed with quantitative reverse transcription-polymerase chain reaction to determine the mRNA expression of the 14-3-3 isoforms. The protein level of 14-3-3 isoforms was measured by Western blot technique in keratome biopsies from patients with psoriasis. Evaluation of dermal and epidermal protein expression was performed by immunofluorescence staining. Increased 14-3-3τ mRNA levels were detected in involved skin from patients with psoriasis, contact dermatitis and BCC. 14-3-3σ mRNA expression was increased in psoriasis and contact dermatitis, but not in BCC. In atopic dermatitis no significant difference between involved and uninvolved skin was found. The expression of the 14-3-3 isoforms was also studied at the protein level in psoriasis. Only 14-3-3τ expression was significantly increased in involved psoriatic skin compared with uninvolved skin. Immunofluorescence staining with 14-3-3τ- and 14-3-3σ-specific antibodies showed localization of both isoforms to the cytoplasm of the keratinocytes in the various skin sections. These results demonstrate a disease specific expression profile of the 14-3-3τ and 14-3-3σ iso-forms. PMID:25386251

  18. 14-3-3 sigma and 14-3-3 zeta plays an opposite role in cell growth inhibition mediated by transforming growth factor-beta 1.

    PubMed

    Hong, Hye-Young; Jeon, Woo-Kwang; Bae, Eun-Jin; Kim, Shin-Tae; Lee, Ho-Jae; Kim, Seong-Jin; Kim, Byung-Chul

    2010-03-01

    The expression of 14-3-3 proteins is dysregulated in various types of cancer. This study was undertaken to investigate the effects of 14-3-3 zeta and 14-3-3 sigma on cell growth inhibition mediated by transforming growth factor-beta 1 (TGF-beta1). Mouse mammary epithelial cells (Eph4) that are transformed with oncogenic c-H-Ras (EpRas) and no longer sensitive to TGF-beta1-mediated growth inhibition displayed increased expression of 14-3-3 zeta and decreased expression of 14-3-3 sigma compared with parental Eph4 cells. Using small interfering RNA-mediated knockdown and overexpression of 14-3-3 sigma or 14-3-3 zeta, we showed that 14-3-3 sigma is required for TGF-beta1-mediated growth inhibition whereas 14-3-3 zeta negatively modulates this growth inhibitory response. Notably, overexpression of 14-3-3 zeta increased the level of Smad3 protein that is phosphorylated at linker regions and cannot mediate the TGF-beta1 growth inhibitory response. Consistent with this finding, mutation of the 14-3-3 zeta phosphorylation sites in Smad3 markedly reduced the 14-3-3 zeta-mediated inhibition of TGF-beta1-induced p15 promoter-reporter activity and cell cycle arrest, suggesting that these residues are critical targets of 14-3-3 zeta in the suppression of TGF-beta1-mediated growth. Taken together, our findings indicate that dysregulation of 14-3-3 sigma or 14-3-3 zeta contributes to TGF-beta1 resistance in cancer cells. PMID:20082218

  19. Ischemia preconditioning protects astrocytes from ischemic injury through 14-3-3γ.

    PubMed

    Pang, Ying; Chai, Chao Rui; Gao, Kai; Jia, Xi Hua; Kong, Jin Ge; Chen, Xiao Qian; Vatcher, Greg; Chen, Jian Guo; Yu, Albert Cheung Hoi

    2015-10-01

    Stroke is a leading cause of death and disability, and new strategies are required to reduce neuronal injury and improve prognosis. Ischemia preconditioning (IPC) is an intrinsic phenomenon that protects cells from subsequent ischemic injury and might provide promising mechanisms for clinical treatment. In this study, primary astrocytes exhibited significantly less cell death than control when exposed to different durations of IPC (15, 30, 60, or 120 min). A 15-min duration was the most effective IPC to protect astrocytes from 8-hr-ischemia injury. The protective mechanisms of IPC involve the upregulation of protective proteins, including 14-3-3γ, and attenuation of malondialdehyde (MDA) content and ATP depletion. 14-3-3γ is an antiapoptotic intracellular protein that was significantly upregulated for up to 84 hr after IPC. In addition, IPC promoted activation of the c-Jun N-terminal kinase (JNK), extracellular signal-related kinase (ERK)-1/2, p38, and protein kinase B (Akt) signaling pathways. When JNK was specifically inhibited with SP600125, the upregulation of 14-3-3γ induced by IPC was almost completely abolished; however, there was no effect on ATP or MDA levels. This suggests that, even though both energy preservation and 14-3-3γ up-regulation were turned on by IPC, they were controlled by different pathways. The ERK1/2, p38, and Akt signaling pathways were not involved in the 14-3-3γ upregulation and energy preservation. These results indicate that IPC could protect astrocytes from ischemia injury by inducing 14-3-3γ and by alleviating energy depletion through different pathways, suggesting multiple protection of IPC and providing new insights into potential stroke therapies. PMID:25711139

  20. Modulation of 14-3-3 interaction with phosphorylated histone H3 by combinatorial modification patterns

    PubMed Central

    Winter, Stefan; Fischle, Wolfgang; Seiser, Christian

    2011-01-01

    Post-translational modifications of histones are determining factors in the global and local regulation of genome activity. Phosphorylation of histone H3 is globally associated with mitotic chromatin compaction but occurs in a much more restricted manner during interphase transcriptional regulation of a limited subset of genes. In the course of gene regulation, serine 10 phosphorylation at histone H3 is targeted to a very small fraction of nucleosomes that is highly susceptible to additional acetylation events. Recently, we and others have identified 14-3-3 as a binding protein that recognizes both phosphorylated serine 10 and phosphorylated serine 28 on histone H3. In vitro, the affinity of 14-3-3 for phosphoserine 10 is weak but becomes significantly increased by additional acetylation of either lysine 9 or lysine 14 on the same histone tail. In contrast, the histone H3S28 site matches elements of 14-3-3 high affinity consensus motifs. This region mediates an initial stronger interaction that is less susceptible to modulation by “auxiliary” modifications. Here we discuss the binding of 14-3-3 proteins to histone H3 in detail and putative biological implications of these interactions. PMID:18418070

  1. 14-3-3{sigma} controls corneal epithelial cell proliferation and differentiation through the Notch signaling pathway

    SciTech Connect

    Xin, Ying; Lu, Qingxian; Li, Qiutang

    2010-02-19

    14-3-3{sigma} (also called stratifin) is specifically expressed in the stratified squamous epithelium and its function was recently shown to be linked to epidermal stratification and differentiation in the skin. In this study, we investigated its role in corneal epithelium cell proliferation and differentiation. We showed that the 14-3-3{sigma} mutation in repeated epilation (Er) mutant mice results in a dominant negative truncated protein. Primary corneal epithelial cells expressing the dominant negative protein failed to undergo high calcium-induced cell cycle arrest and differentiation. We further demonstrated that blocking endogenous 14-3-3{sigma} activity in corneal epithelial cells by overexpressing dominative negative 14-3-3{sigma} led to reduced Notch activity and Notch1/2 transcription. Significantly, expression of the active Notch intracellular domain overcame the block in epithelial cell differentiation in 14-3-3{sigma} mutant-expressing corneal epithelial cells. We conclude that 14-3-3{sigma} is critical for regulating corneal epithelial proliferation and differentiation by regulating Notch signaling activity.

  2. Possible additional roles in mating for Ustilago maydis Rho1 and 14-3-3 homologues

    PubMed Central

    Pham, Cau D

    2010-01-01

    Both the Rho GTPases and 14-3-3 proteins each belong to ubiquitous families of proteins involved in a variety of cellular processes, including cytokinesis, cell polarity, cellular differentiation and apoptosis. In fungi, these components of signaling pathways are involved in cell cycle regulation, cytokinesis and virulence. We study cellular differentiation and pathogenesis for Ustilago maydis, the dimorphic fungal pathogen of maize. We have reported on the interactions of Pdc1, a U. maydis homologue of human 14-3-3ɛ, with Rho1, a small GTP binding protein; these proteins participate in cell polarity and filamentation pathways that include another small G protein, Rac1, and its effector PAK kinase, Cla4. Here we describe additional experiments that explore possible relationships of Pdc1 and Rho1 with another PAK-like kinase pathway and with the a matingtype locus. PMID:20539785

  3. The role of 14-3-3{beta} in transcriptional activation of estrogen receptor {alpha} and its involvement in proliferation of breast cancer cells

    SciTech Connect

    Kim, Yoonseo; Kim, Hyungjin; Jang, Sung-Wuk; Ko, Jesang

    2011-10-14

    Highlights: {yields} 14-3-3{beta} interacts with ER{alpha} and the interaction is Akt-dependent. {yields} 14-3-3{beta} regulates the transcriptional activity of ER{alpha} in a ligand-dependent manner. {yields} 14-3-3{beta} increases expressions of ER{alpha} target genes. {yields} 14-3-3{beta} increases breast cancer cell proliferation. -- Abstract: The estrogen receptor (ER) functions as a transcription factor that mediates the effects of estrogen. ER{alpha}, which plays a crucial role in the development and progression of breast cancer, is activated by estrogen binding, leading to receptor phosphorylation, dimerization, and recruitment of co-activators and chaperons to the estrogen-bound receptor complex. The 14-3-3 proteins bind to target proteins via phosphorylation and influence many cellular events by altering their subcellular localization or acting as a chaperone. However, regulation of ER{alpha} expression and transactivation by the 14-3-3 proteins has not been reported. We demonstrate that 14-3-3{beta} functions as a positive regulator of ER{alpha} through a direct protein-protein interaction in an estrogen-dependent manner. Ectopic expression of 14-3-3{beta} stimulated ER{alpha}-mediated transcriptional activity in MCF-7 breast cancer cells. Enhanced ER{alpha} transcriptional activity due to 14-3-3{beta} increased the expressions of the endogenous ER{alpha} target genes, leading to proliferation of breast cancer cells. We suggest that 14-3-3{beta} has oncogenic potential in breast cancer via binding to ER{alpha} and activation of the transcriptional activity of ER{alpha}.

  4. 14-3-3ζ promotes hepatocellular carcinoma venous metastasis by modulating hypoxia-inducible factor-1α

    PubMed Central

    Shi, Jie; Yu, Hongming; Zhang, Long; Wang, Kang; Liu, Shangrong; Cheng, Shuqun

    2016-01-01

    Portal vein tumor thrombus (PVTT) is a type of intrahepatic metastasis arising from hepatocellular carcinoma (HCC) and is highly correlated with a poor prognosis. Hypoxia is common in solider tumors, including HCC, where it alters the behavior of HCC cells. We asked whether and how hypoxia contributes to PVTT formation. We demonstrated that increased intratumoral hypoxia is strongly associated with PVTT formation in HCC. We also showed that 14-3-3ζ is induced by hypoxia in HCC cells and correlates with PVTT formation in clinical HCC samples. In addition, 14-3-3ζ up-regulates HIF-1α expression by recruiting HDAC4, which prevents HIF-1α acetylation, thereby stabilizing the protein. Under hypoxic conditions in vitro, 14-3-3ζ knockdown inhibits hypoxia-induced HCC invasion by the HIF-1α/EMT pathway. Blockade of 14-3-3ζ in HCC cells reduces PVTT formation and distant lung metastasis in vivo. Moreover, a combination of 14-3-3ζ and HIF-1α expression is more prognostic for HCC patients than either protein alone. These results suggest that the hypoxia/14-3-3ζ/HIF-1α pathway plays an important role in PVTT formation and HCC metastasis. PMID:26910835

  5. 14-3-3-dependent inhibition of the deubiquitinating activity of UBPY and its cancellation in the M phase

    SciTech Connect

    Mizuno, Emi; Kitamura, Naomi; Komada, Masayuki

    2007-10-01

    The deubiquitinating enzyme UBPY, also known as USP8, regulates cargo sorting and membrane traffic at early endosomes. Here we demonstrate the regulatory mechanism of the UBPY catalytic activity. We identified 14-3-3 {epsilon}, {gamma}, and {zeta} as UBPY-binding proteins using co-immunoprecipitation followed by mass spectrometric analysis. The 14-3-3 binding of UBPY was inhibited by mutating the consensus 14-3-3-binding motif RSYS{sup 680}SP, by phosphatase treatment, and by competition with the Ser{sup 680}-phosphorylated RSYS{sup 680}SP peptide. Metabolic labeling with [{sup 32}P]orthophosphate and immunoblotting using antibody against the phosphorylated 14-3-3-binding motif showed that Ser{sup 680} is a major phosphorylation site in UBPY. These results indicated that 14-3-3s bind to the region surrounding Ser{sup 680} in a phosphorylation-dependent manner. The mutation at Ser{sup 680} led to enhanced ubiquitin isopeptidase activity of UBPY toward poly-ubiquitin chains and a cellular substrate, epidermal growth factor receptor, in vitro and in vivo. Moreover, addition of 14-3-3{epsilon} inhibited the UBPY activity in vitro. Finally, UBPY was dephosphorylated at Ser{sup 680} and dissociated from 14-3-3s in the M phase, resulting in enhanced activity of UBPY during cell division. We conclude that UBPY is catalytically inhibited in a phosphorylation-dependent manner by 14-3-3s during the interphase, and this regulation is cancelled in the M phase.

  6. Characterization and small-molecule stabilization of the multisite tandem binding between 14-3-3 and the R domain of CFTR.

    PubMed

    Stevers, Loes M; Lam, Chan V; Leysen, Seppe F R; Meijer, Femke A; van Scheppingen, Daphne S; de Vries, Rens M J M; Carlile, Graeme W; Milroy, Lech G; Thomas, David Y; Brunsveld, Luc; Ottmann, Christian

    2016-03-01

    Cystic fibrosis is a fatal genetic disease, most frequently caused by the retention of the CFTR (cystic fibrosis transmembrane conductance regulator) mutant protein in the endoplasmic reticulum (ER). The binding of the 14-3-3 protein to the CFTR regulatory (R) domain has been found to enhance CFTR trafficking to the plasma membrane. To define the mechanism of action of this protein-protein interaction, we have examined the interaction in vitro. The disordered multiphosphorylated R domain contains nine different 14-3-3 binding motifs. Furthermore, the 14-3-3 protein forms a dimer containing two amphipathic grooves that can potentially bind these phosphorylated motifs. This results in a number of possible binding mechanisms between these two proteins. Using multiple biochemical assays and crystal structures, we show that the interaction between them is governed by two binding sites: The key binding site of CFTR (pS768) occupies one groove of the 14-3-3 dimer, and a weaker, secondary binding site occupies the other binding groove. We show that fusicoccin-A, a natural-product tool compound used in studies of 14-3-3 biology, can stabilize the interaction between 14-3-3 and CFTR by selectively interacting with a secondary binding motif of CFTR (pS753). The stabilization of this interaction stimulates the trafficking of mutant CFTR to the plasma membrane. This definition of the druggability of the 14-3-3-CFTR interface might offer an approach for cystic fibrosis therapeutics. PMID:26888287

  7. Class-Specific Evolution and Transcriptional Differentiation of 14-3-3 Family Members in Mesohexaploid Brassica rapa.

    PubMed

    Chandna, Ruby; Augustine, Rehna; Kanchupati, Praveena; Kumar, Roshan; Kumar, Pawan; Arya, Gulab C; Bisht, Naveen C

    2016-01-01

    14-3-3s are highly conserved, multigene family proteins that have been implicated in modulating various biological processes. The presence of inherent polyploidy and genome complexity has limited the identification and characterization of 14-3-3 proteins from globally important Brassica crops. Through data mining of Brassica rapa, the model Brassica genome, we identified 21 members encoding 14-3-3 proteins namely, BraA.GRF14.a to BraA.GRF14.u. Phylogenetic analysis indicated that B. rapa contains both ε (epsilon) and non-ε 14-3-3 isoforms, having distinct intron-exon structural organization patterns. The non-ε isoforms showed lower divergence rate (Ks < 0.45) compared to ε protein isoforms (Ks > 0.48), suggesting class-specific divergence pattern. Synteny analysis revealed that mesohexaploid B. rapa genome has retained 1-5 orthologs of each Arabidopsis 14-3-3 gene, interspersed across its three fragmented sub-genomes. qRT-PCR analysis showed that 14 of the 21 BraA.GRF14 were expressed, wherein a higher abundance of non-ε transcripts was observed compared to the ε genes, indicating class-specific transcriptional bias. The BraA.GRF14 genes showed distinct expression pattern during plant developmental stages and in response to abiotic stress, phytohormone treatments, and nutrient deprivation conditions. Together, the distinct expression pattern and differential regulation of BraA.GRF14 genes indicated the occurrence of functional divergence of B. rapa 14-3-3 proteins during plant development and stress responses. PMID:26858736

  8. Class-Specific Evolution and Transcriptional Differentiation of 14-3-3 Family Members in Mesohexaploid Brassica rapa

    PubMed Central

    Chandna, Ruby; Augustine, Rehna; Kanchupati, Praveena; Kumar, Roshan; Kumar, Pawan; Arya, Gulab C.; Bisht, Naveen C.

    2016-01-01

    14-3-3s are highly conserved, multigene family proteins that have been implicated in modulating various biological processes. The presence of inherent polyploidy and genome complexity has limited the identification and characterization of 14-3-3 proteins from globally important Brassica crops. Through data mining of Brassica rapa, the model Brassica genome, we identified 21 members encoding 14-3-3 proteins namely, BraA.GRF14.a to BraA.GRF14.u. Phylogenetic analysis indicated that B. rapa contains both ε (epsilon) and non-ε 14-3-3 isoforms, having distinct intron-exon structural organization patterns. The non-ε isoforms showed lower divergence rate (Ks < 0.45) compared to ε protein isoforms (Ks > 0.48), suggesting class-specific divergence pattern. Synteny analysis revealed that mesohexaploid B. rapa genome has retained 1–5 orthologs of each Arabidopsis 14-3-3 gene, interspersed across its three fragmented sub-genomes. qRT-PCR analysis showed that 14 of the 21 BraA.GRF14 were expressed, wherein a higher abundance of non-ε transcripts was observed compared to the ε genes, indicating class-specific transcriptional bias. The BraA.GRF14 genes showed distinct expression pattern during plant developmental stages and in response to abiotic stress, phytohormone treatments, and nutrient deprivation conditions. Together, the distinct expression pattern and differential regulation of BraA.GRF14 genes indicated the occurrence of functional divergence of B. rapa 14-3-3 proteins during plant development and stress responses. PMID:26858736

  9. miR-451 protects against erythroid oxidant stress by repressing 14-3-3zeta.

    PubMed

    Yu, Duonan; dos Santos, Camila O; Zhao, Guowei; Jiang, Jing; Amigo, Julio D; Khandros, Eugene; Dore, Louis C; Yao, Yu; D'Souza, Janine; Zhang, Zhe; Ghaffari, Saghi; Choi, John; Friend, Sherree; Tong, Wei; Orange, Jordan S; Paw, Barry H; Weiss, Mitchell J

    2010-08-01

    The bicistronic microRNA (miRNA) locus miR-144/451 is highly expressed during erythrocyte development, although its physiological roles are poorly understood. We show that miR-144/451 ablation in mice causes mild erythrocyte instability and increased susceptibility to damage after exposure to oxidant drugs. This phenotype is deeply conserved, as miR-451 depletion synergizes with oxidant stress to cause profound anemia in zebrafish embryos. At least some protective activities of miR-451 stem from its ability to directly suppress production of 14-3-3zeta, a phospho-serine/threonine-binding protein that inhibits nuclear accumulation of transcription factor FoxO3, a positive regulator of erythroid anti-oxidant genes. Thus, in miR-144/451(-/-) erythroblasts, 14-3-3zeta accumulates, causing partial relocalization of FoxO3 from nucleus to cytoplasm with dampening of its transcriptional program, including anti-oxidant-encoding genes Cat and Gpx1. Supporting this mechanism, overexpression of 14-3-3zeta in erythroid cells and fibroblasts inhibits nuclear localization and activity of FoxO3. Moreover, shRNA suppression of 14-3-3zeta protects miR-144/451(-/-) erythrocytes against peroxide-induced destruction, and restores catalase activity. Our findings define a novel miRNA-regulated pathway that protects erythrocytes against oxidant stress, and, more generally, illustrate how a miRNA can influence gene expression by altering the activity of a key transcription factor. PMID:20679398

  10. A dual phosphorylation switch controls 14-3-3-dependent cell surface expression of TASK-1

    PubMed Central

    Kilisch, Markus; Lytovchenko, Olga; Arakel, Eric C.; Bertinetti, Daniela; Schwappach, Blanche

    2016-01-01

    ABSTRACT The transport of the K+ channels TASK-1 and TASK-3 (also known as KCNK3 and KCNK9, respectively) to the cell surface is controlled by the binding of 14-3-3 proteins to a trafficking control region at the extreme C-terminus of the channels. The current model proposes that phosphorylation-dependent binding of 14-3-3 sterically masks a COPI-binding motif. However, the direct effects of phosphorylation on COPI binding and on the binding parameters of 14-3-3 isoforms are still unknown. We find that phosphorylation of the trafficking control region prevents COPI binding even in the absence of 14-3-3, and we present a quantitative analysis of the binding of all human 14-3-3 isoforms to the trafficking control regions of TASK-1 and TASK-3. Surprisingly, the affinities of 14-3-3 proteins for TASK-1 are two orders of magnitude lower than for TASK-3. Furthermore, we find that phosphorylation of a second serine residue in the C-terminus of TASK-1 inhibits 14-3-3 binding. Thus, phosphorylation of the trafficking control region can stimulate or inhibit transport of TASK-1 to the cell surface depending on the target serine residue. Our findings indicate that control of TASK-1 trafficking by COPI, kinases, phosphatases and 14-3-3 proteins is highly dynamic. PMID:26743085

  11. A dual phosphorylation switch controls 14-3-3-dependent cell surface expression of TASK-1.

    PubMed

    Kilisch, Markus; Lytovchenko, Olga; Arakel, Eric C; Bertinetti, Daniela; Schwappach, Blanche

    2016-02-15

    The transport of the K(+) channels TASK-1 and TASK-3 (also known as KCNK3 and KCNK9, respectively) to the cell surface is controlled by the binding of 14-3-3 proteins to a trafficking control region at the extreme C-terminus of the channels. The current model proposes that phosphorylation-dependent binding of 14-3-3 sterically masks a COPI-binding motif. However, the direct effects of phosphorylation on COPI binding and on the binding parameters of 14-3-3 isoforms are still unknown. We find that phosphorylation of the trafficking control region prevents COPI binding even in the absence of 14-3-3, and we present a quantitative analysis of the binding of all human 14-3-3 isoforms to the trafficking control regions of TASK-1 and TASK-3. Surprisingly, the affinities of 14-3-3 proteins for TASK-1 are two orders of magnitude lower than for TASK-3. Furthermore, we find that phosphorylation of a second serine residue in the C-terminus of TASK-1 inhibits 14-3-3 binding. Thus, phosphorylation of the trafficking control region can stimulate or inhibit transport of TASK-1 to the cell surface depending on the target serine residue. Our findings indicate that control of TASK-1 trafficking by COPI, kinases, phosphatases and 14-3-3 proteins is highly dynamic. PMID:26743085

  12. Functional identification of a novel 14-3-3 epsilon splicing variant suggests dimerization is not necessary for 14-3-3 epsilon to inhibit UV-induced apoptosis

    SciTech Connect

    Han, Dingding; Ye, Guangming; Liu, Tingting; Chen, Cong; Yang, Xianmei; Wan, Bo; Pan, Yuanwang; Yu, Long

    2010-05-28

    14-3-3 proteins function as a dimer and have been identified to involve in diverse signaling pathways. Here we reported the identification of a novel splicing variant of human 14-3-3 epsilon (14-3-3 epsilon sv), which is derived from a novel exon 1' insertion. The insertion contains a stop codon and leads to a truncated splicing variant of 14-3-3 epsilon. The splicing variant is translated from the exon 2 and results in the deletion of an N-terminal {alpha}-helix which is crucial for the dimerization. Therefore, the 14-3-3 epsilon sv could not form a dimer with 14-3-3 zeta. However, after UV irradiation 14-3-3 epsilon sv could also support cell survival, suggesting monomer of 14-3-3 epsilon is sufficient to protect cell from apoptosis.

  13. Phosphorylation Dependence and Stoichiometry of the Complex Formed by Tyrosine Hydroxylase and 14-3-3γ*

    PubMed Central

    Kleppe, Rune; Rosati, Sara; Jorge-Finnigan, Ana; Alvira, Sara; Ghorbani, Sadaf; Haavik, Jan; Valpuesta, José María; Heck, Albert J. R.; Martinez, Aurora

    2014-01-01

    Phosphorylated tyrosine hydroxylase (TH) can form complexes with 14-3-3 proteins, resulting in enzyme activation and stabilization. Although TH was among the first binding partners identified for these ubiquitous regulatory proteins, the binding stoichiometry and the activation mechanism remain unknown. To address this, we performed native mass spectrometry analyses of human TH (nonphosphorylated or phosphorylated on Ser19 (TH-pS19), Ser40 (TH-pS40), or Ser19 and Ser40 (TH-pS19pS40)) alone and together with 14-3-3γ. Tetrameric TH-pS19 (224 kDa) bound 14-3-3γ (58.3 kDa) with high affinity (Kd = 3.2 nM), generating complexes containing either one (282.4 kDa) or two (340.8 kDa) dimers of 14-3-3. Electron microscopy also revealed one major population of an asymmetric complex, consistent with one TH tetramer and one 14-3-3 dimer, and a minor population of a symmetric complex of one TH tetramer with two 14-3-3 dimers. Lower phosphorylation stoichiometries (0.15–0.54 phosphate/monomer) produced moderate changes in binding kinetics, but native MS detected much less of the symmetric TH:14-3-3γ complex. Interestingly, dephosphorylation of [32P]-TH-pS19 was mono-exponential for low phosphorylation stoichiometries (0.18–0.52), and addition of phosphatase accelerated the dissociation of the TH-pS19:14-3-3γ complex 3- to 4-fold. All together this is consistent with a model in which the pS19 residues in the TH tetramer contribute differently in the association to 14-3-3γ. Complex formation between TH-pS40 and 14-3-3γ was not detected via native MS, and surface plasmon resonance showed that the interaction was very weak. Furthermore, TH-pS19pS40 behaved similarly to TH-pS19 in terms of binding stoichiometry and affinity (Kd = 2.1 nM). However, we found that 14-3-3γ inhibited the phosphorylation rate of TH-pS19 by PKA (3.5-fold) on Ser40. We therefore conclude that Ser40 does not significantly contribute to the binding of 14-3-3γ, and rather has reduced accessibility in

  14. Dysregulated 14-3-3 Family in Peripheral Blood Leukocytes of Patients with Schizophrenia

    PubMed Central

    Qing, Ying; Sun, Liya; Yang, Chao; Jiang, Jie; Yang, Xuhan; Hu, Xiaowen; Cui, Donghong; Xu, Yifeng; He, Lin; Han, Dongmei; Wan, Chunling

    2016-01-01

    The 14-3-3 family, which is composed of seven distinct members in humans, plays important roles in the cell cycle, apoptosis, synaptic plasticity and neuronal differentiation and migration. Previous genetic and post-mortem gene expression studies have linked this family to schizophrenia. However, the direction of gene expression changes in these studies has been inconsistent, and reports of 14-3-3 gene expression in living schizophrenic patients are still lacking. Here, we assessed 14-3-3 gene and protein expression levels in peripheral blood leukocytes from drug-naïve first-episode schizophrenic patients and matched controls. mRNA and protein expression levels were quantified by qRT-PCR and UPLC-MRM/MS, respectively. Expression analysis revealed four downregulated and one upregulated mRNA transcripts as well as five downregulated protein levels of 14-3-3 isoforms in schizophrenia. Moreover, significant positive correlations between 14-3-3 mRNA and protein expression levels were found in schizophrenia, and we also identified negative correlations between ε, θ and ζ isoform expression levels and positive symptoms of schizophrenia. Our results suggest that gene and protein expression levels for the 14-3-3 family are dysregulated in schizophrenia, perhaps owing to specific regulatory mechanisms, and we also suggest that expression of the 14-3-3ε, θ and ζ isoform genes could be useful indicators of disease severity. PMID:27030512

  15. Dissection of Binding between a Phosphorylated Tyrosine Hydroxylase Peptide and 14-3-3ζ: A Complex Story Elucidated by NMR

    PubMed Central

    Hritz, Jozef; Byeon, In-Ja L.; Krzysiak, Troy; Martinez, Aurora; Sklenar, Vladimir; Gronenborn, Angela M.

    2014-01-01

    Human tyrosine hydroxylase activity is regulated by phosphorylation of its N-terminus and by an interaction with the modulator 14-3-3 proteins. We investigated the binding of singly or doubly phosphorylated and thiophosphorylated peptides, comprising the first 50 amino acids of human tyrosine hydroxylase, isoform 1 (hTH1), that contain the critical interaction domain, to 14-3-3ζ, by 31P NMR. Single phosphorylation at S19 generates a high affinity 14-3-3ζ binding epitope, whereas singly S40-phosphorylated peptide interacts with 14-3-3ζ one order-of-magnitude weaker than the S19-phosphorylated peptide. Analysis of the binding data revealed that the 14-3-3ζ dimer and the S19- and S40-doubly phosphorylated peptide interact in multiple ways, with three major complexes formed: 1), a single peptide bound to a 14-3-3ζ dimer via the S19 phosphate with the S40 phosphate occupying the other binding site; 2), a single peptide bound to a 14-3-3ζ dimer via the S19 phosphorous with the S40 free in solution; or 3), a 14-3-3ζ dimer with two peptides bound via the S19 phosphorous to each binding site. Our system and data provide information as to the possible mechanisms by which 14-3-3 can engage binding partners that possess two phosphorylation sites on flexible tails. Whether these will be realized in any particular interacting pair will naturally depend on the details of each system. PMID:25418103

  16. Functional relationship between CABIT, SAM and 14-3-3 binding domains of GAREM1 that play a role in its subcellular localization

    SciTech Connect

    Nishino, Tasuku; Matsunaga, Ryota; Konishi, Hiroaki

    2015-08-21

    GAREM1 (Grb2-associated regulator of Erk/MAPK1) is an adaptor protein that is involved in the epidermal growth factor (EGF) pathway. The nuclear localization of GAREM1 depends on the nuclear localization sequence (NLS), which is located at the N-terminal CABIT (cysteine-containing, all in Themis) domain. Here, we identified 14-3-3ε as a GAREM-binding protein, and its binding site is closely located to the NLS. This 14-3-3 binding site was of the atypical type and independent of GAREM phosphorylation. Moreover, the binding of 14-3-3 had an effect on the nuclear localization of GAREM1. Unexpectedly, we observed that the CABIT domain had intramolecular association with the C-terminal SAM (sterile alpha motif) domain. This association might be inhibited by binding of 14-3-3 at the CABIT domain. Our results demonstrate that the mechanism underlying the nuclear localization of GAREM1 depends on its NLS in the CABIT domain, which is controlled by the binding of 14-3-3 and the C-terminal SAM domain. We suggest that the interplay between 14-3-3, SAM domain and CABIT domain might be responsible for the distribution of GAREM1 in mammalian cells. - Highlights: • 14-3-3ε regulated the nuclear localization of GAREM1 as its binding partner. • The atypical 14-3-3 binding site of GAREM1 is located near the NLS in CABIT domain. • The CABIT domain had intramolecular association with the SAM domain in GAREM1. • Subcellular localization of GAREM1 is affected with its CABIT-SAM interaction.

  17. Clinical implication of 14-3-3 epsilon expression in gastric cancer

    PubMed Central

    Leal, Mariana Ferreira; Calcagno, Danielle Queiroz; Demachki, Sâmia; Assumpção, Paulo Pimentel; Chammas, Roger; Burbano, Rommel Rodríguez; Smith, Marília de Arruda Cardoso

    2012-01-01

    AIM: To evaluate for the first time the protein and mRNA expression of 14-3-3ε in gastric carcinogenesis. METHODS: 14-3-3ε protein expression was determined by western blotting, and mRNA expression was examined by real-time quantitative RT-PCR in gastric tumors and their matched non-neoplastic gastric tissue samples. RESULTS: Authors observed a significant reduction of 14-3-3ε protein expression in gastric cancer (GC) samples compared to their matched non-neoplastic tissue. Reduced levels of 14-3-3ε were also associated with diffuse-type GC and early-onset of this pathology. Our data suggest that reduced 14-3-3ε may have a role in gastric carcinogenesis process. CONCLUSION: Our results reveal that the reduced 14-3-3ε expression in GC and investigation of 14-3-3ε interaction partners may help to elucidate the carcinogenesis process. PMID:22509086

  18. Phosphorylation and 14-3-3 binding of Arabidopsis trehalose-phosphate synthase 5 in response to 2-deoxyglucose.

    PubMed

    Harthill, Jean E; Meek, Sarah E M; Morrice, Nick; Peggie, Mark W; Borch, Jonas; Wong, Barry H C; Mackintosh, Carol

    2006-07-01

    Trehalose-6-phosphate is a 'sugar signal' that regulates plant metabolism and development. The Arabidopsis genome encodes trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphatase (TPP) enzymes. It also encodes class II proteins (TPS isoforms 5-11) that contain both TPS-like and TPP-like domains, although whether these have enzymatic activity is unknown. In this paper, we show that TPS5, 6 and 7 are phosphoproteins that bind to 14-3-3 proteins, by using 14-3-3 affinity chromatography, 14-3-3 overlay assays, and by co-immunoprecipitating TPS5 and 14-3-3 isoforms from cell extracts. GST-TPS5 bound to 14-3-3s after in vitro phosphorylation at Ser22 and Thr49 by either mammalian AMP-activated protein kinase (AMPK) or partially purified plant Snf1-related protein kinase 1 (SnRK1s). Dephosphorylation of TPS5, or mutation of either Ser22 or Thr49, abolished binding to 14-3-3s. Ser22 and Thr49 are both conserved in TPS5, 7, 9 and 10. When GST-TPS5 was expressed in human HEK293 cells, Thr49 was phosphorylated in response to 2-deoxyglucose or phenformin, stimuli that activate the AMPK via the upstream kinase LKB1. 2-deoxyglucose stimulated Thr49 phosphorylation of endogenous TPS5 in Arabidopsis cells, whereas phenformin did not. Moreover, extractable SnRK1 activity was increased in Arabidopsis cells in response to 2-deoxyglucose. The plant kinase was inactivated by dephosphorylation and reactivated by phosphorylation with human LKB1, indicating that elements of the SnRK1/AMPK pathway are conserved in Arabidopsis and human cells. We hypothesize that coordinated phosphorylation and 14-3-3 binding of nitrate reductase (NR), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (F2KP) and class II TPS isoforms mediate responses to signals that activate SnRK1. PMID:16771775

  19. 14-3-3γ Prevents Centrosome Amplification and Neoplastic Progression

    PubMed Central

    Mukhopadhyay, Amitabha; Sehgal, Lalit; Bose, Arunabha; Gulvady, Anushree; Senapati, Parijat; Thorat, Rahul; Basu, Srikanta; Bhatt, Khyati; Hosing, Amol S.; Balyan, Renu; Borde, Lalit; Kundu, Tapas K.; Dalal, Sorab N.

    2016-01-01

    More than 80% of malignant tumors show centrosome amplification and clustering. Centrosome amplification results from aberrations in the centrosome duplication cycle, which is strictly coordinated with DNA-replication-cycle. However, the relationship between cell-cycle regulators and centrosome duplicating factors is not well understood. This report demonstrates that 14-3-3γ localizes to the centrosome and 14-3-3γ loss leads to centrosome amplification. Loss of 14-3-3γ results in the phosphorylation of NPM1 at Thr-199, causing early centriole disjunction and centrosome hyper-duplication. The centrosome amplification led to aneuploidy and increased tumor formation in mice. Importantly, an increase in passage of the 14-3-3γ-knockdown cells led to an increase in the number of cells containing clustered centrosomes leading to the generation of pseudo-bipolar spindles. The increase in pseudo-bipolar spindles was reversed and an increase in the number of multi-polar spindles was observed upon expression of a constitutively active 14-3-3-binding-defective-mutant of cdc25C (S216A) in the 14-3-3γ knockdown cells. The increase in multi-polar spindle formation was associated with decreased cell viability and a decrease in tumor growth. Our findings uncover the molecular basis of regulation of centrosome duplication by 14-3-3γ and inhibition of tumor growth by premature activation of the mitotic program and the disruption of centrosome clustering. PMID:27253419

  20. 14-3-3γ Prevents Centrosome Amplification and Neoplastic Progression.

    PubMed

    Mukhopadhyay, Amitabha; Sehgal, Lalit; Bose, Arunabha; Gulvady, Anushree; Senapati, Parijat; Thorat, Rahul; Basu, Srikanta; Bhatt, Khyati; Hosing, Amol S; Balyan, Renu; Borde, Lalit; Kundu, Tapas K; Dalal, Sorab N

    2016-01-01

    More than 80% of malignant tumors show centrosome amplification and clustering. Centrosome amplification results from aberrations in the centrosome duplication cycle, which is strictly coordinated with DNA-replication-cycle. However, the relationship between cell-cycle regulators and centrosome duplicating factors is not well understood. This report demonstrates that 14-3-3γ localizes to the centrosome and 14-3-3γ loss leads to centrosome amplification. Loss of 14-3-3γ results in the phosphorylation of NPM1 at Thr-199, causing early centriole disjunction and centrosome hyper-duplication. The centrosome amplification led to aneuploidy and increased tumor formation in mice. Importantly, an increase in passage of the 14-3-3γ-knockdown cells led to an increase in the number of cells containing clustered centrosomes leading to the generation of pseudo-bipolar spindles. The increase in pseudo-bipolar spindles was reversed and an increase in the number of multi-polar spindles was observed upon expression of a constitutively active 14-3-3-binding-defective-mutant of cdc25C (S216A) in the 14-3-3γ knockdown cells. The increase in multi-polar spindle formation was associated with decreased cell viability and a decrease in tumor growth. Our findings uncover the molecular basis of regulation of centrosome duplication by 14-3-3γ and inhibition of tumor growth by premature activation of the mitotic program and the disruption of centrosome clustering. PMID:27253419

  1. FTICR-MS analysis of 14-3-3 isoform substrate selection.

    PubMed

    Cardasis, Helene L; Sehnke, Paul C; Laughner, Beth; Eyler, John R; Powell, David H; Ferl, Robert J

    2007-07-01

    The 14-3-3s are a ubiquitous class of eukaryotic proteins that participate in a second regulatory step in many phosphorylation-based signal transduction systems. The Arabidopsis family of 14-3-3 proteins represents a rather large 14-3-3 gene family. The biological motive for such diversity within a single protein family is not yet completely understood. The work presented here utilizes 14-3-3 micro-affinity chromatography in conjunction with Fourier transform ion cyclotron resonance mass spectrometry to survey the substrate sequence selectivity of two Arabidopsis 14-3-3 isoforms that represent the two major subclasses of this protein family. A method was developed to compare the relative binding of eight synthetic phosphopeptide sequences. The degree to which each phosphopeptide bound to either isoform was assigned a relative value, defined here as the binding ratio. The method provided a simple means for visualizing differences in substrate sequence selection among different 14-3-3 isoforms. A reproducible preference for specific phosphopeptide sequences was measured for both isoforms. This binding preference was consistent among the two classes of isoforms, suggesting that any pressure for isoform selectivity must reside outside the central core that interacts with the phosphopeptide sequence of the client. PMID:17569603

  2. Decreased expression of 14-3-3 in Paracoccidioides brasiliensis confirms its involvement in fungal pathogenesis.

    PubMed

    Marcos, Caroline Maria; Silva, Julhiany de Fátima ds; Oliveira, Haroldo Cesar de; Assato, Patrícia Akemi; Singulani, Junya de Lacorte; Lopez, Angela Maria; Tamayo, Diana Patricia; Hernandez-Ruiz, Orville; McEwen, Juan G; Mendes-Giannini, Maria José Soares; Fusco-Almeida, Ana Marisa

    2016-01-01

    The interaction between the fungal pathogen Paracoccidioides brasiliensis and host cells is usually mediated by specific binding events between adhesins on the fungal surface and receptors on the host extracellular matrix or cell surface. One molecule implicated in the P. brasiliensis-host interaction is the 14-3-3 protein. The 14-3-3 protein belongs to a family of conserved regulatory molecules that are expressed in all eukaryotic cells and are involved in diverse cellular functions. Here, we investigated the relevance of the 14-3-3 protein to the virulence of P. brasiliensis. Using antisense RNA technology and Agrobacterium tumefaciens-mediated transformation, we generated a 14-3-3-silenced strain (expression reduced by ˜55%). This strain allowed us to investigate the interaction between 14-3-3 and the host and to correlate the functions of P. brasiliensis 14-3-3 with cellular features, such as morphological characteristics and virulence, that are important for pathogenesis. PMID:26646480

  3. The 14-3-3 gene expression specificity in response to stress is promoter-dependent.

    PubMed

    Aksamit, Anna; Korobczak, Alina; Skala, Jacek; Lukaszewicz, Marcin; Szopa, Jan

    2005-10-01

    Genomic clone coding for the 16R isoform of 14-3-3 proteins from potato plants has recently been described. This paper reports on 20R-gene isolation and analysis, and compares two isoforms. The northern blot analysis of mRNA of the 20R 14-3-3 isoform suggests its similarity to 16R. Vascular tissue-specific expression and age-dependent synthesis in potato leaves has been detected in both promoters. Screening of the potato genomic library using 20R cDNA isoform resulted in identification and isolation of the corresponding gene. This gene contains four exons and three introns. Inspecting the promoter sequence of the 20R isoform revealed several boxes important for the regulation of gene expression. The strongest GUS expression in transgenic potato plants transformed with the uidA reporter gene under the 20R promoter has been found in young leaf and stem vascular tissue, root tips, pollen and ovules. Mature fragments exhibit a significant decrease in GUS staining, which suggests age-dependent promoter activity. The analysis of transgenic plants transformed with 20R-GUS in contrast to 16R-GUS has revealed strong activation of the 20R promoter by metal ions and NaCl. Instead the 16R promoter is strongly affected by virus and salicylic acid treatments. The only factor, which strongly induced both promoters, was abscisic acid. It is thus suggested that promoter domain composition is the main factor differentiating the appearance of 14-3-3 isoforms. PMID:16081528

  4. Identification of 14-3-3β Gene as a Novel miR-152 Target Using a Proteome-based Approach*

    PubMed Central

    Jasinski-Bergner, Simon; Stehle, Franziska; Gonschorek, Evamaria; Kalich, Jana; Schulz, Kristin; Huettelmaier, Stefan; Braun, Juliane; Seliger, Barbara

    2014-01-01

    Recent studies demonstrated that miR-152 overexpression down-regulates the nonclassical human leukocyte antigen (HLA) class I molecule HLA-G in human tumors thereby contributing to their immune surveillance. Using two-dimensional gel electrophoresis followed by MALDI-TOF mass spectrometry, the protein expression profile of HLA-G+, miR-152low cells, and their miR-152-overexpressing (miRhigh) counterparts was compared leading to the identification of 24 differentially expressed proteins. These were categorized according to their function and localization demonstrating for most of them an important role in the initiation and progression of tumors. The novel miR-152 target 14-3-3 protein β/α/YWHAB (14-3-3β) is down-regulated upon miR-152 overexpression, although its overexpression was often found in tumors of distinct origin. The miR-152-mediated reduction of the 14-3-3β expression was accompanied by an up-regulation of BAX protein expression resulting in a pro-apoptotic phenotype. In contrast, the reconstitution of 14-3-3β expression in miR-152high cells increased the expression of the anti-apoptotic BCL2 gene, enhances the proliferative activity in the presence of the cytostatic drug paclitaxel, and causes resistance to apoptosis induced by this drug. By correlating clinical microarray data with the patients' outcome, a link between 14-3-3β and HLA-G expression was found, which could be associated with poor prognosis and overall survival of patients with tumors. Because miR-152 controls both the expression of 14-3-3β and HLA-G, it exerts a dual role in tumor cells by both altering the immunogenicity and the tumorigenicity. PMID:25228695

  5. Loss of ypk1 function causes rapamycin sensitivity, inhibition of translation initiation and synthetic lethality in 14-3-3-deficient yeast.

    PubMed Central

    Gelperin, Daniel; Horton, Lynn; DeChant, Anne; Hensold, Jack; Lemmon, Sandra K

    2002-01-01

    14-3-3 proteins bind to phosphorylated proteins and regulate a variety of cellular activities as effectors of serine/threonine phosphorylation. To define processes requiring 14-3-3 function in yeast, mutants with increased sensitivity to reduced 14-3-3 protein levels were identified by synthetic lethal screening. One mutation was found to be allelic to YPK1, which encodes a Ser/Thr protein kinase. Loss of Ypk function causes hypersensitivity to rapamycin, similar to 14-3-3 mutations and other mutations affecting the TOR signaling pathway in yeast. Similar to treatment with rapamycin, loss of Ypk function disrupted translation, at least in part by causing depletion of eIF4G, a central adaptor protein required for cap-dependent mRNA translation initiation. In addition, Ypk1 as well as eIF4G protein levels were rapidly depleted upon nitrogen starvation, but not during glucose starvation, even though both conditions inhibit translation initiation. These results suggest that Ypk regulates translation initiation in response to nutrient signals, either through the TOR pathway or in a functionally related pathway parallel to TOR. PMID:12196392

  6. Genome-Wide Identification and Expression Analysis of the 14-3-3 Family Genes in Medicago truncatula

    PubMed Central

    Qin, Cheng; Cheng, Linming; Shen, Jingqin; Zhang, Yunhong; Cao, Huimin; Lu, Dan; Shen, Chenjia

    2016-01-01

    The 14-3-3 gene family, which is conserved in eukaryotes, is involved in protein-protein interactions and mediates signal transduction. However, detailed investigations of the 14-3-3 gene family in Medicago truncatula are largely unknown. In this study, the identification and study of M. truncatula 14-3-3-family genes were performed based on the latest M. truncatula genome. In the M. truncatula genome, 10 14-3-3 family genes were identified, and they can be grouped into ε and non-ε groups. An exon-intron analysis showed that the gene structures are conserved in the same group. The protein structure analysis showed that 14-3-3 proteins in M. truncatula are composed of nine typical antiparallel α-helices. The expression patterns of Mt14-3-3 genes indicated that they are expressed in all tissues. Furthermore, the gene expression levels of Mt14-3-3 under hormone treatment and Sinorhizobium meliloti infection showed that the Mt14-3-3 genes were involve in nodule formation. Our findings lay a solid foundation for further functional studies of 14-3-3 in M. truncatula. PMID:27047505

  7. Characterization and subcellular localization of two 14-3-3 genes and their response to abiotic stress in wheat.

    PubMed

    Meng, Xiaodan; Chen, Xin; Wang, Yaying; Xiao, Ruixia; Liu, Hailun; Wang, Xinguo; Ren, Jiangping; Li, Yongchun; Niu, Hongbin; Wang, Xiang; Yin, Jun

    2014-02-01

    In order to investigate biological functions of the 14-3-3 genes and their response to abiotic stress, two cDNAs (designated as Ta14R1 and Ta14R2) encoding putative 14-3-3 proteins were isolated from wheat by PCR and rapid amplification of cDNA end (RACE) technique. The cDNA of Ta14R1 is 999bp and encodes a protein of 262 amino acids, while the cDNA of Ta14R2 is 897bp in length and encodes a protein of 261 amino acids. Transient expression assays using Ta14R1/Ta14R2-GFP fusion constructs indicated that Ta14R1 and Ta14R2 were located in cytoplasm and cell membrane but not in chloroplasts. Real-time quantitative (RT-PCR) analysis revealed that Ta14R1 and Ta14R2 were differentially expressed in wheat tissues and significantly up-regulated in roots and shoots 1d after germination, indicating they may play a role in process of seed germination. The expression of the two genes in roots and leaves were significantly induced by plant hormone ABA, as well as heat, cold and drought treatments, suggesting that the two 14-3-3 genes in wheat may be involved in ABA dependent stress-responding pathway and response to heat, cold and drought stress. PMID:24941745

  8. The pro-inflammatory cytokine 14-3-3ε is a ligand of CD13 in cartilage

    PubMed Central

    Nefla, Meriam; Sudre, Laure; Denat, Guillaume; Priam, Sabrina; Andre-Leroux, Gwenaëlle; Berenbaum, Francis; Jacques, Claire

    2015-01-01

    ABSTRACT Osteoarthritis is a whole-joint disease characterized by the progressive destruction of articular cartilage involving abnormal communication between subchondral bone and cartilage. Our team previously identified 14-3-3ε protein as a subchondral bone soluble mediator altering cartilage homeostasis. The aim of this study was to investigate the involvement of CD13 (also known as aminopeptidase N, APN) in the chondrocyte response to 14-3-3ε. After identifying CD13 in chondrocytes, we knocked down CD13 with small interfering RNA (siRNA) and blocking antibodies in articular chondrocytes. 14-3-3ε-induced MMP-3 and MMP-13 was significantly reduced with CD13 knockdown, which suggests that it has a crucial role in 14-3-3ε signal transduction. Aminopeptidase N activity was identified in chondrocytes, but the activity was unchanged after stimulation with 14-3-3ε. Direct interaction between CD13 and 14-3-3ε was then demonstrated by surface plasmon resonance. Using labeled 14-3-3ε, we also found that 14-3-3ε binds to the surface of chondrocytes in a manner that is dependent on CD13. Taken together, these results suggest that 14-3-3ε might directly bind to CD13, which transmits its signal in chondrocytes to induce a catabolic phenotype similar to that observed in osteoarthritis. The 14-3-3ε–CD13 interaction could be a new therapeutic target in osteoarthritis. PMID:26208633

  9. The pro-inflammatory cytokine 14-3-3ε is a ligand of CD13 in cartilage.

    PubMed

    Nefla, Meriam; Sudre, Laure; Denat, Guillaume; Priam, Sabrina; Andre-Leroux, Gwenaëlle; Berenbaum, Francis; Jacques, Claire

    2015-09-01

    Osteoarthritis is a whole-joint disease characterized by the progressive destruction of articular cartilage involving abnormal communication between subchondral bone and cartilage. Our team previously identified 14-3-3ε protein as a subchondral bone soluble mediator altering cartilage homeostasis. The aim of this study was to investigate the involvement of CD13 (also known as aminopeptidase N, APN) in the chondrocyte response to 14-3-3ε. After identifying CD13 in chondrocytes, we knocked down CD13 with small interfering RNA (siRNA) and blocking antibodies in articular chondrocytes. 14-3-3ε-induced MMP-3 and MMP-13 was significantly reduced with CD13 knockdown, which suggests that it has a crucial role in 14-3-3ε signal transduction. Aminopeptidase N activity was identified in chondrocytes, but the activity was unchanged after stimulation with 14-3-3ε. Direct interaction between CD13 and 14-3-3ε was then demonstrated by surface plasmon resonance. Using labeled 14-3-3ε, we also found that 14-3-3ε binds to the surface of chondrocytes in a manner that is dependent on CD13. Taken together, these results suggest that 14-3-3ε might directly bind to CD13, which transmits its signal in chondrocytes to induce a catabolic phenotype similar to that observed in osteoarthritis. The 14-3-3ε-CD13 interaction could be a new therapeutic target in osteoarthritis. PMID:26208633

  10. The 14-3-3 Gene Function of Cryptococcus neoformans Is Required for its Growth and Virulence.

    PubMed

    Li, Jingbo; Chang, Yun C; Wu, Chun-Hua; Liu, Jennifer; Kwon-Chung, Kyung J; Huang, Sheng-He; Shimada, Hiro; Fante, Rob; Fu, Xiaowei; Jong, Ambrose

    2016-05-28

    Cryptococcus neoformans is a life-threatening pathogenic yeast that causes devastating meningoencephalitis. The mechanism of cryptococcal brain invasion is largely unknown, and recent studies suggest that its extracellular microvesicles may be involved in the invasion process. The 14-3-3 protein is abundant in the extracellular microvesicles of C. neoformans, and the 14-3-3-GFP fusion has been used as the microvesicle's marker. However, the physiological role of 14-3-3 has not been explored. In this report, we have found that C. neoformans contains a single 14-3-3 gene that apparently is an essential gene. To explore the functions of 14-3-3, we substituted the promoter region of the 14-3-3 with the copper-controllable promoter CTR4. The CTR4 regulatory strain showed an enlarged cell size, drastic changes in morphology, and a decrease in the thickness of the capsule under copper-enriched conditions. Furthermore, the mutant cells produced a lower amount of total proteins in their extracellular microvesicles and reduced adhesion to human brain microvascular endothelial cells in vitro. Proteomic analyses of the protein components under 14-3-3-overexpressed and -suppressed conditions revealed that the 14-3-3 function(s) might be associated with the microvesicle biogenesis. Our results support that 14-3-3 has diverse pertinent roles in both physiology and pathogenesis in C. neoformans. Its gene functions are closely relevant to the pathogenesis of this fungus. PMID:26437944

  11. Effects of physical exercise on the P38MAPK/REDD1/14-3-3 pathways in the myocardium of diet-induced obesity rats.

    PubMed

    Pieri, B L S; Souza, D R; Luciano, T F; Marques, S O; Pauli, J R; Silva, A S R; Ropelle, E R; Pinho, R A; Lira, F S; De Souza, C T

    2014-08-01

    Obesity is associated with myocardial insulin resistance and impairment of the mammalian target of rapamycin (mTOR) signaling pathway. The activation of the mTOR cascade by exercise has been largely shown in skeletal muscle, but insufficiently analyzed in myocardial tissue. In addition, little is known regarding the mTOR upstream molecules in the hearts of obese animals and even less about the role of exercise in this process. Thus, the present study was aimed to evaluate the effects of physical exercise on P38 Mitogen-Activated Protein Kinase (P38MAPK) phosphorylation and the REDD1 (regulated in development and DNA damage responses 1) and 14-3-3 protein levels in the myocardium of diet-induced obesity (DIO) rats. After achievement of DIO and insulin resistance, Wistar rats were divided in 2 groups: sedentary obese rats and obese rats performed treadmill running (50-min/day, 5 days per week velocity of 1.0 km/h for 2 months). Forty-eight hours after the final physical exercise, the rats were killed, and the myocardial tissue was removed for Western blot analysis. DIO increased the REDD1 protein levels and reduced the 14-3-3 protein levels and P38MAPK, mTOR, P70S6k (p70 ribosomal S6 protein kinase), and 4EBP1 (4E-binding protein-1) phosphorylation. Interestingly, physical exercise reduced the REDD1 protein levels and increased the 14-3-3 protein levels and P38MAPK, mTOR, P70S6k, and 4EBP1 phosphorylation. Moreover, exercise increased the REDD1/14-3-3 association in the heart. Our results indicate that the phospho-P38MAPK, REDD1, and 14-3-3 protein levels were reduced in the myocardium of obese rats and that physical exercise increased the protein levels of these molecules. PMID:24691733

  12. 14-3-3ζ Mediates Tau Aggregation in Human Neuroblastoma M17 Cells

    PubMed Central

    Li, Tong; Paudel, Hemant K.

    2016-01-01

    Microtubule-associated protein tau is the major component of paired helical filaments (PHFs) associated with the neuropathology of Alzheimer’s disease (AD). Tau in the normal brain binds and stabilizes microtubules. Tau isolated from PHFs is hyperphosphorylated, which prevents it from binding to microtubules. Tau phosphorylation has been suggested to be involved in the development of NFT pathology in the AD brain. Recently, we showed that 14-3-3ζ is bound to tau in the PHFs and when incubated in vitro with 14-3-3ζ, tau formed amorphous aggregates, single-stranded straight filaments, double stranded ribbon-like filaments and PHF-like filaments that displayed close resemblance with corresponding ultrastructures of AD brain. Surprisingly however, phosphorylated and non-phosphorylated tau aggregated in a similar manner, indicating that tau phosphorylation does not affect in vitro tau aggregation (Qureshi et al (2013) Biochemistry 52, 6445–6455). In this study, we have examined the role of tau phosphorylation in tau aggregation in cellular level. We have found that in human M17 neuroblastoma cells, tau phosphorylation by GSK3β or PKA does not cause tau aggregation, but promotes 14-3-3ζ-induced tau aggregation by destabilizing microtubules. Microtubule disrupting drugs also promoted 14-3-3ζ-induced tau aggregation without changing tau phosphorylation in M17 cell. In vitro, when incubated with 14-3-3ζ and microtubules, nonphosphorylated tau bound to microtubules and did not aggregate. Phosphorylated tau on the other hand did not bind to microtubules and aggregated. Our data indicate that microtubule-bound tau is resistant to 14-3-3ζ-induced tau aggregation and suggest that tau phosphorylation promotes tau aggregation in the brain by detaching tau from microtubules and thus making it accessible to 14-3-3ζ. PMID:27548710

  13. 14-3-3ζ Mediates Tau Aggregation in Human Neuroblastoma M17 Cells.

    PubMed

    Li, Tong; Paudel, Hemant K

    2016-01-01

    Microtubule-associated protein tau is the major component of paired helical filaments (PHFs) associated with the neuropathology of Alzheimer's disease (AD). Tau in the normal brain binds and stabilizes microtubules. Tau isolated from PHFs is hyperphosphorylated, which prevents it from binding to microtubules. Tau phosphorylation has been suggested to be involved in the development of NFT pathology in the AD brain. Recently, we showed that 14-3-3ζ is bound to tau in the PHFs and when incubated in vitro with 14-3-3ζ, tau formed amorphous aggregates, single-stranded straight filaments, double stranded ribbon-like filaments and PHF-like filaments that displayed close resemblance with corresponding ultrastructures of AD brain. Surprisingly however, phosphorylated and non-phosphorylated tau aggregated in a similar manner, indicating that tau phosphorylation does not affect in vitro tau aggregation (Qureshi et al (2013) Biochemistry 52, 6445-6455). In this study, we have examined the role of tau phosphorylation in tau aggregation in cellular level. We have found that in human M17 neuroblastoma cells, tau phosphorylation by GSK3β or PKA does not cause tau aggregation, but promotes 14-3-3ζ-induced tau aggregation by destabilizing microtubules. Microtubule disrupting drugs also promoted 14-3-3ζ-induced tau aggregation without changing tau phosphorylation in M17 cell. In vitro, when incubated with 14-3-3ζ and microtubules, nonphosphorylated tau bound to microtubules and did not aggregate. Phosphorylated tau on the other hand did not bind to microtubules and aggregated. Our data indicate that microtubule-bound tau is resistant to 14-3-3ζ-induced tau aggregation and suggest that tau phosphorylation promotes tau aggregation in the brain by detaching tau from microtubules and thus making it accessible to 14-3-3ζ. PMID:27548710

  14. 14-3-3θ is a binding partner of rat Eag1 potassium channels.

    PubMed

    Hsu, Po-Hao; Miaw, Shi-Chuen; Chuang, Chau-Ching; Chang, Pei-Yu; Fu, Ssu-Ju; Jow, Guey-Mei; Chiu, Mei-Miao; Jeng, Chung-Jiuan

    2012-01-01

    The ether-à-go-go (Eag) potassium (K(+)) channel belongs to the superfamily of voltage-gated K(+) channel. In mammals, the expression of Eag channels is neuron-specific but their neurophysiological role remains obscure. We have applied the yeast two-hybrid screening system to identify rat Eag1 (rEag1)-interacting proteins from a rat brain cDNA library. One of the clones we identified was 14-3-3θ, which belongs to a family of small acidic protein abundantly expressed in the brain. Data from in vitro yeast two-hybrid and GST pull-down assays suggested that the direct association with 14-3-3θ was mediated by both the N- and the C-termini of rEag1. Co-precipitation of the two proteins was confirmed in both heterologous HEK293T cells and native hippocampal neurons. Electrophysiological studies showed that over-expression of 14-3-3θ led to a sizable suppression of rEag1 K(+) currents with no apparent alteration of the steady-state voltage dependence and gating kinetics. Furthermore, co-expression with 14-3-3θ failed to affect the total protein level, membrane trafficking, and single channel conductance of rEag1, implying that 14-3-3θ binding may render a fraction of the channel locked in a non-conducting state. Together these data suggest that 14-3-3θ is a binding partner of rEag1 and may modulate the functional expression of the K(+) channel in neurons. PMID:22911758

  15. Identification of 14-3-3 Family in Common Bean and Their Response to Abiotic Stress

    PubMed Central

    Dhaubhadel, Sangeeta; Bian, Shaomin; Li, Xuyan

    2015-01-01

    14-3-3s are a class of conserved regulatory proteins ubiquitously found in eukaryotes, which play important roles in a variety of cellular processes including response to diverse stresses. Although much has been learned about 14-3-3s in several plant species, it remains unknown in common bean. In this study, 9 common bean 14-3-3s (PvGF14s) were identified by exhaustive data mining against the publicly available common bean genomic database. A phylogenetic analysis revealed that each predicted PvGF14 was clustered with two GmSGF14 paralogs from soybean. Both epsilon-like and non-epsilon classes of PvGF14s were found in common bean, and the PvGF14s belonging to each class exhibited similar gene structure. Among 9 PvGF14s, only 8 are transcribed in common bean. Expression patterns of PvGF14s varied depending on tissue type, developmental stage and exposure of plants to stress. A protein-protein interaction study revealed that PvGF14a forms dimer with itself and with other PvGF14 isoforms. This study provides a first comprehensive look at common bean 14-3-3 proteins, a family of proteins with diverse functions in many cellular processes, especially in response to stresses. PMID:26599110

  16. Sporadic Creutzfeldt-Jakob disease diagnostic accuracy is improved by a new CSF ELISA 14-3-3γ assay.

    PubMed

    Leitão, M J; Baldeiras, I; Almeida, M R; Ribeiro, M H; Santos, A C; Ribeiro, M; Tomás, J; Rocha, S; Santana, I; Oliveira, C R

    2016-05-13

    Protein 14-3-3 is a reliable marker of rapid neuronal damage, specifically increased in cerebrospinal fluid (CSF) of sporadic Creutzfeldt-Jakob disease (sCJD) patients. Its detection is usually performed by Western Blot (WB), prone to methodological issues. Our aim was to evaluate the diagnostic performance of a recently developed quantitative enzyme-linked immunosorbent (ELISA) assay for 14-3-3γ, in comparison with WB and other neurodegeneration markers. CSF samples from 145 patients with suspicion of prion disease, later classified as definite sCJD (n=72) or Non-prion diseases (Non-CJD; n=73) comprised our population. 14-3-3 protein was determined by WB and ELISA. Total Tau (t-Tau) and phosphorylated Tau (p-Tau) were also evaluated. Apolipoprotein E gene (ApoE) and prionic protein gene (PRNP) genotyping was assessed. ELISA 14-3-3γ levels were significantly increased in sCJD compared to Non-CJD patients (p<0.001), showing very good accuracy (AUC=0.982; sensitivity=97%; specificity=94%), and matching WB results in 81% of all cases. It strongly correlated with t-Tau and p-Tau (p<0.0001), showing slightly higher specificity (14-3-3 WB - 63%; Tau - 90%; p-Tau/t-Tau ratio - 88%). From WB inconclusive results (n=44), ELISA 14-3-3γ correctly classified 41 patients. Additionally, logistic regression analysis selected ELISA 14-3-3γ as the best single predictive marker for sCJD (overall accuracy=93%). ApoE and PRNP genotypes did not influence ELISA 14-3-3γ levels. Despite specificity for 14-3-3γ isoform, ELISA results not only match WB evaluation but also help discrimination of inconclusive results. Our results therefore reinforce this assay as a single screening test, allowing higher sample throughput and unequivocal results. PMID:26940479

  17. The 14-3-3σ/GSK3β/β-catenin/ZEB1 regulatory loop modulates chemo-sensitivity in human tongue cancer

    PubMed Central

    Xiong, Yan; Yin, Jiang; Li, Nan; Deng, Yingen; Luo, Kai; Zhang, Qiong; Wang, Chengkun; Zhang, Zhijie; Zheng, Guopei; He, Zhimin

    2015-01-01

    Here we demonstrated that chemotherapy induced 14-3-3σ expression in tongue cancer (TC) cells and overexpressed 14-3-3σ sensitized TC cells to chemotherapy especially in multidrug resistant TC (MDR-TC) cells. In agreement, 14-3-3σ knockdown enhanced resistance of TC cells to chemotherapy. Mechanically, we found 14-3-3σ physically bound to GSK3β in protein level and the binding inhibited β-catenin signaling. Coincidentally, chemotherapy as well as 14-3-3σ overexpression led to increase of GSK3β protein level. Increased GSK3β protein sensitized TC cells to chemotherapy. Moreover, deregulation of 14-3-3σ/GSK3β/β-catenin axis led to overexpressed ZEB1 in TC cells, especially in MDR-TC cells. As a negative feedback loop, ZEB1 bond to 14-3-3σ promoter to enhance promoter hypermethylation in TC cells. Promoter hypermethylation resulted into the decrease of 14-3-3σ expression. Importantly, a positive correlation was observed between 14-3-3σ and GSK3β protein expression in TC tissues from patients receiving chemotherapy. High levels of 14-3-3σ and GSK3β were associated with better prognosis in TC patients. PMID:26036631

  18. Transcriptional increase and misexpression of 14-3-3 epsilon in sea urchin embryos exposed to UV-B

    PubMed Central

    Russo, Roberta; Zito, Francesca; Costa, Caterina; Bonaventura, Rosa

    2010-01-01

    Members of the 14-3-3 protein family are involved in many important cellular events, including stress response, survival and apoptosis. Genes of the 14-3-3 family are conserved from plants to humans, and some members are responsive to UV radiation. Here, we report the isolation of the complete cDNA encoding the 14-3-3 epsilon isoform from Paracentrotus lividus sea urchin embryos, referred to as Pl14-3-3ε, and the phylogenetic relationship with other homologues described in different phyla. Pl14-3-3ε mRNA levels were measured by QPCR during development and found to increase from the mesenchyme blastula to the prism stage. In response to UV-B (312 nm) exposure, early stage embryos collected 2 h later showed a 2.3-fold (at 400 J/m2) and a 2.7-fold (at 800 J/m2) increase in Pl14-3-3ε transcript levels compared with controls. The spatial expression of Pl14-3-3ε mRNA, detected by whole mount in situ hybridization in both control and UV-B exposed embryos, harvested at late developmental stages, showed transcripts to be located in the archenteron of gastrula stage and widely distributed in all germ layers, respectively. The Pl14-3-3ε mRNA delocalization parallels the failure in archenteron elongation observed morphologically, as well as the lack of specific endoderm markers, investigated by indirect immuno-fluorescence on whole mount embryos. Results confirm the involvement of 14-3-3ε in the stress response elicited by UV-B and demonstrate, for the first time, its contribution at the transcriptional level in the sea urchin embryo. PMID:20607471

  19. Cyclin Y phosphorylation- and 14-3-3-binding-dependent activation of PCTAIRE-1/CDK16

    PubMed Central

    Shehata, Saifeldin N.; Deak, Maria; Morrice, Nicholas A.; Ohta, Eriko; Hunter, Roger W.; Kalscheuer, Vera M.; Sakamoto, Kei

    2015-01-01

    PCTAIRE-1 [also known as cyclin-dependent kinase 16 (CDK16)] is implicated in various physiological processes such as neurite outgrowth and vesicle trafficking; however, its molecular regulation and downstream targets are largely unknown. Cyclin Y has recently been identified as a key interacting/activating cyclin for PCTAIRE-1; however, the molecular mechanism by which it activates PCTAIRE-1 is undefined. In the present study, we initially performed protein sequence analysis and identified two candidate phosphorylation sites (Ser12 and Ser336) on cyclin Y that might be catalysed by PCTAIRE-1. Although in vitro peptide analysis favoured Ser12 as the candidate phosphorylation site, immunoblot analysis of cell lysates that had been transfected with wild-type (WT) or kinase-inactive (KI) PCTAIRE-1 together with WT or phospho-deficient mutants of cyclin Y suggested Ser336, but not Ser12, as a PCTAIRE-1-dependent phosphorylation site. Monitoring phosphorylation of Ser336 may provide a useful read-out to assess cellular activity of PCTAIRE-1 in vivo; however, a phospho-deficient S336A mutant displayed normal interaction with PCTAIRE-1. Unbiased mass spectrometry and targeted mutagenesis analysis of cyclin Y identified key phosphorylation sites (Ser100 and Ser326) required for 14-3-3 binding. Recombinant WT cyclin Y, but not a S100A/S326A mutant, prepared in COS-1 cells co-purified with 14-3-3 and was able to activate bacterially expressed recombinant PCTAIRE-1 in cell-free assays. Finally, we observed that recently identified PCTAIRE-1 variants found in patients with intellectual disability were unable to interact with cyclin Y, and were inactive enzymes. Collectively, the present work has revealed a new mechanistic insight into activation of PCTAIRE-1, which is mediated through interaction with the phosphorylated form of cyclin Y in complex with 14-3-3. PMID:26205494

  20. Cyclin Y phosphorylation- and 14-3-3-binding-dependent activation of PCTAIRE-1/CDK16.

    PubMed

    Shehata, Saifeldin N; Deak, Maria; Morrice, Nicholas A; Ohta, Eriko; Hunter, Roger W; Kalscheuer, Vera M; Sakamoto, Kei

    2015-08-01

    PCTAIRE-1 [also known as cyclin-dependent kinase 16 (CDK16)] is implicated in various physiological processes such as neurite outgrowth and vesicle trafficking; however, its molecular regulation and downstream targets are largely unknown. Cyclin Y has recently been identified as a key interacting/activating cyclin for PCTAIRE-1; however, the molecular mechanism by which it activates PCTAIRE-1 is undefined. In the present study, we initially performed protein sequence analysis and identified two candidate phosphorylation sites (Ser(12) and Ser(336)) on cyclin Y that might be catalysed by PCTAIRE-1. Although in vitro peptide analysis favoured Ser(12) as the candidate phosphorylation site, immunoblot analysis of cell lysates that had been transfected with wild-type (WT) or kinase-inactive (KI) PCTAIRE-1 together with WT or phospho-deficient mutants of cyclin Y suggested Ser(336), but not Ser(12), as a PCTAIRE-1-dependent phosphorylation site. Monitoring phosphorylation of Ser(336) may provide a useful read-out to assess cellular activity of PCTAIRE-1 in vivo; however, a phospho-deficient S336A mutant displayed normal interaction with PCTAIRE-1. Unbiased mass spectrometry and targeted mutagenesis analysis of cyclin Y identified key phosphorylation sites (Ser(100) and Ser(326)) required for 14-3-3 binding. Recombinant WT cyclin Y, but not a S100A/S326A mutant, prepared in COS-1 cells co-purified with 14-3-3 and was able to activate bacterially expressed recombinant PCTAIRE-1 in cell-free assays. Finally, we observed that recently identified PCTAIRE-1 variants found in patients with intellectual disability were unable to interact with cyclin Y, and were inactive enzymes. Collectively, the present work has revealed a new mechanistic insight into activation of PCTAIRE-1, which is mediated through interaction with the phosphorylated form of cyclin Y in complex with 14-3-3. PMID:26205494

  1. The Silencing of a 14-3-3ɛ Homolog in Tenebrio molitor Leads to Increased Antimicrobial Activity in Hemocyte and Reduces Larval Survivability.

    PubMed

    Seo, Gi Won; Jo, Yong Hun; Seong, Jeong Hwan; Park, Ki Beom; Patnaik, Bharat Bhusan; Tindwa, Hamisi; Kim, Sun-Am; Lee, Yong Seok; Kim, Yu Jung; Han, Yeon Soo

    2016-01-01

    The 14-3-3 family of phosphorylated serine-binding proteins acts as signaling molecules in biological processes such as metabolism, division, differentiation, autophagy, and apoptosis. Herein, we report the requirement of 14-3-3ɛ isoform from Tenebrio molitor (Tm14-3-3ɛ) in the hemocyte antimicrobial activity. The Tm14-3-3ɛ transcript is 771 nucleotides in length and encodes a polypeptide of 256 amino acid residues. The protein has the typical 14-3-3 domain, the nuclear export signal (NES) sequence, and the peptide binding residues. The Tm14-3-3ɛ transcript shows a significant three-fold expression in the hemocyte of T. molitor larvae when infected with Escherichia coli Tm14-3-3ɛ silenced larvae show significantly lower survival rates when infected with E. coli. Under Tm14-3-3ɛ silenced condition, a strong antimicrobial activity is elicited in the hemocyte of the host inoculated with E. coli. This suggests impaired secretion of antimicrobial peptides (AMP) into the hemolymph. Furthermore, a reduction in AMP secretion under Tm14-3-3ɛ silenced condition would be responsible for loss in the capacity to kill bacteria and might explain the reduced survivability of the larvae upon E. coli challenge. This shows that Tm14-3-3ɛ is required to maintain innate immunity in T. molitor by enabling antimicrobial secretion into the hemolymph and explains the functional specialization of the isoform. PMID:27556493

  2. The Silencing of a 14-3-3ɛ Homolog in Tenebrio molitor Leads to Increased Antimicrobial Activity in Hemocyte and Reduces Larval Survivability

    PubMed Central

    Seo, Gi Won; Jo, Yong Hun; Seong, Jeong Hwan; Park, Ki Beom; Patnaik, Bharat Bhusan; Tindwa, Hamisi; Kim, Sun-Am; Lee, Yong Seok; Kim, Yu Jung; Han, Yeon Soo

    2016-01-01

    The 14-3-3 family of phosphorylated serine-binding proteins acts as signaling molecules in biological processes such as metabolism, division, differentiation, autophagy, and apoptosis. Herein, we report the requirement of 14-3-3ɛ isoform from Tenebrio molitor (Tm14-3-3ɛ) in the hemocyte antimicrobial activity. The Tm14-3-3ɛ transcript is 771 nucleotides in length and encodes a polypeptide of 256 amino acid residues. The protein has the typical 14-3-3 domain, the nuclear export signal (NES) sequence, and the peptide binding residues. The Tm14-3-3ɛ transcript shows a significant three-fold expression in the hemocyte of T. molitor larvae when infected with Escherichia coli Tm14-3-3ɛ silenced larvae show significantly lower survival rates when infected with E. coli. Under Tm14-3-3ɛ silenced condition, a strong antimicrobial activity is elicited in the hemocyte of the host inoculated with E. coli. This suggests impaired secretion of antimicrobial peptides (AMP) into the hemolymph. Furthermore, a reduction in AMP secretion under Tm14-3-3ɛ silenced condition would be responsible for loss in the capacity to kill bacteria and might explain the reduced survivability of the larvae upon E. coli challenge. This shows that Tm14-3-3ɛ is required to maintain innate immunity in T. molitor by enabling antimicrobial secretion into the hemolymph and explains the functional specialization of the isoform. PMID:27556493

  3. Regional rescue of spinocerebellar ataxia type 1 phenotypes by 14-3-3epsilon haploinsufficiency in mice underscores complex pathogenicity in neurodegeneration.

    PubMed

    Jafar-Nejad, Paymaan; Ward, Christopher S; Richman, Ronald; Orr, Harry T; Zoghbi, Huda Y

    2011-02-01

    Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disease caused by the expansion of a CAG repeat encoding a polyglutamine tract in Ataxin-1 (ATXN1). Both WT and mutant ATXN1 interact with 14-3-3 proteins, and 14-3-3 overexpression stabilizes ATXN1 levels in cells and increases ATXN1 toxicity in flies. To determine whether reducing 14-3-3 levels might mitigate SCA1 pathogenesis, we bred Sca1(154Q/+) mice to mice lacking one allele of 14-3-3ε. 14-3-3ε haploinsufficiency rescued cerebellar pathology and motor phenotypes but, surprisingly, not weight loss, respiratory dysfunction, or premature lethality. Biochemical studies revealed that reducing 14-3-3ε levels exerted different effects in two brain regions especially vulnerable in SCA1: Although diminishing levels of both WT and mutant ATXN1 in the cerebellum, 14-3-3ε haploinsufficiency did not alter ATXN1 levels in the brainstem. Furthermore, 14-3-3ε haploinsufficiency decreased the incorporation of expanded ATXN1 into its large toxic complexes in the cerebellum but not in the brainstem, and the distribution of ATXN1's small and large native complexes differed significantly between the two regions. These data suggest that distinct pathogenic mechanisms operate in different vulnerable brain regions, adding another level of complexity to SCA1 pathogenesis. PMID:21245341

  4. Suppression of 14-3-3γ-mediated surface expression of ANO1 inhibits cancer progression of glioblastoma cells.

    PubMed

    Lee, Young-Sun; Lee, Jae Kwang; Bae, Yeonju; Lee, Bok-Soon; Kim, Eunju; Cho, Chang-Hoon; Ryoo, Kanghyun; Yoo, Jiyun; Kim, Chul-Ho; Yi, Gwan-Su; Lee, Seok-Geun; Lee, C Justin; Kang, Sang Soo; Hwang, Eun Mi; Park, Jae-Yong

    2016-01-01

    Anoctamin-1 (ANO1) acts as a Ca(2+)-activated Cl(-) channel in various normal tissues, and its expression is increased in several different types of cancer. Therefore, understanding the regulation of ANO1 surface expression is important for determining its physiological and pathophysiological functions. However, the trafficking mechanism of ANO1 remains elusive. Here, we report that segment a (N-terminal 116 amino acids) of ANO1 is crucial for its surface expression, and we identified 14-3-3γ as a binding partner for anterograde trafficking using yeast two-hybrid screening. The surface expression of ANO1 was enhanced by 14-3-3γ, and the Thr9 residue of ANO1 was critical for its interaction with 14-3-3γ. Gene silencing of 14-3-3γ and/or ANO1 demonstrated that suppression of ANO1 surface expression inhibited migration and invasion of glioblastoma cells. These findings provide novel therapeutic implications for glioblastomas, which are associated with poor prognosis. PMID:27212225

  5. Suppression of 14-3-3γ-mediated surface expression of ANO1 inhibits cancer progression of glioblastoma cells

    PubMed Central

    Lee, Young-Sun; Lee, Jae Kwang; Bae, Yeonju; Lee, Bok-Soon; Kim, Eunju; Cho, Chang-Hoon; Ryoo, Kanghyun; Yoo, Jiyun; Kim, Chul-Ho; Yi, Gwan-Su; Lee, Seok-Geun; Lee, C. Justin; Kang, Sang Soo; Hwang, Eun Mi; Park, Jae-Yong

    2016-01-01

    Anoctamin-1 (ANO1) acts as a Ca2+-activated Cl− channel in various normal tissues, and its expression is increased in several different types of cancer. Therefore, understanding the regulation of ANO1 surface expression is important for determining its physiological and pathophysiological functions. However, the trafficking mechanism of ANO1 remains elusive. Here, we report that segment a (N-terminal 116 amino acids) of ANO1 is crucial for its surface expression, and we identified 14-3-3γ as a binding partner for anterograde trafficking using yeast two-hybrid screening. The surface expression of ANO1 was enhanced by 14-3-3γ, and the Thr9 residue of ANO1 was critical for its interaction with 14-3-3γ. Gene silencing of 14-3-3γ and/or ANO1 demonstrated that suppression of ANO1 surface expression inhibited migration and invasion of glioblastoma cells. These findings provide novel therapeutic implications for glioblastomas, which are associated with poor prognosis. PMID:27212225

  6. 14-3-3 eta isoform colocalizes TDP-43 on the coarse granules in the anterior horn cells of patients with sporadic amyotrophic lateral sclerosis.

    PubMed

    Umahara, Takahiko; Uchihara, Toshiki; Shibata, Noriyuki; Nakamura, Ayako; Hanyu, Haruo

    2016-09-01

    The immunolocalization of the 14-3-3 eta isoform in the anterior horn cells (AHCs) of patients with sporadic amyotrophic lateral sclerosis (ALS) and controls was examined. Compared with the immunolocalization of other 14-3-3 isoforms, the immunolocalization of the 14-3-3 eta isoform was either synaptic at the periphery of AHCs, spindle-shaped in neurites, or granular in the cytoplasm. By double labeling with phosphorylated (p-)TDP-43, the transactivation response DNA binding protein of 43kDa (TDP-43) demonstrated frequent colocalization of the 14-3-3 eta isoform in granular structures (90%) and spindle-shaped structures (85.4%), but not in p-TDP-43-positive round inclusions. It is speculated that the 14-3-3 eta isoform is associated with not only a synaptic pathology of ALS but also TDP-positive small lesions in the cytoplasm and neurites. The absence of eta-like immunoreactivity in p-TDP-43-positive large inclusions suggests the restricted relevance of the 14-3-3 eta isoform during ALS pathogenesis to some phases of the p-TDP pathology. PMID:27256400

  7. Oxidative damage of 14-3-3 zeta and gamma isoforms in Alzheimer's disease and cerebral amyloid angiopathy.

    PubMed

    Santpere, G; Puig, B; Ferrer, I

    2007-06-01

    Previous studies have shown oxidative damage resulting from amyloid Abeta exposure to cultured cells and in murine models. A target of oxidation is 14-3-3 which comprises a group of proteins involved in kinase activation and chaperone activity. The present study shows glycoxidative damage, as revealed with mono and bi-dimensional gel electrophoresis and Western blotting, followed by in-gel digestion and mass spectrometry, in the frontal cortex in Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA), a neurodegenerative disease with deposition of Abeta in cerebral blood vessels and in diffuse plaques unaccompanied by intraneuronal hyper-phosphorylated tau deposition. malondialdehyde-lysine (MDA-Lys)-, but not 4-hydroxy-2-nonenal (HNE)-immunoreactive adducts, and N-carboxyethyl-lysine (CEL), but not N-carboxymethyl-lysine (CML)-products, were present in 14-3-3 involving zeta and gamma isoforms in both AD and CAA. These findings demonstrate that 14-3-3 glyco- and lipoxidation occurs in AD and CAA, probably as a direct consequence of Abeta deposition. PMID:17445990

  8. PI 3-kinase-dependent phosphorylation of Plk1–Ser99 promotes association with 14-3-3γ and is required for metaphase–anaphase transition

    PubMed Central

    Kasahara, Kousuke; Goto, Hidemasa; Izawa, Ichiro; Kiyono, Tohru; Watanabe, Nobumoto; Elowe, Sabine; Nigg, Erich A; Inagaki, Masaki

    2013-01-01

    Polo-like kinase 1 (Plk1) controls multiple aspects of mitosis and is activated through its phosphorylation at Thr210. Here we identify Ser99 on Plk1 as a novel mitosis-specific phosphorylation site, which operates independently of Plk1–Thr210 phosphorylation. Plk1–Ser99 phosphorylation creates a docking site for 14-3-3γ, and this interaction stimulates the catalytic activity of Plk1. Knockdown of 14-3-3γ or replacement of wild-type (WT) Plk1 by a Ser99-phospho-blocking mutant leads to a prometaphase/metaphase-like arrest due to the activation of the spindle assembly checkpoint. Inhibition of phosphatidylinositol 3-kinase (PI3K) and Akt significantly reduces the level of Plk1–Ser99 phosphorylation and delays metaphase to anaphase transition. Plk1–Ser99 phosphorylation requires not only Akt activity but also protein(s) associated with Plk1 in a mitosis-specific manner. Therefore, mitotic Plk1 activity is regulated not only by Plk1–Thr210 phosphorylation, but also by Plk1 binding to 14-3-3γ following Plk1–Ser99 phosphorylation downstream of the PI3K–Akt signalling pathway. This novel Plk1 activation pathway controls proper progression from metaphase to anaphase. PMID:23695676

  9. Mechanisms Regulating Protein Localization.

    PubMed

    Bauer, Nicholas C; Doetsch, Paul W; Corbett, Anita H

    2015-10-01

    Cellular functions are dictated by protein content and activity. There are numerous strategies to regulate proteins varying from modulating gene expression to post-translational modifications. One commonly used mode of regulation in eukaryotes is targeted localization. By specifically redirecting the localization of a pool of existing protein, cells can achieve rapid changes in local protein function. Eukaryotic cells have evolved elegant targeting pathways to direct proteins to the appropriate cellular location or locations. Here, we provide a general overview of these localization pathways, with a focus on nuclear and mitochondrial transport, and present a survey of the evolutionarily conserved regulatory strategies identified thus far. We end with a description of several specific examples of proteins that exploit localization as an important mode of regulation. PMID:26172624

  10. 14-3-3-zeta participates in TLR3-mediated TICAM-1 signal-platform formation.

    PubMed

    Funami, Kenji; Matsumoto, Misako; Obuse, Chikashi; Seya, Tsukasa

    2016-05-01

    Recognition of pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs) is important in innate immune signaling. Toll-like receptors (TLRs) are well-characterized PRRs and are pivotal in antiviral and antitumor host defense. TIR domain-containing adaptor molecule 1 (TICAM-1, also called TRIF) is an adapter molecule in TLR3- and TLR4-mediated IRF3 activation, late-phase NF-κB activation and MAPK-mediated AP-1 activation. When a TLR3 ligand is added to TLR3-positive cells, TICAM-1 transiently interacts with TLR3 and forms multimers in the cytosol. However, the precise mechanism of TICAM-1 multimer formation remains unknown. In this study, we identified 14-3-3-zeta as a molecule that functions in TLR3-mediated signaling. Knockdown of 14-3-3-zeta reduced production of type I interferon and inflammatory cytokines, nuclear translocation of IRF3 and phosphorylation of IκB via the TLR3-TICAM-1 pathway. Furthermore, TICAM-1 multimerization by ligand stimulation was prohibited by 14-3-3-zeta knockdown. These results suggest that 14-3-3-zeta is involved in the TLR3-TICAM-1 pathway in promoting multimerization of TICAM-1 for the formation of a TICAM-1 signalosome. PMID:27058640

  11. RNA-processing protein TDP-43 regulates FOXO-dependent protein quality control in stress response.

    PubMed

    Zhang, Tao; Baldie, Gerard; Periz, Goran; Wang, Jiou

    2014-10-01

    Protein homeostasis is critical for cell survival and functions during stress and is regulated at both RNA and protein levels. However, how the cell integrates RNA-processing programs with post-translational protein quality control systems is unknown. Transactive response DNA-binding protein (TARDBP/TDP-43) is an RNA-processing protein that is involved in the pathogenesis of major neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here, we report a conserved role for TDP-43, from C. elegans to mammals, in the regulation of protein clearance via activation of FOXO transcription factors. In response to proteotoxic insults, TDP-43 redistributes from the nucleus to the cytoplasm, promoting nuclear translocation of FOXOs and relieving an inhibition of FOXO activity in the nucleus. The interaction between TDP-43 and the FOXO pathway in mammalian cells is mediated by their competitive binding to 14-3-3 proteins. Consistent with FOXO-dependent protein quality control, TDP-43 regulates the levels of misfolded proteins. Therefore, TDP-43 mediates stress responses and couples the regulation of RNA metabolism and protein quality control in a FOXO-dependent manner. The results suggest that compromising the function of TDP-43 in regulating protein homeostasis may contribute to the pathogenesis of related neurodegenerative diseases. PMID:25329970

  12. Protein Regulation in Signal Transduction.

    PubMed

    Lee, Michael J; Yaffe, Michael B

    2016-01-01

    SUMMARYCells must respond to a diverse, complex, and ever-changing mix of signals, using a fairly limited set of parts. Changes in protein level, protein localization, protein activity, and protein-protein interactions are critical aspects of signal transduction, allowing cells to respond highly specifically to a nearly limitless set of cues and also to vary the sensitivity, duration, and dynamics of the response. Signal-dependent changes in levels of gene expression and protein synthesis play an important role in regulation of protein levels, whereas posttranslational modifications of proteins regulate their degradation, localization, and functional interactions. Protein ubiquitylation, for example, can direct proteins to the proteasome for degradation or provide a signal that regulates their interactions and/or location within the cell. Similarly, protein phosphorylation by specific kinases is a key mechanism for augmenting protein activity and relaying signals to other proteins that possess domains that recognize the phosphorylated residues. PMID:27252361

  13. Genetic and physical interaction of Ssp1 CaMKK and Rad24 14-3-3 during low pH and osmotic stress in fission yeast

    PubMed Central

    Freitag, Silja I.; Wong, Jimson; Young, Paul G.

    2014-01-01

    The Ssp1 calmodulin kinase kinase (CaMKK) is necessary for stress-induced re-organization of the actin cytoskeleton and initiation of growth at the new cell end following division in Schizosaccharomyces pombe. In addition, it regulates AMP-activated kinase and functions in low glucose tolerance. ssp1− cells undergo mitotic delay at elevated temperatures and G2 arrest in the presence of additional stressors. Following hyperosmotic stress, Ssp1-GFP forms transient foci which accumulate at the cell membrane and form a band around the cell circumference, but not co-localizing with actin patches. Hyperosmolarity-induced localization to the cell membrane occurs concomitantly with a reduction of its interaction with the 14-3-3 protein Rad24, but not Rad25 which remains bound to Ssp1. The loss of rad24 in ssp1− cells reduces the severity of hyperosmotic stress response and relieves mitotic delay. Conversely, overexpression of rad24 exacerbates stress response and concomitant cell elongation. rad24− does not impair stress-induced localization of Ssp1 to the cell membrane, however this response is almost completely absent in cells overexpressing rad24. PMID:24451546

  14. Tipping the balance between good and evil: aberrant 14-3-3ζ expression drives oncogenic TGF-β signaling in metastatic breast cancers.

    PubMed

    Morrison, Chevaun D; Schiemann, William P

    2015-01-01

    Transforming growth factor beta (TGF-β) readily suppresses the development of early-stage breast cancers, an activity that gives way to tumor promotion in their late-stage counterparts. The molecular mechanisms underlying this mysterious switch in TGF-β function remain murky. In addressing this conundrum, Xu et al. observed aberrant 14-3-3ζ expression to prevent the formation of tumor-suppressive Smad2/3:p53 complexes, while simultaneously driving the generation of oncogenic Smad2/3:Gli2 complexes. Once formed, Smad2/3:Gli2 complexes stimulate the expression of parathyroid hormone-related protein necessary for breast cancer metastasis to bone. This viewpoint highlights 14-3-3ζ as an essential driver of oncogenic signaling by Smad2/3 and TGF-β in metastatic breast cancers. PMID:26160166

  15. p38- and MK2-dependent signalling promotes stress-induced centriolar satellite remodelling via 14-3-3-dependent sequestration of CEP131/AZI1

    PubMed Central

    Tollenaere, Maxim A. X.; Villumsen, Bine H.; Blasius, Melanie; Nielsen, Julie C.; Wagner, Sebastian A.; Bartek, Jiri; Beli, Petra; Mailand, Niels; Bekker-Jensen, Simon

    2015-01-01

    Centriolar satellites (CS) are small granular structures that cluster in the vicinity of centrosomes. CS are highly susceptible to stress stimuli, triggering abrupt displacement of key CS factors. Here we discover a linear p38-MK2-14-3-3 signalling pathway that specifically targets CEP131 to trigger CS remodelling after cell stress. We identify CEP131 as a substrate of the p38 effector kinase MK2 and pinpoint S47 and S78 as critical MK2 phosphorylation sites in CEP131. Ultraviolet-induced phosphorylation of these residues generates direct binding sites for 14-3-3 proteins, which sequester CEP131 in the cytoplasm to block formation of new CS, thereby leading to rapid depletion of these structures. Mutating S47 and S78 in CEP131 is sufficient to abolish stress-induced CS reorganization, demonstrating that CEP131 is the key regulatory target of MK2 and 14-3-3 in these structures. Our findings reveal the molecular mechanism underlying dynamic CS remodelling to modulate centrosome functions on cell stress. PMID:26616734

  16. The ciliopathy disease protein NPHP9 promotes nuclear delivery and activation of the oncogenic transcriptional regulator TAZ.

    PubMed

    Habbig, Sandra; Bartram, Malte P; Sägmüller, Josef G; Griessmann, Anabel; Franke, Mareike; Müller, Roman-Ulrich; Schwarz, Ricarda; Hoehne, Martin; Bergmann, Carsten; Tessmer, Claudia; Reinhardt, H Christian; Burst, Volker; Benzing, Thomas; Schermer, Bernhard

    2012-12-15

    Nephronophthisis (NPH) is a genetically heterogenous kidney disease and represents the most common genetic cause for end-stage renal disease in children. It is caused by the mutation of genes encoding for the nephrocystin proteins (NPHPs) which localize to primary cilia or centrosomes, classifying this disease as a 'ciliopathy'. Recently, it has been shown that NPHP4 acts as a potent negative regulator of mammalian Hippo signalling by interacting with the Lats protein kinase and controlling the phosphorylation of the oncogenic transcriptional activator TAZ. Here, we demonstrate that NPHP9, another NPH family member, also controls TAZ activity by a distinct mechanism. NPHP9, which is also called NEK8, directly interacted with TAZ and induced nuclear translocation of the TAZ/NPHP9 protein complex. Binding of NPHP9 to TAZ was enhanced in a TAZ mutant that lost its ability to bind 14-3-3, suggesting that 14-3-3 and NPHP9 may compete for TAZ binding, with 14-3-3 favouring cytoplasmic retention and NPHP9 mediating nuclear delivery. Consistently, co-expression of NPHP4, which inhibits TAZ phosphorylation at the 14-3-3 binding site through the inhibition of Lats kinase activity, induced efficient nuclear delivery of the TAZ/NPHP9 protein pair. Consistent with a role for TAZ in controlling proliferation and tumorigenesis, the downregulation of NPHP9 inhibited the TAZ-dependent proliferation of hippo-responsive normal epithelial and also breast cancer cells. As NPHP9 has been shown to be upregulated in breast cancer, these data do not only support a critical role for TAZ/hippo signalling in the pathogenesis of NPH but may also imply a possible role for NPHP9 in TAZ-mediated tumorigenesis. PMID:23026745

  17. Development of a polymerase chain reaction assay for the diagnosis of neosporosis using the Neospora caninum 14-3-3 gene.

    PubMed

    Lally, N C; Jenkins, M C; Dubey, J P

    1996-01-01

    Neospora caninum is a recently described apicomplexan parasite which causes neuromuscular disease in dogs, and abortion and neonatal morbidity in cattle, sheep and horses. Morphological similarities and serological cross-reactivity between N. caninum and the closely related parasite Toxoplasma gondii, have resulted in the frequent misdiagnosis of neosporosis as toxoplasmosis. This report describes the isolation and characterization of an N. caninum cDNA clone encoding a 14-3-3 protein homologue. The 14-3-3 proteins are a class of proteins which show a high degree of amino acid sequence conservation across several eukaryotic taxa. Using less conserved regions of the N. caninum cDNA clone, nested primers were designed for the amplification of a 614-bp N. caninum DNA fragment by the polymerase chain reaction (PCR). The DNA fragment was amplified from N. caninum genomic DNA, but not from T. gondii, Sarcocystis muris, Sarcocystis tenella, or Sarcocystis cruzi genomic DNA. Additionally, the fragment was amplified from DNA prepared from the brains of N. caninum-infected mice, but not from the brain of a mouse infected with T. gondii. These results suggest that this PCR assay may be useful for the diagnosis of neosporosis. PMID:8992315

  18. Akt and 14-3-3 control a PACS-2 homeostatic switch that integrates membrane traffic with TRAIL-induced apoptosis

    PubMed Central

    Aslan, Joseph E.; You, Huihong; Williamson, Danielle M.; Endig, Jessica; Youker, Robert T.; Thomas, Laurel; Shu, Hongjun; Du, Yuhong; Milewski, Robert L.; Brush, Matthew H.; Possemato, Anthony; Sprott, Kam; Fu, Haian; Greis, Kenneth D.; Runckel, Douglas N.; Vogel, Arndt; Thomas, Gary

    2009-01-01

    Summary TRAIL selectively kills diseased cells in vivo, spurring interest in this death ligand as a potential therapeutic. However, many cancer cells are resistant to TRAIL suggesting the mechanism mediating TRAIL-induced apoptosis is complex. Here we identify PACS-2 as an essential TRAIL effector, required for killing tumor cells in vitro and virally infected hepatocytes in vivo. PACS-2 is phosphorylated at Ser437 in vivo and pharmacologic and genetic studies demonstrate Akt is an in vivo Ser437 kinase. Akt cooperates with 14-3-3 to regulate the homeostatic and apoptotic properties of PACS-2 that mediate TRAIL action. Phosphorylated Ser437 binds 14-3-3 with high affinity, which represses PACS-2 apoptotic activity and is required for PACS-2 to mediate trafficking of membrane cargo. TRAIL triggers dephosphorylation of Ser437, reprogramming PACS-2 to promote apoptosis. Together, these studies identify the phosphorylation state of PACS-2 Ser437 as a molecular switch that integrates cellular homeostasis with TRAIL-induced apoptosis. PMID:19481529

  19. Akt and 14-3-3 control a PACS-2 homeostatic switch that integrates membrane traffic with TRAIL-induced apoptosis.

    PubMed

    Aslan, Joseph E; You, Huihong; Williamson, Danielle M; Endig, Jessica; Youker, Robert T; Thomas, Laurel; Shu, Hongjun; Du, Yuhong; Milewski, Robert L; Brush, Matthew H; Possemato, Anthony; Sprott, Kam; Fu, Haian; Greis, Kenneth D; Runckel, Douglas N; Vogel, Arndt; Thomas, Gary

    2009-05-14

    TRAIL selectively kills diseased cells in vivo, spurring interest in this death ligand as a potential therapeutic. However, many cancer cells are resistant to TRAIL, suggesting the mechanism mediating TRAIL-induced apoptosis is complex. Here we identify PACS-2 as an essential TRAIL effector, required for killing tumor cells in vitro and virally infected hepatocytes in vivo. PACS-2 is phosphorylated at Ser437 in vivo, and pharmacologic and genetic studies demonstrate Akt is an in vivo Ser437 kinase. Akt cooperates with 14-3-3 to regulate the homeostatic and apoptotic properties of PACS-2 that mediate TRAIL action. Phosphorylated Ser437 binds 14-3-3 with high affinity, which represses PACS-2 apoptotic activity and is required for PACS-2 to mediate trafficking of membrane cargo. TRAIL triggers dephosphorylation of Ser437, reprogramming PACS-2 to promote apoptosis. Together, these studies identify the phosphorylation state of PACS-2 Ser437 as a molecular switch that integrates cellular homeostasis with TRAIL-induced apoptosis. PMID:19481529

  20. 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

  1. 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

  2. Decreased expression of 14-3-3σ is predictive of poor prognosis for patients with human uterine papillary serous carcinoma.

    PubMed

    Suzuki, Fumihiko; Nagase, Satoru; Suzuki, Kichiya; Oba, Etsuko; Hiroki, Eri; Matsuda, Yukika; Akahira, Jun-Ichi; Nishigori, Hidekazu; Sugiyama, Takashi; Otsuki, Takeo; Yoshinaga, Kousuke; Takano, Tadao; Niikura, Hitoshi; Ito, Kiyoshi; Sasano, Hironobu; Yaegashi, Nobuo

    2013-01-01

    Uterine papillary serous carcinoma (UPSC) morphologically resembles ovarian serous carcinoma and is categorized as a type II endometrial cancer. UPSC comprises about 10% of all types of endometrial cancer and has an aggressive clinical course and a poor prognosis. The 14-3-3σ gene was originally discovered as a p53-inducible gene; its expression is induced by DNA damage in a p53-dependent manner, which leads to G2 arrest and repair of damaged DNA. Moreover, it has been reported that expression of 14-3-3σ is frequently lost in various types of human cancer, including ovarian cancer. We therefore examined the association between 14-3-3σ expression determined by immunohistochemistry and clinical outcomes of 51 patients with UPSC. UPSC was considered positive for 14-3-3σ when > 30% of tumor cells were stained with a specific antibody. Of these patients, 29 (58.7%) showed positive immunoreactivity for 14-3-3σ and 22 (41.3%) had decreased 14-3-3σ staining. Decreased immunoreactivity for 14-3-3σ was associated with stage (P = 0.001) and lymphovascular space involvement (P = 0.005). Moreover, decreased 14-3-3σ expression was an independent risk factor for reduced overall survival (P = 0.0416) in multivariate analysis. Direct bisulfite sequencing was performed to evaluate the methylation status of the 27 CpG islands in the promoter region and first exon of the 14-3-3σ gene. These CpG islands were hypermethylated in 30% of 14-3-3σ-positive UPSC and 80% of 14-3-3σ-negative UPSC, although the difference was not statistically significant. These findings suggest that decreased expression of immunoreactive 14-3-3σ may be a predictor of poor prognosis in patients with UPSC. PMID:24201220

  3. Redox Regulation of Protein Kinases

    PubMed Central

    Truong, Thu H.; Carroll, Kate S.

    2015-01-01

    Protein kinases represent one of the largest families of genes found in eukaryotes. Kinases mediate distinct cellular processes ranging from proliferation, differentiation, survival, and apoptosis. Ligand-mediated activation of receptor kinases can lead to the production of endogenous H2O2 by membrane-bound NADPH oxidases. In turn, H2O2 can be utilized as a secondary messenger in signal transduction pathways. This review presents an overview of the molecular mechanisms involved in redox regulation of protein kinases and its effects on signaling cascades. In the first half, we will focus primarily on receptor tyrosine kinases (RTKs), whereas the latter will concentrate on downstream non-receptor kinases involved in relaying stimulant response. Select examples from the literature are used to highlight the functional role of H2O2 regarding kinase activity, as well as the components involved in H2O2 production and regulation during cellular signaling. In addition, studies demonstrating direct modulation of protein kinases by H2O2 through cysteine oxidation will be emphasized. Identification of these redox-sensitive residues may help uncover signaling mechanisms conserved within kinase subfamilies. In some cases, these residues can even be exploited as targets for the development of new therapeutics. Continued efforts in this field will further basic understanding of kinase redox regulation, and delineate the mechanisms involved in physiologic and pathological H2O2 responses. PMID:23639002

  4. RGS Protein Regulation of Phototransduction

    PubMed Central

    Chen, Ching-Kang Jason

    2015-01-01

    First identified in yeast and worm and later in other species, the physiological importance of regulators of G-protein signaling (RGS) in mammals was first demonstrated at the turn of the century in mouse retinal photoreceptors, in which RGS9 is needed for timely recovery of rod phototransduction. The role of RGS in vision has been established a synapse away in retinal depolarizing bipolar cells (DBCs), where RGS7 and RGS11 work redundantly and in a complex with Gβ5-S as GAPs for Goα in the metabotropic glutamate receptor 6 pathway at DBC dendritic tips. Much less is known on how RGS protein subserves vision in the rest of the visual system. The research into the roles of RGS proteins in vision holds great potential for many exciting new discoveries. PMID:26123301

  5. Echinococcus multilocularis proliferation in mice and respective parasite 14-3-3 gene expression is mainly controlled by an αβ+ CD4+ T-cell-mediated immune response

    PubMed Central

    Dai, Wen Juan; Waldvogel, Andreas; Siles-Lucas, Mar; Gottstein, Bruno

    2004-01-01

    The role of specific B lymphocytes and T-cell populations in the control of experimental Echinococus multilocularis infection was studied in µMT, nude, T-cell receptor (TCR)-β–/–, major histocompatibility complex (MHC)-I–/– and MHC-II–/– mice. At 2 months postinfection, the parasite mass was more than 10 times higher in nude, TCR-β–/– and MHC-II–/– mice than in infected C57BL/6 wild-type (WT) mice, and these T-cell-deficient mice started to die of the high parasite load at this time-point. In contrast, MHC-I–/– and µMT mice exhibited parasite growth rates similar to those found in WT controls. These findings clearly point to the major role that CD4+ αβ+ T cells play in limiting the E. multilocularis proliferation, while CD8+ T and B cells appeared to play a minor role in the control of parasite growth. In the absence of T cells, especially CD4+ or αβ+ T cells, the cellular immune response to infection was impaired, as documented by the lack of hepatic granuloma formation around the parasite and by a decreased splenocyte responsiveness to concanavalin A (Con A) and parasite antigen stimulation. Surprisingly, in T-cell-deficient mice, the ex vivo expression of interferon-γ (IFN-γ) and other inflammatory cytokines (except for interleukin-6) were increased in association with a high parasite load. Thus, the relative protection mediated by CD4+ αβ+ T cells against E. multilocularis infection seemed not be IFN-γ dependent, but rather to rely on the effector's function of CD4+ αβ+ T cells. The local restriction of parasite germinal cell proliferation was reflected by a regulatory effect on the expression of 14-3-3 protein within the parasite tissue in T-cell-deficient mice. These results provide a strong indication that the CD4+ αβ+ T-cell-mediated immune response contributes to the control of the parasite growth and to the regulation of production of the parasite 14-3-3 protein in metacestode tissues. PMID:15196217

  6. Metabolite Regulation of Nuclear Localization of Carbohydrate-response Element-binding Protein (ChREBP): ROLE OF AMP AS AN ALLOSTERIC INHIBITOR.

    PubMed

    Sato, Shogo; Jung, Hunmin; Nakagawa, Tsutomu; Pawlosky, Robert; Takeshima, Tomomi; Lee, Wan-Ru; Sakiyama, Haruhiko; Laxman, Sunil; Wynn, R Max; Tu, Benjamin P; MacMillan, John B; De Brabander, Jef K; Veech, Richard L; Uyeda, Kosaku

    2016-05-13

    The carbohydrate-response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays an essential role in converting excess carbohydrate to fat storage in the liver. In response to glucose levels, ChREBP is regulated by nuclear/cytosol trafficking via interaction with 14-3-3 proteins, CRM-1 (exportin-1 or XPO-1), or importins. Nuclear localization of ChREBP was rapidly inhibited when incubated in branched-chain α-ketoacids, saturated and unsaturated fatty acids, or 5-aminoimidazole-4-carboxamide ribonucleotide. Here, we discovered that protein-free extracts of high fat-fed livers contained, in addition to ketone bodies, a new metabolite, identified as AMP, which specifically activates the interaction between ChREBP and 14-3-3. The crystal structure showed that AMP binds directly to the N terminus of ChREBP-α2 helix. Our results suggest that AMP inhibits the nuclear localization of ChREBP through an allosteric activation of ChREBP/14-3-3 interactions and not by activation of AMPK. AMP and ketone bodies together can therefore inhibit lipogenesis by restricting localization of ChREBP to the cytoplasm during periods of ketosis. PMID:26984404

  7. Endogenous substrates of sphingosine-dependent kinases (SDKs) are chaperone proteins: heat shock proteins, glucose-regulated proteins, protein disulfide isomerase, and calreticulin.

    PubMed

    Megidish, T; Takio, K; Titani, K; Iwabuchi, K; Hamaguchi, A; Igarashi, Y; Hakomori, S

    1999-03-16

    Protein kinases whose activity is detectable only in the presence of sphingosine (Sph) or N,N'-dimethyl-Sph (DMS), but not in the presence of 15 other sphingolipids, phospholipids, and glycerolipids tested (Megidish, T., et al. (1995) Biochem. Biophys. Res. Commun. 216, 739-747), have been termed "sphingosine-dependent kinases" (SDKs). We showed previously that a purified SDK (termed "SDK1") phosphorylates a specific Ser position of adapter/chaperone protein 14-3-3 isoforms beta, eta, and zeta but not tau or sigma (Megidish, T., et al. (1998) J. Biol. Chem. 273, 21834-45). In this study we found the following: (i) other SDKs with different substrate specificities are present in cytosolic and membrane extracts of mouse Balb/c 3T3 (A31) fibroblasts. (ii) The activation of these SDKs is specific to D-erythro-Sph and its N-methyl derivatives, the effect of L-threo-Sph or its N-methyl derivatives is minimal, and nonspecific cationic amphiphiles have no effect at all. An SDK separated as fractions "TN31-33" phosphorylated a 50 kDa substrate which was identified as calreticulin, as well as two endogenous substrates with molecular mass 58 and 55 kDa, both identified as protein disulfide isomerase (PDI). This SDK, which specifically phosphorylates calreticulin and PDI, both molecular chaperones found at high levels in endoplasmic reticulum, is tentatively termed "SDK2". Another SDK activity was copurified with glucose-regulated protein (GRP) and heat shock proteins (HSP). One GRP substrate had the same amino acid sequence as GRP94 (synonym: endoplasmin); another HSP substrate had the same amino acid sequence as mouse HSP86 or HSP84, the analogues of human HSP90. An SDK activity separated and present in "fraction 42" from Q-Sepharose chromatography specifically phosphorylated GRP105 (or GRP94) and HSP68 but did not phosphorylate PDI or 14-3-3. This SDK is clearly different from other SDKs in its substrate specificity and is tentatively termed "SDK3". Interestingly

  8. 14-3-3ζ deficient mice in the BALB/c background display behavioural and anatomical defects associated with neurodevelopmental disorders

    PubMed Central

    Xu, Xiangjun; Jaehne, Emily J.; Greenberg, Zarina; McCarthy, Peter; Saleh, Eiman; Parish, Clare L.; Camera, Daria; Heng, Julian; Haas, Matilda; Baune, Bernhard T.; Ratnayake, Udani; Buuse, Maarten van den; Lopez, Angel F.; Ramshaw, Hayley S.; Schwarz, Quenten

    2015-01-01

    Sequencing and expression analyses implicate 14-3-3ζ as a genetic risk factor for neurodevelopmental disorders such as schizophrenia and autism. In support of this notion, we recently found that 14-3-3ζ−/− mice in the Sv/129 background display schizophrenia-like defects. As epistatic interactions play a significant role in disease pathogenesis we generated a new congenic strain in the BALB/c background to determine the impact of genetic interactions on the 14-3-3ζ−/− phenotype. In addition to replicating defects such as aberrant mossy fibre connectivity and impaired spatial memory, our analysis of 14-3-3ζ−/− BALB/c mice identified enlarged lateral ventricles, reduced synaptic density and ectopically positioned pyramidal neurons in all subfields of the hippocampus. In contrast to our previous analyses, 14-3-3ζ−/− BALB/c mice lacked locomotor hyperactivity that was underscored by normal levels of the dopamine transporter (DAT) and dopamine signalling. Taken together, our results demonstrate that dysfunction of 14-3-3ζ gives rise to many of the pathological hallmarks associated with the human condition. 14-3-3ζ-deficient BALB/c mice therefore provide a novel model to address the underlying biology of structural defects affecting the hippocampus and ventricle, and cognitive defects such as hippocampal-dependent learning and memory. PMID:26207352

  9. Regulating the regulators: serine/arginine-rich proteins under scrutiny.

    PubMed

    Risso, Guillermo; Pelisch, Federico; Quaglino, Ana; Pozzi, Berta; Srebrow, Anabella

    2012-10-01

    Serine/arginine-rich (SR) proteins are among the most studied splicing regulators. They constitute a family of evolutionarily conserved proteins that, apart from their initially identified and deeply studied role in splicing regulation, have been implicated in genome stability, chromatin binding, transcription elongation, mRNA stability, mRNA export and mRNA translation. Remarkably, this list of SR protein activities seems far from complete, as unexpected functions keep being unraveled. An intriguing aspect that awaits further investigation is how the multiple tasks of SR proteins are concertedly regulated within mammalian cells. In this article, we first discuss recent findings regarding the regulation of SR protein expression, activity and accessibility. We dive into recent studies describing SR protein auto-regulatory feedback loops involving different molecular mechanisms such asunproductive splicing, microRNA-mediated regulation and translational repression. In addition, we take into account another step of regulation of SR proteins, presenting new findings about a variety of post-translational modifications by proteomics approaches and how some of these modifications can regulate SR protein sub-cellular localization or stability. Towards the end, we focus in two recently revealed functions of SR proteins beyond mRNA biogenesis and metabolism, the regulation of micro-RNA processing and the regulation of small ubiquitin-like modifier (SUMO) conjugation. PMID:22941908

  10. Decreased expression of 14-3-3 σ, an early event of malignant transformation of respiratory epithelium, also facilitates progression of squamous cell lung cancer

    PubMed Central

    Sun, Nan; Wu, Yongkai; Huang, Bo; Liu, Qian; Dong, Yinan; Ding, Jianqiao; Liu, Yongyu

    2015-01-01

    Background It has been shown that 14-3-3 σ serves as a tumor suppressor gene, and is downregulated in various tumor tissues. However, the role of 14-3-3 σ during the initiation and progression of lung squamous cell carcinoma (SqCC) is not well understood. Methods The expression status of 14-3-3 σ in archival tissue samples from 40 lung SqCC patients (36 with normal bronchia, 19 squamous metaplasia, and 17 dysplasia/carcinoma in situ, in their tissue samples) was examined by immunohistochemical analysis. The proliferation rate and tumor formation ability of the H520 cell transfected with 14-3-3 σ was tested with methyl thiazolyl tetrazolium assay and nude mice subcutaneous injection, respectively. Results In the normal bronchial epithelia, 14-3-3 σ was highly expressed, whereas it was significantly decreased in precancerous and cancerous tissues. Compared with matched invasive cancer tissues, the expression level of 14-3-3 σ in squamous metaplasia was significantly higher (P = 0.049), while that in dysplasia/carcinoma in situ showed no significant changes (P = 0.135). Statistical analysis showed that the expression level of 14-3-3 σ in tumor tissue was associated with the differentiation grade of the tumor (P = 0.001) and the prognosis of the patient (P = 0.003). The overexpression of 14-3-3 σ significantly suppressed the proliferation of H520 cells in vitro and in vivo. Conclusion The inactivation of 14-3-3 σ may be a very early event in tumorigenesis and could facilitate the initiation and progression of lung SqCC in a sustainable way. PMID:26557909

  11. ErbB2, FoxM1 and 14-3-3ζ prime breast cancer cells for invasion in response to ionizing radiation

    PubMed Central

    Kambach, DM; Sodi, VL; Lelkes, PI; Azizkhan-Clifford, J; Reginato, MJ

    2014-01-01

    ErbB2 is frequently highly expressed in premalignant breast cancers, including ductal carcinoma in situ (DCIS); however, little is known about the signals or pathways it contributes to progression into the invasive/malignant state. Radiotherapy is often used to treat early premalignant lesions regardless of ErbB2 status. Here, we show that clinically relevant doses of ionizing radiation (IR)-induce cellular invasion of ErbB2-expressing breast cancer cells, as well as MCF10A cells overexpressing ErbB2. ErbB2-negative breast cancer cells, such as MCF7 and T47D, do not invade following treatment with IR nor do MCF10A cells overexpressing epidermal growth factor receptor. ErbB2 becomes phosphorylated at tyrosine 877 in a dose- and time- dependent manner following exposure to X-rays, and activates downstream signaling cascades including PI3K/Akt. Inhibition of these pathways, as well as inhibition of reactive oxygen species (ROS) with antioxidants, prevents IR-induced invasion. Activation of ErbB2-dependent signaling results in upregulation of the forkhead family transcription factor, FoxM1, and its transcriptional targets, including matrix metalloproteinase 2 (MMP2). Inhibition of FoxM1 by RNA interference prevented induction of invasion by IR, and overexpression of FoxM1 in MCF10A cells was sufficient to promote IR-induced invasion. Moreover, we found that 14-3-3ζ is also upregulated by IR in cancer cells in a ROS-dependent manner, is required for IR-induced invasion in ErbB2-positive breast cancer cells and together with FoxM1 is sufficient for invasion in ErbB2-negative breast cancer cells. Thus, our data show that IR-mediated activation of ErbB2 and induction of 14-3-3ζ collaborate to regulate FoxM1 and promote invasion of breast cancer cells and furthermore, may serve as therapeutic targets to enhance radiosensitivity of breast cancers. PMID:23318431

  12. PreImplantation factor (PIF*) regulates systemic immunity and targets protective regulatory and cytoskeleton proteins.

    PubMed

    Barnea, Eytan R; Hayrabedyan, Soren; Todorova, Krassimira; Almogi-Hazan, Osnat; Or, Reuven; Guingab, Joy; McElhinney, James; Fernandez, Nelson; Barder, Timothy

    2016-07-01

    Secreted by viable embryos, PIF is expressed by the placenta and found in maternal circulation. It promotes implantation and trophoblast invasion, achieving systemic immune homeostasis. Synthetic PIF successfully transposes endogenous PIF features to non-pregnant immune and transplant models. PIF affects innate and activated PBMC cytokines and genes expression. We report that PIF targets similar proteins in CD14+, CD4+ and CD8+ cells instigating integrated immune regulation. PIF-affinity chromatography followed by mass-spectrometry, pathway and heatmap analysis reveals that SET-apoptosis inhibitor, vimentin, myosin-9 and calmodulin are pivotal for immune regulation. PIF acts on macrophages down-stream of LPS (lipopolysaccharide-bacterial antigen) CD14/TLR4/MD2 complex, targeting myosin-9, thymosin-α1 and 14-3-3eta. PIF mainly targets platelet aggregation in CD4+, and skeletal proteins in CD8+ cells. Pathway analysis demonstrates that PIF targets and regulates SET, tubulin, actin-b, and S100 genes expression. PIF targets systemic immunity and has a short circulating half-life. Collectively, PIF targets identified; protective, immune regulatory and cytoskeleton proteins reveal mechanisms involved in the observed efficacy against immune disorders. PMID:26944449

  13. Role of regulator of G protein signaling proteins in bone

    PubMed Central

    Keinan, David; Yang, Shuying; Cohen, Robert E.; Yuan, Xue; Liu, Tongjun; Li, Yi-Ping

    2014-01-01

    Regulators of G protein signaling (RGS) proteins are a family with more than 30 proteins that all contain an RGS domain. In the past decade, increasing evidence has indicated that RGS proteins play crucial roles in the regulation of G protein coupling receptors (GPCR), G proteins, and calcium signaling during cell proliferation, migration, and differentiation in a variety of tissues. In bone, those proteins modulate bone development and remodeling by influencing various signaling pathways such as GPCR-G protein signaling, Wnt, calcium oscillations and PTH. This review summarizes the recent advances in the understanding of the regulation of RGS genes expression, as well as the functions and mechanisms of RGS proteins, especially in regulating GPCR-G protein signaling, Wnt signaling, calcium oscillations signaling and PTH signaling during bone development and remodeling. This review also highlights the regulation of different RGS proteins in osteoblasts, chondrocytes and osteoclasts. The knowledge from the recent advances of RGS study summarized in the review would provide the insights into new therapies for bone diseases. PMID:24389209

  14. Na/H Exchange Regulatory Factor 1, a Novel AKT-associating Protein, Regulates Extracellular Signal-regulated Kinase Signaling through a B-Raf–Mediated Pathway

    PubMed Central

    Wang, Bin; Yang, Yanmei

    2008-01-01

    Na/H exchange regulatory factor 1 (NHERF1) is a scaffolding protein that regulates signaling and trafficking of several G protein-coupled receptors (GPCRs), including the parathyroid hormone receptor (PTH1R). GPCRs activate extracellular signal-regulated kinase (ERK)1/2 through different mechanisms. Here, we characterized NHERF1 regulation of PTH1R-stimulated ERK1/2. Parathyroid hormone (PTH) stimulated ERK1/2 phosphorylation by a protein kinase A (PKA)-dependent, but protein kinase C-, cyclic adenosine 5′-monophosphate-, and Rap1-independent pathway in Chinese hamster ovary cells stably transfected with the PTH1R and engineered to express NHERF1 under the control of tetracycline. NHERF1 blocked PTH-induced ERK1/2 phosphorylation downstream of PKA. This suggested that NHERF1 inhibitory effects on ERK1/2 occur at a postreceptor locus. Forskolin activated ERK1/2, and this effect was blocked by NHERF1. NHERF1 interacted with AKT and inhibited ERK1/2 activation by decreasing the stimulatory effect of 14-3-3 binding to B-Raf, while increasing the inhibitory influence of AKT negative regulation on ERK1/2 activation. This novel regulatory mechanism provides a new model by which cytoplasmic adapter proteins modulate ERK1/2 activation through a receptor-independent mechanism involving B-Raf. PMID:18272783

  15. Regulation of TET Protein Stability by Calpains

    PubMed Central

    Wang, Yu; Zhang, Yi

    2014-01-01

    SUMMARY DNA methylation at the fifth position of cytosine (5mC) is an important epigenetic modification that affects chromatin structure and gene expression. Recent studies have established a critical function of the Ten-eleven translocation (Tet) family of proteins in regulating DNA methylation dynamics. Three Tet genes have been identified in mammals, and they all encode for proteins capable of oxidizing 5mC as part of the DNA demethylation process. While regulation of Tet expression at the transcriptional level is well documented, how TET proteins are regulated at post-translational level is poorly understood. In this study, we report that all three TET proteins are direct substrates of calpains, a family of calcium-dependent proteases. Specifically, calpain1 mediates TET1 and TET2 turnover in mouse ES cells, and calpain2 regulates TET3 level during differentiation. This study provides the first evidence that TET proteins are subject to calpain-mediated degradation. PMID:24412366

  16. CALML5 is a ZNF750- and TINCR-induced protein that binds stratifin to regulate epidermal differentiation

    PubMed Central

    Sun, Bryan K.; Boxer, Lisa D.; Ransohoff, Julia D.; Siprashvili, Zurab; Qu, Kun; Lopez-Pajares, Vanessa; Hollmig, S. Tyler; Khavari, Paul A.

    2015-01-01

    Outward migration of epidermal progenitors occurs with induction of hundreds of differentiation genes, but the identities of all regulators required for this process are unknown. We used laser capture microdissection followed by RNA sequencing to identify calmodulin-like 5 (CALML5) as the most enriched gene in differentiating outer epidermis. CALML5 mRNA was up-regulated by the ZNF750 transcription factor and then stabilized by the long noncoding RNA TINCR. CALML5 knockout impaired differentiation, abolished keratohyalin granules, and disrupted epidermal barrier function. Mass spectrometry identified SFN (stratifin/14-3-3σ) as a CALML5-binding protein. CALML5 interacts with SFN in suprabasal epidermis, cocontrols 13% of late differentiation genes, and modulates interaction of SFN to some of its binding partners. A ZNF750–TINCR–CALML5–SFN network is thus essential for epidermal differentiation. PMID:26545810

  17. Mitochondria-Mediated Protein Regulation Mechanism of Polymorphs-Dependent Inhibition of Nanoselenium on Cancer Cells.

    PubMed

    Wang, Ge; Guo, Yuming; Yang, Gai; Yang, Lin; Ma, Xiaoming; Wang, Kui; Zhu, Lin; Sun, Jiaojiao; Wang, Xiaobing; Zhang, Hua

    2016-01-01

    The present study was (i) to prepare two types of selenium nanoparticles, namely an amorphous form of selenium quantum dots (A-SeQDs) and a crystalline form of selenium quantum dots (C-SeQDs); and (ii) to investigate the nano-bio interactions of A-SeQDs and C-SeQDs in MCF-7, HepG2, HeLa, NIH/3T3, L929 cells and BRL-3A cells. It was found that A-SeQDs could induce the mitochondria-mediated apoptosis, necrosis and death of cells, while C-SeQDs had much weaker effects. This polymorphs-dependent anti-proliferative activity of nano-selenium was scarcely reported. Further investigation demonstrated that A-SeQDs could differentially regulate 61 proteins and several pathways related to stress response, protein synthesis, cell migration and cell cycle, including "p38 MAPK Signaling", "p53 Signaling", "14-3-3-mediated Signaling", "p70S6K Signaling" and "Protein Ubiquitination Pathway". This was the first report to demonstrate the involvement of protein synthesis and post-translational modification pathways in the anti-proliferative activity associated with NMs. Compared with previously fragmentary studies, this study use a nanomics approach combining bioinformatics and proteomics to systematically investigate the nano-bio interactions of selenium nanoparticles in cancer cells. PMID:27514819

  18. Mitochondria-Mediated Protein Regulation Mechanism of Polymorphs-Dependent Inhibition of Nanoselenium on Cancer Cells

    PubMed Central

    Wang, Ge; Guo, Yuming; Yang, Gai; Yang, Lin; Ma, Xiaoming; Wang, Kui; Zhu, Lin; Sun, Jiaojiao; Wang, Xiaobing; Zhang, Hua

    2016-01-01

    The present study was (i) to prepare two types of selenium nanoparticles, namely an amorphous form of selenium quantum dots (A-SeQDs) and a crystalline form of selenium quantum dots (C-SeQDs); and (ii) to investigate the nano-bio interactions of A-SeQDs and C-SeQDs in MCF-7, HepG2, HeLa, NIH/3T3, L929 cells and BRL-3A cells. It was found that A-SeQDs could induce the mitochondria-mediated apoptosis, necrosis and death of cells, while C-SeQDs had much weaker effects. This polymorphs-dependent anti-proliferative activity of nano-selenium was scarcely reported. Further investigation demonstrated that A-SeQDs could differentially regulate 61 proteins and several pathways related to stress response, protein synthesis, cell migration and cell cycle, including “p38 MAPK Signaling”, “p53 Signaling”, “14-3-3-mediated Signaling”, “p70S6K Signaling” and “Protein Ubiquitination Pathway”. This was the first report to demonstrate the involvement of protein synthesis and post-translational modification pathways in the anti-proliferative activity associated with NMs. Compared with previously fragmentary studies, this study use a nanomics approach combining bioinformatics and proteomics to systematically investigate the nano-bio interactions of selenium nanoparticles in cancer cells. PMID:27514819

  19. An unusual arrangement of two 14-3-3-like domains in the SMG5–SMG7 heterodimer is required for efficient nonsense-mediated mRNA decay

    PubMed Central

    Jonas, Stefanie; Weichenrieder, Oliver; Izaurralde, Elisa

    2013-01-01

    The nonsense-mediated mRNA decay (NMD) pathway triggers the rapid degradation of aberrant mRNAs containing premature translation termination codons (PTCs). In metazoans, NMD requires three 14-3-3-like proteins: SMG5, SMG6, and SMG7. These proteins are recruited to PTC-containing mRNAs through the interaction of their 14-3-3-like domains with phosphorylated UPF1, the central NMD effector. Recruitment of SMG5, SMG6, and SMG7 causes NMD target degradation. In this study, we report the crystal structure of the Caenorhabditis elegans SMG5–SMG7 complex. The 14-3-3-like phosphopeptide recognition domains of SMG5 and SMG7 heterodimerize in an unusual perpendicular back-to-back orientation in which the peptide-binding sites face opposite directions. Structure-based mutants and functional assays indicate that the SMG5–SMG7 interaction is conserved and is crucial for efficient NMD in human cells. Notably, we demonstrate that heterodimerization increases the affinity of the SMG5–SMG7 complex for UPF1. Furthermore, we show that the degradative activity of the SMG5–SMG7 complex resides in SMG7 and that the SMG5–SMG7 complex and SMG6 play partially redundant roles in the degradation of aberrant mRNAs. We propose that the SMG5–SMG7 complex binds to phosphorylated UPF1 with high affinity and recruits decay factors to the mRNA target through SMG7, thus promoting target degradation. PMID:23348841

  20. FET proteins regulate lifespan and neuronal integrity

    PubMed Central

    Therrien, Martine; Rouleau, Guy A.; Dion, Patrick A.; Parker, J. Alex

    2016-01-01

    The FET protein family includes FUS, EWS and TAF15 proteins, all of which have been linked to amyotrophic lateral sclerosis, a fatal neurodegenerative disease affecting motor neurons. Here, we show that a reduction of FET proteins in the nematode Caenorhabditis elegans causes synaptic dysfunction accompanied by impaired motor phenotypes. FET proteins are also involved in the regulation of lifespan and stress resistance, acting partially through the insulin/IGF-signalling pathway. We propose that FET proteins are involved in the maintenance of lifespan, cellular stress resistance and neuronal integrity. PMID:27117089

  1. Regulation of cardiac C-protein phosphorylation

    SciTech Connect

    Titus, F.L.

    1985-01-01

    Molecular mechanisms of cardiac sympathetic and parasympathetic responses were addressed by studying subcellular changes in protein phosphorylation, cAMP-dependent protein kinase activity and protein phosphatase activity in frog hearts. B-adrenergic agonists increased and muscarinic cholinergic agonists decreased (/sup 32/P)phosphate incorporation into C-protein, a thick filament component. Regulation of protein phosphatase activity by Iso and methacholine (MCh) was assayed using extracts of drug treated frog hearts and (/sup 32/P)phospho-C-protein as substrate. Total phosphatase activity decreased 21% in extracts from hearts perfused with 0.1 ..mu..M Iso and 17% in hearts exposed to Iso plus 1 ..mu..M methacholine. This decrease reflected decreased phosphatase-2A activity. No changes in total phosphatase activity were measurable in broken cells treated with Iso or MCh. The results suggest adrenergic stimulation changes contractile activity in frog hearts by activating cAMP-dependent protein kinase associated with particulate cellular elements and inactivating soluble protein phosphatase-2A. This is the first demonstration of coordinated regulation of these enzymes by B-adrenergic agonists favoring phosphorylation of effector proteins. Coordinated regulation by methacholine in the presence of Iso was not observed.

  2. Induction of activation-induced cytidine deaminase-targeting adaptor 14-3-3γ is mediated by NF-κB-dependent recruitment of CFP1 to the 5'-CpG-3'-rich 14-3-3γ promoter and is sustained by E2A.

    PubMed

    Mai, Thach; Pone, Egest J; Li, Guideng; Lam, Tonika S; Moehlman, J'aime; Xu, Zhenming; Casali, Paolo

    2013-08-15

    Class switch DNA recombination (CSR) crucially diversifies Ab biologic effector functions. 14-3-3γ specifically binds to the 5'-AGCT-3' repeats in the IgH locus switch (S) regions. By interacting directly with the C-terminal region of activation-induced cytidine deaminase (AID), 14-3-3γ targets this enzyme to S regions to mediate CSR. In this study, we showed that 14-3-3γ was expressed in germinal center B cells in vivo and induced in B cells by T-dependent and T-independent primary CSR-inducing stimuli in vitro in humans and mice. Induction of 14-3-3γ was rapid, peaking within 3 h of stimulation by LPSs, and sustained over the course of AID and CSR induction. It was dependent on recruitment of NF-κB to the 14-3-3γ gene promoter. The NF-κB recruitment enhanced the occupancy of the CpG island within the 14-3-3γ promoter by CFP1, a component of the COMPASS histone methyltransferase complex, and promoter-specific enrichment of histone 3 lysine 4 trimethylation (H3K4me3), which is indicative of open chromatin state and marks transcription-competent promoters. NF-κB also potentiated the binding of B cell lineage-specific factor E2A to an E-box motif located immediately downstream of the two closely-spaced transcription start sites for sustained 14-3-3γ expression and CSR induction. Thus, 14-3-3γ induction in CSR is enabled by the CFP1-mediated H3K4me3 enrichment in the promoter, dependent on NF-κB and sustained by E2A. PMID:23851690

  3. Amifostine alleviates radiation-induced lethal small bowel damage via promotion of 14-3-3σ-mediated nuclear p53 accumulation.

    PubMed

    Huang, Eng-Yen; Wang, Feng-Sheng; Chen, Yu-Min; Chen, Yi-Fan; Wang, Chung-Chi; Lin, I-Hui; Huang, Yu-Jie; Yang, Kuender D

    2014-10-30

    Amifostine (AM) is a radioprotector that scavenges free radicals and is used in patients undergoing radiotherapy. p53 has long been implicated in cell cycle arrest for cellular repair after radiation exposure. We therefore investigated the protective p53-dependent mechanism of AM on small bowel damage after lethal whole-abdominal irradiation (WAI). AM increased both the survival rate of rats and crypt survival following lethal 18 Gy WAI. The p53 inhibitor PFT-α compromised AM-mediated effects when administered prior to AM administration. AM significantly increased clonogenic survival in IEC-6 cells expressing wild type p53 but not in p53 knockdown cells. AM significantly increased p53 nuclear accumulation and p53 tetramer expression before irradiation through the inhibition of p53 degradation. AM inhibited p53 interactions with MDM2 but enhanced p53 interactions with 14-3-3σ. Knockdown of 14-3-3σ also compromised the effect of AM on clonogenic survival and p53 nuclear accumulation in IEC-6 cells. For the first time, our data reveal that AM alleviates lethal small bowel damage through the induction of 14-3-3σ and subsequent accumulation of p53. Enhancement of the p53/14-3-3σ interaction results in p53 tetramerization in the nucleus that rescues lethal small bowel damage. PMID:25230151

  4. Protein Kinase C-mediated Phosphorylation and Activation of PDE3A Regulate cAMP Levels in Human Platelets*S⃞

    PubMed Central

    Hunter, Roger W.; MacKintosh, Carol; Hers, Ingeborg

    2009-01-01

    The elevation of [cAMP]i is an important mechanism of platelet inhibition and is regulated by the opposing activity of adenylyl cyclase and phosphodiesterase (PDE). In this study, we demonstrate that a variety of platelet agonists, including thrombin, significantly enhance the activity of PDE3A in a phosphorylation-dependent manner. Stimulation of platelets with the PAR-1 agonist SFLLRN resulted in rapid and transient phosphorylation of PDE3A on Ser312, Ser428, Ser438, Ser465, and Ser492, in parallel with the PKC (protein kinase C) substrate, pleckstrin. Furthermore, phosphorylation and activation of PDE3A required the activation of PKC, but not of PI3K/PKB, mTOR/p70S6K, or ERK/RSK. Activation of PKC by phorbol esters also resulted in phosphorylation of the same PDE3A sites in a PKC-dependent, PKB-independent manner. This was further supported by the finding that IGF-1, which strongly activates PI3K/PKB, but not PKC, did not regulate PDE3A. Platelet activation also led to a PKC-dependent association between PDE3A and 14-3-3 proteins. In contrast, cAMP-elevating agents such as PGE1 and forskolin-induced phosphorylation of Ser312 and increased PDE3A activity, but did not stimulate 14-3-3 binding. Finally, complete antagonism of PGE1-evoked cAMP accumulation by thrombin required both Gi and PKC activation. Together, these results demonstrate that platelet activation stimulates PKC-dependent phosphorylation of PDE3A on Ser312, Ser428, Ser438, Ser465, and Ser492 leading to a subsequent increase in cAMP hydrolysis and 14-3-3 binding. PMID:19261611

  5. [RGS proteins (regulators of G protein signaling) and their roles in regulation of immune response].

    PubMed

    Lewandowicz, Anna M; Kowalski, Marek L; Pawliczak, Rafał

    2004-01-01

    RGS proteins (Regulators of G-protein Signaling) comprise a protein family responsible for regulating G proteins. By enhancing the GTPase activity of the a subunit, they speed up the reconstruction of the heterotrimeric structure of G protein, thus inhibiting its signal transduction. Sst2 protein in yeast Saccharomyces cervisiae, FlbA in fungus Aspergillus nidulans, and Egl-10 in the nematode Caenorhabditis elegans are the first native G regulators with GTPase activity (GAPs:--GTPase-activating proteins). The existence of over 30 RGS human proteins has been confirmed thus far, and they have been grouped and classified into six subfamilies. In immunocompetent cells, RGS proteins are entangled in a complicate net of different interrelating signal pathways. They are connected with B- and T-cell chemokine susceptibility, efficient T cell proliferation, and the regulation of B cell maturation. They also take an essential part in inflammation. High hopes are held for drugs, which handle would be RGS proteins and which would further provide the possibility of modifying the pharmacokinetics of drugs acting through G protein- coupled receptors. The aim of this review is to discuss the new RGS protein family and explain the potential involvement of RGS proteins in the modulation of the immune response PMID:15459549

  6. EH domain proteins regulate cardiac membrane protein targeting

    PubMed Central

    Gudmundsson, Hjalti; Hund, Thomas J.; Wright, Patrick J.; Kline, Crystal F.; Snyder, Jedidiah S.; Qian, Lan; Koval, Olha M.; Cunha, Shane R.; George, Manju; Rainey, Mark A.; Kashef, Farshid E.; Dun, Wen; Boyden, Penelope A.; Anderson, Mark E.; Band, Hamid; Mohler, Peter J.

    2010-01-01

    Rationale Cardiac membrane excitability is tightly regulated by an integrated network of membrane-associated ion channels, transporters, receptors, and signaling molecules. Membrane protein dynamics in health and disease are maintained by a complex ensemble of intracellular targeting, scaffolding, recycling, and degradation pathways. Surprisingly, despite decades of research linking dysfunction in membrane protein trafficking with human cardiovascular disease, essentially nothing is known regarding the molecular identity or function of these intracellular targeting pathways in excitable cardiomyocytes. Objective We sought to discover novel pathways for membrane protein targeting in primary cardiomyocytes. Methods and Results We report the initial characterization of a large family of membrane trafficking proteins in human heart. We employed a tissue-wide screen for novel ankyrin-associated trafficking proteins and identified four members of a unique Eps15 homology (EH) domain-containing protein family (EHD1, EHD2, EHD3, EHD4) that serve critical roles in endosome-based membrane protein targeting in other cell types. We show that EHD1-4 directly associate with ankyrin, provide the first information on the expression and localization of these molecules in primary cardiomyocytes, and demonstrate that EHD1-4 are co-expressed with ankyrin-B in the myocyte perinuclear region. Notably, the expression of multiple EHD proteins is increased in animal models lacking ankyrin-B, and EHD3-deficient cardiomyocytes display aberrant ankyrin-B localization and selective loss of Na/Ca exchanger expression and function. Finally, we report significant modulation of EHD expression following myocardial infarction, suggesting that these proteins may play a key role in regulating membrane excitability in normal and diseased heart. Conclusions Our findings identify and characterize a new class of cardiac trafficking proteins, define the first group of proteins associated with the ankyrin

  7. Molecular Characterization of the 14-3-3 Gene Family in Brachypodium distachyon L. Reveals High Evolutionary Conservation and Diverse Responses to Abiotic Stresses

    PubMed Central

    Cao, Hui; Xu, Yuxing; Yuan, Linlin; Bian, Yanwei; Wang, Lihui; Zhen, Shoumin; Hu, Yingkao; Yan, Yueming

    2016-01-01

    The 14-3-3 gene family identified in all eukaryotic organisms is involved in a wide range of biological processes, particularly in resistance to various abiotic stresses. Here, we performed the first comprehensive study on the molecular characterization, phylogenetics, and responses to various abiotic stresses of the 14-3-3 gene family in Brachypodium distachyon L. A total of seven 14-3-3 genes from B. distachyon and 120 from five main lineages among 12 species were identified, which were divided into five well-conserved subfamilies. The molecular structure analysis showed that the plant 14-3-3 gene family is highly evolutionarily conserved, although certain divergence had occurred in different subfamilies. The duplication event investigation revealed that segmental duplication seemed to be the predominant form by which the 14-3-3 gene family had expanded. Moreover, seven critical amino acids were detected, which may contribute to functional divergence. Expression profiling analysis showed that BdGF14 genes were abundantly expressed in the roots, but showed low expression in the meristems. All seven BdGF14 genes showed significant expression changes under various abiotic stresses, including heavy metal, phytohormone, osmotic, and temperature stresses, which might play important roles in responses to multiple abiotic stresses mainly through participating in ABA-dependent signaling and reactive oxygen species-mediated MAPK cascade signaling pathways. In particular, BdGF14 genes generally showed upregulated expression in response to multiple stresses of high temperature, heavy metal, abscisic acid (ABA), and salicylic acid (SA), but downregulated expression under H2O2, NaCl, and polyethylene glycol (PEG) stresses. Meanwhile, dynamic transcriptional expression analysis of BdGF14 genes under longer treatments with heavy metals (Cd2+, Cr3+, Cu2+, and Zn2+) and phytohormone (ABA) and recovery revealed two main expression trends in both roots and leaves: up-down and up

  8. Regulation of Axonal Transport by Protein Kinases.

    PubMed

    Gibbs, Katherine L; Greensmith, Linda; Schiavo, Giampietro

    2015-10-01

    The intracellular transport of organelles, proteins, lipids, and RNA along the axon is essential for neuronal function and survival. This process, called axonal transport, is mediated by two classes of ATP-dependent motors, kinesins, and cytoplasmic dynein, which carry their cargoes along microtubule tracks. Protein kinases regulate axonal transport through direct phosphorylation of motors, adapter proteins, and cargoes, and indirectly through modification of the microtubule network. The misregulation of axonal transport by protein kinases has been implicated in the pathogenesis of several nervous system disorders. Here, we review the role of protein kinases acting directly on axonal transport and discuss how their deregulation affects neuronal function, paving the way for the exploitation of these enzymes as novel drug targets. PMID:26410600

  9. Redox regulation of protein damage in plasma.

    PubMed

    Griffiths, Helen R; Dias, Irundika H K; Willetts, Rachel S; Devitt, Andrew

    2014-01-01

    The presence and concentrations of modified proteins circulating in plasma depend on rates of protein synthesis, modification and clearance. In early studies, the proteins most frequently analysed for damage were those which were more abundant in plasma (e.g. albumin and immunoglobulins) which exist at up to 10 orders of magnitude higher concentrations than other plasma proteins e.g. cytokines. However, advances in analytical techniques using mass spectrometry and immuno-affinity purification methods, have facilitated analysis of less abundant, modified proteins and the nature of modifications at specific sites is now being characterised. The damaging reactive species that cause protein modifications in plasma principally arise from reactive oxygen species (ROS) produced by NADPH oxidases (NOX), nitric oxide synthases (NOS) and oxygenase activities; reactive nitrogen species (RNS) from myeloperoxidase (MPO) and NOS activities; and hypochlorous acid from MPO. Secondary damage to proteins may be caused by oxidized lipids and glucose autooxidation. In this review, we focus on redox regulatory control of those enzymes and processes which control protein maturation during synthesis, produce reactive species, repair and remove damaged plasma proteins. We have highlighted the potential for alterations in the extracellular redox compartment to regulate intracellular redox state and, conversely, for intracellular oxidative stress to alter the cellular secretome and composition of extracellular vesicles. Through secreted, redox-active regulatory molecules, changes in redox state may be transmitted to distant sites. PMID:24624332

  10. Regulation of Mutant p53 Protein Expression

    PubMed Central

    Vijayakumaran, Reshma; Tan, Kah Hin; Miranda, Panimaya Jeffreena; Haupt, Sue; Haupt, Ygal

    2015-01-01

    For several decades, p53 has been detected in cancer biopsies by virtue of its high protein expression level which is considered indicative of mutation. Surprisingly, however, mouse genetic studies revealed that mutant p53 is inherently labile, similar to its wild type (wt) counterpart. Consistently, in response to stress conditions, both wt and mutant p53 accumulate in cells. While wt p53 returns to basal level following recovery from stress, mutant p53 remains stable. In part, this can be explained in mutant p53-expressing cells by the lack of an auto-regulatory loop with Mdm2 and other negative regulators, which are pivotal for wt p53 regulation. Further, additional protective mechanisms are acquired by mutant p53, largely mediated by the co-chaperones and their paralogs, the stress-induced heat shock proteins. Consequently, mutant p53 is accumulated in cancer cells in response to chronic stress and this accumulation is critical for its oncogenic gain of functions (GOF). Building on the extensive knowledge regarding wt p53, the regulation of mutant p53 is unraveling. In this review, we describe the current understanding on the major levels at which mutant p53 is regulated. These include the regulation of p53 protein levels by microRNA and by enzymes controlling p53 proteasomal degradation. PMID:26734569

  11. SUMOylation regulates the SNF1 protein kinase

    PubMed Central

    Simpson-Lavy, Kobi J.; Johnston, Mark

    2013-01-01

    The AMP-activated protein kinase (AMPK) is a major stress sensor of mammalian cells. AMPK’s homolog in the yeast Saccharomyces cerevisiae, the SNF1 protein kinase, is a central regulator of carbon metabolism that inhibits the Snf3/Rgt2-Rgt1 glucose sensing pathway and activates genes involved in respiration. We present evidence that glucose induces modification of the Snf1 catalytic subunt of SNF1 with the small ubiquitin-like modifier protein SUMO, catalyzed by the SUMO (E3) ligase Mms21. Our results suggest that SUMOylation of Snf1 inhibits its function in two ways: by interaction of SUMO attached to lysine 549 with a SUMO-interacting sequence motif located near the active site of Snf1, and by targeting Snf1 for destruction via the Slx5-Slx8 (SUMO-directed) ubiquitin ligase. These findings reveal another way SNF1 function is regulated in response to carbon source. PMID:24108357

  12. 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.

  13. Dynamic regulation of lipid-protein interactions.

    PubMed

    Martfeld, Ashley N; Rajagopalan, Venkatesan; Greathouse, Denise V; Koeppe, Roger E

    2015-09-01

    We review the importance of helix motions for the function of several important categories of membrane proteins and for the properties of several model molecular systems. For voltage-gated potassium or sodium channels, sliding, tilting and/or rotational movements of the S4 helix accompanied by a swapping of cognate side-chain ion-pair interactions regulate the channel gating. In the seven-helix G protein-coupled receptors, exemplified by the rhodopsins, collective helix motions serve to activate the functional signaling. Peptides which initially associate with lipid-bilayer membrane surfaces may undergo dynamic transitions from surface-bound to tilted-transmembrane orientations, sometimes accompanied by changes in the molecularity, formation of a pore or, more generally, the activation of biological function. For single-span membrane proteins, such as the tyrosine kinases, an interplay between juxtamembrane and transmembrane domains is likely to be crucial for the regulation of dimer assembly that in turn is associated with the functional responses to external signals. Additionally, we note that experiments with designed single-span transmembrane helices offer fundamental insights into the molecular features that govern protein-lipid interactions. This article is part of a Special Issue entitled: Lipid-protein interactions. PMID:25666872

  14. Regulation of Pluripotency by RNA Binding Proteins

    PubMed Central

    Ye, Julia; Blelloch, Robert

    2015-01-01

    Establishment, maintenance, and exit from pluripotency require precise coordination of a cell’s molecular machinery. Substantial headway has been made in deciphering many aspects of this elaborate system, particularly with respect to epigenetics, transcription, and noncoding RNAs. Less attention has been paid to posttranscriptional regulatory processes such as alternative splicing, RNA processing and modification, nuclear export, regulation of transcript stability, and translation. Here, we introduce the RNA binding proteins that enable the posttranscriptional regulation of gene expression, summarizing current and ongoing research on their roles at different regulatory points and discussing how they help script the fate of pluripotent stem cells. PMID:25192462

  15. Regulation of gene transcription by Polycomb proteins

    PubMed Central

    Aranda, Sergi; Mas, Gloria; Di Croce, Luciano

    2015-01-01

    The Polycomb group (PcG) of proteins defines a subset of factors that physically associate and function to maintain the positional identity of cells from the embryo to adult stages. PcG has long been considered a paradigmatic model for epigenetic maintenance of gene transcription programs. Despite intensive research efforts to unveil the molecular mechanisms of action of PcG proteins, several fundamental questions remain unresolved: How many different PcG complexes exist in mammalian cells? How are PcG complexes targeted to specific loci? How does PcG regulate transcription? In this review, we discuss the diversity of PcG complexes in mammalian cells, examine newly identified modes of recruitment to chromatin, and highlight the latest insights into the molecular mechanisms underlying the function of PcGs in transcription regulation and three-dimensional chromatin conformation. PMID:26665172

  16. 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.

  17. Intramembrane proteolysis in regulated protein trafficking.

    PubMed

    Lemberg, Marius K

    2011-09-01

    Regulated intramembrane proteolysis is an evolutionarily conserved mechanism by which membrane-anchored bioactive molecules are released from cellular membranes. In eukaryotic cells, intramembrane proteases are found in different cellular organelles ranging from the endosomal system to mitochondria and chloroplasts. These proteases function in diverse processes such as transcription control, regulated growth factor secretion and recently even a role in the control of mitophagy has been suggested. Genomic annotation has predicted 13 different intramembrane proteases in humans. Apart from few studied examples, very little is known about their function. This review describes emerging principles of how intramembrane proteases contribute to the regulation of cellular protein trafficking in eukaryotic cells and raises the important question of how their activity is controlled. PMID:21585636

  18. 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

  19. 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

  20. Quantitative Proteomics of Caveolin-1-regulated Proteins

    PubMed Central

    Dávalos, Alberto; Fernández-Hernando, Carlos; Sowa, Grzegorz; Derakhshan, Behrad; Lin, Michelle I.; Lee, Ji Y.; Zhao, Hongyu; Luo, Ruiyan; Colangelo, Christopher; Sessa, William C.

    2010-01-01

    Caveolae are organelles abundant in the plasma membrane of many specialized cells including endothelial cells (ECs), epithelial cells, and adipocytes, and in these cells, caveolin-1 (Cav-1) is the major coat protein essential for the formation of caveolae. To identify proteins that require Cav-1 for stable incorporation into membrane raft domains, a quantitative proteomics analysis using isobaric tagging for relative and absolute quantification was performed on rafts isolated from wild-type and Cav-1-deficient mice. In three independent experiments, 117 proteins were consistently identified in membrane rafts with the largest differences in the levels of Cav-2 and in the caveola regulatory proteins Cavin-1 and Cavin-2. Because the lung is highly enriched in ECs, we validated and characterized the role of the newly described protein Cavin-1 in several cardiovascular tissues and in ECs. Cavin-1 was highly expressed in ECs lining blood vessels and in cultured ECs. Knockdown of Cavin-1 reduced the levels of Cav-1 and -2 and weakly influenced the formation of high molecular weight oligomers containing Cav-1 and -2. Cavin-1 silencing enhanced basal nitric oxide release from ECs but blocked proangiogenic phenotypes such as EC proliferation, migration, and morphogenesis in vitro. Thus, these data support an important role of Cavin-1 as a regulator of caveola function in ECs. PMID:20585024

  1. The FAD-dependent glycerol-3-phosphate dehydrogenase of Giardia duodenalis: an unconventional enzyme that interacts with the g14-3-3 and it is a target of the antitumoral compound NBDHEX

    PubMed Central

    Lalle, Marco; Camerini, Serena; Cecchetti, Serena; Finelli, Renata; Sferra, Gabriella; Müller, Joachim; Ricci, Giorgio; Pozio, Edoardo

    2015-01-01

    The flagellated protozoan Giardia duodenalis is a worldwide parasite causing giardiasis, an acute and chronic diarrheal disease. Metabolism in G. duodenalis has a limited complexity thus making metabolic enzymes ideal targets for drug development. However, only few metabolic pathways (i.e., carbohydrates) have been described so far. Recently, the parasite homolog of the mitochondrial-like glycerol-3-phosphate dehydrogenase (gG3PD) has been identified among the interactors of the g14-3-3 protein. G3PD is involved in glycolysis, electron transport, glycerophospholipids metabolism, and hyperosmotic stress response, and is emerging as promising target in tumor treatment. In this work, we demonstrate that gG3PD is a functional flavoenzyme able to convert glycerol-3-phosphate into dihydroxyacetone phosphate and that its activity and the intracellular glycerol level increase during encystation. Taking advantage of co-immunoprecipitation assays and deletion mutants, we provide evidence that gG3PD and g14-3-3 interact at the trophozoite stage, the intracellular localization of gG3PD is stage dependent and it partially co-localizes with mitosomes during cyst development. Finally, we demonstrate that the gG3PD activity is affected by the antitumoral compound 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol, that results more effective in vitro at killing G. duodenalis trophozoites than the reference drug metronidazole. Overall, our results highlight the involvement of gG3PD in processes crucial for the parasite survival thus proposing this enzyme as target for novel antigiardial interventions. PMID:26082764

  2. The usefulness of S100P, mesothelin, fascin, prostate stem cell antigen, and 14-3-3 sigma in diagnosing pancreatic adenocarcinoma in cytological specimens obtained by endoscopic ultrasound guided fine-needle aspiration.

    PubMed

    Dim, Daniel C; Jiang, Feng; Qiu, Qi; Li, Ting; Darwin, Peter; Rodgers, William H; Peng, Hong Qi

    2014-03-01

    Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) of the pancreas is an efficient and minimally invasive procedure for the diagnosis and staging of pancreatic adenocarcinoma. Because of some limitations of EUS-FNA in diagnosis of well-differentiated or early stage cancers, the purpose of this study is to assess the added benefit of immunohistochemistry. We studied five proteins overexpressed in pancreatic adenocarcinoma, namely, prostate stem cell antigen, fascin, 14-3-3 sigma, mesothelin and S100P utilizing immunohistochemistry on paraffin sections from cellblocks obtained by EUS-FNA. Sixty-two cases of EUS-FNA of the pancreas that had follow-up histological and/or clinical diagnosis and sufficient material in cell blocks were included. Using histological diagnosis and/or clinical outcome as the reference standard, EUS-FNA shows the highest sensitivity (95%) and specificity (91%) and is superior to any marker in this study. Among five antibodies, S100P reveals the best diagnostic characters showing 90% of sensitivity and 67% of specificity. Fascin shows high specificity (92%) but low sensitivity (38%). Mesothelin has a moderate sensitivity (74%) and low specificity (33%), PSCA and 14-3-3 show high sensitivity but zero specificity. S100P and mesothelin were useful in nine indeterminate cases. S100P correctly predicted six of seven cancers and one of one without cancer and mesothelin correctly diagnosed five of seven cancers and one of two noncancers in this group. EUS-FNA cytomorphology is superior to any of the immunohistochemical markers used in this study. Use of S100P and mesothelin in cytologically borderline cases can increase the diagnostic accuracy in this group. PMID:21538952

  3. Organization and Regulation of Mitochondrial Protein Synthesis.

    PubMed

    Ott, Martin; Amunts, Alexey; Brown, Alan

    2016-06-01

    Mitochondria are essential organelles of endosymbiotic origin that are responsible for oxidative phosphorylation within eukaryotic cells. Independent evolution between species has generated mitochondrial genomes that are extremely diverse, with the composition of the vestigial genome determining their translational requirements. Typically, translation within mitochondria is restricted to a few key subunits of the oxidative phosphorylation complexes that are synthesized by dedicated ribosomes (mitoribosomes). The dramatically rearranged mitochondrial genomes, the limited set of transcripts, and the need for the synthesized proteins to coassemble with nuclear-encoded subunits have had substantial consequences for the translation machinery. Recent high-resolution cryo-electron microscopy has revealed the effect of coevolution on the mitoribosome with the mitochondrial genome. In this review, we place the new structural information in the context of the molecular mechanisms of mitochondrial translation and focus on the novel ways protein synthesis is organized and regulated in mitochondria. PMID:26789594

  4. The actin binding protein adseverin regulates osteoclastogenesis.

    PubMed

    Hassanpour, Siavash; Jiang, Hongwei; Wang, Yongqiang; Kuiper, Johannes W P; Glogauer, Michael

    2014-01-01

    Adseverin (Ads), a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin) strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesis (OCG). Ads is induced during OCG downstream of RANK-ligand (RANKL) stimulation and is highly expressed in mature osteoclasts. The D5 isoform of Ads is not involved in regulating OCG, as its expression is not induced in response to RANKL. Three clonal Ads knockdown RAW264.7 (RAW) macrophage cell lines with varying degrees of Ads expression and OCG deficiency were generated. The most drastic OCG defect was noted in the clonal cell line with the greatest degree of Ads knockdown as indicated by a lack of TRAcP staining and multinucleation. RNAi mediated knockdown of Ads in osteoclast precursors resulted in distinct morphological changes characterized by altered F-actin distribution and increased filopodia formation. Ads knockdown precursor cells experienced enhanced migration while fusion of knockdown precursors cells was limited. Transient reintroduction of de novo Ads back into the knockdown system was capable of rescuing TRAcP expression but not osteoclast multinucleation most likely due to the transient nature of Ads expression. This preliminary study allows us to conclude that Ads is a RANKL induced early regulator of OCG with a potential role in pre-osteoclast differentiation and fusion. PMID:25275604

  5. The Actin Binding Protein Adseverin Regulates Osteoclastogenesis

    PubMed Central

    Wang, Yongqiang; Kuiper, Johannes W. P.; Glogauer, Michael

    2014-01-01

    Adseverin (Ads), a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin) strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesis (OCG). Ads is induced during OCG downstream of RANK-ligand (RANKL) stimulation and is highly expressed in mature osteoclasts. The D5 isoform of Ads is not involved in regulating OCG, as its expression is not induced in response to RANKL. Three clonal Ads knockdown RAW264.7 (RAW) macrophage cell lines with varying degrees of Ads expression and OCG deficiency were generated. The most drastic OCG defect was noted in the clonal cell line with the greatest degree of Ads knockdown as indicated by a lack of TRAcP staining and multinucleation. RNAi mediated knockdown of Ads in osteoclast precursors resulted in distinct morphological changes characterized by altered F-actin distribution and increased filopodia formation. Ads knockdown precursor cells experienced enhanced migration while fusion of knockdown precursors cells was limited. Transient reintroduction of de novo Ads back into the knockdown system was capable of rescuing TRAcP expression but not osteoclast multinucleation most likely due to the transient nature of Ads expression. This preliminary study allows us to conclude that Ads is a RANKL induced early regulator of OCG with a potential role in pre-osteoclast differentiation and fusion. PMID:25275604

  6. Exercise regulation of intestinal tight junction proteins.

    PubMed

    Zuhl, Micah; Schneider, Suzanne; Lanphere, Katherine; Conn, Carole; Dokladny, Karol; Moseley, Pope

    2014-06-01

    Gastrointestinal distress, such as diarrhoea, cramping, vomiting, nausea and gastric pain are common among athletes during training and competition. The mechanisms that cause these symptoms are not fully understood. The stress of heat and oxidative damage during exercise causes disruption to intestinal epithelial cell tight junction proteins resulting in increased permeability to luminal endotoxins. The endotoxin moves into the blood stream leading to a systemic immune response. Tight junction integrity is altered by the phosphoylation state of the proteins occludin and claudins, and may be regulated by the type of exercise performed. Prolonged exercise and high-intensity exercise lead to an increase in key phosphorylation enzymes that ultimately cause tight junction dysfunction, but the mechanisms are different. The purpose of this review is to (1) explain the function and physiology of tight junction regulation, (2) discuss the effects of prolonged and high-intensity exercise on tight junction permeability leading to gastrointestinal distress and (3) review agents that may increase or decrease tight junction integrity during exercise. PMID:23134759

  7. A FRET-based study reveals site-specific regulation of spindle position checkpoint proteins at yeast centrosomes.

    PubMed

    Gryaznova, Yuliya; Koca Caydasi, Ayse; Malengo, Gabriele; Sourjik, Victor; Pereira, Gislene

    2016-01-01

    The spindle position checkpoint (SPOC) is a spindle pole body (SPB, equivalent of mammalian centrosome) associated surveillance mechanism that halts mitotic exit upon spindle mis-orientation. Here, we monitored the interaction between SPB proteins and the SPOC component Bfa1 by FRET microscopy. We show that Bfa1 binds to the scaffold-protein Nud1 and the γ-tubulin receptor Spc72. Spindle misalignment specifically disrupts Bfa1-Spc72 interaction by a mechanism that requires the 14-3-3-family protein Bmh1 and the MARK/PAR-kinase Kin4. Dissociation of Bfa1 from Spc72 prevents the inhibitory phosphorylation of Bfa1 by the polo-like kinase Cdc5. We propose Spc72 as a regulatory hub that coordinates the activity of Kin4 and Cdc5 towards Bfa1. In addition, analysis of spc72∆ cells shows that a mitotic-exit-promoting dominant signal, which is triggered upon elongation of the spindle into the bud, overrides the SPOC. Our data reinforce the importance of daughter-cell-associated factors and centrosome-based regulations in mitotic exit and SPOC control. PMID:27159239

  8. A FRET-based study reveals site-specific regulation of spindle position checkpoint proteins at yeast centrosomes

    PubMed Central

    Gryaznova, Yuliya; Caydasi, Ayse Koca; Malengo, Gabriele; Sourjik, Victor; Pereira, Gislene

    2016-01-01

    The spindle position checkpoint (SPOC) is a spindle pole body (SPB, equivalent of mammalian centrosome) associated surveillance mechanism that halts mitotic exit upon spindle mis-orientation. Here, we monitored the interaction between SPB proteins and the SPOC component Bfa1 by FRET microscopy. We show that Bfa1 binds to the scaffold-protein Nud1 and the γ-tubulin receptor Spc72. Spindle misalignment specifically disrupts Bfa1-Spc72 interaction by a mechanism that requires the 14-3-3-family protein Bmh1 and the MARK/PAR-kinase Kin4. Dissociation of Bfa1 from Spc72 prevents the inhibitory phosphorylation of Bfa1 by the polo-like kinase Cdc5. We propose Spc72 as a regulatory hub that coordinates the activity of Kin4 and Cdc5 towards Bfa1. In addition, analysis of spc72∆ cells shows that a mitotic-exit-promoting dominant signal, which is triggered upon elongation of the spindle into the bud, overrides the SPOC. Our data reinforce the importance of daughter-cell-associated factors and centrosome-based regulations in mitotic exit and SPOC control. DOI: http://dx.doi.org/10.7554/eLife.14029.001 PMID:27159239

  9. Ion transport proteins anchor and regulate the cytoskeleton.

    PubMed

    Denker, Sheryl P; Barber, Diane L

    2002-04-01

    Structurally diverse ion transport proteins anchor the spectrin-actin cytoskeleton to the plasma membrane by binding directly to linker proteins of the ankyrin and protein 4.1 families. Cytoskeletal anchoring regulates cell shape and restricts the activity of ion transport proteins to specialised membrane domains. New directions are being forged by recent findings that localised anchoring by ion transport proteins regulates the ordered assembly of actin filaments and the actin-dependent processes of cell adhesion and motility. PMID:11891121

  10. The insulator protein CTCF regulates Drosophila steroidogenesis

    PubMed Central

    Fresán, Ujué; Cuartero, Sergi; O'Connor, Michael B.; Espinàs, M. Lluisa

    2015-01-01

    ABSTRACT The steroid hormone ecdysone is a central regulator of insect development. In this report we show that CTCF expression in the prothoracic gland is required for full transcriptional activation of the Halloween genes spookier, shadow and noppera-bo, which encode ecdysone biosynthetic enzymes, and for proper timing of ecdysone-responsive gene expression. Loss of CTCF results in delayed and less synchronized larval development that can only be rescued by feeding larvae with both, the steroid hormone 20-hydroxyecdysone and cholesterol. Moreover, CTCF-knockdown in prothoracic gland cells leads to increased lipid accumulation. In conclusion, the insulator protein CTCF is required for Halloween gene expression and cholesterol homeostasis in ecdysone-producing cells controlling steroidogenesis. PMID:25979705

  11. Regulation of osteogenic proteins by chondrocytes.

    PubMed

    Chubinskaya, Susan; Kuettner, Klaus E

    2003-09-01

    The purpose of this review is to summarize the current scientific knowledge of bone morphogenetic proteins (BMPs) in adult articular cartilage. We specifically focus on adult cartilage, since one of the major potential applications of the members of the BMP family may be a repair of adult tissue after trauma and/or disease. After reviewing cartilage physiology and BMPs, we analyze the data on the role of recombinant BMPs as anabolic agents in tissue formation and restoration in different in vitro and in vivo models following with the endogenous expression of BMPs and factors that regulate their expression. We also discuss recent transgenic modifications of BMP genes and subsequent effect on cartilage matrix synthesis. We found that the most studied BMPs in adult articular cartilage are BMP-7 and BMP-2 as well as transforming growth factor-beta (TGF-beta). There are a number of contradicting reports for some of these growth factors, since different models, animals, doses, time points, culture conditions and devices were used. However, regardless of the experimental conditions, only BMP-7 or osteogenic protein-1 (OP-1) exhibits the most convincing effects. It is the only BMP studied thus far in adult cartilage that demonstrates strong anabolic activity in vitro and in vivo with and without serum. OP-1 stimulates the synthesis of the majority of cartilage extracellular matrix proteins in adult articular chondrocytes derived from different species and of different age. OP-1 counteracts the degenerative effect of numerous catabolic mediators; it is also expressed in adult human, bovine, rabbit and goat articular cartilage. This review reveals the importance of the exploration of the BMPs in the cartilage field and highlights their significance for clinical applications in the treatment of cartilage-related diseases. PMID:12798347

  12. An Overview of Chromatin-Regulating Proteins in Cells

    PubMed Central

    Zhang, Pingyu; Torres, Keila; Liu, Xiuping; Liu, Chang-gong; Pollock, Raphael E.

    2016-01-01

    In eukaryotic cells, gene expressions on chromosome DNA are orchestrated by a dynamic chromosome structure state that is largely controlled by chromatin-regulating proteins, which regulate chromatin structures, release DNA from the nucleosome, and activate or suppress gene expression by modifying nucleosome histones or mobilizing DNA-histone structure. The two classes of chromatin- regulating proteins are 1) enzymes that modify histones through methylation, acetylation, phosphorylation, adenosine diphosphate–ribosylation, glycosylation, sumoylation, or ubiquitylation and 2) enzymes that remodel DNA-histone structure with energy from ATP hydrolysis. Chromatin-regulating proteins, which modulate DNA-histone interaction, change chromatin conformation, and increase or decrease the binding of functional DNA-regulating protein complexes, have major functions in nuclear processes, including gene transcription and DNA replication, repair, and recombination. This review provides a general overview of chromatin-regulating proteins, including their classification, molecular functions, and interactions with the nucleosome in eukaryotic cells. PMID:26796306

  13. TRIM-NHL proteins take on miRNA regulation.

    PubMed

    Loedige, Inga; Filipowicz, Witold

    2009-03-01

    The TRIM-NHL family of proteins is conserved among metazoans and has been shown to regulate cell proliferation and development. In this issue, Hammell et al. (2009) and Schwamborn et al. (2009) identify two members of this protein family, NHL-2 in worms and TRIM32 in mice, as positive regulators of microRNA function. PMID:19269362

  14. Bcl-2 family proteins: master regulators of cell survival.

    PubMed

    Hatok, Jozef; Racay, Peter

    2016-08-01

    The most prominent function of proteins of the Bcl-2 family is regulation of the initiation of intrinsic (mitochondrial) pathways of apoptosis. However, recent research has revealed that in addition to regulation of mitochondrial apoptosis, proteins of the Bcl-2 family play important roles in regulating other cellular pathways with a strong impact on cell survival like autophagy, endoplasmic reticulum (ER) stress response, intracellular calcium dynamics, cell cycle progression, mitochondrial dynamics and energy metabolism. This review summarizes the recent knowledge about functions of Bcl-2 family proteins that are related to cell survival. PMID:27505095

  15. Functional phosphoproteomic analysis reveals cold-shock domain protein A to be a Bcr-Abl effector-regulating proliferation and transformation in chronic myeloid leukemia

    PubMed Central

    Sears, D; Luong, P; Yuan, M; Nteliopoulos, G; Man, Y K S; Melo, J V; Basu, S

    2010-01-01

    One proposed strategy to suppress the proliferation of imatinib-resistant cells in chronic myeloid leukemia (CML) is to inhibit key proteins downstream of Bcr-Abl. The PI3K/Akt pathway is activated by Bcr-Abl and is specifically required for the growth of CML cells. To identify targets of this pathway, we undertook a proteomic screen and identified several proteins that differentially bind 14-3-3, dependent on Bcr-Abl kinase activity. An siRNA screen of candidates selected by bioinformatics analysis reveals cold-shock domain protein A (CSDA), shown previously to regulate cell cycle progression in epithelial cells, to be a positive regulator of proliferation in a CML cell line. We show that Akt can phosphorylate the serine 134 residue of CSDA but, downstream of Bcr-Abl activity, this modification is mediated through the activation of MEK/p90 ribosomal S6 kinase (RSK) signaling. Inhibition of RSK, similarly to treatment with imatinib, blocked proliferation specifically in Bcr-Abl-positive leukemia cell lines, as well as cells from CML patients. Furthermore, these primary CML cells showed an increase in CSDA phosphorylation. Expression of a CSDA phospho-deficient mutant resulted in the decrease of Bcr-Abl-dependent transformation in Rat1 cells. Our results support a model whereby phosphorylation of CSDA downstream of Bcr-Abl enhances proliferation in CML cells to drive leukemogenesis. PMID:21368869

  16. MTBreg: The Database of Conditionally Regulated Proteins in Mycobacterium Tuberculosis

    DOE Data Explorer

    Kaufman, Markus; Pal, Debnath; Eisenberg, David

    Proteins up- and down- regulated in Mycobacterium tuberculosis grown under conditions mimicking infection are included in this database. It also includes information on proteins that are regulated by selected transcription factors or other regulatory proteins. The literature data provided here is complimentary to the databases provided by Michael Strong that include recent TB computational functional linkages and the Prolinks Database by Peter Bowers. The experimental condition, the experimental dataset and a literature reference will be displayed, including links to the computationally linked proteins in the Prolinks Database and the entry in the Mycobacterium tuberculosis Structural Genomics Database.[Copied from information at http://www.doe-mbi.ucla.edu/Services/MTBreg/

  17. Copper Delivery to Chloroplast Proteins and its Regulation

    PubMed Central

    Aguirre, Guadalupe; Pilon, Marinus

    2016-01-01

    Copper is required for photosynthesis in chloroplasts of plants because it is a cofactor of plastocyanin, an essential electron carrier in the thylakoid lumen. Other chloroplast copper proteins are copper/zinc superoxide dismutase and polyphenol oxidase, but these proteins seem to be dispensable under conditions of low copper supply when transcripts for these proteins undergo microRNA-mediated down regulation. Two ATP-driven copper transporters function in tandem to deliver copper to chloroplast compartments. This review seeks to summarize the mechanisms of copper delivery to chloroplast proteins and its regulation. We also delineate some of the unanswered questions that still remain in this field. PMID:26793223

  18. ASIC proteins regulate smooth muscle cell migration.

    PubMed

    Grifoni, Samira C; Jernigan, Nikki L; Hamilton, Gina; Drummond, Heather A

    2008-03-01

    The purpose of the present study was to investigate Acid Sensing Ion Channel (ASIC) protein expression and importance in cellular migration. We recently demonstrated that Epithelial Na(+)Channel (ENaC) proteins are required for vascular smooth muscle cell (VSMC) migration; however, the role of the closely related ASIC proteins has not been addressed. We used RT-PCR and immunolabeling to determine expression of ASIC1, ASIC2, ASIC3 and ASIC4 in A10 cells. We used small interference RNA to silence individual ASIC expression and determine the importance of ASIC proteins in wound healing and chemotaxis (PDGF-bb)-initiated migration. We found ASIC1, ASIC2, and ASIC3, but not ASIC4, expression in A10 cells. ASIC1, ASIC2, and ASIC3 siRNA molecules significantly suppressed expression of their respective proteins compared to non-targeting siRNA (RISC) transfected controls by 63%, 44%, and 55%, respectively. Wound healing was inhibited by 10, 20, and 26% compared to RISC controls following suppression of ASIC1, ASIC2, and ASIC3, respectively. Chemotactic migration was inhibited by 30% and 45%, respectively, following suppression of ASIC1 and ASIC3. ASIC2 suppression produced a small, but significant, increase in chemotactic migration (4%). Our data indicate that ASIC expression is required for normal migration and may suggest a novel role for ASIC proteins in cellular migration. PMID:17936312

  19. Regulation of mitochondrial protein import by cytosolic kinases.

    PubMed

    Schmidt, Oliver; Harbauer, Angelika B; Rao, Sanjana; Eyrich, Beate; Zahedi, René P; Stojanovski, Diana; Schönfisch, Birgit; Guiard, Bernard; Sickmann, Albert; Pfanner, Nikolaus; Meisinger, Chris

    2011-01-21

    Mitochondria import a large number of nuclear-encoded proteins via membrane-bound transport machineries; however, little is known about regulation of the preprotein translocases. We report that the main protein entry gate of mitochondria, the translocase of the outer membrane (TOM complex), is phosphorylated by cytosolic kinases-in particular, casein kinase 2 (CK2) and protein kinase A (PKA). CK2 promotes biogenesis of the TOM complex by phosphorylation of two key components, the receptor Tom22 and the import protein Mim1, which in turn are required for import of further Tom proteins. Inactivation of CK2 decreases the levels of the TOM complex and thus mitochondrial protein import. PKA phosphorylates Tom70 under nonrespiring conditions, thereby inhibiting its receptor activity and the import of mitochondrial metabolite carriers. We conclude that cytosolic kinases exert stimulatory and inhibitory effects on biogenesis and function of the TOM complex and thus regulate protein import into mitochondria. PMID:21215441

  20. Multilevel regulation of protein protein interactions in biological circuitry

    NASA Astrophysics Data System (ADS)

    Beckett, Dorothy

    2005-06-01

    Protein-protein interactions are central to biology and, in this 'post-genomic era', prediction of these interactions has become the goal of many computational efforts. Close inspection of even relatively simple biological regulatory circuitry reveals multiple levels of control of the contributing protein interactions. The fundamental probability that an interaction will occur under a given set of conditions is difficult to predict because the relationship between structure and energy is not known. Layered on this basic difficulty are allosteric control mechanisms involving post-translational modification or small ligand binding. In addition, many biological processes involve multiple protein-protein interactions, some of which may be cooperative or even competitive. Finally, although the emphasis in predicting protein interactions is based on equilibrium thermodynamic principles, kinetics can be a major controlling feature in these systems. This complexity reinforces the necessity of performing detailed quantitative studies of the component interactions of complex biological regulatory systems. Results of such studies will help us to bridge the gap between our knowledge of structure and our understanding of functional biology.

  1. Tor1 regulates protein solubility in Saccharomyces cerevisiae

    PubMed Central

    Peters, Theodore W.; Rardin, Matthew J.; Czerwieniec, Gregg; Evani, Uday S.; Reis-Rodrigues, Pedro; Lithgow, Gordon J.; Mooney, Sean D.; Gibson, Bradford W.; Hughes, Robert E.

    2012-01-01

    Accumulation of insoluble protein in cells is associated with aging and aging-related diseases; however, the roles of insoluble protein in these processes are uncertain. The nature and impact of changes to protein solubility during normal aging are less well understood. Using quantitative mass spectrometry, we identify 480 proteins that become insoluble during postmitotic aging in Saccharomyces cerevisiae and show that this ensemble of insoluble proteins is similar to those that accumulate in aging nematodes. SDS-insoluble protein is present exclusively in a nonquiescent subpopulation of postmitotic cells, indicating an asymmetrical distribution of this protein. In addition, we show that nitrogen starvation of young cells is sufficient to cause accumulation of a similar group of insoluble proteins. Although many of the insoluble proteins identified are known to be autophagic substrates, induction of macroautophagy is not required for insoluble protein formation. However, genetic or chemical inhibition of the Tor1 kinase is sufficient to promote accumulation of insoluble protein. We conclude that target of rapamycin complex 1 regulates accumulation of insoluble proteins via mechanisms acting upstream of macroautophagy. Our data indicate that the accumulation of proteins in an SDS-insoluble state in postmitotic cells represents a novel autophagic cargo preparation process that is regulated by the Tor1 kinase. PMID:23097491

  2. Coevolution of RAC Small GTPases and their Regulators GEF Proteins

    PubMed Central

    Jiménez-Sánchez, Alejandro

    2016-01-01

    RAC proteins are small GTPases involved in important cellular processes in eukaryotes, and their deregulation may contribute to cancer. Activation of RAC proteins is regulated by DOCK and DBL protein families of guanine nucleotide exchange factors (GEFs). Although DOCK and DBL proteins act as GEFs on RAC proteins, DOCK and DBL family members are evolutionarily unrelated. To understand how DBL and DOCK families perform the same function on RAC proteins despite their unrelated primary structure, phylogenetic analyses of the RAC, DBL, and DOCK families were implemented, and interaction patterns that may suggest a coevolutionary process were searched. Interestingly, while RAC and DOCK proteins are very well conserved in humans and among eukaryotes, DBL proteins are highly divergent. Moreover, correlation analyses of the phylogenetic distances of RAC and GEF proteins and covariation analyses between residues in the interacting domains showed significant coevolution rates for both RAC–DOCK and RAC–DBL interactions. PMID:27226705

  3. Minimalist Design of Allosterically Regulated Protein Catalysts.

    PubMed

    Makhlynets, O V; Korendovych, I V

    2016-01-01

    Nature facilitates chemical transformations with exceptional selectivity and efficiency. Despite a tremendous progress in understanding and predicting protein function, the overall problem of designing a protein catalyst for a given chemical transformation is far from solved. Over the years, many design techniques with various degrees of complexity and rational input have been developed. Minimalist approach to protein design that focuses on the bare minimum requirements to achieve activity presents several important advantages. By focusing on basic physicochemical properties and strategic placing of only few highly active residues one can feasibly evaluate in silico a very large variety of possible catalysts. In more general terms minimalist approach looks for the mere possibility of catalysis, rather than trying to identify the most active catalyst possible. Even very basic designs that utilize a single residue introduced into nonenzymatic proteins or peptide bundles are surprisingly active. Because of the inherent simplicity of the minimalist approach computational tools greatly enhance its efficiency. No complex calculations need to be set up and even a beginner can master this technique in a very short time. Here, we present a step-by-step protocol for minimalist design of functional proteins using basic, easily available, and free computational tools. PMID:27586334

  4. Protein import into plant mitochondria: signals, machinery, processing, and regulation.

    PubMed

    Murcha, Monika W; Kmiec, Beata; Kubiszewski-Jakubiak, Szymon; Teixeira, Pedro F; Glaser, Elzbieta; Whelan, James

    2014-12-01

    The majority of more than 1000 proteins present in mitochondria are imported from nuclear-encoded, cytosolically synthesized precursor proteins. This impressive feat of transport and sorting is achieved by the combined action of targeting signals on mitochondrial proteins and the mitochondrial protein import apparatus. The mitochondrial protein import apparatus is composed of a number of multi-subunit protein complexes that recognize, translocate, and assemble mitochondrial proteins into functional complexes. While the core subunits involved in mitochondrial protein import are well conserved across wide phylogenetic gaps, the accessory subunits of these complexes differ in identity and/or function when plants are compared with Saccharomyces cerevisiae (yeast), the model system for mitochondrial protein import. These differences include distinct protein import receptors in plants, different mechanistic operation of the intermembrane protein import system, the location and activity of peptidases, the function of inner-membrane translocases in linking the outer and inner membrane, and the association/regulation of mitochondrial protein import complexes with components of the respiratory chain. Additionally, plant mitochondria share proteins with plastids, i.e. dual-targeted proteins. Also, the developmental and cell-specific nature of mitochondrial biogenesis is an aspect not observed in single-celled systems that is readily apparent in studies in plants. This means that plants provide a valuable model system to study the various regulatory processes associated with protein import and mitochondrial biogenesis. PMID:25324401

  5. Proteomic analysis of differentially expressed proteins in Penaeus monodon hemocytes after Vibrio harveyi infection

    PubMed Central

    2010-01-01

    Background Viral and bacterial diseases can cause mass mortalities in commercial shrimp aquaculture. In contrast to studies on the antiviral response, the responses of shrimps to bacterial infections by high throughput techniques have been reported only at the transcriptional level and not at the translational level. In this study, a proteomic analysis of shrimp hemocytes to identify differentially expressed proteins in response to a luminous bacterium Vibrio harveyi was evaluated for its feasibility and is reported for the first time. Results The two-dimensional gel electrophoresis (2-DE) patterns of the hemocyte proteins from the unchallenged and V. harveyi challenged shrimp, Penaeus monodon, at 24 and 48 h post infection were compared. From this, 27 differentially expressed protein spots, and a further 12 weakly to non-differentially regulated control spots, were selected for further analyses by the LC-ESI-MS/MS. The 21 differentially expressed proteins that could be identified by homologous annotation were comprised of proteins that are directly involved in the host defense responses, such as hemocyanin, prophenoloxidase, serine proteinase-like protein, heat shock protein 90 and alpha-2-macroglobulin, and those involved in signal transduction, such as the14-3-3 protein epsilon and calmodulin. Western blot analysis confirmed the up-regulation of hemocyanin expression upon bacterial infection. The expression of the selected proteins which were the representatives of the down-regulated proteins (the 14-3-3 protein epsilon and alpha-2-macroglobulin) and of the up-regulated proteins (hemocyanin) was further assessed at the transcription level using real-time RT-PCR. Conclusions This work suggests the usefulness of a proteomic approach to the study of shrimp immunity and revealed hemocyte proteins whose expression were up regulated upon V. harveyi infection such as hemocyanin, arginine kinase and down regulated such as alpha-2-macroglobulin, calmodulin and 14-3-3

  6. Claudins, dietary milk proteins, and intestinal barrier regulation.

    PubMed

    Kotler, Belinda M; Kerstetter, Jane E; Insogna, Karl L

    2013-01-01

    The family of claudin proteins plays an important role in regulating the intestinal barrier by modulating the permeability of tight junctions. The impact of dietary protein on claudin biology has not been studied extensively. Whey proteins have been reported to improve intestinal barrier function, but their mechanism of action is not clear. Recent studies, however, have demonstrated increased intestinal claudin expression in response to milk protein components. Reviewed here are new findings suggesting that whey-protein-derived transforming growth factor β transcriptionally upregulates claudin-4 expression via a Smad-4-dependent pathway. These and other data, including limited clinical studies, are summarized below and, in the aggregate, suggest a therapeutic role for whey protein in diseases of intestinal barrier dysfunction, perhaps, in part, by regulating claudin expression. PMID:23282252

  7. Regulation of the gibberellin pathway by auxin and DELLA proteins.

    PubMed

    O'Neill, Damian P; Davidson, Sandra E; Clarke, Victoria C; Yamauchi, Yukika; Yamaguchi, Shinjiro; Kamiya, Yuji; Reid, James B; Ross, John J

    2010-10-01

    The synthesis and deactivation of bioactive gibberellins (GA) are regulated by auxin and by GA signalling. The effect of GA on its own pathway is mediated by DELLA proteins. Like auxin, the DELLAs promote GA synthesis and inhibit its deactivation. Here, we investigate the relationships between auxin and DELLA regulation of the GA pathway in stems, using a pea double mutant that is deficient in DELLA proteins. In general terms our results demonstrate that auxin and DELLAs independently regulate the GA pathway, contrary to some previous suggestions. The extent to which DELLA regulation was able to counteract the effects of auxin regulation varied from gene to gene. For Mendel's LE gene (PsGA3ox1) no counteraction was observed. However, for another synthesis gene, a GA 20-oxidase, the effect of auxin was weak and in WT plants appeared to be completely over-ridden by DELLA regulation. For a key GA deactivation (2-oxidase) gene, PsGA2ox1, the up-regulation induced by auxin deficiency was reduced to some extent by DELLA regulation. A second pea 2-oxidase gene, PsGA2ox2, was up-regulated by auxin, in a DELLA-independent manner. In Arabidopsis also, one 2-oxidase gene was down-regulated by auxin while another was up-regulated. Monitoring the metabolism pattern of GA(20) showed that in Arabidopsis, as in pea, auxin can promote the accumulation of bioactive GA. PMID:20706734

  8. Piezo Proteins: Regulators of Mechanosensation and Other Cellular Processes*

    PubMed Central

    Bagriantsev, Sviatoslav N.; Gracheva, Elena O.; Gallagher, Patrick G.

    2014-01-01

    Piezo proteins have recently been identified as ion channels mediating mechanosensory transduction in mammalian cells. Characterization of these channels has yielded important insights into mechanisms of somatosensation, as well as other mechano-associated biologic processes such as sensing of shear stress, particularly in the vasculature, and regulation of urine flow and bladder distention. Other roles for Piezo proteins have emerged, some unexpected, including participation in cellular development, volume regulation, cellular migration, proliferation, and elongation. Mutations in human Piezo proteins have been associated with a variety of disorders including hereditary xerocytosis and several syndromes with muscular contracture as a prominent feature. PMID:25305018

  9. The regulation and turnover of mitochondrial uncoupling proteins

    PubMed Central

    Azzu, Vian; Jastroch, Martin; Divakaruni, Ajit S; Brand, Martin D

    2010-01-01

    Uncoupling proteins (UCP1, UCP2 and UCP3) are important in regulating cellular fuel metabolism and as attenuators of reactive oxygen species production, through strong or mild uncoupling. The generic function and broad tissue distribution of the uncoupling protein family means that they are increasingly implicated in a range of pathophysiological processes including obesity, insulin resistance and diabetes mellitus, neurodegeneration, cardiovascular disease, immunity and cancer. The significant recent progress describing the turnover of novel uncoupling proteins, as well as current views on the physiological roles and regulation of UCPs, is outlined. PMID:20211596

  10. Regulation of Genome Architecture and Function by Polycomb Proteins.

    PubMed

    Entrevan, Marianne; Schuettengruber, Bernd; Cavalli, Giacomo

    2016-07-01

    Polycomb group (PcG) proteins dynamically define cellular identities through the epigenetic repression of key developmental regulatory genes. PcG proteins are recruited to specific regulatory elements to modify the chromatin surrounding them. In addition, they regulate the organization of their target genes in the 3D space of the nucleus, and this regulatory function of the 3D genome architecture is involved in cell differentiation and the maintenance of cellular memory. In this review we discuss recent advances in our understanding of how PcG proteins are recruited to chromatin to induce local and global changes in chromosome conformation and regulate their target genes. PMID:27198635

  11. Rab proteins: The key regulators of intracellular vesicle transport

    SciTech Connect

    Bhuin, Tanmay; Roy, Jagat Kumar

    2014-10-15

    Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes. - Highlights: • Rab proteins regulate different signalling pathways. • Deregulation of Rabs is the fundamental causes of a variety of human diseases. • This paper gives potential directions in developing therapeutic targets. • This paper also gives ample directions for modulating pathways central to normal physiology. • These are the huge challenges for drug discovery and delivery in near future.

  12. mir-29 regulates Mcl-1 protein expression and apoptosis.

    PubMed

    Mott, J L; Kobayashi, S; Bronk, S F; Gores, G J

    2007-09-13

    Cellular expression of Mcl-1, an anti-apoptotic Bcl-2 family member, is tightly regulated. Recently, Bcl-2 expression was shown to be regulated by microRNAs, small endogenous RNA molecules that regulate protein expression through sequence-specific interaction with messenger RNA. By analogy, we reasoned that Mcl-1 expression may also be regulated by microRNAs. We chose human immortalized, but non-malignant, H69 cholangiocyte and malignant KMCH cholangiocarcinoma cell lines for these studies, because Mcl-1 is dysregulated in cells with the malignant phenotype. By in silico analysis, we identified a putative target site in the Mcl-1 mRNA for the mir-29 family, and found that mir-29b was highly expressed in cholangiocytes. Interestingly, mir-29b was downregulated in malignant cells, consistent with Mcl-1 protein upregulation. Enforced mir-29b expression reduced Mcl-1 protein expression in KMCH cells. This effect was direct, as mir-29b negatively regulated the expression of an Mcl-1 3' untranslated region (UTR)-based reporter construct. Enforced mir-29b expression reduced Mcl-1 cellular protein levels and sensitized the cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity. Transfection of non-malignant cells (that express high levels of mir-29) with a locked-nucleic acid antagonist of mir-29b increased Mcl-1 levels and reduced TRAIL-mediated apoptosis. Thus mir-29 is an endogenous regulator of Mcl-1 protein expression, and thereby, apoptosis. PMID:17404574

  13. mir-29 Regulates Mcl-1 Protein Expression and Apoptosis

    PubMed Central

    Mott, Justin L.; Kobayashi, Shogo; Bronk, Steven F.; Gores, Gregory J.

    2008-01-01

    Cellular expression of Mcl-1, an anti-apoptotic Bcl-2 family member, is tightly regulated. Recently, Bcl-2 expression was shown to be regulated by microRNAs, small endogenous RNA molecules that regulate protein expression through sequence-specific interaction with messenger RNA. By analogy, we reasoned that Mcl-1 expression may also be regulated by microRNAs. We chose human immortalized, but non-malignant, H69 cholangiocyte and malignant KMCH cholangiocarcinoma cell lines for these studies because Mcl-1 is dysregulated in cells with the malignant phenotype. In silico analysis identified a putative target site in the Mcl-1 mRNA for the mir-29 family, and we found that mir-29b was highly expressed in cholangiocytes. Interestingly, mir-29b was downregulated in malignant cells, consistent with Mcl-1 protein upregulation. Enforced mir-29b expression reduced Mcl-1 protein expression in KMCH cells. This effect was direct, as mir-29b negatively regulated expression of an Mcl-1 3’ untranslated region (UTR)-based reporter construct. Enforced mir-29b expression reduced Mcl-1 cellular protein levels and sensitized the cancer cells to TRAIL cytotoxicity. Transfection of non-malignant cells (that express high levels of mir-29) with a locked-nucleic acid antagonist of mir-29b increased Mcl-1 levels and reduced TRAIL-mediated apoptosis. Thus mir-29 is an endogenous regulator of Mcl-1 protein expression and, thereby, apoptosis. PMID:17404574

  14. Combinatorial H3K9acS10ph histone modification in IgH locus S regions targets 14-3-3 adaptors and AID to specify antibody class-switch DNA recombination.

    PubMed

    Li, Guideng; White, Clayton A; Lam, Tonika; Pone, Egest J; Tran, Daniel C; Hayama, Ken L; Zan, Hong; Xu, Zhenming; Casali, Paolo

    2013-11-14

    Class-switch DNA recombination (CSR) is central to the antibody response, in that it changes the immunoglobulin heavy chain (IgH) constant region, thereby diversifying biological effector functions of antibodies. The activation-induced cytidine deaminase (AID)-centered CSR machinery excises and rejoins DNA between an upstream (donor) and a downstream (acceptor) S region, which precede the respective constant region DNA. AID is stabilized on S regions by 14-3-3 adaptors. These adaptors display a high affinity for 5'-AGCT-3' repeats, which recur in all S regions. However, how 14-3-3, AID, and the CSR machinery target exclusively the donor and acceptor S regions is poorly understood. Here, we show that histone methyltransferases and acetyltransferases are induced by CD40 or Toll-like receptor signaling and catalyze H3K4me3 and H3K9ac/K14ac histone modifications, which are enriched in S regions but do not specify the S region targets of CSR. By contrast, the combinatorial H3K9acS10ph modification specifically marks the S regions set to recombine and directly recruits 14-3-3 adaptors for AID stabilization there. Inhibition of the enzymatic activity of GCN5 and PCAF histone acetyltransferases reduces H3K9acS10ph in S regions, 14-3-3 and AID stabilization, and CSR. Thus, H3K9acS10ph is a histone code that is "written" specifically in S regions and is "read" by 14-3-3 adaptors to target AID for CSR as an important biological outcome. PMID:24209747

  15. Light-Inducible Gene Regulation with Engineered Zinc Finger Proteins

    PubMed Central

    Polstein, Lauren R.; Gersbach, Charles A.

    2014-01-01

    The coupling of light-inducible protein-protein interactions with gene regulation systems has enabled the control of gene expression with light. In particular, heterodimer protein pairs from plants can be used to engineer a gene regulation system in mammalian cells that is reversible, repeatable, tunable, controllable in a spatiotemporal manner, and targetable to any DNA sequence. This system, Light-Inducible Transcription using Engineered Zinc finger proteins (LITEZ), is based on the blue light-induced interaction of GIGANTEA and the LOV domain of FKF1 that drives the localization of a transcriptional activator to the DNA-binding site of a highly customizable engineered zinc finger protein. This chapter provides methods for modifying LITEZ to target new DNA sequences, engineering a programmable LED array to illuminate cell cultures, and using the modified LITEZ system to achieve spatiotemporal control of transgene expression in mammalian cells. PMID:24718797

  16. Antemortem stress regulates protein acetylation and glycolysis in postmortem muscle.

    PubMed

    Li, Zhongwen; Li, Xin; Wang, Zhenyu; Shen, Qingwu W; Zhang, Dequan

    2016-07-01

    Although exhaustive research has established that preslaughter stress is a major factor contributing to pale, soft, exudative (PSE) meat, questions remain regarding the biochemistry of postmortem glycolysis. In this study, the influence of preslaughter stress on protein acetylation in relationship to glycolysis was studied. The data show that antemortem swimming significantly enhanced glycolysis and the total acetylated proteins in postmortem longissimus dorsi (LD) muscle of mice. Inhibition of protein acetylation by histone acetyltransferase (HAT) inhibitors eliminated stress induced increase in glycolysis. Inversely, antemortem injection of histone deacetylase (HDAC) inhibitors, trichostatin A (TSA) and nicotinamide (NAM), further increased protein acetylation early postmortem and the glycolysis. These data provide new insight into the biochemistry of postmortem glycolysis by showing that protein acetylation regulates glycolysis, which may participate in the regulation of preslaughter stress on glycolysis in postmortem muscle. PMID:26920270

  17. PRDM Proteins: Molecular Mechanisms in Signal Transduction and Transcriptional Regulation.

    PubMed

    Di Zazzo, Erika; De Rosa, Caterina; Abbondanza, Ciro; Moncharmont, Bruno

    2013-01-01

    PRDM (PRDI-BF1 and RIZ homology domain containing) protein family members are characterized by the presence of a PR domain and a variable number of Zn-finger repeats. Experimental evidence has shown that the PRDM proteins play an important role in gene expression regulation, modifying the chromatin structure either directly, through the intrinsic methyltransferase activity, or indirectly through the recruitment of chromatin remodeling complexes. PRDM proteins have a dual action: they mediate the effect induced by different cell signals like steroid hormones and control the expression of growth factors. PRDM proteins therefore have a pivotal role in the transduction of signals that control cell proliferation and differentiation and consequently neoplastic transformation. In this review, we describe pathways in which PRDM proteins are involved and the molecular mechanism of their transcriptional regulation. PMID:24832654

  18. PRDM Proteins: Molecular Mechanisms in Signal Transduction and Transcriptional Regulation

    PubMed Central

    Di Zazzo, Erika; De Rosa, Caterina; Abbondanza, Ciro; Moncharmont, Bruno

    2013-01-01

    PRDM (PRDI-BF1 and RIZ homology domain containing) protein family members are characterized by the presence of a PR domain and a variable number of Zn-finger repeats. Experimental evidence has shown that the PRDM proteins play an important role in gene expression regulation, modifying the chromatin structure either directly, through the intrinsic methyltransferase activity, or indirectly through the recruitment of chromatin remodeling complexes. PRDM proteins have a dual action: they mediate the effect induced by different cell signals like steroid hormones and control the expression of growth factors. PRDM proteins therefore have a pivotal role in the transduction of signals that control cell proliferation and differentiation and consequently neoplastic transformation. In this review, we describe pathways in which PRDM proteins are involved and the molecular mechanism of their transcriptional regulation. PMID:24832654

  19. Regulation of cilia assembly, disassembly, and length by protein phosphorylation.

    PubMed

    Cao, Muqing; Li, Guihua; Pan, Junmin

    2009-01-01

    The exact mechanism by which cells are able to assemble, regulate, and disassemble cilia or flagella is not yet completely understood. Recent studies in several model systems, including Chlamydomonas, Tetrahymena, Leishmania, Caenorhabditis elegans, and mammals, provide increasing biochemical and genetic evidence that phosphorylation of multiple protein kinases plays a key role in cilia assembly, disassembly, and length regulation. Members of several protein kinase families--including aurora kinases, never in mitosis A (NIMA)-related protein kinases, mitogen-activated protein (MAP) kinases, and a novel cyclin-dependent protein kinase--are involved in the ciliary regulation process. Among the newly identified protein kinase substrates are Chlamydomonas kinesin-13 (CrKinesin13), a microtubule depolymerizer, and histone deacetylase 6 (HDAC6), a microtubule deacetylase. Chlamydomonas aurora/Ipl1p-like protein kinase (CALK) and CrKinesin13 are two proteins that undergo phosphorylation changes correlated with flagellar assembly or disassembly. CALK becomes phosphorylated when flagella are lost, whereas CrKinesin13 is phosphorylated when new flagella are assembled. Conversely, suppressing CrKinesin13 expression results in cells with shorter flagella. PMID:20362099

  20. A Fungal Family of Transcriptional Regulators: the Zinc Cluster Proteins

    PubMed Central

    MacPherson, Sarah; Larochelle, Marc; Turcotte, Bernard

    2006-01-01

    The trace element zinc is required for proper functioning of a large number of proteins, including various enzymes. However, most zinc-containing proteins are transcription factors capable of binding DNA and are named zinc finger proteins. They form one of the largest families of transcriptional regulators and are categorized into various classes according to zinc-binding motifs. This review focuses on one class of zinc finger proteins called zinc cluster (or binuclear) proteins. Members of this family are exclusively fungal and possess the well-conserved motif CysX2CysX6CysX5-12CysX2CysX6-8Cys. The cysteine residues bind to two zinc atoms, which coordinate folding of the domain involved in DNA recognition. The first- and best-studied zinc cluster protein is Gal4p, a transcriptional activator of genes involved in the catabolism of galactose in the budding yeast Saccharomyces cerevisiae. Since the discovery of Gal4p, many other zinc cluster proteins have been characterized; they function in a wide range of processes, including primary and secondary metabolism and meiosis. Other roles include regulation of genes involved in the stress response as well as pleiotropic drug resistance, as demonstrated in budding yeast and in human fungal pathogens. With the number of characterized zinc cluster proteins growing rapidly, it is becoming more and more apparent that they are important regulators of fungal physiology. PMID:16959962

  1. Sch9 regulates intracellular protein ubiquitination by controlling stress responses.

    PubMed

    Qie, Beibei; Lyu, Zhou; Lyu, Lei; Liu, Jun; Gao, Xuejie; Liu, Yanyan; Duan, Wei; Zhang, Nianhui; Du, Linfang; Liu, Ke

    2015-08-01

    Protein ubiquitination and the subsequent degradation are important means by which aberrant proteins are removed from cells, a key requirement for long-term survival. In this study, we found that the overall level of ubiquitinated proteins dramatically decreased as yeast cell grew from log to stationary phase. Deletion of SCH9, a gene encoding a key protein kinase for longevity control, decreased the level of ubiquitinated proteins in log phase and this effect could be reversed by restoring Sch9 function. We demonstrate here that the decrease of ubiquitinated proteins in sch9Δ cells in log phase is not caused by changes in ubiquitin expression, proteasome activity, or autophagy, but by enhanced expression of stress response factors and a decreased level of oxidative stress. Our results revealed for the first time how Sch9 regulates the level of ubiquitinated proteins and provides new insight into how Sch9 controls longevity. PMID:26087116

  2. Sch9 regulates intracellular protein ubiquitination by controlling stress responses

    PubMed Central

    Qie, Beibei; Lyu, Zhou; Lyu, Lei; Liu, Jun; Gao, Xuejie; Liu, Yanyan; Duan, Wei; Zhang, Nianhui; Du, Linfang; Liu, Ke

    2015-01-01

    Protein ubiquitination and the subsequent degradation are important means by which aberrant proteins are removed from cells, a key requirement for long-term survival. In this study, we found that the overall level of ubiquitinated proteins dramatically decreased as yeast cell grew from log to stationary phase. Deletion of SCH9, a gene encoding a key protein kinase for longevity control, decreased the level of ubiquitinated proteins in log phase and this effect could be reversed by restoring Sch9 function. We demonstrate here that the decrease of ubiquitinated proteins in sch9Δ cells in log phase is not caused by changes in ubiquitin expression, proteasome activity, or autophagy, but by enhanced expression of stress response factors and a decreased level of oxidative stress. Our results revealed for the first time how Sch9 regulates the level of ubiquitinated proteins and provides new insight into how Sch9 controls longevity. PMID:26087116

  3. Regulation of bacterial RecA protein function.

    PubMed

    Cox, Michael M

    2007-01-01

    The RecA protein is a recombinase functioning in recombinational DNA repair in bacteria. RecA is regulated at many levels. The expression of the recA gene is regulated within the SOS response. The activity of the RecA protein itself is autoregulated by its own C-terminus. RecA is also regulated by the action of other proteins. To date, these include the RecF, RecO, RecR, DinI, RecX, RdgC, PsiB, and UvrD proteins. The SSB protein also indirectly affects RecA function by competing for ssDNA binding sites. The RecO and RecR, and possibly the RecF proteins, all facilitate RecA loading onto SSB-coated ssDNA. The RecX protein blocks RecA filament extension, and may have other effects on RecA activity. The DinI protein stabilizes RecA filaments. The RdgC protein binds to dsDNA and blocks RecA access to dsDNA. The PsiB protein, encoded by F plasmids, is uncharacterized, but may inhibit RecA in some manner. The UvrD helicase removes RecA filaments from RecA. All of these proteins function in a network that determines where and how RecA functions. Additional regulatory proteins may remain to be discovered. The elaborate regulatory pattern is likely to be reprised for RecA homologues in archaeans and eukaryotes. PMID:17364684

  4. PDZ Protein Regulation of G Protein-Coupled Receptor Trafficking and Signaling Pathways.

    PubMed

    Dunn, Henry A; Ferguson, Stephen S G

    2015-10-01

    G protein-coupled receptors (GPCRs) contribute to the regulation of every aspect of human physiology and are therapeutic targets for the treatment of numerous diseases. As a consequence, understanding the myriad of mechanisms controlling GPCR signaling and trafficking is essential for the development of new pharmacological strategies for the treatment of human pathologies. Of the many GPCR-interacting proteins, postsynaptic density protein of 95 kilodaltons, disc large, zona occludens-1 (PDZ) domain-containing proteins appear most abundant and have similarly been implicated in disease mechanisms. PDZ proteins play an important role in regulating receptor and channel protein localization within synapses and tight junctions and function to scaffold intracellular signaling protein complexes. In the current study, we review the known functional interactions between PDZ domain-containing proteins and GPCRs and provide insight into the potential mechanisms of action. These PDZ domain-containing proteins include the membrane-associated guanylate-like kinases [postsynaptic density protein of 95 kilodaltons; synapse-associated protein of 97 kilodaltons; postsynaptic density protein of 93 kilodaltons; synapse-associated protein of 102 kilodaltons; discs, large homolog 5; caspase activation and recruitment domain and membrane-associated guanylate-like kinase domain-containing protein 3; membrane protein, palmitoylated 3; calcium/calmodulin-dependent serine protein kinase; membrane-associated guanylate kinase protein (MAGI)-1, MAGI-2, and MAGI-3], Na(+)/H(+) exchanger regulatory factor proteins (NHERFs) (NHERF1, NHERF2, PDZ domain-containing kidney protein 1, and PDZ domain-containing kidney protein 2), Golgi-associated PDZ proteins (Gα-binding protein interacting protein, C-terminus and CFTR-associated ligand), PDZ domain-containing guanine nucleotide exchange factors (GEFs) 1 and 2, regulator of G protein signaling (RGS)-homology-RhoGEFs (PDZ domain-containing RhoGEF and

  5. Wheat seed proteins regulated by imbibition independent of dormancy status.

    PubMed

    Park, Seokhoon; Rampitsch, Christof; Humphreys, Gavin D; Ayele, Belay T

    2013-01-01

    Seed dormancy is an important trait in wheat (Trticum aestivum L.) and it can be released by germination-stimulating treatments such as after-ripening. Previously, we identified proteins specifically associated with after-ripening mediated developmental switches of wheat seeds from the state of dormancy to germination. Here, we report seed proteins that exhibited imbibition induced co-regulation in both dormant and after-ripened seeds of wheat, suggesting that the expression of these specific proteins/protein isoforms is not associated with the maintenance or release of seed dormancy in wheat. PMID:24084602

  6. Introns regulate the production of ribosomal proteins by modulating splicing of duplicated ribosomal protein genes.

    PubMed

    Petibon, Cyrielle; Parenteau, Julie; Catala, Mathieu; Elela, Sherif Abou

    2016-05-01

    Most budding yeast introns exist in the many duplicated ribosomal protein genes (RPGs) and it has been posited that they remain there to modulate the expression of RPGs and cell growth in response to stress. However, the mechanism by which introns regulate the expression of RPGs and their impact on the synthesis of ribosomal proteins remain unclear. In this study, we show that introns determine the ratio of ribosomal protein isoforms through asymmetric paralog-specific regulation of splicing. Exchanging the introns and 3' untranslated regions of the duplicated RPS9 genes altered the splicing efficiency and changed the ratio of the ribosomal protein isoforms. Mutational analysis of the RPS9 genes indicated that splicing is regulated by variations in the intron structure and the 3' untranslated region. Together these data suggest that preferential splicing of duplicated RPGs provides a means for adjusting the ratio of different ribosomal protein isoforms, while maintaining the overall expression level of each ribosomal protein. PMID:26945043

  7. Role of Regulators of G Protein Signaling Proteins in Bone Physiology and Pathophysiology

    PubMed Central

    Jules, Joel; Yang, Shuying; Chen, Wei; Li, Yi-Ping

    2016-01-01

    Regulators of G protein signaling (RGS) proteins enhance the intrinsic GTPase activity of α subunits of the heterotrimeric G protein complex of G protein-coupled receptors (GPCRs) and thereby inactivate signal transduction initiated by GPCRs. The RGS family consists of nearly 37 members with a conserved RGS homology domain which is critical for their GTPase accelerating activity. RGS proteins are expressed in most tissues, including heart, lung, brain, kidney, and bone and play essential roles in many physiological and pathological processes. In skeletal development and bone homeostasis as well as in many bone disorders, RGS proteins control the functions of various GPCRs, including the parathyroid hormone receptor type 1 and calcium-sensing receptor and also regulate various critical signaling pathways, such as Wnt and calcium oscillations. This chapter will discuss the current findings on the roles of RGS proteins in regulating signaling of key GPCRs in skeletal development and bone homeostasis. We also will examine the current updates of RGS proteins’ regulation of calcium oscillations in bone physiology and highlight the roles of RGS proteins in selected bone pathological disorders. Despite the recent advances in bone and mineral research, RGS proteins remain understudied in the skeletal system. Further understanding of the roles of RGS proteins in bone should not only provide great insights into the molecular basis of various bone diseases but also generate great therapeutic drug targets for many bone diseases. PMID:26123302

  8. DPF2 regulates OCT4 protein level and nuclear distribution.

    PubMed

    Liu, Chao; Zhang, Dijuan; Shen, Yuxian; Tao, Xiaofang; Liu, Lihua; Zhong, Yongwang; Fang, Shengyun

    2015-12-01

    The amount of transcription factor OCT4 is strictly regulated. A tight regulation of OCT4 levels is crucial for mammalian embryonic development and oncogenesis. However, the mechanisms underlying regulation of OCT4 protein expression and nuclear distribution are largely unknown. Here, we report that DPF2, a plant homeodomain (PHD) finger protein, is upregulated during H9 cell differentiation induced by retinoic acid. Endogenous interaction between DPF2 and OCT4 in P19 cells was revealed by an immunoprecipitation assay. GST-pull down assay proved that OCT4 protein in H9 cells and recombinant OCT4 can precipitate with DPF2 in vitro. In vitro ubiquitination assay demonstrated DPF2 might serve as an E3 ligase. Knock down of dpf2 using siRNA increased OCT4 protein level and stability in P19 cells. DPF2 siRNAs also up-regulates OCT4 but not NANOG in H9 cells. However, RA fails to downregulates OCT4 protein level in cells infected by lenitviruses containing DPF2 siRNA. Moreover, overexpression of both DPF2 and OCT4 in 293 cells proved the DPF2-OCT4 interaction. DPF2 but not PHD2 mutant DPF2 enhanced ubiquitination and degradation of OCT4 in 293 cells co-expressed DPF2 and OCT4. Both wild type DPF2 and PHD2 mutant DPF2 redistributes nuclear OCT4 without affecting DPF2-OCT4 interaction. Further analysis indicated that DPF2 decreases monomeric and mono-ubiquitinated OCT4, assembles poly-ubiquitin chains on OCT4 mainly through Ub-K48 linkage. These findings contribute to an understanding of how OCT4 protein level and nuclear distribution is regulated by its associated protein. PMID:26417682

  9. 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

  10. G-proteins in etiolated Avena seedlings. Possible phytochrome regulation.

    PubMed

    Romero, L C; Sommer, D; Gotor, C; Song, P S

    1991-05-01

    The molecular mechanism of light signal transduction in plants mediated by the photosensor phytochrome is not well understood. The possibility that phytochrome initiates the signal transduction chain by modulating a G-protein-like receptor is examined in the present work. Etiolated Avena seedlings contain G-proteins as examined in terms of the binding of GTP as well as by cross-reaction with mammalian G-protein antibodies. The binding of GTP was regulated in vivo by red/far-red light. The possible involvement of G-proteins in the phytochrome-mediated signal transduction in etiolated Avena seedlings has been implicated from the study of the light regulated expression of the Cab and phy genes. PMID:1903719

  11. Protein phosphatases and their regulation in the control of mitosis

    PubMed Central

    Mochida, Satoru; Hunt, Tim

    2012-01-01

    Cell cycle transitions depend on protein phosphorylation and dephosphorylation. The discovery of cyclin-dependent kinases (CDKs) and their mode of activation by their cyclin partners explained many important aspects of cell cycle control. As the cell cycle is basically a series of recurrences of a defined set of events, protein phosphatases must obviously be as important as kinases. However, our knowledge about phosphatases lags well behind that of kinases. We still do not know which phosphatase(s) is/are truly responsible for dephosphorylating CDK substrates, and we know very little about whether and how protein phosphatases are regulated. Here, we summarize our present understanding of the phosphatases that are important in the control of the cell cycle and pose the questions that need to be answered as regards the regulation of protein phosphatases. PMID:22482124

  12. G protein-coupled receptors and the regulation of autophagy

    PubMed Central

    Wauson, Eric M.; Dbouk, Hashem A.; Ghosh, Anwesha B.; Cobb, Melanie H.

    2014-01-01

    Autophagy is an important catabolic cellular process that eliminates damaged and unnecessary cytoplasmic proteins and organelles. Basal autophagy occurs during normal physiological conditions, but the activity of this process can be significantly altered in human diseases. Thus, defining the regulatory inputs and signals that control autophagy is essential. Nutrients are key modulators of autophagy. While autophagy is generally accepted to be regulated in a cell autonomous fashion, recent studies suggest nutrients can modulate autophagy in a systemic manner by inducing the secretion of hormones and neurotransmitters that regulate G protein-coupled receptors (GPCRs). Emerging studies show that GPCRs also regulate autophagy by directly detecting extracellular nutrients. We review the role of GPCRs in autophagy regulation, highlighting their potential as therapeutic drug targets. PMID:24751357

  13. Established and emerging fluorescence-based assays for G-protein function: heterotrimeric G-protein alpha subunits and regulator of G-protein signaling (RGS) proteins.

    PubMed

    Kimple, Randall J; Jones, Miller B; Shutes, Adam; Yerxa, Benjamin R; Siderovski, David P; Willard, Francis S

    2003-06-01

    Heterotrimeric G-proteins are molecular switches that couple serpentine receptors to intracellular effector pathways and the regulation of cell physiology. Ligand-bound receptors cause G-protein alpha subunits to bind guanosine 5'-triphosphate (GTP) and activate effector pathways. Signal termination is facilitated by the intrinsic GTPase activity of G-protein alpha subunits. Regulators of G-protein signaling (RGS) proteins accelerate the GTPase activity of the G-protein alpha subunit, and thus negatively regulate G-protein-mediated signal transduction. In vitro biochemical assays of heterotrimeric G-proteins commonly include measurements of nucleotide binding, GTPase activity, and interaction with RGS proteins. However, the conventional assays for most of these processes involve radiolabeled guanine nucleotide analogues and scintillation counting. In this article, we focus on fluorescence-based methodologies to study heterotrimeric G-protein alpha subunit regulation in vitro. Furthermore, we consider the potential of such techniques in high-throughput screening and drug discovery. PMID:12769684

  14. Protein S-glutathiolation: Redox-sensitive regulation of protein function

    PubMed Central

    Hill, Bradford G.; Bhatnagar, Aruni

    2011-01-01

    Reversible protein S-glutathiolation has emerged as an important mechanism of post-translational modification. Under basal conditions several proteins remain adducted to glutathione, and physiological glutathiolation of proteins has been shown to regulate protein function. Enzymes that promote glutathiolation (e.g., glutathione-S-transferase-P) or those that remove glutathione from proteins (e.g., glutaredoxin) have been identified. Modification by glutathione has been shown to affect protein catalysis, ligand binding, oligomerization and protein-protein interactions. Conditions associated with oxidative or nitrosative stress, such as ischemia-reperfusion, hypertension and tachycardia increase protein glutathiolation via changes in the glutathione redox status (GSH/GSSG) or through the formation of sulfenic acid (SOH) or nitrosated (SNO) cysteine intermediates. These “activated” thiols promote reversible S-glutathiolation of key proteins involved in cell signaling, energy production, ion transport, and cell death. Hence, S-glutathiolation is ideally suited for integrating and mounting fine-tuned responses to changes in the redox state. S-glutathiolation also provides a temporary glutathione “cap” to protect protein thiols from irreversible oxidation and it could be an important mechanism of protein “encryption” to maintain proteins in a functionally silent state until they are needed during conditions of stress. Current evidence suggests that the glutathiolation-deglutathiolation cycle integrates and interacts with other post-translational mechanisms to regulate signal transduction, metabolism, inflammation, and apoptosis. PMID:21784079

  15. Sex Hormones Regulate Cytoskeletal Proteins Involved in Brain Plasticity

    PubMed Central

    Hansberg-Pastor, Valeria; González-Arenas, Aliesha; Piña-Medina, Ana Gabriela; Camacho-Arroyo, Ignacio

    2015-01-01

    In the brain of female mammals, including humans, a number of physiological and behavioral changes occur as a result of sex hormone exposure. Estradiol and progesterone regulate several brain functions, including learning and memory. Sex hormones contribute to shape the central nervous system by modulating the formation and turnover of the interconnections between neurons as well as controlling the function of glial cells. The dynamics of neuron and glial cells morphology depends on the cytoskeleton and its associated proteins. Cytoskeletal proteins are necessary to form neuronal dendrites and dendritic spines, as well as to regulate the diverse functions in astrocytes. The expression pattern of proteins, such as actin, microtubule-associated protein 2, Tau, and glial fibrillary acidic protein, changes in a tissue-specific manner in the brain, particularly when variations in sex hormone levels occur during the estrous or menstrual cycles or pregnancy. Here, we review the changes in structure and organization of neurons and glial cells that require the participation of cytoskeletal proteins whose expression and activity are regulated by estradiol and progesterone. PMID:26635640

  16. Sex Hormones Regulate Cytoskeletal Proteins Involved in Brain Plasticity.

    PubMed

    Hansberg-Pastor, Valeria; González-Arenas, Aliesha; Piña-Medina, Ana Gabriela; Camacho-Arroyo, Ignacio

    2015-01-01

    In the brain of female mammals, including humans, a number of physiological and behavioral changes occur as a result of sex hormone exposure. Estradiol and progesterone regulate several brain functions, including learning and memory. Sex hormones contribute to shape the central nervous system by modulating the formation and turnover of the interconnections between neurons as well as controlling the function of glial cells. The dynamics of neuron and glial cells morphology depends on the cytoskeleton and its associated proteins. Cytoskeletal proteins are necessary to form neuronal dendrites and dendritic spines, as well as to regulate the diverse functions in astrocytes. The expression pattern of proteins, such as actin, microtubule-associated protein 2, Tau, and glial fibrillary acidic protein, changes in a tissue-specific manner in the brain, particularly when variations in sex hormone levels occur during the estrous or menstrual cycles or pregnancy. Here, we review the changes in structure and organization of neurons and glial cells that require the participation of cytoskeletal proteins whose expression and activity are regulated by estradiol and progesterone. PMID:26635640

  17. Structural Biology and Regulation of Protein Import into the Nucleus.

    PubMed

    Christie, Mary; Chang, Chiung-Wen; Róna, Gergely; Smith, Kate M; Stewart, Alastair G; Takeda, Agnes A S; Fontes, Marcos R M; Stewart, Murray; Vértessy, Beáta G; Forwood, Jade K; Kobe, Bostjan

    2016-05-22

    Proteins are translated in the cytoplasm, but many need to access the nucleus to perform their functions. Understanding how these nuclear proteins are transported through the nuclear envelope and how the import processes are regulated is therefore an important aspect of understanding cell function. Structural biology has played a key role in understanding the molecular events during the transport processes and their regulation, including the recognition of nuclear targeting signals by the corresponding receptors. Here, we review the structural basis of the principal nuclear import pathways and the molecular basis of their regulation. The pathways involve transport factors that are members of the β-karyopherin family, which can bind cargo directly (e.g., importin-β, transportin-1, transportin-3, importin-13) or through adaptor proteins (e.g., importin-α, snurportin-1, symportin-1), as well as unrelated transport factors such as Hikeshi, involved in the transport of heat-shock proteins, and NTF2, involved in the transport of RanGDP. Solenoid proteins feature prominently in these pathways. Nuclear transport factors recognize nuclear targeting signals on the cargo proteins, including the classical nuclear localization signals, recognized by the adaptor importin-α, and the PY nuclear localization signals, recognized by transportin-1. Post-translational modifications, particularly phosphorylation, constitute key regulatory mechanisms operating in these pathways. PMID:26523678

  18. Eosinophil granule cationic proteins regulate the classical pathway of complement.

    PubMed Central

    Weiler, J M; Edens, R E; Bell, C S; Gleich, G J

    1995-01-01

    Major basic protein, the primary constituent of eosinophil granules, regulates the alternative and classical pathways of complement. Major basic protein and other eosinophil granule cationic proteins, which are important in mediating tissue damage in allergic disease, regulate the alternative pathway by interfering with C3b interaction with factor B to assemble an alternative pathway C3 convertase. In the present study, eosinophil peroxidase, eosinophil cationic protein and eosinophil-derived neurotoxin, as well as major basic protein, were examined for capacity to regulate the classical pathway. Eosinophil peroxidase, eosinophil cationic protein and major basic protein inhibited formation of cell-bound classical pathway C3 convertase (EAC1,4b,2a), causing 50% inhibition of complement-mediated lysis at about 0.19, 0.75 and 0.5 micrograms/10(7) cellular intermediates, respectively. Eosinophil-derived neurotoxin had no activity on this pathway of complement. The eosinophil granule proteins were examined for activity on the formation of the membrane attack complex. Major basic protein and eosinophil cationic protein had no activity on terminal lysis. In contrast, eosinophil peroxidase inhibited lysis of EAC1,4b,2a,3b,5b, but had only minimal activity on later events in complement lysis. These polycations were then examined to determine the site(s) at which they regulated the early classical pathway. Eosinophil granule polycationic proteins: (1) reduced the Zmax at all time points but had only minimal effect on the Tmax during the formation of the classical pathway C3 convertase (EAC1,4b,2a); (2) inhibited formation of EAC1,4b,2a proportional to C4 but independent of C2 concentration; (3) inhibited fluid phase formation of C1,4b,2a, as reflected by a decrease in C1-induced consumption of C2 over time; and (4) inhibited C1 activity over time without a direct effect on either C4 or C2. These observations suggest that polycations regulate the early classical pathway by

  19. Exocyst proteins in cytokinesis: Regulation by Rab11.

    PubMed

    Neto, Hélia; Balmer, Gemma; Gould, Gwyn

    2013-11-01

    The Exocyst is an octameric protein complex comprised of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 subunits.(1, 2) This complex was first identified in budding yeast where it acts to target vesicles to the bud tip and the cleavage furrow.(3) Here, we show that all Exocyst subunits are required for cytokinesis in mammalian cells. We further show that a subset of Exocyst proteins are differentially regulated by Rab11, consistent with recent studies implicating Rab11 vesicles in Exocyst protein trafficking. PMID:24563720

  20. Lil3 Assembles with Proteins Regulating Chlorophyll Synthesis in Barley

    PubMed Central

    Gargano, Daniela; Furnes, Clemens; Reisinger, Veronika; Arnold, Janine; Kmiec, Karol; Eichacker, Lutz Andreas

    2015-01-01

    The light-harvesting-like (LIL) proteins are a family of membrane proteins that share a chlorophyll a/b-binding motif with the major light-harvesting antenna proteins of oxygenic photoautotrophs. LIL proteins have been associated with the regulation of tetrapyrrol biosynthesis, and plant responses to light-stress. Here, it was found in a native PAGE approach that chlorophyllide, and chlorophyllide plus geranylgeraniolpyrophosphate trigger assembly of Lil3 in three chlorine binding fluorescent protein bands, termed F1, F2, and F3. It is shown that light and chlorophyllide trigger accumulation of protochlorophyllide-oxidoreductase, and chlorophyll synthase in band F3. Chlorophyllide and chlorophyll esterified to geranylgeraniol were identified as basis of fluorescence recorded from band F3. A direct interaction between Lil3, CHS and POR was confirmed in a split ubiquitin assay. In the presence of light or chlorophyllide, geranylgeraniolpyrophosphate was shown to trigger a loss of the F3 band and accumulation of Lil3 and geranylgeranyl reductase in F1 and F2. No direct interaction between Lil3 and geranylgeraniolreductase was identified in a split ubiquitin assay; however, accumulation of chlorophyll esterified to phytol in F1 and F2 corroborated the enzymes assembly. Chlorophyll esterified to phytol and the reaction center protein psbD of photosystem II were identified to accumulate together with psb29, and APX in the fluorescent band F2. Data show that Lil3 assembles with proteins regulating chlorophyll synthesis in etioplasts from barley (Hordeum vulgare L.). PMID:26172838

  1. CRK proteins selectively regulate T cell migration into inflamed tissues

    PubMed Central

    Huang, Yanping; Clarke, Fiona; Karimi, Mobin; Roy, Nathan H.; Williamson, Edward K.; Okumura, Mariko; Mochizuki, Kazuhiro; Chen, Emily J.H.; Park, Tae-Ju; Debes, Gudrun F.; Zhang, Yi; Curran, Tom; Kambayashi, Taku; Burkhardt, Janis K.

    2015-01-01

    Effector T cell migration into inflamed sites greatly exacerbates tissue destruction and disease severity in inflammatory diseases, including graft-versus-host disease (GVHD). T cell migration into such sites depends heavily on regulated adhesion and migration, but the signaling pathways that coordinate these functions downstream of chemokine receptors are largely unknown. Using conditional knockout mice, we found that T cells lacking the adaptor proteins CRK and CRK-like (CRKL) exhibit reduced integrin-dependent adhesion, chemotaxis, and diapedesis. Moreover, these two closely related proteins exhibited substantial functional redundancy, as ectopic expression of either protein rescued defects in T cells lacking both CRK and CRKL. We determined that CRK proteins coordinate with the RAP guanine nucleotide exchange factor C3G and the adhesion docking molecule CASL to activate the integrin regulatory GTPase RAP1. CRK proteins were required for effector T cell trafficking into sites of inflammation, but not for migration to lymphoid organs. In a murine bone marrow transplantation model, the differential migration of CRK/CRKL-deficient T cells resulted in efficient graft-versus-leukemia responses with minimal GVHD. Together, the results from our studies show that CRK family proteins selectively regulate T cell adhesion and migration at effector sites and suggest that these proteins have potential as therapeutic targets for preventing GVHD. PMID:25621495

  2. Binding-regulated click ligation for selective detection of proteins.

    PubMed

    Cao, Ya; Han, Peng; Wang, Zhuxin; Chen, Weiwei; Shu, Yongqian; Xiang, Yang

    2016-04-15

    Herein, a binding-regulated click ligation (BRCL) strategy for endowing selective detection of proteins is developed with the incorporation of small-molecule ligand and clickable DNA probes. The fundamental principle underlying the strategy is the regulating capability of specific protein-ligand binding against the ligation between clickable DNA probes, which could efficiently combine the detection of particular protein with enormous DNA-based sensing technologies. In this work, the feasibly of the BRCL strategy is first verified through agarose gel electrophoresis and electrochemical impedance spectroscopy measurements, and then confirmed by transferring it to a nanomaterial-assisted fluorescence assay. Significantly, the BRCL strategy-based assay is able to respond to target protein with desirable selectivity, attributing to the specific recognition between small-molecule ligand and its target. Further experiments validate the general applicability of the sensing method by tailoring the ligand toward different proteins (i.e., avidin and folate receptor), and demonstrate its usability in complex biological samples. To our knowledge, this work pioneers the practice of click chemistry in probing specific small-molecule ligand-protein binding, and therefore may pave a new way for selective detection of proteins. PMID:26599478

  3. Protein-dependent regulation of feeding and metabolism.

    PubMed

    Morrison, Christopher D; Laeger, Thomas

    2015-05-01

    Free-feeding animals often face complex nutritional choices that require the balancing of competing nutrients, but the mechanisms driving macronutrient-specific food intake are poorly defined. A large number of behavioral studies indicate that both the quantity and quality of dietary protein can markedly influence food intake and metabolism, and that dietary protein intake may be prioritized over energy intake. This review focuses on recent progress in defining the mechanisms underlying protein-specific feeding. Considering the evidence that protein powerfully regulates both food intake and metabolism, uncovering these protein-specific mechanisms may reveal new molecular targets for the treatment of obesity and diabetes while also offering a more complete understanding of how dietary factors shape both food intake and food choice. PMID:25771038

  4. Control of Striatal Signaling by G Protein Regulators

    PubMed Central

    Xie, Keqiang; Martemyanov, Kirill A.

    2011-01-01

    Signaling via heterotrimeric G proteins plays a crucial role in modulating the responses of striatal neurons that ultimately shape core behaviors mediated by the basal ganglia circuitry, such as reward valuation, habit formation, and movement coordination. Activation of G protein-coupled receptors (GPCRs) by extracellular signals activates heterotrimeric G proteins by promoting the binding of GTP to their α subunits. G proteins exert their effects by influencing the activity of key effector proteins in this region, including ion channels, second messenger enzymes, and protein kinases. Striatal neurons express a staggering number of GPCRs whose activation results in the engagement of downstream signaling pathways and cellular responses with unique profiles but common molecular mechanisms. Studies over the last decade have revealed that the extent and duration of GPCR signaling are controlled by a conserved protein family named regulator of G protein signaling (RGS). RGS proteins accelerate GTP hydrolysis by the α subunits of G proteins, thus promoting deactivation of GPCR signaling. In this review, we discuss the progress made in understanding the roles of RGS proteins in controlling striatal G protein signaling and providing integration and selectivity of signal transmission. We review evidence on the formation of a macromolecular complex between RGS proteins and other components of striatal signaling pathways, their molecular regulatory mechanisms and impacts on GPCR signaling in the striatum obtained from biochemical studies and experiments involving genetic mouse models. Special emphasis is placed on RGS9-2, a member of the RGS family that is highly enriched in the striatum and plays critical roles in drug addiction and motor control. PMID:21852966

  5. Erythrocyte Protein 4.1 Binds and Regulates Myosin

    NASA Astrophysics Data System (ADS)

    Pasternack, Gary R.; Racusen, Richard H.

    1989-12-01

    Myosin was recently identified in erythrocytes and was shown to partition both with membrane and cytosolic fractions, suggesting that it may be loosely bound to membranes [Fowler, V. M., Davis, J. Q. & Bennett, V. (1985) J. Cell Biol. 100, 47-55, and Wong, A. J., Kiehart, D. P. & Pollard, T. D. (1985) J. Biol. Chem. 260, 46-49]; however, the molecular basis for this binding was unclear. The present studies employed immobilized monomeric myosin to examine the interaction of myosin with erythrocyte protein 4.1. In human erythrocytes, protein 4.1 binds to integral membrane proteins and mediates spectrin-actin assembly. Protein 4.1 binds to rabbit skeletal muscle myosin with a Kd = 140 nM and a stoichiometry consistent with 1:1 binding. Heavy meromyosin competes for protein 4.1 binding with Ki = 36-54 nM; however, the S1 fragment (the myosin head) competes less efficiently. Affinity chromatography of partial chymotryptic digests of protein 4.1 on immobilized myosin identified a 10-kDa domain of protein 4.1 as the myosin-binding site. In functional studies, protein 4.1 partially inhibited the actin-activated Mg2+-ATPase activity of rabbit skeletal muscle myosin with Ki = 51 nM. Liver cytosolic and erythrocyte myosins preactivated with myosin light-chain kinase were similarly inhibited by protein 4.1. These studies show that protein 4.1 binds, modulates, and thus may regulate myosin. This interaction might serve to generate the contractile forces involved in Mg2+-ATP-dependent shape changes in erythrocytes and may additionally serve as a model for myosin organization and regulation in non-muscle cells.

  6. MICAL-Family Proteins: Complex Regulators of the Actin Cytoskeleton

    PubMed Central

    Giridharan, Sai Srinivas Panapakkam

    2014-01-01

    Abstract Significance: The molecules interacting with CasL (MICAL) family members participate in a multitude of activities, including axonal growth cone repulsion, membrane trafficking, apoptosis, and bristle development in flies. An interesting feature of MICAL proteins is the presence of an N-terminal flavo-mono-oxygenase domain. This mono-oxygenase domain generates redox potential with which MICALs can either oxidize proteins or produce reactive oxygen species (ROS). Actin is one such protein that is affected by MICAL function, leading to dramatic cytoskeletal rearrangements. This review describes the MICAL-family members, and discusses their mechanisms of actin-binding and regulation of actin cytoskeleton organization. Recent Advances: Recent studies show that MICALs directly induce oxidation of actin molecules, leading to actin depolymerization. ROS production by MICALs also causes oxidation of collapsin response mediator protein-2, a microtubule assembly promoter, which subsequently undergoes phosphorylation. Critical Issues: MICAL proteins oxidize proteins through two mechanisms: either directly by oxidizing methionine residues or indirectly via the production of ROS. It remains unclear whether MICAL proteins employ both mechanisms or whether the activity of MICAL-family proteins might vary with different substrates. Future Directions: The identification of additional substrates oxidized by MICAL will shed new light on MICAL protein function. Additional directions include expanding studies toward the MICAL-like homologs that lack flavin adenine dinucleotide domains and oxidation activity. Antioxid. Redox Signal. 20, 2059–2073. PMID:23834433

  7. Lipid droplet meets a mitochondrial protein to regulate adipocyte lipolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In response to adrenergic stimulation, adipocytes undergo protein kinase A (PKA)-stimulated lipolysis. A key PKA target in this context is perilipin 1, a major regulator of lipolysis on lipid droplets (LDs). A study published in this issue of The EMBO Journal (Pidoux et al, 2011) identifies optic at...

  8. The Up-Regulation of Ribosomal Proteins Further Regulates Protein Expression Profile in Female Schistosoma japonicum after Pairing

    PubMed Central

    Sun, Jun; Li, Chen; Wang, Suwen

    2015-01-01

    Background Pairing of Schistosoma males and females leads to and maintains female sexual maturation. However, the mechanism by which pairing facilitates sexual maturation of females is not clear. An increasing body of evidence suggests that ribosomal proteins have regulatory rather than constitutive roles in protein translation. Methodology/Principal Findings To investigate the effect of ribosome regulation on female sex maturation, Solexa and iTRAQ techniques were used to analyze the relationship between ribosomal gene or protein expression and sexual development of Schistosoma females. In the present study, considerably higher number of ribosomal genes or proteins were found to be differentially expressed in paired 23-day-old females. Moreover, mature female-specific proteins associated with egg production, such as ferritin-1 heavy chain and superoxide dismutase, were selectively highly expressed in paired females, rather than higher level of protein synthesis of all transcripts compared with those in unpaired 23-day-old females. Furthermore, other developmental stages were utilized to investigate different expression pattern of ribosomal proteins in females by analysing 18-day-old female schistosomula from single- or double-sex infections to determine the relationship between ribosomal protein expression pattern and development. Results showed that undeveloped 18-day-old females from single- and double-sex infections, as well as 23-day-old unpaired females, possessed similar ribosomal protein expression patterns, which were distinct from those in 23-day-old paired females. Conclusions/Significance Our findings reveal that the pairing of females and males triggers a specialized ribosomal protein expression profile which further regulates the protein profile for sexual maturation in Schistosoma japonicum, based on its gene expression profile. PMID:26070205

  9. Protein Phosphorylation: A Major Switch Mechanism for Metabolic Regulation.

    PubMed

    Humphrey, Sean J; James, David E; Mann, Matthias

    2015-12-01

    Metabolism research is undergoing a renaissance because many diseases are increasingly recognized as being characterized by perturbations in intracellular metabolic regulation. Metabolic changes can be conferred through changes to the expression of metabolic enzymes, the concentrations of substrates or products that govern reaction kinetics, or post-translational modification (PTM) of the proteins that facilitate these reactions. On the 60th anniversary since its discovery, reversible protein phosphorylation is widely appreciated as an essential PTM regulating metabolism. With the ability to quantitatively measure dynamic changes in protein phosphorylation on a global scale - hereafter referred to as phosphoproteomics - we are now entering a new era in metabolism research, with mass spectrometry (MS)-based proteomics at the helm. PMID:26498855

  10. BCL-2 family proteins as regulators of mitochondria metabolism.

    PubMed

    Gross, Atan

    2016-08-01

    The BCL-2 family proteins are major regulators of apoptosis, and one of their major sites of action are the mitochondria. Mitochondria are the cellular hubs for metabolism and indeed selected BCL-2 family proteins also possess roles related to mitochondria metabolism and dynamics. Here we discuss the link between mitochondrial metabolism/dynamics and the fate of stem cells, with an emphasis on the role of the BID-MTCH2 pair in regulating this link. We also discuss the possibility that BCL-2 family proteins act as metabolic sensors/messengers coming on and off of mitochondria to "sample" the cytosol and provide the mitochondria with up-to-date metabolic information. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi. PMID:26827940

  11. Emerging roles of zinc finger proteins in regulating adipogenesis

    PubMed Central

    Wei, Shengjuan; Zhang, Lifan; Zhou, Xiang; Du, Min; Jiang, Zhihua; Hausman, Gary J.; Bergen, Werner G.; Zan, Linsen; Dodson, Michael V.

    2014-01-01

    Proteins containing the zinc finger domain(s) are named zinc finger proteins (ZFPs), which are one of the largest classes of transcription factors in eukaryotic genomes. A large number of ZFPs have been studied and many of them were found to be involved regulating normal growth and development of cells and tissues through diverse signal transduction pathways. Recent studies revealed that a small but increasing number of ZFPs could function as key transcriptional regulators involved in adipogenesis. As the prevalence of obesity and metabolic disorders, the investigation of molecular regulatory mechanisms of adipocyte development must be more completely understood to develop novel and long term impact strategies for ameliorating obesity. In this review, we discuss recent work which has documented that ZFPs are important functional contributors to the regulation of adipogenesis. Taken altogether these data lead to the conclusion that ZFPs may become promising targets to combat human obesity. PMID:23760207

  12. Regulation of bone morphogenetic proteins in early embryonic development

    NASA Astrophysics Data System (ADS)

    Yamamoto, Yukiyo; Oelgeschläger, Michael

    2004-11-01

    Bone morphogenetic proteins (BMPs), a large subgroup of the TGF-β family of secreted growth factors, control fundamental events in early embryonic development, organogenesis and adult tissue homeostasis. The plethora of dose-dependent cellular processes regulated by BMP signalling demand a tight regulation of BMP activity. Over the last decade, a number of proteins have been identified that bind BMPs in the extracellular space and regulate the interaction of BMPs with their cognate receptors, including the secreted BMP antagonist Chordin. In the early vertebrate embryo, the localized secretion of BMP antagonists from the dorsal blastopore lip establishes a functional BMP signalling gradient that is required for the determination of the dorsoventral or back to belly body axis. In particular, inhibition of BMP activity is essential for the formation of neural tissue in the development of vertebrate and invertebrate embryos. Here we review recent studies that have provided new insight into the regulation of BMP signalling in the extracellular space. In particular, we discuss the recently identified Twisted gastrulation protein that modulates, in concert with metalloproteinases of the Tolloid family, the interaction of Chordin with BMP and a family of proteins that share structural similarities with Chordin in the respective BMP binding domains. In addition, genetic and functional studies in zebrafish and frog provide compelling evidence that the secreted protein Sizzled functionally interacts with the Chd BMP pathway, despite being expressed ventrally in the early gastrula-stage embryo. These intriguing discoveries may have important implications, not only for our current concept of early embryonic patterning, but also for the regulation of BMP activity at later developmental stages and tissue homeostasis in the adult.

  13. Regulation of thrombosis and vascular function by protein methionine oxidation.

    PubMed

    Gu, Sean X; Stevens, Jeff W; Lentz, Steven R

    2015-06-18

    Redox biology is fundamental to both normal cellular homeostasis and pathological states associated with excessive oxidative stress. Reactive oxygen species function not only as signaling molecules but also as redox regulators of protein function. In the vascular system, redox reactions help regulate key physiologic responses such as cell adhesion, vasoconstriction, platelet aggregation, angiogenesis, inflammatory gene expression, and apoptosis. During pathologic states, altered redox balance can cause vascular cell dysfunction and affect the equilibrium between procoagulant and anticoagulant systems, contributing to thrombotic vascular disease. This review focuses on the emerging role of a specific reversible redox reaction, protein methionine oxidation, in vascular disease and thrombosis. A growing number of cardiovascular and hemostatic proteins are recognized to undergo reversible methionine oxidation, in which methionine residues are posttranslationally oxidized to methionine sulfoxide. Protein methionine oxidation can be reversed by the action of stereospecific enzymes known as methionine sulfoxide reductases. Calcium/calmodulin-dependent protein kinase II is a prototypical methionine redox sensor that responds to changes in the intracellular redox state via reversible oxidation of tandem methionine residues in its regulatory domain. Several other proteins with oxidation-sensitive methionine residues, including apolipoprotein A-I, thrombomodulin, and von Willebrand factor, may contribute to vascular disease and thrombosis. PMID:25900980

  14. Regulation of thrombosis and vascular function by protein methionine oxidation

    PubMed Central

    Gu, Sean X.; Stevens, Jeff W.

    2015-01-01

    Redox biology is fundamental to both normal cellular homeostasis and pathological states associated with excessive oxidative stress. Reactive oxygen species function not only as signaling molecules but also as redox regulators of protein function. In the vascular system, redox reactions help regulate key physiologic responses such as cell adhesion, vasoconstriction, platelet aggregation, angiogenesis, inflammatory gene expression, and apoptosis. During pathologic states, altered redox balance can cause vascular cell dysfunction and affect the equilibrium between procoagulant and anticoagulant systems, contributing to thrombotic vascular disease. This review focuses on the emerging role of a specific reversible redox reaction, protein methionine oxidation, in vascular disease and thrombosis. A growing number of cardiovascular and hemostatic proteins are recognized to undergo reversible methionine oxidation, in which methionine residues are posttranslationally oxidized to methionine sulfoxide. Protein methionine oxidation can be reversed by the action of stereospecific enzymes known as methionine sulfoxide reductases. Calcium/calmodulin-dependent protein kinase II is a prototypical methionine redox sensor that responds to changes in the intracellular redox state via reversible oxidation of tandem methionine residues in its regulatory domain. Several other proteins with oxidation-sensitive methionine residues, including apolipoprotein A-I, thrombomodulin, and von Willebrand factor, may contribute to vascular disease and thrombosis. PMID:25900980

  15. Regulator of G Protein Signaling 2: A Versatile Regulator of Vascular Function

    PubMed Central

    Osei-Owusu, Patrick; Blumer, Kendall J.

    2016-01-01

    Regulators of G protein signaling (RGS) proteins of the B/R4 family are widely expressed in the cardiovascular system where their role in fine tuning G protein signaling is critical to maintaining homeostasis. Among members of this family, RGS2 and RGS5 have been shown to play key roles in cardiac and smooth muscle function by tightly regulating signaling pathways that are activated through Gq/11 and Gi/o classes of heterotrimeric G proteins. This chapter reviews accumulating evidence supporting a key role for RGS2 in vascular function and the implication of changes in RGS2 function and/or expression in the pathogenesis of blood pressure disorders, particularly hypertension. With such understanding, RGS2 and the signaling pathways it controls may emerge as novel targets for developing next-generation anti-hypertensive drugs/agents. PMID:26123303

  16. The interactome of a PTB domain-containing adapter protein, Odin, revealed by SILAC

    PubMed Central

    Zhong, Jun; Chaerkady, Raghothama; Kandasamy, Kumaran; Gucek, Marjan; Cole, Robert N.; Pandey, Akhilesh

    2011-01-01

    Signal transduction pathways are tightly controlled by positive and negative regulators. We have previously identified Odin (also known as ankyrin repeat and sterile alpha motif domain containing 1A; gene symbol AKNS1A) as a negative regulator of growth factor signaling; however, the mechanisms through which Odin regulates these pathways remain to be elucidated. To determine how Odin negatively regulates growth factor signaling, we undertook a proteomic approach to systematically identify proteins that interact with Odin using the SILAC strategy. In this study, we identified 18 molecules that were specifically associated in a protein complex with Odin. Our study established that the complete family of 14-3-3 proteins occur in a protein complex with Odin, which is also supported by earlier reports that identified a few members of the 14-3-3 family as Odin interactors. Among the novel protein interactors of Odin were CD2-associated protein, SH3 domain kinase binding protein 1 and DAB2 interacting protein. We confirmed 8 of the eighteen interactions identified in the Odin protein complex by co-immunoprecipitation experiments. Finally, a literature-based network analysis revealed that Odin interacting partners are involved in various cellular processes, some of which are key molecules in regulating receptor endocytosis. PMID:21081186

  17. Dynamic regulation of macroautophagy by distinctive, ubiquitin-like proteins

    PubMed Central

    Klionsky, Daniel J.; Schulman, Brenda A.

    2014-01-01

    Autophagy complements the ubiquitin-proteasome system in mediating protein turnover. Whereas the proteasome degrades individual proteins modified with ubiquitin chains, autophagy degrades many proteins and organelles en masse. Macromolecules destined for autophagic degradation are “selected” through sequestration within a specialized double-membrane compartment termed the “phagophore”, the precursor to an “autophagosome”, and then hydrolyzed in a lysosome/vacuole-dependent manner. Notably, a pair of distinctive ubiquitin-like proteins (UBLs), Atg8 and Atg12, regulate degradation by autophagy in unique ways, by controlling autophagosome biogenesis and recruitment of specific cargos during selective autophagy. Here we review structural mechanisms underlying functions and conjugation of these UBLs that are specialized to provide interaction platforms linked to phagophore membranes. PMID:24699082

  18. Differential regulation of oligodendrocyte markers by glucocorticoids: Post-transcriptional regulation of both proteolipid protein and myelin basic protein and transcriptional regulation of glycerol phosphate dehydrogenase

    SciTech Connect

    Kumar, S.; Cole, R.; Chiappelli, F.; De Vellis, J. )

    1989-09-01

    During neonatal development glucocorticoids potentiate oligodendrocyte differentiation and myelinogenesis by regulating the expression of myelin basic protein, proteolipid protein, and glycerol phosphate dehydrogenase. The actual locus at which hydrocortisone exerts its developmental influence on glial physiology is, however, not well understood. Gycerol phosphate dehydrogenase is glucocorticoid-inducible in oligodendrocytes at all stages of development both in vivo and in vitro. In newborn rat cerebral cultures, between 9 and 15 days in vitro, a 2- to 3-fold increase in myelin basic protein and proteolipid protein mRNA levels occurs in oligodendrocytes within 12 hr of hydrocortisone treatment. Immunostaining demonstrates that this increase in mRNAs is followed by a 2- to 3-fold increase in the protein levels within 24 hr. In vitro transcription assays performed with oligodendrocyte nuclei show an 11-fold increase in the transcriptional activity of glycerol phosphate dehydrogenase in response to hydrocortisone but no increase in transcription of myelin basic protein or proteolipid protein. These results indicate that during early myelinogeneis, glucocorticoids influence the expression of key oligodendroglial markers by different processes: The expression of glycerol phosphate dehydrogenase is regulated at the transcriptional level, whereas the expression of myelin basic protein and proteolipid protein is modulated via a different, yet uncharacterized, mechanism involving post-transcriptional regulation.

  19. Wrecked regulation of intrinsically disordered proteins in diseases: pathogenicity of deregulated regulators

    PubMed Central

    Uversky, Vladimir N.

    2014-01-01

    Biologically active proteins without stable tertiary structure are common in all known proteomes. Functions of these intrinsically disordered proteins (IDPs) are typically related to regulation, signaling, and control. Cellular levels of these important regulators are tightly regulated by a variety mechanisms ranging from firmly controlled expression to precisely targeted degradation. Functions of IDPs are controlled by binding to specific partners, alternative splicing, and posttranslational modifications among other means. In the norm, right amounts of precisely activated IDPs have to be present in right time at right places. Wrecked regulation brings havoc to the ordered world of disordered proteins, leading to protein misfolding, misidentification, and missignaling that give rise to numerous human diseases, such as cancer, cardiovascular disease, neurodegenerative diseases, and diabetes. Among factors inducing pathogenic transformations of IDPs are various cellular mechanisms, such as chromosomal translocations, damaged splicing, altered expression, frustrated posttranslational modifications, aberrant proteolytic degradation, and defective trafficking. This review presents some of the aspects of deregulated regulation of IDPs leading to human diseases. PMID:25988147

  20. Regulation of Bone Morphogenetic Protein Signaling by ADP-ribosylation.

    PubMed

    Watanabe, Yukihide; Papoutsoglou, Panagiotis; Maturi, Varun; Tsubakihara, Yutaro; Hottiger, Michael O; Heldin, Carl-Henrik; Moustakas, Aristidis

    2016-06-10

    We previously established a mechanism of negative regulation of transforming growth factor β signaling mediated by the nuclear ADP-ribosylating enzyme poly-(ADP-ribose) polymerase 1 (PARP1) and the deribosylating enzyme poly-(ADP-ribose) glycohydrolase (PARG), which dynamically regulate ADP-ribosylation of Smad3 and Smad4, two central signaling proteins of the pathway. Here we demonstrate that the bone morphogenetic protein (BMP) pathway can also be regulated by the opposing actions of PARP1 and PARG. PARG positively contributes to BMP signaling and forms physical complexes with Smad5 and Smad4. The positive role PARG plays during BMP signaling can be neutralized by PARP1, as demonstrated by experiments where PARG and PARP1 are simultaneously silenced. In contrast to PARG, ectopic expression of PARP1 suppresses BMP signaling, whereas silencing of endogenous PARP1 enhances signaling and BMP-induced differentiation. The two major Smad proteins of the BMP pathway, Smad1 and Smad5, interact with PARP1 and can be ADP-ribosylated in vitro, whereas PARG causes deribosylation. The overall outcome of this mode of regulation of BMP signal transduction provides a fine-tuning mechanism based on the two major enzymes that control cellular ADP-ribosylation. PMID:27129221

  1. ORMDL proteins regulate ceramide levels during sterile inflammation.

    PubMed

    Cai, Lin; Oyeniran, Clement; Biswas, Debolina D; Allegood, Jeremy; Milstien, Sheldon; Kordula, Tomasz; Maceyka, Michael; Spiegel, Sarah

    2016-08-01

    The bioactive sphingolipid metabolite, ceramide, regulates physiological processes important for inflammation and elevated levels of ceramide have been implicated in IL-1-mediated events. Although much has been learned about ceramide generation by activation of sphingomyelinases in response to IL-1, the contribution of the de novo pathway is not completely understood. Because yeast ORM1 and ORM2 proteins negatively regulate ceramide levels through inhibition of serine palmitoyltransferase, the first committed step in ceramide biosynthesis, we examined the functions of individual mammalian ORM orthologs, ORM (yeast)-like (ORMDL)1-3, in regulation of ceramide levels. In HepG2 liver cells, downregulation of ORMDL3 markedly increased the ceramide precursors, dihydrosphingosine and dihydroceramide, primarily from de novo biosynthesis based on [U-(13)C]palmitate incorporation into base-labeled and dual-labeled dihydroceramides, whereas downregulation of each isoform increased dihydroceramides [(13)C]labeled in only the amide-linked fatty acid. IL-1 and the IL-6 family cytokine, oncostatin M, increased dihydroceramide and ceramide levels in HepG2 cells and concomitantly decreased ORMDL proteins. Moreover, during irritant-induced sterile inflammation in mice leading to induction of the acute-phase response, which is dependent on IL-1, expression of ORMDL proteins in the liver was strongly downregulated and accompanied by increased ceramide levels in the liver and accumulation in the blood. Together, our results suggest that ORMDLs may be involved in regulation of ceramides during IL-1-mediated sterile inflammation. PMID:27313060

  2. Dynamics of adenylate cyclase regulation via heterotrimeric G-proteins.

    PubMed

    Milde, Markus; Werthmann, Ruth C; von Hayn, Kathrin; Bünemann, Moritz

    2014-04-01

    A wide variety of G-protein-coupled receptors either activate or inhibit ACs (adenylate cyclases), thereby regulating cellular cAMP levels and consequently inducing proper physiological responses. Stimulatory and inhibitory G-proteins interact directly with ACs, whereas G(q)-coupled receptors exert their effects primarily via Ca2+. Using the FRET-based cAMP sensor Epac1 (exchange protein directly activated by cAMP 1)-cAMPS (adenosine 3',5'-cyclic monophosphorothioate), we studied cAMP levels in single living VSMCs (vascular smooth muscle cells) or HUVECs (human umbilical vein endothelial cells) with subsecond temporal resolution. Stimulation of purinergic (VSMCs) or thrombin (HUVECs) receptors rapidly decreased cAMP levels in the presence of the β-adrenergic agonist isoprenaline via a rise in Ca2+ and subsequent inhibition of AC5 and AC6. Specifically in HUVECs, we observed that, in the continuous presence of thrombin, cAMP levels climbed slowly after the initial decline with a delay of a little less than 1 min. The underlying mechanism includes phospholipase A2 activity and cyclo-oxygenase-mediated synthesis of prostaglandins. We studied further the dynamics of the inhibition of ACs via G(i)-proteins utilizing FRET imaging to resolve interactions between fluorescently labelled G(i)-proteins and AC5. FRET between Gα(i1) and AC5 developed at much lower concentration of agonist compared with the overall G(i)-protein activity. We found the dissociation of Gα(i1) subunits and AC5 to occur slower than the G(i)-protein deactivation. This led us to the conclusion that AC5, by binding active Gα(i1), interferes with G-protein deactivation and reassembly and thereby might sensitize its own regulation. PMID:24646224

  3. Protein phosphorylation and regulation of adaptive responses in bacteria.

    PubMed Central

    Stock, J B; Ninfa, A J; Stock, A M

    1989-01-01

    Bacteria continuously adapt to changes in their environment. Responses are largely controlled by signal transduction systems that contain two central enzymatic components, a protein kinase that uses adenosine triphosphate to phosphorylate itself at a histidine residue and a response regulator that accepts phosphoryl groups from the kinase. This conserved phosphotransfer chemistry is found in a wide range of bacterial species and operates in diverse systems to provide different regulatory outputs. The histidine kinases are frequently membrane receptor proteins that respond to environmental signals and phosphorylate response regulators that control transcription. Four specific regulatory systems are discussed in detail: chemotaxis in response to attractant and repellent stimuli (Che), regulation of gene expression in response to nitrogen deprivation (Ntr), control of the expression of enzymes and transport systems that assimilate phosphorus (Pho), and regulation of outer membrane porin expression in response to osmolarity and other culture conditions (Omp). Several additional systems are also examined, including systems that control complex developmental processes such as sporulation and fruiting-body formation, systems required for virulent infections of plant or animal host tissues, and systems that regulate transport and metabolism. Finally, an attempt is made to understand how cross-talk between parallel phosphotransfer pathways can provide a global regulatory curcuitry. PMID:2556636

  4. Regulation of heartbeat by G protein-coupled ion channels.

    PubMed

    Brown, A M

    1990-12-01

    The coupling of ion channels to receptors by G proteins is the subject of this American Physiological Society Walter B. Cannon Memorial "Physiology in Perspective" Lecture. This subject is particularly appropriate because it includes a molecular explanation of a homeostatic mechanism involving the autonomic nervous system and the latter subject preoccupied Dr. Cannon during most of his career. With the use of reconstitution methods, we and others have shown that heterotrimeric guanine nucleotide-binding (G) proteins couple receptors to ion channels by both membrane-delimited, direct pathways and cytoplasmic second messenger pathways. Furthermore, one set of receptors may be coupled to as many as three different sets of ion channels to form networks. Dual G protein pathways lead to the prediction of biphasic ion current responses in cell signaling, and this prediction was confirmed. In sinoatrial pacemaker cells, the pacemaking hyperpolarization-activated inward current (If) is directly regulated by the G proteins Gs and Go, and the two can act simultaneously. This could explain the classical observation that vagal inhibition of heart rate is greater during sympathetic stimulation. Because deactivation of the muscarinic response occurs much faster than the G protein alpha-subunit hydrolyzes guanosine 5'-triphosphate, we looked for accessory cellular factors. A surprising result was that the small monomeric ras G protein blocked the muscarinic pathway. The significance of this observation is unknown, but it appears that small and large G proteins may interact in ion channel signaling pathways. PMID:1701981

  5. Regulation of the retinoblastoma proteins by the human herpesviruses

    PubMed Central

    Hume, Adam J; Kalejta, Robert F

    2009-01-01

    Viruses are obligate intracellular parasites that alter the environment of infected cells in order to replicate more efficiently. One way viruses achieve this is by modulating cell cycle progression. The main regulators of progression out of G0, through G1, and into S phase are the members of the retinoblastoma (Rb) family of tumor suppressors. Rb proteins repress the transcription of genes controlled by the E2F transcription factors. Because the expression of E2F-responsive genes is required for cell cycle progression into the S phase, Rb arrests the cell cycle in G0/G1. A number of viral proteins directly target Rb family members for inactivation, presumably to create an environment more hospitable for viral replication. Such viral proteins include the extensively studied oncoproteins E7 (from human papillomavirus), E1A (from adenovirus), and the large T (tumor) antigen (from simian virus 40). Elucidating how these three viral proteins target and inactivate Rb has proven to be an invaluable approach to augment our understanding of both normal cell cycle progression and carcinogenesis. In addition to these proteins, a number of other virally-encoded inactivators of the Rb family have subsequently been identified including a surprising number encoded by human herpesviruses. Here we review how the human herpesviruses modulate Rb function during infection, introduce the individual viral proteins that directly or indirectly target Rb, and speculate about what roles Rb modulation by these proteins may play in viral replication, pathogenesis, and oncogenesis. PMID:19146698

  6. Regulation of the protein stability of EMT transcription factors

    PubMed Central

    Díaz, VM; Viñas-Castells, R; García de Herreros, A

    2014-01-01

    The epithelial to mesenchymal transition (EMT) consists of a rapid change of cell phenotype, characterized by the loss of epithelial characteristics and the acquisition of a more invasive phenotype. Transcription factors regulating EMT (Snail, Twist and Zeb) are extremely labile proteins, rapidly degraded by the proteasome system. In this review we analyze the current mechanisms controlling degradation of EMT transcription factors, focusing on the role of new E3 ubiquitin-ligases involved in EMT. We also summarize the regulation of the stability of these EMT transcription factors, specially observed in different stress conditions, such as hypoxia, chemotherapeutic drugs, oxidative stress or γ-irradiation. PMID:25482633

  7. HOX13 proteins: the molecular switcher in Hoxd bimodal regulation.

    PubMed

    Ros, Marian A

    2016-05-15

    The striking correlation between the genomic arrangement of Hox genes and their temporal and spatial pattern of expression during embryonic development has been a source of fascination since its discovery. This correspondence has been used as a privileged example in the investigation of the connection between genomic architecture and function. In this issue of Genes & Development, Beccari and colleagues (pp. 1172-1186) make a big step forward in understanding Hox gene regulation during limb development by showing the pivotal role of HOXA13 and HOXD13 proteins in the transition from a proximal to a distal type of Hoxd transcriptional regulation. PMID:27222515

  8. Post-translational regulation and modifications of flavivirus structural proteins.

    PubMed

    Roby, Justin A; Setoh, Yin Xiang; Hall, Roy A; Khromykh, Alexander A

    2015-07-01

    Flaviviruses are a group of single-stranded, positive-sense RNA viruses that generally circulate between arthropod vectors and susceptible vertebrate hosts, producing significant human and veterinary disease burdens. Intensive research efforts have broadened our scientific understanding of the replication cycles of these viruses and have revealed several elegant and tightly co-ordinated post-translational modifications that regulate the activity of viral proteins. The three structural proteins in particular - capsid (C), pre-membrane (prM) and envelope (E) - are subjected to strict regulatory modifications as they progress from translation through virus particle assembly and egress. The timing of proteolytic cleavage events at the C-prM junction directly influences the degree of genomic RNA packaging into nascent virions. Proteolytic maturation of prM by host furin during Golgi transit facilitates rearrangement of the E proteins at the virion surface, exposing the fusion loop and thus increasing particle infectivity. Specific interactions between the prM and E proteins are also important for particle assembly, as prM acts as a chaperone, facilitating correct conformational folding of E. It is only once prM/E heterodimers form that these proteins can be secreted efficiently. The addition of branched glycans to the prM and E proteins during virion transit also plays a key role in modulating the rate of secretion, pH sensitivity and infectivity of flavivirus particles. The insights gained from research into post-translational regulation of structural proteins are beginning to be applied in the rational design of improved flavivirus vaccine candidates and make attractive targets for the development of novel therapeutics. PMID:25711963

  9. An oxygen-regulated switch in the protein synthesis machinery

    PubMed Central

    Uniacke, James; Holterman, Chet E.; Lachance, Gabriel; Franovic, Aleksandra; Jacob, Mathieu D.; Fabian, Marc R.; Payette, Josianne; Holcik, Martin; Pause, Arnim; Lee, Stephen

    2016-01-01

    SUMMARY Protein synthesis involves the translation of ribonucleic acid information into proteins, the building blocks of life. The initial step of protein synthesis consists of the eukaryotic translation initiation factor 4E (eIF4E) binding to the 7-methylguanosine (m7-GpppG) 5′cap of mRNAs1,2. Low oxygen tension (hypoxia) represses cap-mediated translation by sequestering eIF4E through mammalian target of rapamycin (mTOR)-dependent mechanisms3–6. While the internal ribosome entry site is an alternative translation initiation mechanism, this pathway alone cannot account for the translational capacity of hypoxic cells7,8. This raises a fundamental question in biology as to how proteins are synthesized in periods of oxygen scarcity and eIF4E inhibition9. Here, we uncover an oxygen-regulated translation initiation complex that mediates selective cap-dependent protein synthesis. Hypoxia stimulates the formation of a complex that includes the oxygen-regulated hypoxia-inducible factor 2α (HIF-2α), the RNA binding protein RBM4 and the cap-binding eIF4E2, an eIF4E homologue. PAR-CLIP10 analysis identified an RNA hypoxia response element (rHRE) that recruits this complex to a wide array mRNAs, including the epidermal growth factor receptor (EGFR). Once assembled at the rHRE, HIF-2α/RBM4/eIF4E2 captures the 5′cap and targets mRNAs to polysomes for active translation thereby evading hypoxia-induced repression of protein synthesis. These findings demonstrate that cells have evolved a program whereby oxygen tension switches the basic translation initiation machinery. PMID:22678294

  10. Temperature-Regulated Protein Synthesis by Leptospira interrogans

    PubMed Central

    Nally, Jarlath E.; Timoney, John F.; Stevenson, Brian

    2001-01-01

    Leptospira interrogans is an important mammalian pathogen. Transmission from an environmental source requires adaptations to a range of new environmental conditions in the organs and tissues of the infected host. Since many pathogenic bacteria utilize temperature to discern their environment and regulate the synthesis of appropriate proteins, we investigated the effects of temperature on protein synthesis in L. interrogans. Bacteria were grown for several days after culture temperatures were shifted from 30 to 37°C. Triton X-114 cellular fractionation identified several proteins of the cytoplasm, periplasm, and outer membrane for which synthesis was dependent on the culture temperature. Synthesis of a cytoplasmic protein of 20 kDa was switched off at 37°C, whereas synthesis of a 66-kDa periplasmic protein was increased at the higher temperature. Increased synthesis of a 25-kDa outer membrane protein was observed when the organisms were shifted from 30 to 37°C. A 36-kDa protein synthesized at 30 but not at 37°C was identified as LipL36, an outer membrane lipoprotein. In contrast, expression of another lipoprotein, LipL41, was the same at either temperature. Immunoblotting with convalescent equine sera revealed that some proteins exhibiting thermoregulation of synthesis elicited antibody responses during infection. Our results show that sera from horses which aborted as a result of naturally acquired infection with L. interrogans serovar pomona type kennewicki recognize periplasmic and outer membrane proteins which are differentially synthesized in response to temperature and which therefore may be important in the host-pathogen interaction during infection. PMID:11119530

  11. Redox regulation by reversible protein S-thiolation in bacteria

    PubMed Central

    Loi, Vu Van; Rossius, Martina; Antelmann, Haike

    2015-01-01

    Low molecular weight (LMW) thiols function as thiol-redox buffers to maintain the reduced state of the cytoplasm. The best studied LMW thiol is the tripeptide glutathione (GSH) present in all eukaryotes and Gram-negative bacteria. Firmicutes bacteria, including Bacillus and Staphylococcus species utilize the redox buffer bacillithiol (BSH) while Actinomycetes produce the related redox buffer mycothiol (MSH). In eukaryotes, proteins are post-translationally modified to S-glutathionylated proteins under conditions of oxidative stress. S-glutathionylation has emerged as major redox-regulatory mechanism in eukaryotes and protects active site cysteine residues against overoxidation to sulfonic acids. First studies identified S-glutathionylated proteins also in Gram-negative bacteria. Advances in mass spectrometry have further facilitated the identification of protein S-bacillithiolations and S-mycothiolation as BSH- and MSH-mixed protein disulfides formed under oxidative stress in Firmicutes and Actinomycetes, respectively. In Bacillus subtilis, protein S-bacillithiolation controls the activities of the redox-sensing OhrR repressor and the methionine synthase MetE in vivo. In Corynebacterium glutamicum, protein S-mycothiolation was more widespread and affected the functions of the maltodextrin phosphorylase MalP and thiol peroxidase (Tpx). In addition, novel bacilliredoxins (Brx) and mycoredoxins (Mrx1) were shown to function similar to glutaredoxins in the reduction of BSH- and MSH-mixed protein disulfides. Here we review the current knowledge about the functions of the bacterial thiol-redox buffers glutathione, bacillithiol, and mycothiol and the role of protein S-thiolation in redox regulation and thiol protection in model and pathogenic bacteria. PMID:25852656

  12. Regulation of protein degradation in muscle by calcium

    NASA Technical Reports Server (NTRS)

    Zeman, Richard J.; Kameyama, Tsuneo; Matsumoto, Kazue; Bernstein, Paul; Etlinger, Joseph D.

    1985-01-01

    Calcium-dependent regulation of intracellular protein degradation was studied in isolated rat skeletal muscles incubated in vitro in the presence of a large variety of agents known to affect calcium movement and distribution. The effect of different classes of protease inhibitors was tested to determine the responsible proteolytic systems involved in calcium-dependent degradation. The results suggest that nonlysosomal leupetin- and E-64-c-sensitive proteases are resposible for calcium-dependent proteolysis in muscle.

  13. The protein kinase LKB1 negatively regulates bone morphogenetic protein receptor signaling

    PubMed Central

    Raja, Erna; Edlund, Karolina; Kahata, Kaoru; Zieba, Agata; Morén, Anita; Watanabe, Yukihide; Voytyuk, Iryna; Botling, Johan; Söderberg, Ola; Micke, Patrick; Pyrowolakis, George; Heldin, Carl-Henrik; Moustakas, Aristidis

    2016-01-01

    The protein kinase LKB1 regulates cell metabolism and growth and is implicated in intestinal and lung cancer. Bone morphogenetic protein (BMP) signaling regulates cell differentiation during development and tissue homeostasis. We demonstrate that LKB1 physically interacts with BMP type I receptors and requires Smad7 to promote downregulation of the receptor. Accordingly, LKB1 suppresses BMP-induced osteoblast differentiation and affects BMP signaling in Drosophila wing longitudinal vein morphogenesis. LKB1 protein expression and Smad1 phosphorylation analysis in a cohort of non-small cell lung cancer patients demonstrated a negative correlation predominantly in a subset enriched in adenocarcinomas. Lung cancer patient data analysis indicated strong correlation between LKB1 loss-of-function mutations and high BMP2 expression, and these two events further correlated with expression of a gene subset functionally linked to apoptosis and migration. This new mechanism of BMP receptor regulation by LKB1 has ramifications in physiological organogenesis and disease. PMID:26701726

  14. Differential regulation of actin microfilaments by human MICAL proteins

    PubMed Central

    Giridharan, Sai Srinivas Panapakkam; Rohn, Jennifer L.; Naslavsky, Naava; Caplan, Steve

    2012-01-01

    The Drosophila melanogaster MICAL protein is essential for the neuronal growth cone machinery that functions through plexin- and semaphorin-mediated axonal signaling. Drosophila MICAL is also involved in regulating myofilament organization and synaptic structures, and serves as an actin disassembly factor downstream of plexin-mediated axonal repulsion. In mammalian cells there are three known isoforms, MICAL1, MICAL2 and MICAL3, as well as the MICAL-like proteins MICAL-L1 and MICAL-L2, but little is known of their function, and information comes almost exclusively from neural cells. In this study we show that in non-neural cells human MICALs are required for normal actin organization, and all three MICALs regulate actin stress fibers. Moreover, we provide evidence that the generation of reactive oxygen species by MICAL proteins is crucial for their actin-regulatory function. However, although MICAL1 is auto-inhibited by its C-terminal coiled-coil region, MICAL2 remains constitutively active and affects stress fibers. These data suggest differential but complementary roles for MICAL1 and MICAL2 in actin microfilament regulation. PMID:22331357

  15. Chromatin-regulating proteins as targets for cancer therapy

    PubMed Central

    Oike, Takahiro; Ogiwara, Hideaki; Amornwichet, Napapat; Nakano, Takashi; Kohno, Takashi

    2014-01-01

    Chromatin-regulating proteins represent a large class of novel targets for cancer therapy. In the context of radiotherapy, acetylation and deacetylation of histones by histone acetyltransferases (HATs) and histone deacetylases (HDACs) play important roles in the repair of DNA double-strand breaks generated by ionizing irradiation, and are therefore attractive targets for radiosensitization. Small-molecule inhibitors of HATs (garcinol, anacardic acid and curcumin) and HDACs (vorinostat, sodium butyrate and valproic acid) have been shown to sensitize cancer cells to ionizing irradiation in preclinical models, and some of these molecules are being tested in clinical trials, either alone or in combination with radiotherapy. Meanwhile, recent large-scale genome analyses have identified frequent mutations in genes encoding chromatin-regulating proteins, especially in those encoding subunits of the SWI/SNF chromatin-remodeling complex, in various human cancers. These observations have driven researchers toward development of targeted therapies against cancers carrying these mutations. DOT1L inhibition in MLL-rearranged leukemia, EZH2 inhibition in EZH2-mutant or MLL-rearranged hematologic malignancies and SNF5-deficient tumors, BRD4 inhibition in various hematologic malignancies, and BRM inhibition in BRG1-deficient tumors have demonstrated promising anti-tumor effects in preclinical models, and these strategies are currently awaiting clinical application. Overall, the data collected so far suggest that targeting chromatin-regulating proteins is a promising strategy for tomorrow's cancer therapy, including radiotherapy and molecularly targeted chemotherapy. PMID:24522270

  16. A Repressor Protein Complex Regulates Leaf Growth in Arabidopsis

    PubMed Central

    Gonzalez, Nathalie; Pauwels, Laurens; Baekelandt, Alexandra; De Milde, Liesbeth; Van Leene, Jelle; Besbrugge, Nienke; Heyndrickx, Ken S.; Pérez, Amparo Cuéllar; Durand, Astrid Nagels; De Clercq, Rebecca; Van De Slijke, Eveline; Vanden Bossche, Robin; Eeckhout, Dominique; Gevaert, Kris; Vandepoele, Klaas; De Jaeger, Geert; Goossens, Alain; Inzé, Dirk

    2015-01-01

    Cell number is an important determinant of final organ size. In the leaf, a large proportion of cells are derived from the stomatal lineage. Meristemoids, which are stem cell-like precursor cells, undergo asymmetric divisions, generating several pavement cells adjacent to the two guard cells. However, the mechanism controlling the asymmetric divisions of these stem cells prior to differentiation is not well understood. Here, we characterized PEAPOD (PPD) proteins, the only transcriptional regulators known to negatively regulate meristemoid division. PPD proteins interact with KIX8 and KIX9, which act as adaptor proteins for the corepressor TOPLESS. D3-type cyclin encoding genes were identified among direct targets of PPD2, being negatively regulated by PPDs and KIX8/9. Accordingly, kix8 kix9 mutants phenocopied PPD loss-of-function producing larger leaves resulting from increased meristemoid amplifying divisions. The identified conserved complex might be specific for leaf growth in the second dimension, since it is not present in Poaceae (grasses), which also lack the developmental program it controls. PMID:26232487

  17. Control of protein function through regulated protein degradation: biotechnological and biomedical applications.

    PubMed

    Nagpal, Jyotsna; Tan, Ju Lin; Truscott, Kaye N; Heras, Begoña; Dougan, David A

    2013-01-01

    Targeted protein degradation is crucial for the correct function and maintenance of a cell. In bacteria, this process is largely performed by a handful of ATP-dependent machines, which generally consist of two components - an unfoldase and a peptidase. In some cases, however, substrate recognition by the protease may be regulated by specialized delivery factors (known as adaptor proteins). Our detailed understanding of how these machines are regulated to prevent uncontrolled degradation within a cell has permitted the identification of novel antimicrobials that dysregulate these machines, as well as the development of tunable degradation systems that have applications in biotechnology. Here, we focus on the physiological role of the ClpP peptidase in bacteria, its role as a novel antibiotic target and the use of protein degradation as a biotechnological approach to artificially control the expression levels of a protein of interest. PMID:23920496

  18. RNA structures regulating ribosomal protein biosynthesis in bacilli.

    PubMed

    Deiorio-Haggar, Kaila; Anthony, Jon; Meyer, Michelle M

    2013-07-01

    In Bacilli, there are three experimentally validated ribosomal-protein autogenous regulatory RNAs that are not shared with E. coli. Each of these RNAs forms a unique secondary structure that interacts with a ribosomal protein encoded by a downstream gene, namely S4, S15, and L20. Only one of these RNAs that interacts with L20 is currently found in the RNA Families Database. We created, or modified, existing structural alignments for these three RNAs and used them to perform homology searches. We have determined that each structure exhibits a narrow phylogenetic distribution, mostly relegated to the Firmicute class Bacilli. This work, in conjunction with other similar work, demonstrates that there are most likely many non-homologous RNA regulatory elements regulating ribosomal protein biosynthesis that still await discovery and characterization in other bacterial species. PMID:23611891

  19. RNA structures regulating ribosomal protein biosynthesis in bacilli

    PubMed Central

    Deiorio-Haggar, Kaila; Anthony, Jon; Meyer, Michelle M.

    2013-01-01

    In Bacilli, there are three experimentally validated ribosomal-protein autogenous regulatory RNAs that are not shared with E. coli. Each of these RNAs forms a unique secondary structure that interacts with a ribosomal protein encoded by a downstream gene, namely S4, S15, and L20. Only one of these RNAs that interacts with L20 is currently found in the RNA Families Database. We created, or modified, existing structural alignments for these three RNAs and used them to perform homology searches. We have determined that each structure exhibits a narrow phylogenetic distribution, mostly relegated to the Firmicute class Bacilli. This work, in conjunction with other similar work, demonstrates that there are most likely many non-homologous RNA regulatory elements regulating ribosomal protein biosynthesis that still await discovery and characterization in other bacterial species. PMID:23611891

  20. Transcriptional regulation of decreased protein synthesis during skeletal muscle unloading

    NASA Technical Reports Server (NTRS)

    Howard, G.; Steffen, J. M.; Geoghegan, T. E.

    1989-01-01

    The regulatory role of transcriptional alterations in unloaded skeletal muscles was investigated by determining levels of total muscle RNA and mRNA fractions in soleus, gastrocnemius, and extensor digitorum longus (EDL) of rats subjected to whole-body suspension for up to 7 days. After 7 days, total RNA and mRNA contents were lower in soleus and gastrocnemius, compared with controls, but the concentrations of both RNAs per g muscle were unaltered. Alpha-actin mRNA (assessed by dot hybridization) was significantly reduced in soleus after 1, 3, and 7 days of suspension and in gastrocnemius after 3 and 7 days, but was unchanged in EDL. Protein synthesis directed by RNA extracted from soleus and EDL indicated marked alteration in mRNAs coding for several small proteins. Results suggest that altered transcription and availability of specific mRNAs contribute significantly to the regulation of protein synthesis during skeletal muscle unloading.

  1. Protein-Tyrosine Phosphatase 1B Substrates and Metabolic Regulation

    PubMed Central

    Bakke, Jesse; Haj, Fawaz G.

    2014-01-01

    Metabolic homeostasis requires integration of complex signaling networks which, when deregulated, contribute to metabolic syndrome and related disorders. Protein-tyrosine phosphatase 1B (PTP1B) has emerged as a key regulator of signaling networks that are implicated in metabolic diseases such as obesity and type 2 diabetes. In this review, we examine mechanisms that regulate PTP1B-substrate interaction, enzymatic activity and experimental approaches to identify PTP1B substrates. We then highlight findings that implicate PTP1B in metabolic regulation. In particular, insulin and leptin signaling are discussed as well as recently identified PTP1B substrates that are involved in endoplasmic reticulum stress response, cell-cell communication, energy balance and vesicle trafficking. In summary, PTP1B exhibits exquisite substrate specificity and is an outstanding pharmaceutical target for obesity and type 2 diabetes. PMID:25263014

  2. Heat Shock Protein 90 regulates encystation in Entamoeba

    PubMed Central

    Singh, Meetali; Sharma, Shalini; Bhattacharya, Alok; Tatu, Utpal

    2015-01-01

    Enteric protozoan Entamoeba histolytica is a major cause of debilitating diarrheal infection worldwide with high morbidity and mortality. Even though the clinical burden of this parasite is very high, this infection is categorized as a neglected disease. Parasite is transmitted through feco-oral route and exhibit two distinct stages namely – trophozoites and cysts. Mechanism and regulation of encystation is not clearly understood. Previous studies have established the role of Heat shock protein 90 (Hsp90) in regulating stage transition in various protozoan parasites like Giardia, Plasmodium, Leishmania, and Toxoplasma. Our study for the first time reports that Hsp90 plays a crucial role in life cycle of Entamoeba as well. We identify Hsp90 to be a negative regulator of encystation in Entamoeba. We also show that Hsp90 inhibition interferes with the process of phagocytosis in Entamoeba. Overall, we show that Hsp90 plays an important role in virulence and transmission of Entamoeba. PMID:26528271

  3. Function and Regulation of Heterotrimeric G Proteins during Chemotaxis

    PubMed Central

    Kamp, Marjon E.; Liu, Youtao; Kortholt, Arjan

    2016-01-01

    Chemotaxis, or directional movement towards an extracellular gradient of chemicals, is necessary for processes as diverse as finding nutrients, the immune response, metastasis and wound healing. Activation of G-protein coupled receptors (GPCRs) is at the very base of the chemotactic signaling pathway. Chemotaxis starts with binding of the chemoattractant to GPCRs at the cell-surface, which finally leads to major changes in the cytoskeleton and directional cell movement towards the chemoattractant. Many chemotaxis pathways that are directly regulated by Gβγ have been identified and studied extensively; however, whether Gα is just a handle that regulates the release of Gβγ or whether Gα has its own set of distinct chemotactic effectors, is only beginning to be understood. In this review, we will discuss the different levels of regulation in GPCR signaling and the downstream pathways that are essential for proper chemotaxis. PMID:26784171

  4. Regulation of cholesterol esterification by protein kinase C

    SciTech Connect

    Jeng, I.; Dills, C.; Klemm, N.; Wu, C.

    1986-03-05

    They have recently identified acyl-CoA cholesterol acyltransferase as the key enzyme for cholesterol esterification in the central nervous system. They found that the activity of glial acyl-CoA cholesterol acyltransferase could be controlled by a phosphorylation-dephosphorylation mechanism. However, repeated attempts to identify cyclic AMP as the bioregulator for this reaction failed. Recently, they have studied the possible involvement of protein kinase C in the regulation of glial cholesterol esterification. Phorbol-12-myristate 13-acetate (PMA) can activate cellular cholesterol esterification in a complex, time-dependent manner. Phorbol analogues inactive toward protein kinase C are also ineffective in this assay. Furthermore, oleoyl-acetyl-glycerol mimics the effect of PMA, confirming the proposal that protein kinase C mediates the effect of these compounds and that the natural bioregulator is probably diacylglycerol. Receptor-mediated polyphosphatidyl-inositol cleavage often produces diacylglycerol and inositol triphosphate. The synergic effects of these two compounds are known to be necessary to elicit other biological responses. Their preliminary studies using calcium ionophore A23187 indicates that Ca/sup + +/ is not required for cellular cholesterol esterification. In sum, glial cholesterol esterification is probably regulated by a calcium-independent and protein kinase C-dependent reaction.

  5. 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

  6. Small Molecule Proteostasis Regulators for Protein Conformational Diseases

    PubMed Central

    Calamini, Barbara; Silva, Maria Catarina; Madoux, Franck; Hutt, Darren M.; Khanna, Shilpi; Chalfant, Monica A.; Saldanha, Sanjay A.; Hodder, Peter; Tait, Bradley D.; Garza, Dan; Balch, William E.; Morimoto, Richard I.

    2011-01-01

    Protein homeostasis (proteostasis) is essential for cellular and organismal health. Stress, aging, and the chronic expression of misfolded proteins, however, challenge the proteostasis machinery and the vitality of the cell. Enhanced expression of molecular chaperones, regulated by heat shock transcription factor-1 (HSF-1), has been shown to restore proteostasis in a variety of conformational disease models, suggesting a promising therapeutic approach. We describe the results of a ∼900,000 small molecule screen that identified novel classes of small molecule proteostasis regulators (PRs) that induce HSF-1-dependent chaperone expression and restore protein folding in multiple conformational disease models. The beneficial effects to proteome stability are mediated by HSF-1, DAF-16/FOXO, SKN-1/Nrf-2, and the chaperone machinery through mechanisms that are distinct from current known small molecule activators of the HSR. We suggest that modulation of the proteostasis network by PRs represents a promising therapeutic approach for the treatment of a variety of protein conformational diseases. PMID:22198733

  7. [cAMP cascade in regulation of protein glycosylation].

    PubMed

    Surman, Magdalena; Janik, Marcelina

    2014-01-01

    O- and N-glycosylation are the most common and complex of the post-translational modifications. Both are enzymatic processes and it was suggested that both could be regulated by cAMP cascade at the early stages. N-glycosylation starts with the formation of lipid-linked oligosaccharides and this process is catalysed by crucial glycosyltransferase - dolichol phosphate mannose synthase. The results of several studies strongly suggest that the cAMP acting through a cAMP-dependent protein kinase A-mediated protein phosphorylation/dephosphorylation cycle may modulate activation of this enzyme. It was shown that cAMP can also up regulate another enzyme involved in phosphodolichole synthesis - cis-prenyltransferase. The mechanism acting here is the alteration of the rate of its gene expression. cAMP cascade is also involved in regulation of O-glycosylation since phosphorylation of human glutamine:fructose-6-phosphate amidotransferase results in depletion of O-GlcNAc structure formation. These observation suggested an important role of GPCRs and their ligand in regulation of N- and O-glycan synthesis. PMID:26263760

  8. Proteomic analysis of differentially expressed proteins in the lymphoid organ of Vibrio harveyi-infected Penaeus monodon.

    PubMed

    Chaikeeratisak, Vorrapon; Somboonwiwat, Kunlaya; Wang, Hao-Ching; Lo, Chu Fang; Tassanakajon, Anchalee

    2012-05-01

    The protein expression profiles of the lymphoid organ, taken from mock and systemic Vibrio harveyi-infected Penaeus monodon at 6 and 48 h post infection, were revealed. The considerable changes in the expression level of several proteins were observed between the mock and V. harveyi-infected shrimps. From 30 analyzed protein spots with 27 differentially expressed, 21 were known proteins with the most common of these being cytoskeleton proteins (33%) which were all down-regulated upon systemic bacterial infection. Other six proteins including four proteins that are involved in the shrimp immunity (alpha-2-macroglobulin, transglutaminase, heat shock protein 1 and hemocyanin subunit Y), and two proteins that are involved in metabolism (triosephosphate isomerase) and cell signaling (14-3-3 like protein), displayed significantly decreased expression levels. There was, however, an increase in the expression level of the ATP synthase beta subunit, a protein involved in energy balance. Transcription levels of ATP synthase beta subunit and 14-3-3 like protein were up- and down-regulated, respectively, in accord with the observed protein expression levels, but the alpha-2-macroglobulin transcript levels were significantly increased in contrast to the decreased protein expression levels. Interestingly, partial gene silencing of ATP synthase beta subunit revealed a high cumulative mortality of the knockdown shrimps (73.3%) and a dramatic reduction of the total hemocyte numbers in the survival shrimps. These altered proteins are likely to play essential roles in shrimp defense against the pathogenic bacterium V. harveyi. PMID:22302389

  9. Introns regulate the production of ribosomal proteins by modulating splicing of duplicated ribosomal protein genes

    PubMed Central

    Petibon, Cyrielle; Parenteau, Julie; Catala, Mathieu; Elela, Sherif Abou

    2016-01-01

    Most budding yeast introns exist in the many duplicated ribosomal protein genes (RPGs) and it has been posited that they remain there to modulate the expression of RPGs and cell growth in response to stress. However, the mechanism by which introns regulate the expression of RPGs and their impact on the synthesis of ribosomal proteins remain unclear. In this study, we show that introns determine the ratio of ribosomal protein isoforms through asymmetric paralog-specific regulation of splicing. Exchanging the introns and 3′ untranslated regions of the duplicated RPS9 genes altered the splicing efficiency and changed the ratio of the ribosomal protein isoforms. Mutational analysis of the RPS9 genes indicated that splicing is regulated by variations in the intron structure and the 3′ untranslated region. Together these data suggest that preferential splicing of duplicated RPGs provides a means for adjusting the ratio of different ribosomal protein isoforms, while maintaining the overall expression level of each ribosomal protein. PMID:26945043

  10. Regulation of coat protein polymerization by the scaffolding protein of bacteriophage P22

    SciTech Connect

    Fuller, M.T.; King, J.

    1980-10-01

    In the morphogenesis of double stranded DNA phages, a precursor protein shell empty of DNA is first assembled and then filled with DNA. The assembly of the correctly dimensioned precursor shell (procapsid) of Salmonella bacteriophage P22 requires the interaction of some 420 coat protein subunits with approx. 200 scaffolding protein subunits to form a double shelled particle with the scaffolding protein on the inside. In the course of DNA packaging, all of the scaffolding protein subunits exit from the procapsid and participate in further rounds of procapsid assembly. To study the mechanism of shell assembly we have purified the coat and scaffolding protein subunits by selective dissociation of isolated procapsids. Both proteins can be obtained as soluble sununits in Tris buffer at near neutral pH. The coat protein sedimented in sucrose gradients as a roughly spherical monomer, while the scaffolding protein sedimented as if it were an elongated monomer. When the two proteins were mixed together in 1.5 M guanidine hydrochloride and dialyzed back to buffer at room temperature, procapsids formed which were very similar in morphology, sedimentation behavior, and protein composition to procapsids formed in vivo. Incubation of either protein alone under the same conditions did not yield any large structures. We interpret these results to mean that the assembly of the shell involves a switching of both proteins from their nonaggregating to their aggregating forms through their mutual interaction. The results are discussed in terms of the general problem of self-regulated assembly and the control of protein polymerization in morphogenesis.

  11. Regulation of the epithelial sodium channel by accessory proteins.

    PubMed Central

    Gormley, Kelly; Dong, Yanbin; Sagnella, Giuseppe A

    2003-01-01

    The epithelial sodium channel (ENaC) is of fundamental importance in the control of sodium fluxes in epithelial cells. Modulation of sodium reabsorption through the distal nephron ENaC is an important component in the overall control of sodium balance, blood volume and thereby of blood pressure. This is clearly demonstrated by rare genetic disorders of sodium-channel activity (Liddle's syndrome and pseudohypoaldosteronism type 1), associated with contrasting effects on blood pressure. The mineralocorticoid aldosterone is a well-established modulator of sodium-channel activity. Considerable insight has now been gained into the intracellular signalling pathways linking aldosterone-mediated changes in gene transcription with changes in ion transport. Activating pathways include aldosterone-induced proteins and especially the serum- and glucocorticoid-inducible kinase (SGK) and the small G-protein, K-Ras 2A. Targeting of the ENaC for endocytosis and degradation is now emerging as a major mechanism for the down-regulation of channel activity. Several proteins acting in concert are an intrinsic part of this process but Nedd4 (neural precursor cell expressed developmentally down-regulated 4) is of central importance. Other mechanisms known to interact with ENaC and affect sodium transport include channel-activating protease 1 (CAP-1), a membrane-anchored protein, and the cystic fibrosis transmembrane regulator. The implications of research on accessory factors controlling ENaC activity are wide-ranging. Understanding cellular mechanisms controlling ENaC activity may provide a more detailed insight not only of ion-channel abnormalities in cystic fibrosis but also of the link between abnormal renal sodium transport and essential hypertension. PMID:12460120

  12. Regulation of Sp1 by cell cycle related proteins

    PubMed Central

    Tapias, Alicia; Ciudad, Carlos J.; Roninson, Igor B.; Noé, Véronique

    2009-01-01

    Sp1 transcription factor regulates the expression of multiple genes, including the Sp1 gene itself. We analyzed the ability of different cell cycle regulatory proteins to interact with Sp1 and to affect Sp1 promoter activity. Using an antibody array, we observed that CDK4, SKP2, Rad51, BRCA2 and p21 could interact with Sp1 and we confirmed these interactions by co-immunoprecipitation. CDK4, SKP2, Rad51, BRCA2 and p21 also activated the Sp1 promoter. Among the known Sp1-interacting proteins, E2F-DP1, Cyclin D1, Stat3 and Rb activated the Sp1 promoter, whereas p53 and NFκB inhibited it. The proteins that regulated Sp1 gene expression were shown by positive chromatin immunoprecipitation to be bound to the Sp1 promoter. Moreover, SKP2, BRCA2, p21, E2F-DP1, Stat3, Rb, p53 and NFκB had similar effects on an artificial promoter containing only Sp1 binding sites. Transient transfections of CDK4, Rad51, E2F-DP1, p21 and Stat3 increased mRNA expression from the endogenous Sp1 gene in HeLa cells whereas overexpression of NFκB, and p53 decreased Sp1 mRNA levels. p21 expression from a stably integrated inducible promoter in HT1080 cells activated Sp1 expression at the promoter and mRNA levels, but at the same time it decreased Sp1 protein levels due to the activation of Sp1 degradation. The observed multiple effects of cell cycle regulators on Sp1 suggest that Sp1 may be a key mediator of cell cycle associated changes in gene expression. PMID:18769160

  13. Regulation of protein synthesis during sea urchin early development

    SciTech Connect

    Kelso, L.C.

    1989-01-01

    Fertilization of the sea urchin egg results in a 20-40 fold increase in the rate of protein synthesis. The masked message hypothesis proposes that mRNAs are masked or unavailable for translation in the egg. We devised an in vivo assay to test this hypothesis. Our results show that masked mRNAs limit protein synthesis in the unfertilized egg. In addition, we show that protein synthesis is also regulated at the level of translational machinery. Following fertilization is a period of rapid cell divisions. This period, known as the rapid cleavage stage, is characterized by the transient synthesis of a novel set of proteins. The synthesis of these proteins is programmed by maternal mRNAs stored in the unfertilized egg. To study the behavior of these mRNAs, we prepared a cDNA library from polysomal poly (A+) RNA from 2-hour embryos. ({sup 32}P) labeled probes, prepared from the cDNA library, were used to monitor the levels of individual mRNAs in polysomes at fertilization and during early development.

  14. Chapter Two - Heterotrimeric G Protein Ubiquitination as a Regulator of G Protein Signaling.

    PubMed

    Torres, M

    2016-01-01

    Ubiquitin-mediated regulation of G proteins has been known for over 20 years as a result of discoveries made independently in yeast and vertebrate model systems for pheromone and photoreception, respectively. Since that time, several details underlying the cause and effect of G protein ubiquitination have been determined-including the initiating signals, responsible enzymes, trafficking pathways, and their effects on protein structure, function, interactions, and cell signaling. The collective body of evidence suggests that Gα subunits are the primary targets of ubiquitination. However, longstanding and recent results suggest that Gβ and Gγ subunits are also ubiquitinated, in some cases impacting cell polarization-a process essential for chemotaxis and polarized cell growth. More recently, evidence from mass spectrometry (MS)-based proteomics coupled with advances in PTM bioinformatics have revealed that protein families representing G protein subunits contain several structural hotspots for ubiquitination-most of which have not been investigated for a functional role in signal transduction. Taken together, our knowledge and understanding of heterotrimeric G protein ubiquitination as a regulator of G protein signaling-despite 20 years of research-is still emerging. PMID:27378755

  15. Protein stability regulators screening assay (Pro-SRSA): protein degradation meets the CRISPR-Cas9 library.

    PubMed

    Wu, Yuanzhong; Kang, Tiebang

    2016-01-01

    The regulation of protein stability is a fundamental issue for biophysical processes, but there has not previously been a convenient and unbiased method of identifying regulators of protein stability. However, as reported in the article entitled "A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A," recently published in Cell Discovery, our team developed a protein stability regulators screening assay (Pro-SRSA) by combining the whole-genome clustered regularly interspaced short palindromic repeats Cas9 (CRISPR-Cas9) library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen for regulators of protein stability. Based on our findings, we are confident that this efficient and unbiased screening method at the genome scale will be used by researchers worldwide to identify regulators of protein stability. PMID:27357860

  16. Regulation of protein glycosylation and sorting by the Golgi matrix proteins GRASP55/65

    PubMed Central

    Xiang, Yi; Zhang, Xiaoyan; Nix, David B.; Katoh, Toshihiko; Aoki, Kazuhiro; Tiemeyer, Michael; Wang, Yanzhuang

    2013-01-01

    The Golgi receives the entire output of newly synthesized cargo from the endoplasmic reticulum (ER), processes it in the stack largely through modification of bound oligosaccharides, and sorts it in the trans-Golgi network (TGN). GRASP65 and GRASP55, two proteins localized to the Golgi stack and early secretory pathway, mediate processes including Golgi stacking, Golgi ribbon linking, and unconventional secretion. Previously we have shown that GRASP depletion in cells disrupts Golgi stack formation. Here we report that knockdown of the GRASP proteins, alone or combined, accelerates protein trafficking through the Golgi membranes but also has striking negative effects on protein glycosylation and sorting. These effects are not caused by Golgi ribbon unlinking, unconventional secretion, or ER stress. We propose that GRASP55/65 are negative regulators of exocytic transport and that this slowdown helps to ensure more complete protein glycosylation in the Golgi stack and proper sorting at the TGN. PMID:23552074

  17. Studying allosteric regulation in metal sensor proteins using computational methods.

    PubMed

    Chakravorty, Dhruva K; Merz, Kenneth M

    2014-01-01

    In this chapter, we describe advances made in understanding the mechanism of allosteric regulation of DNA operator binding in the ArsR/SmtB family of metal-sensing proteins using computational methods. The paradigm, zinc-sensing transcriptional repressor Staphylococcus aureus CzrA represents an excellent model system to understand how metal sensor proteins maintain cellular metal homeostasis. Here, we discuss studies that helped to characterize a metal ion-mediated hydrogen-bonding pathway (HBP) that plays a dominant role in the allosteric mechanism of DNA operator binding in these proteins. The chapter discusses computational methods used to provide a molecular basis for the large conformational motions and allosteric coupling free energy (~6kcal/mol) associated with Zn(II) binding in CzrA. We present an accurate and convenient means by which to include metal ions in the nuclear magnetic resonance (NMR) structure determination process using molecular dynamics (MD) constrained by NMR-derived data. The method provides a realistic and physically viable description of the metal-binding site(s) and has potentially broad applicability in the structure determination of metal ion-bound proteins, protein folding, and metal template protein-design studies. Finally, our simulations provide strong support for a proposed HBP that physically connects the metal-binding residue, His97, to the DNA-binding interface through the αR helix that is present only in the Zn(II)-bound state. We find the interprotomer hydrogen bond interaction to be significantly stronger (~8kcal/mol) at functional allosteric metal-binding sites compared to the apo proteins. This interaction works to overcome the considerable disorder at these hydrogen-bonding sites in apo protein and functions as a "switch" to lock in a weak DNA-binding conformation once metal is bound. This interaction is found to be considerably weaker in nonresponsive metal-binding sites. These findings suggest a conserved functional

  18. JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships.

    PubMed

    Zeke, András; Misheva, Mariya; Reményi, Attila; Bogoyevitch, Marie A

    2016-09-01

    The c-Jun N-terminal kinases (JNKs), as members of the mitogen-activated protein kinase (MAPK) family, mediate eukaryotic cell responses to a wide range of abiotic and biotic stress insults. JNKs also regulate important physiological processes, including neuronal functions, immunological actions, and embryonic development, via their impact on gene expression, cytoskeletal protein dynamics, and cell death/survival pathways. Although the JNK pathway has been under study for >20 years, its complexity is still perplexing, with multiple protein partners of JNKs underlying the diversity of actions. Here we review the current knowledge of JNK structure and isoforms as well as the partnerships of JNKs with a range of intracellular proteins. Many of these proteins are direct substrates of the JNKs. We analyzed almost 100 of these target proteins in detail within a framework of their classification based on their regulation by JNKs. Examples of these JNK substrates include a diverse assortment of nuclear transcription factors (Jun, ATF2, Myc, Elk1), cytoplasmic proteins involved in cytoskeleton regulation (DCX, Tau, WDR62) or vesicular transport (JIP1, JIP3), cell membrane receptors (BMPR2), and mitochondrial proteins (Mcl1, Bim). In addition, because upstream signaling components impact JNK activity, we critically assessed the involvement of signaling scaffolds and the roles of feedback mechanisms in the JNK pathway. Despite a clarification of many regulatory events in JNK-dependent signaling during the past decade, many other structural and mechanistic insights are just beginning to be revealed. These advances open new opportunities to understand the role of JNK signaling in diverse physiological and pathophysiological states. PMID:27466283

  19. Autopalmitoylation of TEAD Proteins Regulates Transcriptional Output of Hippo Pathway

    PubMed Central

    Chan, PuiYee; Han, Xiao; Zheng, Baohui; DeRan, Michael; Yu, Jianzhong; Jarugumilli, Gopala K.; Deng, Hua; Pan, Duojia; Luo, Xuelian; Wu, Xu

    2016-01-01

    TEA domain (TEAD) transcription factors bind to the co-activator YAP/TAZ, and regulate the transcriptional output of Hippo pathway, playing critical roles in organ size control and tumorigenesis. Protein S-palmitoylation attaches fatty acid (palmitate) to cysteine residues, and regulates protein trafficking, membrane localization and signaling activities. Using activity-based chemical probes, we discovered that human TEADs possess intrinsic palmitoylating enzyme-like activities, and undergo autopalmitoylation at evolutionarily conserved cysteine residues under physiological conditions. We determined the crystal structures of lipid-bound TEADs, and found that the lipid chain of palmitate inserts into a conserved deep hydrophobic pocket. Strikingly, palmitoylation is required for TEAD’s binding to YAP/TAZ, but dispensable for the binding to Vgll4 tumor suppressor. In addition, palmitoylation does not alter TEAD’s localization. Moreover, TEAD palmitoylation-deficient mutants impaired TAZ-mediated muscle differentiation in vitro, and Yorkie-mediated tissue overgrowth in Drosophila in vivo. Our study directly linked autopalmitoylation to the transcriptional regulation of Hippo pathway. PMID:26900866

  20. Regulation of RAG-2 protein expression in avian thymocytes.

    PubMed Central

    Ferguson, S E; Accavitti, M A; Wang, D D; Chen, C L; Thompson, C B

    1994-01-01

    The recombinase-activating genes, RAG-1 and RAG-2, have been shown to be necessary to initiate the process of V(D)J recombination during the ontogeny of lymphocytes. While much is known about the end products of this rearrangement process, little is known about the function or regulation of the components of the recombinase system. To this end, we have generated a monoclonal antibody to the chicken RAG-2 protein. Chicken thymocytes were found to express high levels of RAG-2, part of which is phosphorylated. Within thymocytes, RAG-2 is expressed primarily within the nucleus. RAG-2 protein levels are high in the CD4- CD8- and CD4+ CD8+ immature thymocytes but absent at the single-positive CD4+ CD8- or CD4- CD8+ stage of thymocyte development. Mitogenic stimulation of thymocytes with phorbol myristate acetate and ionomycin results in down-regulation of RAG-2 expression. Consistent with these data, in vivo levels of RAG-2 are markedly lower in proliferating thymocytes than in smaller, G0/G1 cells. Down-regulation of RAG-2 expression appears to occur before cells enter S phase, suggesting that RAG-2 function may be limited to noncycling cells. Images PMID:7935443

  1. Type One Protein Phosphatase 1 and Its Regulatory Protein Inhibitor 2 Negatively Regulate ABA Signaling

    PubMed Central

    Zhao, Yang; Xie, Shaojun; Batelli, Giorgia; Wang, Bangshing; Duan, Cheng-Guo; Wang, Xingang; Xing, Lu; Lei, Mingguang; Yan, Jun; Zhu, Xiaohong; Zhu, Jian-Kang

    2016-01-01

    The phytohormone abscisic acid (ABA) regulates plant growth, development and responses to biotic and abiotic stresses. The core ABA signaling pathway consists of three major components: ABA receptor (PYR1/PYLs), type 2C Protein Phosphatase (PP2C) and SNF1-related protein kinase 2 (SnRK2). Nevertheless, the complexity of ABA signaling remains to be explored. To uncover new components of ABA signal transduction pathways, we performed a yeast two-hybrid screen for SnRK2-interacting proteins. We found that Type One Protein Phosphatase 1 (TOPP1) and its regulatory protein, At Inhibitor-2 (AtI-2), physically interact with SnRK2s and also with PYLs. TOPP1 inhibited the kinase activity of SnRK2.6, and this inhibition could be enhanced by AtI-2. Transactivation assays showed that TOPP1 and AtI-2 negatively regulated the SnRK2.2/3/6-mediated activation of the ABA responsive reporter gene RD29B, supporting a negative role of TOPP1 and AtI-2 in ABA signaling. Consistent with these findings, topp1 and ati-2 mutant plants displayed hypersensitivities to ABA and salt treatments, and transcriptome analysis of TOPP1 and AtI-2 knockout plants revealed an increased expression of multiple ABA-responsive genes in the mutants. Taken together, our results uncover TOPP1 and AtI-2 as negative regulators of ABA signaling. PMID:26943172

  2. Regulation of secretory transport by protein kinase D-mediated phosphorylation of the ceramide transfer protein.

    PubMed

    Fugmann, Tim; Hausser, Angelika; Schöffler, Patrik; Schmid, Simone; Pfizenmaier, Klaus; Olayioye, Monilola A

    2007-07-01

    Protein kinase D (PKD) has been identified as a crucial regulator of secretory transport at the trans-Golgi network (TGN). Recruitment and activation of PKD at the TGN is mediated by the lipid diacylglycerol, a pool of which is generated by sphingomyelin synthase from ceramide and phosphatidylcholine. The nonvesicular transfer of ceramide from the endoplasmic reticulum to the Golgi complex is mediated by the lipid transfer protein CERT (ceramide transport). In this study, we identify CERT as a novel in vivo PKD substrate. Phosphorylation on serine 132 by PKD decreases the affinity of CERT toward its lipid target phosphatidylinositol 4-phosphate at Golgi membranes and reduces ceramide transfer activity, identifying PKD as a regulator of lipid homeostasis. We also show that CERT, in turn, is critical for PKD activation and PKD-dependent protein cargo transport to the plasma membrane. Thus, the interdependence of PKD and CERT is key to the maintenance of Golgi membrane integrity and secretory transport. PMID:17591919

  3. Regulation of secretory transport by protein kinase D–mediated phosphorylation of the ceramide transfer protein

    PubMed Central

    Fugmann, Tim; Hausser, Angelika; Schöffler, Patrik; Schmid, Simone; Pfizenmaier, Klaus; Olayioye, Monilola A.

    2007-01-01

    Protein kinase D (PKD) has been identified as a crucial regulator of secretory transport at the trans-Golgi network (TGN). Recruitment and activation of PKD at the TGN is mediated by the lipid diacylglycerol, a pool of which is generated by sphingomyelin synthase from ceramide and phosphatidylcholine. The nonvesicular transfer of ceramide from the endoplasmic reticulum to the Golgi complex is mediated by the lipid transfer protein CERT (ceramide transport). In this study, we identify CERT as a novel in vivo PKD substrate. Phosphorylation on serine 132 by PKD decreases the affinity of CERT toward its lipid target phosphatidylinositol 4-phosphate at Golgi membranes and reduces ceramide transfer activity, identifying PKD as a regulator of lipid homeostasis. We also show that CERT, in turn, is critical for PKD activation and PKD-dependent protein cargo transport to the plasma membrane. Thus, the interdependence of PKD and CERT is key to the maintenance of Golgi membrane integrity and secretory transport. PMID:17591919

  4. The RHOX Homeodomain Proteins Regulate the Expression of Insulin and Other Metabolic Regulators in the Testis*

    PubMed Central

    MacLean, James A.; Hu, Zhiying; Welborn, Joshua P.; Song, Hye-Won; Rao, Manjeet K.; Wayne, Chad M.; Wilkinson, Miles F.

    2013-01-01

    Defects in cellular metabolism have been widely implicated in causing male infertility, but there has been little progress in understanding the underlying mechanism. Here we report that several key metabolism genes are regulated in the testis by Rhox5, the founding member of a large X-linked homeobox gene cluster. Among these Rhox5-regulated genes are insulin 2 (Ins2), resistin (Retn), and adiponectin (Adipoq), all of which encode secreted proteins that have profound and wide-ranging effects on cellular metabolism. The ability of Rhox5 to regulate their levels in the testis has the potential to dictate metabolism locally in this organ, given the existence of the blood-testes barrier. We demonstrate that Ins2 is a direct target of Rhox5 in Sertoli cells, and we show that this regulation is physiologically significant, because Rhox5-null mice fail to up-regulate Ins2 expression during the first wave of spermatogenesis and have insulin-signaling defects. We identify other Rhox family members that induce Ins2 transcription, define protein domains and homeodomain amino acid residues crucial for this property, and demonstrate that this regulation is conserved. Rhox5-null mice also exhibit altered expression of other metabolism genes, including those encoding the master transcriptional regulators of metabolism, PPARG and PPARGC1A, as well as SCD1, the rate-limiting enzyme for fatty acid metabolism. These results, coupled with the known roles of RHOX5 and its target metabolism genes in spermatogenesis in vivo, lead us to propose a model in which RHOX5 is a central transcription factor that promotes the survival of male germ cells via its effects on cellular metabolism. PMID:24121513

  5. Regulation of RNA binding proteins in trypanosomatid protozoan parasites.

    PubMed

    Romaniuk, María Albertina; Cervini, Gabriela; Cassola, Alejandro

    2016-02-26

    Posttranscriptional mechanisms have a critical role in the overall outcome of gene expression. These mechanisms are especially relevant in protozoa from the genus Trypanosoma, which is composed by death threatening parasites affecting people in Sub-saharan Africa or in the Americas. In these parasites the classic view of regulation of transcription initiation to modulate the products of a given gene cannot be applied. This is due to the presence of transcription start sites that give rise to long polycistronic units that need to be processed costranscriptionally by trans-splicing and polyadenylation to give mature monocistronic mRNAs. Posttranscriptional mechanisms such as mRNA degradation and translational repression are responsible for the final synthesis of the required protein products. In this context, RNA-binding proteins (RBPs) in trypanosomes have a relevant role as modulators of mRNA abundance and translational repression by associating to the 3' untranslated regions in mRNA. Many different RBPs have been proposed to modulate cohorts of mRNAs in trypanosomes. However, the current understanding of their functions lacks a dynamic view on the different steps at which these RBPs are regulated. Here, we discuss different evidences to propose regulatory events for different RBPs in these parasites. These events vary from regulated developmental expression, to biogenesis of cytoplasmic ribonucleoprotein complexes in the nucleus, and condensation of RBPs and mRNA into large cytoplasmic granules. Finally, we discuss how newly identified posttranslational modifications of RBPs and mRNA metabolism-related proteins could have an enormous impact on the modulation of mRNA abundance. To understand these modifications is especially relevant in these parasites due to the fact that the enzymes involved could be interesting targets for drug therapy. PMID:26981203

  6. Regulation of RNA binding proteins in trypanosomatid protozoan parasites

    PubMed Central

    Romaniuk, María Albertina; Cervini, Gabriela; Cassola, Alejandro

    2016-01-01

    Posttranscriptional mechanisms have a critical role in the overall outcome of gene expression. These mechanisms are especially relevant in protozoa from the genus Trypanosoma, which is composed by death threatening parasites affecting people in Sub-saharan Africa or in the Americas. In these parasites the classic view of regulation of transcription initiation to modulate the products of a given gene cannot be applied. This is due to the presence of transcription start sites that give rise to long polycistronic units that need to be processed costranscriptionally by trans-splicing and polyadenylation to give mature monocistronic mRNAs. Posttranscriptional mechanisms such as mRNA degradation and translational repression are responsible for the final synthesis of the required protein products. In this context, RNA-binding proteins (RBPs) in trypanosomes have a relevant role as modulators of mRNA abundance and translational repression by associating to the 3’ untranslated regions in mRNA. Many different RBPs have been proposed to modulate cohorts of mRNAs in trypanosomes. However, the current understanding of their functions lacks a dynamic view on the different steps at which these RBPs are regulated. Here, we discuss different evidences to propose regulatory events for different RBPs in these parasites. These events vary from regulated developmental expression, to biogenesis of cytoplasmic ribonucleoprotein complexes in the nucleus, and condensation of RBPs and mRNA into large cytoplasmic granules. Finally, we discuss how newly identified posttranslational modifications of RBPs and mRNA metabolism-related proteins could have an enormous impact on the modulation of mRNA abundance. To understand these modifications is especially relevant in these parasites due to the fact that the enzymes involved could be interesting targets for drug therapy. PMID:26981203

  7. Ribozymes, riboswitches and beyond: regulation of gene expression without proteins

    PubMed Central

    Serganov, Alexander; Patel, Dinshaw J.

    2015-01-01

    Although various functions of RNA are carried out in conjunction with proteins, some catalytic RNAs, or ribozymes, which contribute to a range of cellular processes, require little or no assistance from proteins. Furthermore, the discovery of metabolite-sensing riboswitches and other types of RNA sensors has revealed RNA-based mechanisms that cells use to regulate gene expression in response to internal and external changes. Structural studies have shown how these RNAs can carry out a range of functions. In addition, the contribution of ribozymes and riboswitches to gene expression is being revealed as far more widespread than was previously appreciated. These findings have implications for understanding how cellular functions might have evolved from RNA-based origins. PMID:17846637

  8. RCAN 1 and 3 proteins regulate thymic positive selection.

    PubMed

    Serrano-Candelas, Eva; Alemán-Muench, Germán; Solé-Sánchez, Sònia; Aubareda, Anna; Martínez-Høyer, Sergio; Adán, Jaume; Aranguren-Ibáñez, Álvaro; Pritchard, Melanie A; Soldevila, Gloria; Pérez-Riba, Mercè

    2015-05-01

    Cooperation between calcineurin (CN)-NFATc and RAF-MEK-ERK signaling pathways is essential in thymocyte positive selection. It is known that the Regulators of Calcineurin (RCAN) proteins can act either facilitating or suppressing CN-dependent signaling events. Here, we show that RCAN genes are expressed in lymphoid tissues, and address the role of RCAN proteins in T cell development. Overexpression of human RCAN3 and RCAN1 can modulate T cell development by increasing positive selection-related surface markers, as well as the "Erk(hi) competence state" in double positive thymocytes, a characteristic molecular signature of positive selection, without affecting CN activity. We also found that RCAN1/3 interact with RAF kinases and CN in a non-exclusive manner. Our data suggests that the balance of RCAN interactions with CN and/or RAF kinases may influence T cell positive selection. PMID:25783055

  9. Ribosomal protein S6 kinase 1 signaling regulates mammalian lifespan

    PubMed Central

    Selman, Colin; Tullet, Jennifer M.A.; Wieser, Daniela; Irvine, Elaine; Lingard, Steven J.; Choudhury, Agharul I.; Claret, Marc; Al-Qassab, Hind; Carmignac, Danielle; Ramadani, Faruk; Woods, Angela; Robinson, Iain C.A.; Schuster, Eugene; Batterham, Rachel L.; Kozma, Sara C.; Thomas, George; Carling, David; Okkenhaug, Klaus; Thornton, Janet M.; Partridge, Linda; Gems, David; Withers, Dominic J.

    2016-01-01

    Caloric restriction (CR) protects against aging and disease but the mechanisms by which this affects mammalian lifespan are unclear. We show in mice that deletion of the nutrient-responsive mTOR (mammalian target of rapamycin) signaling pathway component ribosomal S6 protein kinase 1 (S6K1) led to increased lifespan and resistance to age-related pathologies such as bone, immune and motor dysfunction and loss of insulin sensitivity. Deletion of S6K1 induced gene expression patterns similar to those seen in CR or with pharmacological activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), a conserved regulator of the metabolic response to CR. Our results demonstrate that S6K1 influences healthy mammalian lifespan, and suggest therapeutic manipulation of S6K1 and AMPK might mimic CR and provide broad protection against diseases of aging. PMID:19797661

  10. Redox regulation of protein folding in the mitochondrial intermembrane space

    PubMed Central

    Koehler, Carla M.; Tienson, Heather L.

    2014-01-01

    Protein translocation pathways to the mitochondrial matrix and inner membrane have been well characterized. However, translocation into the intermembrane space, which was thought to be simply a modification of the traditional translocation pathways, is complex. The mechanism by which a subset of intermembrane space proteins, those with disulfide bonds, are translocated has been largely unknown until recently. Specifically, the intermembrane space proteins with disulfide bonds are imported via the mitochondrial intermembrane space assembly (MIA) pathway. Substrates are imported via a disulfide exchange relay with two components Mia40 and Erv1. This new breakthrough has resulted in novel concepts for assembly of proteins in the intermembrane space, suggesting that this compartment may be similar to that of the endoplasmic reticulum and the prokaryotic periplasm. As a better understanding of this pathway emerges, new paradigms for thiol-disulfide exchange mechanisms may be developed. Given that the intermembrane space is important for disease processes including apoptosis and neurodegeneration, new roles in regulation by oxidation–reduction chemistry seem likely to be relevant. PMID:18761382

  11. Cluster size regulates protein sorting in the immunological synapse

    PubMed Central

    Hartman, Niña C.; Nye, Jeffrey A.; Groves, Jay T.

    2009-01-01

    During antigen recognition by T cells, signaling molecules on the T cell engage ligands on the antigen-presenting cell and organize into spatially distinctive patterns. These are collectively known as the immunological synapse (IS). Causal relationships between large-scale spatial organization and signal transduction have previously been established. Although it is known that receptor transport during IS formation is driven by actin polymerization, the mechanisms by which different proteins become spatially sorted remain unclear. These sorting processes contribute a facet of signal regulation; thus their elucidation is important for ultimately understanding signal transduction through the T cell receptor. Here we investigate protein cluster size as a sorting mechanism using the hybrid live T cell−supported membrane system. The clustering state of the co-stimulatory molecule lymphocyte function-associated antigen-1 (LFA-1) is modulated, either by direct antibody crosslinking or by crosslinking its intercellular adhesion molecule-1 ligand on the supported bilayer. In a mature IS, native LFA-1 generally localizes into a peripheral ring surrounding a central T cell receptor cluster. Higher degrees of LFA-1 clustering, induced by either method, result in progressively more central localization, with the most clustered species fully relocated to the central zone. These results demonstrate that cluster size directly influences protein spatial positioning in the T cell IS. We discuss a sorting mechanism, based on frictional coupling to the actin cytoskeleton, that is consistent with these observations and is, in principle, extendable to all cell surface proteins in the synapse. PMID:19622735

  12. The ubiquitin–protein ligase Itch regulates p73 stability

    PubMed Central

    Rossi, Mario; De Laurenzi, Vincenzo; Munarriz, Eliana; Green, Douglas R; Liu, Yun-Cai; Vousden, Karen H; Cesareni, Gianni; Melino, Gerry

    2005-01-01

    p73, a member of the p53 family of transcription factors, is upregulated in response to DNA damage, inducing cell cycle arrest and apoptosis. Besides indications that this p73 response is post-transcriptional, little is known about the underlying molecular mechanisms of p73 protein degradation. Ubiquitination and proteasomal-dependent degradation of p53 are regulated by its transcriptional target MDM2. However, unlike p53, p73 binds to, but is not degraded by, MDM2. Here we describe the binding of p73 to Itch, a Hect ubiquitin–protein ligase. Itch selectively binds and ubiquitinates p73 but not p53; this results in the rapid proteasome-dependent degradation of p73. Upon DNA damage Itch itself is downregulated, allowing p73 protein levels to rise and thus interfere with p73 function. In conclusion, we have identified a key mechanism in the control of p73 protein levels both in normal as well as in stress conditions. PMID:15678106

  13. Quantitative phosphoproteomics of protein kinase SnRK1 regulated protein phosphorylation in Arabidopsis under submergence.

    PubMed

    Cho, Hsing-Yi; Wen, Tuan-Nan; Wang, Ying-Tsui; Shih, Ming-Che

    2016-04-01

    SNF1 RELATED PROTEIN KINASE 1 (SnRK1) is proposed to be a central integrator of the plant stress and energy starvation signalling pathways. We observed that the Arabidopsis SnRK1.1 dominant negative mutant (SnRK1.1 (K48M) ) had lower tolerance to submergence than the wild type, suggesting that SnRK1.1-dependent phosphorylation of target proteins is important in signalling pathways triggered by submergence. We conducted quantitative phosphoproteomics and found that the phosphorylation levels of 57 proteins increased and the levels of 27 proteins decreased in Col-0 within 0.5-3h of submergence. Among the 57 proteins with increased phosphorylation in Col-0, 38 did not show increased phosphorylation levels in SnRK1.1 (K48M) under submergence. These proteins are involved mainly in sugar and protein synthesis. In particular, the phosphorylation of MPK6, which is involved in regulating ROS responses under abiotic stresses, was disrupted in the SnRK1.1 (K48M) mutant. In addition, PTP1, a negative regulator of MPK6 activity that directly dephosphorylates MPK6, was also regulated by SnRK1.1. We also showed that energy conservation was disrupted in SnRK1.1 (K48M) , mpk6, and PTP1 (S7AS8A) under submergence. These results reveal insights into the function of SnRK1 and the downstream signalling factors related to submergence. PMID:27029354

  14. Quantitative phosphoproteomics of protein kinase SnRK1 regulated protein phosphorylation in Arabidopsis under submergence

    PubMed Central

    Cho, Hsing-Yi; Wen, Tuan-Nan; Wang, Ying-Tsui; Shih, Ming-Che

    2016-01-01

    SNF1 RELATED PROTEIN KINASE 1 (SnRK1) is proposed to be a central integrator of the plant stress and energy starvation signalling pathways. We observed that the Arabidopsis SnRK1.1 dominant negative mutant (SnRK1.1 K48M) had lower tolerance to submergence than the wild type, suggesting that SnRK1.1-dependent phosphorylation of target proteins is important in signalling pathways triggered by submergence. We conducted quantitative phosphoproteomics and found that the phosphorylation levels of 57 proteins increased and the levels of 27 proteins decreased in Col-0 within 0.5–3h of submergence. Among the 57 proteins with increased phosphorylation in Col-0, 38 did not show increased phosphorylation levels in SnRK1.1 K48M under submergence. These proteins are involved mainly in sugar and protein synthesis. In particular, the phosphorylation of MPK6, which is involved in regulating ROS responses under abiotic stresses, was disrupted in the SnRK1.1 K48M mutant. In addition, PTP1, a negative regulator of MPK6 activity that directly dephosphorylates MPK6, was also regulated by SnRK1.1. We also showed that energy conservation was disrupted in SnRK1.1 K48M, mpk6, and PTP1 S7AS8A under submergence. These results reveal insights into the function of SnRK1 and the downstream signalling factors related to submergence. PMID:27029354

  15. PAT proteins, an ancient family of lipid droplet proteins that regulate cellular lipid stores.

    PubMed

    Bickel, Perry E; Tansey, John T; Welte, Michael A

    2009-06-01

    The PAT family of lipid droplet proteins includes 5 members in mammals: perilipin, adipose differentiation-related protein (ADRP), tail-interacting protein of 47 kDa (TIP47), S3-12, and OXPAT. Members of this family are also present in evolutionarily distant organisms, including insects, slime molds and fungi. All PAT proteins share sequence similarity and the ability to bind intracellular lipid droplets, either constitutively or in response to metabolic stimuli, such as increased lipid flux into or out of lipid droplets. Positioned at the lipid droplet surface, PAT proteins manage access of other proteins (lipases) to the lipid esters within the lipid droplet core and can interact with cellular machinery important for lipid droplet biogenesis. Genetic variations in the gene for the best-characterized of the mammalian PAT proteins, perilipin, have been associated with metabolic phenotypes, including type 2 diabetes mellitus and obesity. In this review, we discuss how the PAT proteins regulate cellular lipid metabolism both in mammals and in model organisms. PMID:19375517

  16. Transcriptional Regulation by Protein Kinase A in Cryptococcus neoformans

    PubMed Central

    Hu, Guanggan; Steen, Barbara R; Lian, Tianshun; Sham, Anita P; Tam, Nicola; Tangen, Kristin L; Kronstad, James W

    2007-01-01

    A defect in the PKA1 gene encoding the catalytic subunit of cyclic adenosine 5′-monophosphate (cAMP)–dependent protein kinase A (PKA) is known to reduce capsule size and attenuate virulence in the fungal pathogen Cryptococcus neoformans. Conversely, loss of the PKA regulatory subunit encoded by pkr1 results in overproduction of capsule and hypervirulence. We compared the transcriptomes between the pka1 and pkr1 mutants and a wild-type strain, and found that PKA influences transcript levels for genes involved in cell wall synthesis, transport functions such as iron uptake, the tricarboxylic acid cycle, and glycolysis. Among the myriad of transcriptional changes in the mutants, we also identified differential expression of ribosomal protein genes, genes encoding stress and chaperone functions, and genes for secretory pathway components and phospholipid synthesis. The transcriptional influence of PKA on these functions was reminiscent of the linkage between transcription, endoplasmic reticulum stress, and the unfolded protein response in Saccharomyces cerevisiae. Functional analyses confirmed that the PKA mutants have a differential response to temperature stress, caffeine, and lithium, and that secretion inhibitors block capsule production. Importantly, we also found that lithium treatment limits capsule size, thus reinforcing potential connections between this virulence trait and inositol and phospholipid metabolism. In addition, deletion of a PKA-regulated gene, OVA1, revealed an epistatic relationship with pka1 in the control of capsule size and melanin formation. OVA1 encodes a putative phosphatidylethanolamine-binding protein that appears to negatively influence capsule production and melanin accumulation. Overall, these findings support a role for PKA in regulating the delivery of virulence factors such as the capsular polysaccharide to the cell surface and serve to highlight the importance of secretion and phospholipid metabolism as potential targets for

  17. PREFACE: Physics approaches to protein interactions and gene regulation Physics approaches to protein interactions and gene regulation

    NASA Astrophysics Data System (ADS)

    Nussinov, Ruth; Panchenko, Anna R.; Przytycka, Teresa

    2011-06-01

    networks have been identified, including scale free distribution of the vertex degree, network motifs, and modularity, to name a few. These studies of network organization require the network to be as complete as possible, which given the limitations of experimental techniques is not currently the case. Therefore, experimental procedures for detecting biomolecular interactions should be complemented by computational approaches. The paper by Lees et al provides a review of computational methods, integrating multiple independent sources of data to infer physical and functional protein-protein interaction networks. One of the important aspects of protein interactions that should be accounted for in the prediction of protein interaction networks is that many proteins are composed of distinct domains. Protein domains may mediate protein interactions while proteins and their interaction networks may gain complexity through gene duplication and expansion of existing domain architectures via domain rearrangements. The latter mechanisms have been explored in detail in the paper by Cohen-Gihon et al. Protein-protein interactions are not the only component of the cell's interactome. Regulation of cell activity can be achieved at the level of transcription and involve a transcription factor—DNA binding which typically requires recognition of a specific DNA sequence motif. Chip-Chip and the more recent Chip-Seq technologies allow in vivo identification of DNA binding sites and, together with novel in vitro approaches, provide data necessary for deciphering the corresponding binding motifs. Such information, complemented by structures of protein-DNA complexes and knowledge of the differences in binding sites among homologs, opens the door to constructing predictive binding models. The paper by Persikov and Singh provides an example of such a model in the Cys2His2 zinc finger family. Recent studies have indicated that the presence of such binding motifs is, however, neither necessary

  18. Photoreactive synthetic regulator of protein function and methods of use thereof

    DOEpatents

    Trauner, Dirk; Isacoff, Ehud Y; Kramer, Richard H; Banghart, Matthew R; Fortin, Doris L; Mourot, Alexandre

    2015-03-31

    The present disclosure provides a photoreactive synthetic regulator of protein function. The present disclosure further provides a light-regulated polypeptide that includes a subject synthetic regulator. Also provided are cells and membranes comprising a subject light-regulated polypeptide. The present disclosure further provides methods of modulating protein function, involving use of light.

  19. Scaffold Proteins Regulating Extracellular Regulated Kinase Function in Cardiac Hypertrophy and Disease

    PubMed Central

    Liang, Yan; Sheikh, Farah

    2016-01-01

    The mitogen activated protein kinase (MAPK)-extracellular regulated kinase 1/2 (ERK1/2) pathway is a central downstream signaling pathway that is activated in cardiac muscle cells during mechanical and agonist-mediated hypertrophy. Studies in genetic mouse models deficient in ERK-associated MAPK components pathway have further reinforced a direct role for this pathway in stress-induced cardiac hypertrophy and disease. However, more recent studies have highlighted that these signaling pathways may exert their regulatory functions in a more compartmentalized manner in cardiac muscle. Emerging data has uncovered specific MAPK scaffolding proteins that tether MAPK/ERK signaling specifically at the sarcomere and plasma membrane in cardiac muscle and show that deficiencies in these scaffolding proteins alter ERK activity and phosphorylation, which are then critical in altering the cardiac myocyte response to stress-induced hypertrophy and disease progression. In this review, we provide insights on ERK-associated scaffolding proteins regulating cardiac myofilament function and their impact on cardiac hypertrophy and disease. PMID:26973524

  20. Regulation of Vaccinia Virus E3 Protein by Small Ubiquitin-Like Modifier Proteins ▿ †

    PubMed Central

    González-Santamaría, José; Campagna, Michela; García, María Angel; Marcos-Villar, Laura; González, Dolores; Gallego, Pedro; Lopitz-Otsoa, Fernando; Guerra, Susana; Rodríguez, Manuel S.; Esteban, Mariano; Rivas, Carmen

    2011-01-01

    The vaccinia virus (VACV) E3 protein is essential for virulence and has antiapoptotic activity and the ability to impair the host innate immune response. Here we demonstrate that E3 interacts with SUMO1 through a small ubiquitin-like modifier (SUMO)-interacting motif (SIM). SIM integrity is required for maintaining the stability of the viral protein and for the covalent conjugation of E3 to SUMO1 or SUMO2, a modification that has a negative effect on the E3 transcriptional transactivation of the p53-upregulated modulator of apoptosis (PUMA) and APAF-1 genes. We also demonstrate that E3 is ubiquitinated, a modification that does not destabilize the wild-type protein but triggers the degradation of an E3-ΔSIM mutant. This report constitutes the first demonstration of the important roles that both SUMO and ubiquitin play in the regulation of the VACV protein E3. PMID:21957283

  1. Role of basic leucine zipper proteins in transcriptional regulation of the steroidogenic acute regulatory protein gene

    PubMed Central

    Manna, Pulak R.; Dyson, Matthew T.; Stocco, Douglas M.

    2016-01-01

    The regulation of steroidogenic acute regulatory protein (StAR) gene transcription by cAMP-dependent mechanisms occurs in the absence of a consensus cAMP response element (CRE, TGACGTGA). This regulation is coordinated by multiple transcription factors that bind to sequence-specific elements located approximately 150 bp upstream of the transcription start site. Among the proteins that bind within this region, the basic leucine zipper (bZIP) family of transcription factors, i.e. CRE binding protein (CREB)/CRE modulator (CREM)/activating transcription factor (ATF), activator protein 1 (AP-1; Fos/Jun), and CCAAT enhancer binding protein β (C/EBPβ), interact with an overlapping region (−81/−72 bp) in the StAR promoter, mediate stimulus-transcription coupling of cAMP signaling and play integral roles in regulating StAR gene expression. These bZIP proteins are structurally similar and bind to DNA sequences as dimers; however, they exhibit discrete transcriptional activities, interact with several transcription factors and other properties that contribute in their regulatory functions. The 5′-flanking −81/−72 bp region of the StAR gene appears to function as a key element within a complex cAMP response unit by binding to different bZIP members, and the StAR promoter displays variable states of cAMP responsivity contingent upon the occupancy of these cis-elements with these transcription factors. The expression and activities of CREB/CREM/ATF, Fos/Jun and C/EBPβ have been demonstrated to be mediated by a plethora of extracellular signals, and the phosphorylation of these proteins at several Ser and Thr residues allows recruitment of the transcriptional coactivator CREB binding protein (CBP) or its functional homolog p300 to the StAR promoter. This review will focus on the current level of understanding of the roles of selective bZIP family proteins within the complex series of processes involved in regulating StAR gene transcription. PMID:19150388

  2. Interaction of 5-aza-2'-deoxycytidine and depsipeptide on antineoplastic activity and activation of 14-3-3sigma, E-cadherin and tissue inhibitor of metalloproteinase 3 expression in human breast carcinoma cells.

    PubMed

    Gagnon, Jacynthe; Shaker, Sepideh; Primeau, Mélanie; Hurtubise, Annie; Momparler, Richard L

    2003-03-01

    Genes that suppress tumorigenesis can be silenced by epigenetic events, such as aberrant DNA methylation and modification of chromatin structure. Inhibitors of DNA methylase and histone deacetylase (HDAC) can potentially reverse these events. The aim of this study was to determine the in vitro antineoplastic activity of 5-aza-2'-deoxycytidine (5-AZA-CdR), a potent inhibitor of DNA methylase, in combination with depsipeptide (depsi), an inhibitor of HDAC, on human breast carcinoma cells. We observed a synergistic antineoplastic interaction between 5-AZA-CdR and depsi in their capacity to inhibit colony formation of Hs578T and MCF-7 breast carcinoma cells. In order to understand the molecular mechanism of this interaction, we investigated the effect of these drugs on the activation of the 14-3-3sigma, E-cadherin and tissue inhibitor of metalloproteinase 3 (TIMP3) cancer-related genes, which were reported to be silenced by aberrant methylation in many breast tumor cell lines. 14-3-3sigma was reported to produce G cell cycle arrest following DNA damage. E-cadherin and TIMP3 function as suppressors of tumor metastasis. Semi-quantitative RT-PCR was used to determine the effect of the co-administration of 5-AZA-CdR and depsi on four breast carcinoma cell lines for the reactivation of these genes. We observed a synergistic activation of E-cadherin by the combination in Hs578T, MDA-MB-231 and MDA-MB-435 tumor cells. For 14-3-3sigma, we demonstrated an additive to synergistic activation by the combination for Hs578T and MDA-MB-435 tumor cells, respectively. In the MCF-7 tumor cells, the drug combination produced a synergistic activation of TIMP3. The association between the synergistic antineoplastic activity and the synergistic activation of the target genes in this study suggests that the mechanism of anticancer activity of 5-AZA-CdR, in combination with depsi, is probably related to their enhanced activation of different types of tumor suppressor genes that have been

  3. Regulation of polar auxin transport by protein and lipid kinases.

    PubMed

    Armengot, Laia; Marquès-Bueno, Maria Mar; Jaillais, Yvon

    2016-07-01

    The directional transport of auxin, known as polar auxin transport (PAT), allows asymmetric distribution of this hormone in different cells and tissues. This system creates local auxin maxima, minima, and gradients that are instrumental in both organ initiation and shape determination. As such, PAT is crucial for all aspects of plant development but also for environmental interaction, notably in shaping plant architecture to its environment. Cell to cell auxin transport is mediated by a network of auxin carriers that are regulated at the transcriptional and post-translational levels. Here we review our current knowledge on some aspects of the 'non-genomic' regulation of auxin transport, placing an emphasis on how phosphorylation by protein and lipid kinases controls the polarity, intracellular trafficking, stability, and activity of auxin carriers. We describe the role of several AGC kinases, including PINOID, D6PK, and the blue light photoreceptor phot1, in phosphorylating auxin carriers from the PIN and ABCB families. We also highlight the function of some receptor-like kinases (RLKs) and two-component histidine kinase receptors in PAT, noting that there are probably RLKs involved in co-ordinating auxin distribution yet to be discovered. In addition, we describe the emerging role of phospholipid phosphorylation in polarity establishment and intracellular trafficking of PIN proteins. We outline these various phosphorylation mechanisms in the context of primary and lateral root development, leaf cell shape acquisition, as well as root gravitropism and shoot phototropism. PMID:27242371

  4. 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

  5. Regulation of polar auxin transport by protein and lipid kinases

    PubMed Central

    Jaillais, Yvon

    2016-01-01

    The directional transport of auxin, known as polar auxin transport, allows asymmetric distribution of this hormone in different cells and tissues. This system creates local auxin maxima, minima and gradients that are instrumental in both organ initiation and shape determination. As such, polar auxin transport is crucial for all aspects of plant development but also for environmental interaction, notably in shaping plant architecture to its environment. Cell-to-cell auxin transport is mediated by a network of auxin carriers that are regulated at the transcriptional and post-translational levels. Here we review our current knowledge on some aspects of the ‘non-genomic’ regulation of auxin transport, putting an emphasis on how phosphorylation by protein and lipid kinases controls the polarity, intracellular trafficking, stability and activity of auxin carriers. We describe the role of several AGC kinases, including PINOID, D6PK and the blue light photoreceptor phot1, in phosphorylating auxin carriers from the PIN and ABCB families. We also highlight the function of some Receptor-Like Kinases (RLK) and two-component histidine kinase receptors in polar auxin transport, noticing that there are likely RLKs involved in coordinating auxin distribution yet to be discovered. In addition, we describe the emerging role of phospholipid phosphorylation in polarity establishment and intracellular trafficking of PIN proteins. We outline these various phosphorylation mechanisms in the context of primary and lateral root development, leaf cell shape acquisition as well as root gravitropism and shoot phototropism. PMID:27242371

  6. Protein-protein interactions among ion channels regulate ion transport in the kidney.

    PubMed

    Boulpaep, E

    2009-01-01

    Epithelial ion transport in various organs has long been known to be controlled by extracellular agonists acting via membrane receptors or by intracellular messengers. Evidence is mounting for regulation of transport by direct interaction among membrane proteins or between a membrane transport protein and membrane-attached proteins. The membrane protein CFTR (Cystic Fibrosis Transmembrane Regulator) is widely expressed along the length of the nephron, but its role as a chloride channel does not appear to be critical for renal handling of salt and water. It is well established that the inward rectifying K channels (ROMK = Kir 1.1) in the thick ascending limb of Henle and in principal cells of the collecting duct are inhibited by millimolar concentrations of cytosolic Mg-ATP. However, the mechanism of this inhibition has been an enigma. We propose that the ATP-Binding Cassette (ABC) protein CFTR is a cofactor for Kir 1.1 regulation. Indeed, Mg-ATP sensitivity of Kir 1.1 is completely absent in two different mouse models of cystic fibrosis. In addition, the open-closed state of CFTR appears to provide a molecular gating switch that prevents or facilitates the ATP sensing of Kir 1.1. Does Mg-ATP sensing by the CFTR- Kir 1.1 complex play a role in coupling metabolism to ion transport? Physiological intracellular ATP concentrations in tubule cells are in the millimolar range, a saturating concentration for the gating of Kir 1.1 by Mg-ATP. Therefore, Kir 1.1 channels would be closed and unable to contribute to regulation of potassium secretion unless some other process modulated the CFTR-dependent ATP-sensitivity of Kir 1.1. The third component of the metabolic sensor-effector complex for Kir 1.1 regulation is most likely the AMP-regulated serine-threonine kinase, AMP kinase (AMPK). Changing levels in AMP rather than in ATP constitute the metabolic signal "sensed" by tubule cells. Because AMPK inhibits CFTR by modulating CFTR channel gating, we propose that renal K

  7. Oxysterol-related-binding-protein related Protein-2 (ORP2) regulates cortisol biosynthesis and cholesterol homeostasis.

    PubMed

    Escajadillo, Tamara; Wang, Hongxia; Li, Linda; Li, Donghui; Sewer, Marion B

    2016-05-15

    Oxysterol binding protein-related protein 2 (ORP2) is a lipid binding protein that has been implicated in various cellular processes, including lipid sensing, cholesterol efflux, and endocytosis. We recently identified ORP2 as a member of a protein complex that regulates glucocorticoid biosynthesis. Herein, we examine the effect of silencing ORP2 on adrenocortical function and show that the ORP2 knockdown cells exhibit reduced amounts of multiple steroid metabolites, including progesterone, 11-deoxycortisol, and cortisol, but have increased concentrations of androgens, and estrogens. Moreover, silencing ORP2 suppresses the expression of most proteins required for cortisol production and reduces the expression of steroidogenic factor 1 (SF1). ORP2 silencing also increases cellular cholesterol, concomitant with decreased amounts of 22-hydroxycholesterol and 7-ketocholesterol, two molecules that have been shown to bind to ORP2. Further, we show that ORP2 binds to liver X receptor (LXR) and is required for nuclear LXR expression. LXR and ORP2 are recruited to the CYP11B1 promoter in response to cAMP signaling. Additionally, ORP2 is required for the expression of other LXR target genes, including ABCA1 and the LDL receptor (LDLR). In summary, we establish a novel role for ORP2 in regulating steroidogenic capacity and cholesterol homeostasis in the adrenal cortex. PMID:26992564

  8. Functional Reconstitution of an Atypical G Protein Heterotrimer and Regulator of G Protein Signaling Protein (RGS1) from Arabidopsis thaliana*

    PubMed Central

    Jones, Janice C.; Temple, Brenda R. S.; Jones, Alan M.; Dohlman, Henrik G.

    2011-01-01

    It has long been known that animal heterotrimeric Gαβγ proteins are activated by cell-surface receptors that promote GTP binding to the Gα subunit and dissociation of the heterotrimer. In contrast, the Gα protein from Arabidopsis thaliana (AtGPA1) can activate itself without a receptor or other exchange factor. It is unknown how AtGPA1 is regulated by Gβγ and the RGS (regulator of G protein signaling) protein AtRGS1, which is comprised of an RGS domain fused to a receptor-like domain. To better understand the cycle of G protein activation and inactivation in plants, we purified and reconstituted AtGPA1, full-length AtRGS1, and two putative Gβγ dimers. We show that the Arabidopsis Gα protein binds to its cognate Gβγ dimer directly and in a nucleotide-dependent manner. Although animal Gβγ dimers inhibit GTP binding to the Gα subunit, AtGPA1 retains fast activation in the presence of its cognate Gβγ dimer. We show further that the full-length AtRGS1 protein accelerates GTP hydrolysis and thereby counteracts the fast nucleotide exchange rate of AtGPA1. Finally, we show that AtGPA1 is less stable in complex with GDP than in complex with GTP or the Gβγ dimer. Molecular dynamics simulations and biophysical studies reveal that altered stability is likely due to increased dynamic motion in the N-terminal α-helix and Switch II of AtGPA1. Thus, despite profound differences in the mechanisms of activation, the Arabidopsis G protein is readily inactivated by its cognate RGS protein and forms a stable, GDP-bound, heterotrimeric complex similar to that found in animals. PMID:21325279

  9. Plant TRAF Proteins Regulate NLR Immune Receptor Turnover.

    PubMed

    Huang, Shuai; Chen, Xuejin; Zhong, Xionghui; Li, Meng; Ao, Kevin; Huang, Jianhua; Li, Xin

    2016-02-10

    In animals, Tumor necrosis factor receptor-associated factor (TRAF) proteins are molecular adaptors that regulate innate and adaptive immunity, development, and abiotic stress responses. Although gene families encoding TRAF domain-containing proteins exhibit enriched diversity in higher plants, their biological roles are poorly defined. Here, we report the identification of two redundant TRAF proteins, Mutant, snc1-enhancing 13 (MUSE13) and MUSE14, that contribute to the turnover of nucleotide-binding domain and leucine-rich repeat-containing (NLR) immune receptors SNC1 and RPS2. Loss of both MUSE13 and MUSE14 leads to enhanced pathogen resistance, NLR accumulation, and autoimmunity, while MUSE13 overexpression results in reduced NLR levels and activity. In planta, MUSE13 associates with SNC1, RPS2, and the E3 ubiquitin ligase SCF(CPR1). Taken together, we speculate that MUSE13 and MUSE14 associate with the SCF E3 ligase complex to form a plant-type TRAFasome, which modulates ubiquitination and subsequent degradation of NLR immune sensors to maintain their homeostasis. PMID:26867179

  10. Perilipin-related protein regulates lipid metabolism in C. elegans

    PubMed Central

    Chughtai, Ahmed Ali; Kaššák, Filip; Kostrouchová, Markéta; Novotný, Jan Philipp; Krause, Michael W.; Kostrouch, Zdenek

    2015-01-01

    Perilipins are lipid droplet surface proteins that contribute to fat metabolism by controlling the access of lipids to lipolytic enzymes. Perilipins have been identified in organisms as diverse as metazoa, fungi, and amoebas but strikingly not in nematodes. Here we identify the protein encoded by the W01A8.1 gene in Caenorhabditis elegans as the closest homologue and likely orthologue of metazoan perilipin. We demonstrate that nematode W01A8.1 is a cytoplasmic protein residing on lipid droplets similarly as human perilipins 1 and 2. Downregulation or elimination of W01A8.1 affects the appearance of lipid droplets resulting in the formation of large lipid droplets localized around the dividing nucleus during the early zygotic divisions. Visualization of lipid containing structures by CARS microscopy in vivo showed that lipid-containing structures become gradually enlarged during oogenesis and relocate during the first zygotic division around the dividing nucleus. In mutant embryos, the lipid containing structures show defective intracellular distribution in subsequent embryonic divisions and become gradually smaller during further development. In contrast to embryos, lipid-containing structures in enterocytes and in epidermal cells of adult animals are smaller in mutants than in wild type animals. Our results demonstrate the existence of a perilipin-related regulation of fat metabolism in nematodes and provide new possibilities for functional studies of lipid metabolism. PMID:26357594

  11. Regulation of Nuclear Localization of Signaling Proteins by Cytokinin

    SciTech Connect

    Kieber, J.J.

    2010-05-01

    Cytokinins are a class of mitogenic plant hormones that play an important role in most aspects of plant development, including shoot and root growth, vascular and photomorphogenic development and leaf senescence. A model for cytokinin perception and signaling has emerged that is similar to bacterial two-component phosphorelays. In this model, binding of cytokinin to the extracellular domain of the Arabidopsis histidine kinase (AHKs) receptors induces autophosphorylation within the intracellular histidine-kinase domain. The phosphoryl group is subsequently transferred to cytosolic Arabidopsis histidine phosphotransfer proteins (AHPs), which have been suggested to translocate to the nucleus in response to cytokinin treatment, where they then transfer the phosphoryl group to nuclear-localized response regulators (Type-A and Type-B ARRs). We examined the effects of cytokinin on AHP subcellular localization in Arabidopsis and, contrary to expectations, the AHPs maintained a constant nuclear/cytosolic distribution following cytokinin treatment. Furthermore, mutation of the conserved phosphoacceptor histidine residue of the AHP, as well as disruption of multiple cytokinin signaling elements, did not affect the subcellular localization of the AHP proteins. Finally, we present data indicating that AHPs maintain a nuclear/cytosolic distribution by balancing active transport into and out of the nucleus. Our findings suggest that the current models indicating relocalization of AHP protein into the nucleus in response to cytokinin are incorrect. Rather, AHPs actively maintain a consistent nuclear/cytosolic distribution regardless of the status of the cytokinin response pathway.

  12. Regulation of G protein-coupled receptor export trafficking

    PubMed Central

    Dong, Chunmin; Filipeanu, Catalin M.; Duvernay, Matthew T.; Wu, Guangyu

    2007-01-01

    G protein-coupled receptors (GPCRs) constitute a superfamily of cell-surface receptors which share a common topology of seven transmembrane domains and modulate a variety of cell functions through coupling to heterotrimeric G proteins by responding to a vast array of stimuli. The magnitude of cellular response elicited by a given signal is dictated by the level of GPCR expression at the plasma membrane, which is the balance of elaborately regulated endocytic and exocytic trafficking. This review will cover recent advances in understanding the molecular mechanism underlying anterograde transport of the newly synthesized GPCRs from the endoplasmic reticulum (ER) through the Golgi to the plasma membrane. We will focus on recently identified motifs involved in GPCR exit from the ER and the Golgi, GPCR folding in the ER and the rescue of misfolded receptors from within, GPCR-interacting proteins that modulate receptor cell-surface targeting, pathways that mediate GPCR traffic, and the functional role of export in controlling GPCR signaling. PMID:17074298

  13. Expression of Tyrosine Hydroxylase is Negatively Regulated Via Prion Protein.

    PubMed

    da Luz, Marcio Henrique Mello; Glezer, Isaias; Xavier, Andre Machado; da Silva, Marcelo Alberti Paiva; Pino, Jessica Monteiro Volejnik; Zamith, Thiago Panaro; Vieira, Taynara Fernanda; Antonio, Bruno Brito; Antunes, Hanna Karen Moreira; Martins, Vilma Regina; Lee, Kil Sun

    2016-07-01

    Cellular prion protein (PrP(C)) is a glycoprotein of the plasma membrane that plays pleiotropic functions by interacting with multiple signaling complexes at the cell surface. Recently, a number of studies have reported the involvement of PrP(C) in dopamine metabolism and signaling, including its interactions with tyrosine hydroxylase (TH) and dopamine receptors. However, the outcomes reported by independent studies are still debatable. Therefore in this study, we investigated the effects of PrP(C) on the TH expression during the differentiation of N2a cells with dibutyryl-cAMP, a well-known cAMP analog that activates TH transcription. Upon differentiation, TH was induced with concomitant reduction of PrP(C) at protein level, but not at mRNA level. shRNA-mediated PrP(C) reduction increased the basal level of TH at both mRNA and protein levels without dibutyryl-cAMP treatment. This phenotype was reversed by re-expression of PrP(C). PrP(C) knockdown also potentiated the effect of dibutyryl-cAMP on TH expression. Our findings suggest that PrP(C) has suppressive effects on TH expression. As a consequence, altered PrP(C) functions may affect the regulation of dopamine metabolism and related neurological disorders. PMID:26975317

  14. The acute phase protein haptoglobin regulates host immunity

    PubMed Central

    Huntoon, Kristin M.; Wang, Yanping; Eppolito, Cheryl A.; Barbour, Karen W.; Berger, Franklin G.; Shrikant, Protul A.; Baumann, Heinz

    2008-01-01

    The contribution of acute phase plasma proteins to host immune responses remains poorly characterized. To better understand the role of the acute phase reactant and major hemoglobin-binding protein haptoglobin (Hp) on the function of immune cells, we generated Hp-deficient C57BL/6J mice. These mice exhibit stunted development of lymphoid organs associated with lower counts of mature T and B cells in the blood and secondary lymphoid compartments. Moreover, these mice show markedly reduced adaptive immune responses as represented by reduced accumulation of IgG antibody after immunization with adjuvant and nominal antigen, abrogation of Th1-dominated delayed-type hypersensitivity reaction, loss of mitogenic responses mounted by T cells, and reduced T cell responses conveyed by APCs. Collectively, these defects are in agreement with the observations that Hp-deficient mice are not capable of generating a recall response or deterring a Salmonella infection as well as failing to generate tumor antigen-specific responses. The administration of Hp to lymphocytes in tissue culture partially ameliorates these functional defects, lending further support to our contention that the acute phase response protein Hp has the ability to regulate immune cell responses and host immunity. The phenotype of Hp-deficient mice suggests a major regulatory activity for Hp in supporting proliferation and functional differentiation of B and T cells as part of homeostasis and in response to antigen stimulation. PMID:18436583

  15. Regulation of ABC Transporter Function Via Phosphorylation by Protein Kinases

    PubMed Central

    Stolarczyk, Elzbieta I.; Reiling, Cassandra J.; Paumi, Christian M.

    2011-01-01

    ATP-binding cassette (ABC) transporters are multispanning membrane proteins that utilize ATP to move a broad range of substrates across cellular membranes. ABC transporters are involved in a number of human disorders and diseases [1]. Overexpression of a subset of the transporters has been closely linked to multidrug resistance in both bacteria and viruses and in cancer. A poorly understood and important aspect of ABC transporter biology is the role of phosphorylation as a mechanism to regulate transporter function. In this review, we summarize the current literature addressing the role of phosphorylation in regulating ABC transporter function. A comprehensive list of all the phosphorylation sites that have been identified for the human ABC transporters is presented, and we discuss the role of individual kinases in regulating transporter function. We address the potential pitfalls and difficulties associated with identifying phosphorylation sites and the corresponding kinase(s), and we discuss novel techniques that may circumvent these problems. We conclude by providing a brief perspective on studying ABC transporter phosphorylation. PMID:21118091

  16. Regulation of alternative splicing in Drosophila by 56 RNA binding proteins

    DOE PAGESBeta

    Brooks, Angela N.; Duff, Michael O.; May, Gemma; Yang, Li; Bolisetty, Mohan; Landolin, Jane; Wan, Ken; Sandler, Jeremy; Booth, Benjamin W.; Celniker, Susan E.; et al

    2015-08-20

    Alternative splicing is regulated by RNA binding proteins (RBPs) that recognize pre-mRNA sequence elements and activate or repress adjacent exons. Here, we used RNA interference and RNA-seq to identify splicing events regulated by 56 Drosophila proteins, some previously unknown to regulate splicing. Nearly all proteins affected alternative first exons, suggesting that RBPs play important roles in first exon choice. Half of the splicing events were regulated by multiple proteins, demonstrating extensive combinatorial regulation. We observed that SR and hnRNP proteins tend to act coordinately with each other, not antagonistically. We also identified a cross-regulatory network where splicing regulators affected themore » splicing of pre-mRNAs encoding other splicing regulators. In conclusion, this large-scale study substantially enhances our understanding of recent models of splicing regulation and provides a resource of thousands of exons that are regulated by 56 diverse RBPs.« less

  17. Regulation of alternative splicing in Drosophila by 56 RNA binding proteins

    PubMed Central

    Brooks, Angela N.; Duff, Michael O.; May, Gemma; Yang, Li; Bolisetty, Mohan; Landolin, Jane; Wan, Ken; Sandler, Jeremy; Booth, Benjamin W.; Celniker, Susan E.; Graveley, Brenton R.; Brenner, Steven E.

    2015-01-01

    Alternative splicing is regulated by RNA binding proteins (RBPs) that recognize pre-mRNA sequence elements and activate or repress adjacent exons. Here, we used RNA interference and RNA-seq to identify splicing events regulated by 56 Drosophila proteins, some previously unknown to regulate splicing. Nearly all proteins affected alternative first exons, suggesting that RBPs play important roles in first exon choice. Half of the splicing events were regulated by multiple proteins, demonstrating extensive combinatorial regulation. We observed that SR and hnRNP proteins tend to act coordinately with each other, not antagonistically. We also identified a cross-regulatory network where splicing regulators affected the splicing of pre-mRNAs encoding other splicing regulators. This large-scale study substantially enhances our understanding of recent models of splicing regulation and provides a resource of thousands of exons that are regulated by 56 diverse RBPs. PMID:26294686

  18. Regulation of alternative splicing in Drosophila by 56 RNA binding proteins

    SciTech Connect

    Brooks, Angela N.; Duff, Michael O.; May, Gemma; Yang, Li; Bolisetty, Mohan; Landolin, Jane; Wan, Ken; Sandler, Jeremy; Booth, Benjamin W.; Celniker, Susan E.; Graveley, Brenton R.; Brenner, Steven E.

    2015-08-20

    Alternative splicing is regulated by RNA binding proteins (RBPs) that recognize pre-mRNA sequence elements and activate or repress adjacent exons. Here, we used RNA interference and RNA-seq to identify splicing events regulated by 56 Drosophila proteins, some previously unknown to regulate splicing. Nearly all proteins affected alternative first exons, suggesting that RBPs play important roles in first exon choice. Half of the splicing events were regulated by multiple proteins, demonstrating extensive combinatorial regulation. We observed that SR and hnRNP proteins tend to act coordinately with each other, not antagonistically. We also identified a cross-regulatory network where splicing regulators affected the splicing of pre-mRNAs encoding other splicing regulators. In conclusion, this large-scale study substantially enhances our understanding of recent models of splicing regulation and provides a resource of thousands of exons that are regulated by 56 diverse RBPs.

  19. Regulation of blood-testis barrier by actin binding proteins and protein kinases.

    PubMed

    Li, Nan; Tang, Elizabeth I; Cheng, C Yan

    2016-03-01

    The blood-testis barrier (BTB) is an important ultrastructure in the testis, since the onset of meiosis and spermiogenesis coincides with the establishment of a functional barrier in rodents and humans. It is also noted that a delay in the assembly of a functional BTB following treatment of neonatal rats with drugs such as diethylstilbestrol or adjudin also delays the first wave of spermiation. While the BTB is one of the tightest blood-tissue barriers, it undergoes extensive remodeling, in particular, at stage VIII of the epithelial cycle to facilitate the transport of preleptotene spermatocytes connected in clones across the immunological barrier. Without this timely transport of preleptotene spermatocytes derived from type B spermatogonia, meiosis will be arrested, causing aspermatogenesis. Yet the biology and regulation of the BTB remains largely unexplored since the morphological studies in the 1970s. Recent studies, however, have shed new light on the biology of the BTB. Herein, we critically evaluate some of these findings, illustrating that the Sertoli cell BTB is regulated by actin-binding proteins (ABPs), likely supported by non-receptor protein kinases, to modulate the organization of actin microfilament bundles at the site. Furthermore, microtubule-based cytoskeleton is also working in concert with the actin-based cytoskeleton to confer BTB dynamics. This timely review provides an update on the unique biology and regulation of the BTB based on the latest findings in the field, focusing on the role of ABPs and non-receptor protein kinases. PMID:26628556

  20. Keap1-Independent Regulation of Nrf2 Activity by Protein Acetylation and a BET Bromodomain Protein

    PubMed Central

    Chatterjee, Nirmalya; Tian, Min; Spirohn, Kerstin; Boutros, Michael; Bohmann, Dirk

    2016-01-01

    Mammalian BET proteins comprise a family of bromodomain-containing epigenetic regulators with complex functions in chromatin organization and gene regulation. We identified the sole member of the BET protein family in Drosophila, Fs(1)h, as an inhibitor of the stress responsive transcription factor CncC, the fly ortholog of Nrf2. Fs(1)h physically interacts with CncC in a manner that requires the function of its bromodomains and the acetylation of CncC. Treatment of cultured Drosophila cells or adult flies with fs(1)h RNAi or with the BET protein inhibitor JQ1 de-represses CncC transcriptional activity and engages protective gene expression programs. The mechanism by which Fs(1)h inhibits CncC function is distinct from the canonical mechanism that stimulates Nrf2 function by abrogating Keap1-dependent proteasomal degradation. Consistent with the independent modes of CncC regulation by Keap1 and Fs(1)h, combinations of drugs that can specifically target these pathways cause a strong synergistic and specific activation of protective CncC- dependent gene expression and boosts oxidative stress resistance. This synergism might be exploitable for the design of combinatorial therapies to target diseases associated with oxidative stress or inflammation. PMID:27233051

  1. Regulation of blood-testis barrier by actin binding proteins and protein kinases

    PubMed Central

    Li, Nan; Tang, Elizabeth I.; Cheng, C. Yan

    2016-01-01

    The blood-testis barrier (BTB) is an important ultrastructure in the testis since the onset of spermatogenesis coincides with the establishment of a functional barrier in rodents and humans. It is also noted that a delay in the assembly of a functional BTB following treatment of neonatal rats with drugs such as diethylstilbestrol or adjudin also delays the first wave of spermiation. While the BTB is one of the tightest blood-tissue barriers, it undergoes extensive remodeling, in particular at stage VIII of the epithelial cycle to facilitate the transport of preleptotene spermatocytes connected in clones across the immunological barrier. Without this timely transport of preleptotene spermatocytes derived from type B spermatogonia, meiosis will be arrested, causing aspermatogenesis. Yet the biology and regulation of the BTB remains largely unexplored since the morphological studies in the 1970s. Recent studies, however, have shed new light on the biology of the BTB. Herein, we critically evaluate some of these findings, illustrating that the Sertoli cell BTB is regulated by actin binding proteins (ABPs), likely supported by non-receptor protein kinases, to modulate the organization of actin microfilament bundles at the site. Furthermore, microtubule (MT)-based cytoskeleton is also working in concert with the actin-based cytoskeleton to confer BTB dynamics. This timely review provides an update on the unique biology and regulation of the BTB based on the latest findings in the field, focusing on the role of ABPs and non-receptor protein kinases. PMID:26628556

  2. Second messenger-dependent protein kinases and protein synthesis regulate endogenous secretin receptor responsiveness

    PubMed Central

    Ghadessy, Roxana S; Kelly, Eamonn

    2002-01-01

    The present study investigated the role of second messenger-dependent protein kinase A (PKA) and C (PKC) in the regulation of endogenous secretin receptor responsiveness in NG108-15 mouse neuroblastoma×rat glioma hybrid cells. In whole cell cyclic AMP accumulation studies, activation of PKC either by phorbol 12-myristate 13-acetate (PMA) or by purinoceptor stimulation using uridine 5′-triphosphate (UTP) decreased secretin receptor responsiveness. PKC activation also inhibited forskolin-stimulated cyclic AMP accumulation but did not affect cyclic AMP responses mediated by the prostanoid-IP receptor agonist iloprost, or the A2 adenosine receptor agonist 5′-(N-ethylcarboxamido) adenosine (NECA). In additivity experiments, saturating concentrations of secretin and iloprost were found to be additive in terms of cyclic AMP accumulation, whereas saturating concentrations of NECA and iloprost together were not. This suggests compartmentalization of Gs-coupling components in NG108-15 cells and possible heterologous regulation of secretin receptor responsiveness at the level of adenylyl cyclase activation. Cells exposed to the PKA inhibitor H-89, exhibited a time-dependent increase in secretin receptor responsiveness compared to control cells. This effect was selective since cyclic AMP responses to forskolin, iloprost and NECA were not affected by H-89 treatment. Furthermore, treatment with the protein synthesis inhibitor cycloheximide produced a time-dependent increase in secretin receptor responsiveness. Together these results indicate that endogenous secretin receptor responsiveness is regulated by PKC, PKA and protein neosynthesis in NG108-15 cells. PMID:11959806

  3. Saccharomyces cerevisiae Tti2 Regulates PIKK Proteins and Stress Response.

    PubMed

    Hoffman, Kyle S; Duennwald, Martin L; Karagiannis, Jim; Genereaux, Julie; McCarton, Alexander S; Brandl, Christopher J

    2016-01-01

    The TTT complex is composed of the three essential proteins Tel2, Tti1, and Tti2 The complex is required to maintain steady state levels of phosphatidylinositol 3-kinase-related kinase (PIKK) proteins, including mTOR, ATM/Tel1, ATR/Mec1, and TRRAP/Tra1, all of which serve as regulators of critical cell signaling pathways. Due to their association with heat shock proteins, and with newly synthesized PIKK peptides, components of the TTT complex may act as cochaperones. Here, we analyze the consequences of depleting the cellular level of Tti2 in Saccharomyces cerevisiae We show that yeast expressing low levels of Tti2 are viable under optimal growth conditions, but the cells are sensitive to a number of stress conditions that involve PIKK pathways. In agreement with this, depleting Tti2 levels decreased expression of Tra1, Mec1, and Tor1, affected their localization and inhibited the stress responses in which these molecules are involved. Tti2 expression was not increased during heat shock, implying that it does not play a general role in the heat shock response. However, steady state levels of Hsp42 increase when Tti2 is depleted, and tti2L187P has a synthetic interaction with exon 1 of the human Huntingtin gene containing a 103 residue polyQ sequence, suggesting a general role in protein quality control. We also find that overexpressing Hsp90 or its cochaperones is synthetic lethal when Tti2 is depleted, an effect possibly due to imbalanced stoichiometry of a complex required for PIKK assembly. These results indicate that Tti2 does not act as a general chaperone, but may have a specialized function in PIKK folding and/or complex assembly. PMID:27172216

  4. Saccharomyces cerevisiae Tti2 Regulates PIKK Proteins and Stress Response

    PubMed Central

    Hoffman, Kyle S.; Duennwald, Martin L.; Karagiannis, Jim; Genereaux, Julie; McCarton, Alexander S.; Brandl, Christopher J.

    2016-01-01

    The TTT complex is composed of the three essential proteins Tel2, Tti1, and Tti2. The complex is required to maintain steady state levels of phosphatidylinositol 3-kinase-related kinase (PIKK) proteins, including mTOR, ATM/Tel1, ATR/Mec1, and TRRAP/Tra1, all of which serve as regulators of critical cell signaling pathways. Due to their association with heat shock proteins, and with newly synthesized PIKK peptides, components of the TTT complex may act as cochaperones. Here, we analyze the consequences of depleting the cellular level of Tti2 in Saccharomyces cerevisiae. We show that yeast expressing low levels of Tti2 are viable under optimal growth conditions, but the cells are sensitive to a number of stress conditions that involve PIKK pathways. In agreement with this, depleting Tti2 levels decreased expression of Tra1, Mec1, and Tor1, affected their localization and inhibited the stress responses in which these molecules are involved. Tti2 expression was not increased during heat shock, implying that it does not play a general role in the heat shock response. However, steady state levels of Hsp42 increase when Tti2 is depleted, and tti2L187P has a synthetic interaction with exon 1 of the human Huntingtin gene containing a 103 residue polyQ sequence, suggesting a general role in protein quality control. We also find that overexpressing Hsp90 or its cochaperones is synthetic lethal when Tti2 is depleted, an effect possibly due to imbalanced stoichiometry of a complex required for PIKK assembly. These results indicate that Tti2 does not act as a general chaperone, but may have a specialized function in PIKK folding and/or complex assembly. PMID:27172216

  5. Auxin acts independently of DELLA proteins in regulating gibberellin levels.

    PubMed

    Reid, James B; Davidson, Sandra E; Ross, John J

    2011-03-01

    Shoot elongation is a vital process for plant development and productivity, in both ecological and economic contexts. Auxin and bioactive gibberellins (GAs), such as GA1, play critical roles in the control of elongation, along with environmental and endogenous factors, including other hormones such as the brassinosteroids. The effect of auxins, such as indole-3-acetic acid (IAA), is at least in part mediated by its effect on GA metabolism, since auxin up-regulates biosynthesis genes such as GA 3-oxidase and GA 20-oxidase and down regulates GA catabolism genes such as GA 2-oxidases, leading to elevated levels of bioactive GA 1. In our recent paper, we have provided evidence that this action of IAA is largely independent of DELLA proteins, the negative regulators of GA action, since the auxin effects are still present in the DELLA-deficient la cry-s genotype of pea. This was a crucial issue to resolve, since like auxin, the DELLAs also promote GA 1 synthesis and inhibit its deactivation. DELLAs are deactivated by GA, and thereby mediate a feedback system by which bioactive GA regulates its own level. However, our recent results, in themselves, do not show the generality of the auxin-GA relationship across species and phylogenetic groups or across different tissue types and responses. Further, they do not touch on the ecological benefits of the auxin-GA interaction. These issues are discussed below as well as the need for the development of suitable experimental systems to allow this process to be examined. PMID:21358281

  6. Regulation of NADPH Oxidase in Vascular Endothelium: The Role of Phospholipases, Protein Kinases, and Cytoskeletal Proteins

    PubMed Central

    Pendyala, Srikanth; Usatyuk, Peter V.; Gorshkova, Irina A.; Garcia, Joe G.N.

    2009-01-01

    The generation of reactive oxygen species (ROS) in the vasculature plays a major role in the genesis of endothelial cell (EC) activation and barrier function. Of the several potential sources of ROS in the vasculature, the endothelial NADPH oxidase family of proteins is a major contributor of ROS associated with lung inflammation, ischemia/reperfusion injury, sepsis, hyperoxia, and ventilator-associated lung injury. The NADPH oxidase in lung ECs has most of the components found in phagocytic oxidase, and recent studies show the expression of several homologues of Nox proteins in vascular cells. Activation of NADPH oxidase of nonphagocytic vascular cells is complex and involves assembly of the cytosolic (p47phox, p67phox, and Rac1) and membrane-associated components (Noxes and p22phox). Signaling pathways leading to NADPH oxidase activation are not completely defined; however, they do appear to involve the cytoskeleton and posttranslation modification of the components regulated by protein kinases, protein phosphatases, and phospholipases. Furthermore, several key components regulating NADPH oxidase recruitment, assembly, and activation are enriched in lipid microdomains to form a functional signaling platform. Future studies on temporal and spatial localization of Nox isoforms will provide new insights into the role of NADPH oxidase–derived ROS in the pathobiology of lung diseases. Antioxid. Redox Signal. 11, 841–860. PMID:18828698

  7. The bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system: regulation by protein phosphorylation and phosphorylation-dependent protein-protein interactions.

    PubMed

    Deutscher, Josef; Aké, Francine Moussan Désirée; Derkaoui, Meriem; Zébré, Arthur Constant; Cao, Thanh Nguyen; Bouraoui, Houda; Kentache, Takfarinas; Mokhtari, Abdelhamid; Milohanic, Eliane; Joyet, Philippe

    2014-06-01

    The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions. It catalyzes the transport and phosphorylation of a variety of sugars and sugar derivatives but also carries out numerous regulatory functions related to carbon, nitrogen, and phosphate metabolism, to chemotaxis, to potassium transport, and to the virulence of certain pathogens. For these different regulatory processes, the signal is provided by the phosphorylation state of the PTS components, which varies according to the availability of PTS substrates and the metabolic state of the cell. PEP acts as phosphoryl donor for enzyme I (EI), which, together with HPr and one of several EIIA and EIIB pairs, forms a phosphorylation cascade which allows phosphorylation of the cognate carbohydrate bound to the membrane-spanning EIIC. HPr of firmicutes and numerous proteobacteria is also phosphorylated in an ATP-dependent reaction catalyzed by the bifunctional HPr kinase/phosphorylase. PTS-mediated regulatory mechanisms are based either on direct phosphorylation of the target protein or on phosphorylation-dependent interactions. For regulation by PTS-mediated phosphorylation, the target proteins either acquired a PTS domain by fusing it to their N or C termini or integrated a specific, conserved PTS regulation domain (PRD) or, alternatively, developed their own specific sites for PTS-mediated phosphorylation. Protein-protein interactions can occur with either phosphorylated or unphosphorylated PTS components and can either stimulate or inhibit the function of the target proteins. This large variety of signal transduction mechanisms allows the PTS to regulate numerous proteins and to form a vast regulatory network responding to the phosphorylation state of various PTS components. PMID:24847021

  8. The Bacterial Phosphoenolpyruvate:Carbohydrate Phosphotransferase System: Regulation by Protein Phosphorylation and Phosphorylation-Dependent Protein-Protein Interactions

    PubMed Central

    Aké, Francine Moussan Désirée; Derkaoui, Meriem; Zébré, Arthur Constant; Cao, Thanh Nguyen; Bouraoui, Houda; Kentache, Takfarinas; Mokhtari, Abdelhamid; Milohanic, Eliane; Joyet, Philippe

    2014-01-01

    SUMMARY The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions. It catalyzes the transport and phosphorylation of a variety of sugars and sugar derivatives but also carries out numerous regulatory functions related to carbon, nitrogen, and phosphate metabolism, to chemotaxis, to potassium transport, and to the virulence of certain pathogens. For these different regulatory processes, the signal is provided by the phosphorylation state of the PTS components, which varies according to the availability of PTS substrates and the metabolic state of the cell. PEP acts as phosphoryl donor for enzyme I (EI), which, together with HPr and one of several EIIA and EIIB pairs, forms a phosphorylation cascade which allows phosphorylation of the cognate carbohydrate bound to the membrane-spanning EIIC. HPr of firmicutes and numerous proteobacteria is also phosphorylated in an ATP-dependent reaction catalyzed by the bifunctional HPr kinase/phosphorylase. PTS-mediated regulatory mechanisms are based either on direct phosphorylation of the target protein or on phosphorylation-dependent interactions. For regulation by PTS-mediated phosphorylation, the target proteins either acquired a PTS domain by fusing it to their N or C termini or integrated a specific, conserved PTS regulation domain (PRD) or, alternatively, developed their own specific sites for PTS-mediated phosphorylation. Protein-protein interactions can occur with either phosphorylated or unphosphorylated PTS components and can either stimulate or inhibit the function of the target proteins. This large variety of signal transduction mechanisms allows the PTS to regulate numerous proteins and to form a vast regulatory network responding to the phosphorylation state of various PTS components. PMID:24847021

  9. Shoc2/Sur8 Protein Regulates Neurite Outgrowth

    PubMed Central

    Leon, Gonzalo; Sanchez-Ruiloba, Lucia; Perez-Rodriguez, Andrea; Gragera, Teresa; Martinez, Natalia; Hernandez, Silvia; Anta, Berta; Calero, Olga; Garcia-Dominguez, Carlota A.; Dura, Lara M.; Peña-Jimenez, Daniel; Castro, Judit; Zarich, Natasha; Sanchez-Gomez, Pilar; Calero, Miguel; Iglesias, Teresa; Oliva, Jose L.; Rojas, Jose M.

    2014-01-01

    The Shoc2 protein has been implicated in the positive regulation of the Ras-ERK pathway by increasing the functional binding interaction between Ras and Raf, leading to increased ERK activity. Here we found that Shoc2 overexpression induced sustained ERK phosphorylation, notably in the case of EGF stimulation, and Shoc2 knockdown inhibited ERK activation. We demonstrate that ectopic overexpression of human Shoc2 in PC12 cells significantly promotes neurite extension in the presence of EGF, a stimulus that induces proliferation rather than differentiation in these cells. Finally, Shoc2 depletion reduces both NGF-induced neurite outgrowth and ERK activation in PC12 cells. Our data indicate that Shoc2 is essential to modulate the Ras-ERK signaling outcome in cell differentiation processes involved in neurite outgrowth. PMID:25514808

  10. Werner syndrome protein positively regulates XRCC4-like factor transcription

    PubMed Central

    LIU, DONGYUN; DENG, XIAOLI; YUAN, CHONGZHEN; CHEN, LIN; CONG, YUSHENG; XU, XINGZHI

    2014-01-01

    XRCC4-like factor (XLF) is involved in non-homologous end joining-mediated repair of DNA double-strand breaks (DSBs). Mutations in the WRN gene results in the development of Werner syndrome (WS), a rare autosomal recessive disorder characterized by premature ageing and genome instability. In the present study, it was identified that XLF protein levels were lower in WRN-deficient fibroblasts, compared with normal fibroblasts. Depletion of WRN in HeLa cells led to a decrease of XLF mRNA and its promoter activity. Chromatin immunoprecipitation assays demonstrated that WRN was associated with the XLF promoter. Depletion of XLF in normal human fibroblasts increased the percentage of β-galactosidase (β-gal) staining-positive cells, indicating acceleration in cellular senescence. Taken together, the results suggest that XLF is a transcriptional target of WRN and may be involved in the regulation of cellular senescence. PMID:24626809

  11. Pbx Homeodomain Proteins: TALEnted regulators of Limb Patterning and Outgrowth

    PubMed Central

    Capellini, Terence D.; Zappavigna, Vincenzo; Selleri, Licia

    2011-01-01

    Limb development has long provided an excellent model for understanding the genetic principles driving embryogenesis. Studies utilizing chick and mouse have led to new insights into limb patterning and morphogenesis. Recent research has centered on the regulatory networks underlying limb development. Here, we discuss the hierarchical, overlapping, and iterative roles of Pbx family members in appendicular development that have emerged from genetic analyses in the mouse. Pbx genes are essential in determining limb bud positioning, early bud formation, limb axes establishment and coordination, and patterning and morphogenesis of most elements of the limb and girdle. Pbx proteins directly regulate critical effectors of limb and girdle development, including morphogen-encoding genes like Shh in limb posterior mesoderm, and transcription factor-encoding genes like Alx1 in pre-scapular domains. Interestingly, at least in limb buds, Pbx appear to act not only as Hox cofactors, but also in the upstream control of 5' HoxA/D gene expression. PMID:21416555

  12. Regulation of G Protein-Coupled Receptors by Allosteric Ligands

    PubMed Central

    2013-01-01

    Topographically distinct, druggable, allosteric sites may be present on all G protein-coupled receptors (GPCRs). As such, targeting these sites with synthetic small molecules offers an attractive approach to develop receptor-subtype selective chemical leads for the development of novel therapies. A crucial part of drug development is to understand the acute and chronic effects of such allosteric modulators at their corresponding GPCR target. Key regulatory processes including cell-surface delivery, endocytosis, recycling, and down-regulation tightly control the number of receptors at the surface of the cell. As many GPCR therapeutics will be administered chronically, understanding how such ligands modulate these regulatory pathways forms an essential part of the characterization of novel GPCR ligands. This is true for both orthosteric and allosteric ligands. In this Review, we summarize our current understanding of GPCR regulatory processes with a particular focus on the effects and implications of allosteric targeting of GPCRs. PMID:23398684

  13. The Polarity Protein Pals1 Regulates Radial Sorting of Axons

    PubMed Central

    Zollinger, Daniel R.; Chang, Kae-Jiun; Baalman, Kelli; Kim, Seonhee

    2015-01-01

    Myelin is essential for rapid and efficient action potential propagation in vertebrates. However, the molecular mechanisms regulating myelination remain incompletely characterized. For example, even before myelination begins in the PNS, Schwann cells must radially sort axons to form 1:1 associations. Schwann cells then ensheathe and wrap axons, and establish polarized, subcellular domains, including apical and basolateral domains, paranodes, and Schmidt-Lanterman incisures. Intriguingly, polarity proteins, such as Pals1/Mpp5, are highly enriched in some of these domains, suggesting that they may regulate the polarity of Schwann cells and myelination. To test this, we generated mice with Schwann cells and oligodendrocytes that lack Pals1. During early development of the PNS, Pals1-deficient mice had impaired radial sorting of axons, delayed myelination, and reduced nerve conduction velocities. Although myelination and conduction velocities eventually recovered, polyaxonal myelination remained a prominent feature of adult Pals1-deficient nerves. Despite the enrichment of Pals1 at paranodes and incisures of control mice, nodes of Ranvier and paranodes were unaffected in Pals1-deficient mice, although we measured a significant increase in the number of incisures. As in other polarized cells, we found that Pals1 interacts with Par3 and loss of Pals1 reduced levels of Par3 in Schwann cells. In the CNS, loss of Pals1 affected neither myelination nor the establishment of polarized membrane domains. These results demonstrate that Schwann cells and oligodendrocytes use distinct mechanisms to control their polarity, and that radial sorting in the PNS is a key polarization event that requires Pals1. SIGNIFICANCE STATEMENT This paper reveals the role of the canonical polarity protein Pals1 in radial sorting of axons by Schwann cells. Radial sorting is essential for efficient and proper myelination and is disrupted in some types of congenital muscular dystrophy. PMID:26203142

  14. 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

  15. SLy1 regulates T-cell proliferation during Listeria monocytogenes infection in a Foxo1-dependent manner.

    PubMed

    Schäll, Daniel; Schmitt, Fee; Reis, Bernhard; Brandt, Simone; Beer-Hammer, Sandra

    2015-11-01

    Infection of mice with Listeria monocytogenes results in a strong T-cell response that is critical for an efficient defense. Here, we demonstrate that the adapter protein SLy1 (SH3-domain protein expressed in Lymphocytes 1) is essential for the generation of a fully functional T-cell response. The lack of SLy1 leads to reduced survival rates of infected mice. The increased susceptibility of SLy1 knock-out (KO) mice was caused by reduced proliferation of differentiated T cells. Ex vivo analyses of isolated SLy1 KO T cells displayed a dysregulation of Forkhead box protein O1 shuttling after TCR signaling, which resulted in an increased expression of cell cycle inhibiting genes, and therefore, reduced expansion of the T-cell population. Forkhead box protein O1 shuttles to the cytoplasm after phosphorylation in a protein complex including 14-3-3 proteins. Interestingly, we observed a similar regulation for the adapter protein SLy1, where TCR stimulation results in SLy1 phosphorylation and SLy1 export to the cytoplasm. Moreover, immunoprecipitation analyses revealed a binding of SLy1 to 14-3-3 proteins. Altogether, this study describes SLy1 as an immunoregulatory protein, which is involved in the generation of adaptive immune responses during L. monocytogenes infection, and provides a model of how SLy1 regulates T-cell proliferation. PMID:26306874

  16. DELLA proteins regulate arbuscule formation in arbuscular mycorrhizal symbiosis

    PubMed Central

    Floss, Daniela S.; Levy, Julien G.; Lévesque-Tremblay, Véronique; Pumplin, Nathan; Harrison, Maria J.

    2013-01-01

    Most flowering plants are able to form endosymbioses with arbuscular mycorrhizal fungi. In this mutualistic association, the fungus colonizes the root cortex and establishes elaborately branched hyphae, called arbuscules, within the cortical cells. Arbuscule development requires the cellular reorganization of both symbionts, and the resulting symbiotic interface functions in nutrient exchange. A plant symbiosis signaling pathway controls the development of the symbiosis. Several components of the pathway have been identified, but transcriptional regulators that control downstream pathways for arbuscule formation are still unknown. Here we show that DELLA proteins, which are repressors of gibberellic acid (GA) signaling and function at the nexus of several signaling pathways, are required for arbuscule formation. Arbuscule formation is severely impaired in a Medicago truncatula Mtdella1/Mtdella2 double mutant; GA treatment of wild-type roots phenocopies the della double mutant, and a dominant DELLA protein (della1-Δ18) enables arbuscule formation in the presence of GA. Ectopic expression of della1-Δ18 suggests that DELLA activity in the vascular tissue and endodermis is sufficient to enable arbuscule formation in the inner cortical cells. In addition, expression of della1-Δ18 restores arbuscule formation in the symbiosis signaling pathway mutant cyclops/ipd3, indicating an intersection between DELLA and symbiosis signaling for arbuscule formation. GA signaling also influences arbuscule formation in monocots, and a Green Revolution wheat variety carrying dominant DELLA alleles shows enhanced colonization but a limited growth response to arbuscular mycorrhizal symbiosis. PMID:24297892

  17. Phosphoinositides Regulate Ciliary Protein Trafficking to Modulate Hedgehog Signaling

    PubMed Central

    Roberson, Elle C.; Garcia, Galo; Abedin, Monika; Schurmans, Stéphane; Inoue, Takanari; Reiter, Jeremy F.

    2015-01-01

    SUMMARY Primary cilia interpret vertebrate Hedgehog (Hh) signals. Why cilia are essential for signaling is unclear. One possibility is that some forms of signaling require a distinct membrane lipid composition, found at cilia. We found that the ciliary membrane contains a particular phosphoinositide, PI(4)P, whereas a different phosphoinositide, PI(4,5)P2, is restricted to the membrane of the ciliary base. This distribution is created by Inpp5e, a ciliary phosphoinositide 5-phosphatase. Without Inpp5e, ciliary PI(4,5)P2 levels are elevated and Hh signaling is disrupted. Inpp5e limits the ciliary levels of inhibitors of Hh signaling, including Gpr161 and the PI(4,5)P2-binding protein Tulp3. Increasing ciliary PI(4,5)P2 levels or conferring the ability to bind PI(4)P on Tulp3 increases the ciliary localization of Tulp3. Lowering Tulp3 in cells lacking Inpp5e reduces ciliary Gpr161 levels and restores Hh signaling. Therefore, Inpp5e regulates ciliary membrane phosphoinositide composition, and Tulp3 reads out ciliary phosphoinositides to control ciliary protein localization, enabling Hh signaling. PMID:26305592

  18. Hipk proteins dually regulate Wnt/Wingless signal transduction.

    PubMed

    Verheyen, Esther M; Swarup, Sharan; Lee, Wendy

    2012-01-01

    The Wnt/Wingless (Wg) pathway is an evolutionarily conserved signaling system that is used reiteratively, both spatially and temporally, to control the development of multicellular animals. The stability of cytoplasmic β-catenin/Armadillo, the transcriptional effector of the pathway, is controlled by sequential N-terminal phosphorylation and ubiquitination that targets it for proteasome-mediated degradation. Orthologous members of the Homeodomain-interacting protein kinase family from Drosophila to vertebrates have been implicated in the regulation of Wnt/Wingless signaling. In Drosophila, as a consequence of Hipk activity, cells accumulate stabilized Armadillo that directs the expression of Wg-specific target genes. Hipk promotes the stabilization of Armadillo by inhibiting its ubiquitination (and hence subsequent degradation) by the SCF(Slimb) E3 ubiquitin ligase complex. Vertebrate Hipk2 impedes β-catenin ubiquitination to promote its stability and the Wnt signal in a mechanism that is functionally conserved. Moreover, we describe here that Hipk proteins have a role independent of their effect on β-catenin/Armadillo stability to enhance Wnt/Wingless signaling. PMID:22634475

  19. Ribosomal protein S14 negatively regulates c-Myc activity.

    PubMed

    Zhou, Xiang; Hao, Qian; Liao, Jun-Ming; Liao, Peng; Lu, Hua

    2013-07-26

    The ribosomal gene RPS14 is associated with the cancer-prone 5q-syndrome, which is caused by an interstitial deletion of the long arm of human chromosome 5. Previously, we found that ribosomal protein S14 (RPS14) binds to and inactivates MDM2, consequently leading to p53-dependent cell-cycle arrest and growth inhibition. However, it remains elusive whether RPS14 regulates cell proliferation in a p53-independent manner. Here, we show that RPS14 interacts with the Myc homology box II (MBII) and the C-terminal basic helix-loop-helix leucine zipper (bHLH-LZ) domains of the oncoprotein c-Myc. Further, RPS14 inhibited c-Myc transcriptional activity by preventing the recruitment of c-Myc and its cofactor, TRRAP, to the target gene promoters, as thus suppressing c-Myc-induced cell proliferation. Also, siRNA-mediated RPS14 depletion elevated c-Myc transcriptional activity determined by its target gene, Nucleolin, expression. Interestingly, RPS14 depletion also resulted in the induction of c-Myc mRNA and subsequent protein levels. Consistent with this, RPS14 promoted c-Myc mRNA turnover through an Argonaute 2 (Ago2)- and microRNA-mediated pathway. Taken together, our study demonstrates that RPS14 negates c-Myc functions by directly inhibiting its transcriptional activity and mediating its mRNA degradation via miRNA. PMID:23775087

  20. Cardiac myosin binding protein C regulates postnatal myocyte cytokinesis.

    PubMed

    Jiang, Jianming; Burgon, Patrick G; Wakimoto, Hiroko; Onoue, Kenji; Gorham, Joshua M; O'Meara, Caitlin C; Fomovsky, Gregory; McConnell, Bradley K; Lee, Richard T; Seidman, J G; Seidman, Christine E

    2015-07-21

    Homozygous cardiac myosin binding protein C-deficient (Mybpc(t/t)) mice develop dramatic cardiac dilation shortly after birth; heart size increases almost twofold. We have investigated the mechanism of cardiac enlargement in these hearts. Throughout embryogenesis myocytes undergo cell division while maintaining the capacity to pump blood by rapidly disassembling and reforming myofibrillar components of the sarcomere throughout cell cycle progression. Shortly after birth, myocyte cell division ceases. Cardiac MYBPC is a thick filament protein that regulates sarcomere organization and rigidity. We demonstrate that many Mybpc(t/t) myocytes undergo an additional round of cell division within 10 d postbirth compared with their wild-type counterparts, leading to increased numbers of mononuclear myocytes. Short-hairpin RNA knockdown of Mybpc3 mRNA in wild-type mice similarly extended the postnatal window of myocyte proliferation. However, adult Mybpc(t/t) myocytes are unable to fully regenerate the myocardium after injury. MYBPC has unexpected inhibitory functions during postnatal myocyte cytokinesis and cell cycle progression. We suggest that human patients with homozygous MYBPC3-null mutations develop dilated cardiomyopathy, coupled with myocyte hyperplasia (increased cell number), as observed in Mybpc(t/t) mice. Human patients, with heterozygous truncating MYBPC3 mutations, like mice with similar mutations, have hypertrophic cardiomyopathy. However, the mechanism leading to hypertrophic cardiomyopathy in heterozygous MYBPC3(+/-) individuals is myocyte hypertrophy (increased cell size), whereas the mechanism leading to cardiac dilation in homozygous Mybpc3(-/-) mice is primarily myocyte hyperplasia. PMID:26153423

  1. Cardiac myosin binding protein C regulates postnatal myocyte cytokinesis

    PubMed Central

    Jiang, Jianming; Burgon, Patrick G.; Wakimoto, Hiroko; Onoue, Kenji; Gorham, Joshua M.; O’Meara, Caitlin C.; Fomovsky, Gregory; McConnell, Bradley K.; Lee, Richard T.; Seidman, J. G.; Seidman, Christine E.

    2015-01-01

    Homozygous cardiac myosin binding protein C-deficient (Mybpct/t) mice develop dramatic cardiac dilation shortly after birth; heart size increases almost twofold. We have investigated the mechanism of cardiac enlargement in these hearts. Throughout embryogenesis myocytes undergo cell division while maintaining the capacity to pump blood by rapidly disassembling and reforming myofibrillar components of the sarcomere throughout cell cycle progression. Shortly after birth, myocyte cell division ceases. Cardiac MYBPC is a thick filament protein that regulates sarcomere organization and rigidity. We demonstrate that many Mybpct/t myocytes undergo an additional round of cell division within 10 d postbirth compared with their wild-type counterparts, leading to increased numbers of mononuclear myocytes. Short-hairpin RNA knockdown of Mybpc3 mRNA in wild-type mice similarly extended the postnatal window of myocyte proliferation. However, adult Mybpct/t myocytes are unable to fully regenerate the myocardium after injury. MYBPC has unexpected inhibitory functions during postnatal myocyte cytokinesis and cell cycle progression. We suggest that human patients with homozygous MYBPC3-null mutations develop dilated cardiomyopathy, coupled with myocyte hyperplasia (increased cell number), as observed in Mybpct/t mice. Human patients, with heterozygous truncating MYBPC3 mutations, like mice with similar mutations, have hypertrophic cardiomyopathy. However, the mechanism leading to hypertrophic cardiomyopathy in heterozygous MYBPC3+/− individuals is myocyte hypertrophy (increased cell size), whereas the mechanism leading to cardiac dilation in homozygous Mybpc3−/− mice is primarily myocyte hyperplasia. PMID:26153423

  2. Regulation of glutamate metabolism by protein kinases in mycobacteria.

    PubMed

    O'Hare, Helen M; Durán, Rosario; Cerveñansky, Carlos; Bellinzoni, Marco; Wehenkel, Anne Marie; Pritsch, Otto; Obal, Gonzalo; Baumgartner, Jens; Vialaret, Jérome; Johnsson, Kai; Alzari, Pedro M

    2008-12-01

    Protein kinase G of Mycobacterium tuberculosis has been implicated in virulence and in regulation of glutamate metabolism. Here we show that this kinase undergoes a pattern of autophosphorylation that is distinct from that of other M. tuberculosis protein kinases characterized to date and we identify GarA as a substrate for phosphorylation by PknG. Autophosphorylation of PknG has little effect on kinase activity but promotes binding to GarA, an interaction that is also detected in living mycobacteria. PknG phosphorylates GarA at threonine 21, adjacent to the residue phosphorylated by PknB (T22), and these two phosphorylation events are mutually exclusive. Like the homologue OdhI from Corynebacterium glutamicum, the unphosphorylated form of GarA is shown to inhibit alpha-ketoglutarate decarboxylase in the TCA cycle. Additionally GarA is found to bind and modulate the activity of a large NAD(+)-specific glutamate dehydrogenase with an unusually low affinity for glutamate. Previous reports of a defect in glutamate metabolism caused by pknG deletion may thus be explained by the effect of unphosphorylated GarA on these two enzyme activities, which may also contribute to the attenuation of virulence. PMID:19019160

  3. Senescence Regulation by the p53 Protein Family

    PubMed Central

    Qian, Yingjuan; Chen, Xinbin

    2013-01-01

    p53, a guardian of the genome, exerts its tumor suppression activity by regulating a large number of downstream targets involved in cell cycle arrest, DNA repair, apoptosis, and cellular senescence. Although p53-mediated apoptosis is able to kill cancer cells, a role for cellular senescence in p53-dependent tumor suppression is becoming clear. Mouse studies showed that activation of p53-induced premature senescence promotes tumor regression in vivo. However, p53-mediated cellular senescence also leads to aging-related phenotypes, such as tissue atrophy, stem cell depletion, and impaired wound healing. In addition, several p53 isoforms and two p53 homologs, p63 and p73, have been shown to play a role in cellular senescence and/or aging. Importantly, p53, p63, and p73 are necessary for the maintenance of adult stem cells. Therefore, understanding the dual role the p53 protein family in cancer and aging is critical to solve cancer and longevity in the future. In this chapter, we provide an overview on how p53, p63, p73, and their isoforms regulate cellular senescence and aging. PMID:23296650

  4. Interacting Protein Kinases Involved in the Regulation of Flagellar Length

    PubMed Central

    Erdmann, Maja; Scholz, Anne; Melzer, Inga M.; Schmetz, Christel; Wiese, Martin

    2006-01-01

    A striking difference of the life stages of the protozoan parasite Leishmania is a long flagellum in the insect stage promastigotes and a rudimentary organelle in the mammalian amastigotes. LmxMKK, a mitogen-activated protein (MAP) kinase kinase from Leishmania mexicana, is required for growth of a full-length flagellum. We identified LmxMPK3, a MAP kinase homologue, with a similar expression pattern as LmxMKK being not detectable in amastigotes, up-regulated during the differentiation to promastigotes, constantly expressed in promastigotes, and shut down during the differentiation to amastigotes. LmxMPK3 null mutants resemble the LmxMKK knockouts with flagella reduced to one-fifth of the wild-type length, stumpy cell bodies, and vesicles and membrane fragments in the flagellar pocket. A constitutively activated recombinant LmxMKK activates LmxMPK3 in vitro. Moreover, LmxMKK is likely to be directly involved in the phosphorylation of LmxMPK3 in vivo. Finally, LmxMPK3 is able to phosphorylate LmxMKK, indicating a possible feedback regulation. This is the first time that two interacting components of a signaling cascade have been described in the genus Leishmania. Moreover, we set the stage for the analysis of reversible phosphorylation in flagellar morphogenesis. PMID:16467378

  5. Regulation of peptidoglycan synthesis by outer membrane proteins

    PubMed Central

    Typas, Athanasios; Banzhaf, Manuel; van den Berg van Saparoea, Bart; Verheul, Jolanda; Biboy, Jacob; Nichols, Robert J.; Zietek, Matylda; Beilharz, Katrin; Kannenberg, Kai; von Rechenberg, Moritz; Breukink, Eefjan; den Blaauwen, Tanneke; Gross, Carol A.; Vollmer, Waldemar

    2011-01-01

    Summary Growth of the meshlike peptidoglycan (PG) sacculus located between the bacterial inner and outer membranes (OM) is tightly regulated to ensure cellular integrity, maintain cell shape and orchestrate division. Cytoskeletal elements direct placement and activity of PG synthases from inside the cell but precise spatiotemporal control over this process is poorly understood. We demonstrate that PG synthases are also controlled from outside the sacculus. Two OM lipoproteins, LpoA and LpoB, are essential for the function respectively of PBP1A and PBP1B, the major E. coli bifunctional PG synthases. Each Lpo protein binds specifically to its cognate PBP and stimulates its transpeptidase activity, thereby facilitating attachment of new PG to the sacculus. LpoB shows partial septal localization and our data suggest that th