Science.gov

Sample records for phosphorylation regulates directional

  1. Paxillin-kinase-linker tyrosine phosphorylation regulates directional cell migration.

    PubMed

    Yu, Jianxin A; Deakin, Nicholas O; Turner, Christopher E

    2009-11-01

    Directed cell migration requires the coordination of growth factor and cell adhesion signaling and is of fundamental importance during embryonic development, wound repair, and pathological conditions such as tumor metastasis. Herein, we demonstrate that the ArfGAP, paxillin-kinase-linker (PKL/GIT2), is tyrosine phosphorylated in response to platelet-derived growth factor (PDGF) stimulation, in an adhesion dependent manner and is necessary for directed cell migration. Using a combination of pharmacological inhibitors, knockout cells and kinase mutants, FAK, and Src family kinases were shown to mediate PDGF-dependent PKL tyrosine phosphorylation. In fibroblasts, expression of a PKL mutant lacking the principal tyrosine phosphorylation sites resulted in loss of wound-induced cell polarization as well as directional migration. PKL phosphorylation was necessary for PDGF-stimulated PKL binding to the focal adhesion protein paxillin and expression of paxillin or PKL mutants defective in their respective binding motifs recapitulated the polarization defects. RNA interference or expression of phosphorylation mutants of PKL resulted in disregulation of PDGF-stimulated Rac1 and PAK activities, reduction of Cdc42 and Erk signaling, as well as mislocalization of betaPIX. Together these studies position PKL as an integral component of growth factor and cell adhesion cross-talk signaling, controlling the development of front-rear cell polarity and directional cell migration.

  2. A PP6-type phosphatase holoenzyme directly regulates PIN phosphorylation and auxin efflux in Arabidopsis.

    PubMed

    Dai, Mingqiu; Zhang, Chen; Kania, Urszula; Chen, Fang; Xue, Qin; McCray, Tyra; Li, Gang; Qin, Genji; Wakeley, Michelle; Terzaghi, William; Wan, Jianmin; Zhao, Yunde; Xu, Jian; Friml, Jirí; Deng, Xing Wang; Wang, Haiyang

    2012-06-01

    The directional transport of the phytohormone auxin depends on the phosphorylation status and polar localization of PIN-FORMED (PIN) auxin efflux proteins. While PINIOD (PID) kinase is directly involved in the phosphorylation of PIN proteins, the phosphatase holoenzyme complexes that dephosphorylate PIN proteins remain elusive. Here, we demonstrate that mutations simultaneously disrupting the function of Arabidopsis thaliana FyPP1 (for Phytochrome-associated serine/threonine protein phosphatase1) and FyPP3, two homologous genes encoding the catalytic subunits of protein phosphatase6 (PP6), cause elevated accumulation of phosphorylated PIN proteins, correlating with a basal-to-apical shift in subcellular PIN localization. The changes in PIN polarity result in increased root basipetal auxin transport and severe defects, including shorter roots, fewer lateral roots, defective columella cells, root meristem collapse, abnormal cotyledons (small, cup-shaped, or fused cotyledons), and altered leaf venation. Our molecular, biochemical, and genetic data support the notion that FyPP1/3, SAL (for SAPS DOMAIN-LIKE), and PP2AA proteins (RCN1 [for ROOTS CURL IN NAPHTHYLPHTHALAMIC ACID1] or PP2AA1, PP2AA2, and PP2AA3) physically interact to form a novel PP6-type heterotrimeric holoenzyme complex. We also show that FyPP1/3, SAL, and PP2AA interact with a subset of PIN proteins and that for SAL the strength of the interaction depends on the PIN phosphorylation status. Thus, an Arabidopsis PP6-type phosphatase holoenzyme acts antagonistically with PID to direct auxin transport polarity and plant development by directly regulating PIN phosphorylation.

  3. Phosphorylation regulates the Star-PAP-PIPKIα interaction and directs specificity toward mRNA targets.

    PubMed

    Mohan, Nimmy; Sudheesh, A P; Francis, Nimmy; Anderson, Richard; Laishram, Rakesh S

    2015-08-18

    Star-PAP is a nuclear non-canonical poly(A) polymerase (PAP) that shows specificity toward mRNA targets. Star-PAP activity is stimulated by lipid messenger phosphatidyl inositol 4,5 bisphoshate (PI4,5P2) and is regulated by the associated Type I phosphatidylinositol-4-phosphate 5-kinase that synthesizes PI4,5P2 as well as protein kinases. These associated kinases act as coactivators of Star-PAP that regulates its activity and specificity toward mRNAs, yet the mechanism of control of these interactions are not defined. We identified a phosphorylated residue (serine 6, S6) on Star-PAP in the zinc finger region, the domain required for PIPKIα interaction. We show that S6 is phosphorylated by CKIα within the nucleus which is required for Star-PAP nuclear retention and interaction with PIPKIα. Unlike the CKIα mediated phosphorylation at the catalytic domain, Star-PAP S6 phosphorylation is insensitive to oxidative stress suggesting a signal mediated regulation of CKIα activity. S6 phosphorylation together with coactivator PIPKIα controlled select subset of Star-PAP target messages by regulating Star-PAP-mRNA association. Our results establish a novel role for phosphorylation in determining Star-PAP target mRNA specificity and regulation of 3'-end processing.

  4. Phosphorylation regulates human OCT4.

    PubMed

    Brumbaugh, Justin; Hou, Zhonggang; Russell, Jason D; Howden, Sara E; Yu, Pengzhi; Ledvina, Aaron R; Coon, Joshua J; Thomson, James A

    2012-05-08

    The transcription factor OCT4 is fundamental to maintaining pluripotency and self-renewal. To better understand protein-level regulation of OCT4, we applied liquid chromatography-MS to identify 14 localized sites of phosphorylation, 11 of which were previously unknown. Functional analysis of two sites, T234 and S235, suggested that phosphorylation within the homeobox region of OCT4 negatively regulates its activity by interrupting sequence-specific DNA binding. Mutating T234 and S235 to mimic constitutive phosphorylation at these sites reduces transcriptional activation from an OCT4-responsive reporter and decreases reprogramming efficiency. We also cataloged 144 unique phosphopeptides on known OCT4 interacting partners, including SOX2 and SALL4, that copurified during immunoprecipitation. These proteins were enriched for phosphorylation at motifs associated with ERK signaling. Likewise, OCT4 harbored several putative ERK phosphorylation sites. Kinase assays confirmed that ERK2 phosphorylated these sites in vitro, providing a direct link between ERK signaling and the transcriptional machinery that governs pluripotency.

  5. Phosphorylation regulates human OCT4

    PubMed Central

    Brumbaugh, Justin; Russell, Jason D.; Howden, Sara E.; Yu, Pengzhi; Ledvina, Aaron R.; Coon, Joshua J.; Thomson, James A.

    2012-01-01

    The transcription factor OCT4 is fundamental to maintaining pluripotency and self-renewal. To better understand protein-level regulation of OCT4, we applied liquid chromatography–MS to identify 14 localized sites of phosphorylation, 11 of which were previously unknown. Functional analysis of two sites, T234 and S235, suggested that phosphorylation within the homeobox region of OCT4 negatively regulates its activity by interrupting sequence-specific DNA binding. Mutating T234 and S235 to mimic constitutive phosphorylation at these sites reduces transcriptional activation from an OCT4-responsive reporter and decreases reprogramming efficiency. We also cataloged 144 unique phosphopeptides on known OCT4 interacting partners, including SOX2 and SALL4, that copurified during immunoprecipitation. These proteins were enriched for phosphorylation at motifs associated with ERK signaling. Likewise, OCT4 harbored several putative ERK phosphorylation sites. Kinase assays confirmed that ERK2 phosphorylated these sites in vitro, providing a direct link between ERK signaling and the transcriptional machinery that governs pluripotency. PMID:22474382

  6. Phosphorylation regulates mycobacterial proteasome.

    PubMed

    Anandan, Tripti; Han, Jaeil; Baun, Heather; Nyayapathy, Seeta; Brown, Jacob T; Dial, Rebekah L; Moltalvo, Juan A; Kim, Min-Seon; Yang, Seung Hwan; Ronning, Donald R; Husson, Robert N; Suh, Joowon; Kang, Choong-Min

    2014-09-01

    Mycobacterium tuberculosis possesses a proteasome system that is required for the microbe to resist elimination by the host immune system. Despite the importance of the proteasome in the pathogenesis of tuberculosis, the molecular mechanisms by which proteasome activity is controlled remain largely unknown. Here, we demonstrate that the α-subunit (PrcA) of the M. tuberculosis proteasome is phosphorylated by the PknB kinase at three threonine residues (T84, T202, and T178) in a sequential manner. Furthermore, the proteasome with phosphorylated PrcA enhances the degradation of Ino1, a known proteasomal substrate, suggesting that PknB regulates the proteolytic activity of the proteasome. Previous studies showed that depletion of the proteasome and the proteasome-associated proteins decreases resistance to reactive nitrogen intermediates (RNIs) but increases resistance to hydrogen peroxide (H2O2). Here we show that PknA phosphorylation of unprocessed proteasome β-subunit (pre-PrcB) and α-subunit reduces the assembly of the proteasome complex and thereby enhances the mycobacterial resistance to H2O2 and that H2O2 stress diminishes the formation of the proteasome complex in a PknA-dependent manner. These findings indicate that phosphorylation of the M. tuberculosis proteasome not only modulates proteolytic activity of the proteasome, but also affects the proteasome complex formation contributing to the survival of M. tuberculosis under oxidative stress conditions.

  7. A PP6-Type Phosphatase Holoenzyme Directly Regulates PIN Phosphorylation and Auxin Efflux in Arabidopsis[C][W

    PubMed Central

    Dai, Mingqiu; Zhang, Chen; Kania, Urszula; Chen, Fang; Xue, Qin; Mccray, Tyra; Li, Gang; Qin, Genji; Wakeley, Michelle; Terzaghi, William; Wan, Jianmin; Zhao, Yunde; Xu, Jian; Friml, Jiří; Deng, Xing Wang; Wang, Haiyang

    2012-01-01

    The directional transport of the phytohormone auxin depends on the phosphorylation status and polar localization of PIN-FORMED (PIN) auxin efflux proteins. While PINIOD (PID) kinase is directly involved in the phosphorylation of PIN proteins, the phosphatase holoenzyme complexes that dephosphorylate PIN proteins remain elusive. Here, we demonstrate that mutations simultaneously disrupting the function of Arabidopsis thaliana FyPP1 (for Phytochrome-associated serine/threonine protein phosphatase1) and FyPP3, two homologous genes encoding the catalytic subunits of protein phosphatase6 (PP6), cause elevated accumulation of phosphorylated PIN proteins, correlating with a basal-to-apical shift in subcellular PIN localization. The changes in PIN polarity result in increased root basipetal auxin transport and severe defects, including shorter roots, fewer lateral roots, defective columella cells, root meristem collapse, abnormal cotyledons (small, cup-shaped, or fused cotyledons), and altered leaf venation. Our molecular, biochemical, and genetic data support the notion that FyPP1/3, SAL (for SAPS DOMAIN-LIKE), and PP2AA proteins (RCN1 [for ROOTS CURL IN NAPHTHYLPHTHALAMIC ACID1] or PP2AA1, PP2AA2, and PP2AA3) physically interact to form a novel PP6-type heterotrimeric holoenzyme complex. We also show that FyPP1/3, SAL, and PP2AA interact with a subset of PIN proteins and that for SAL the strength of the interaction depends on the PIN phosphorylation status. Thus, an Arabidopsis PP6-type phosphatase holoenzyme acts antagonistically with PID to direct auxin transport polarity and plant development by directly regulating PIN phosphorylation. PMID:22715043

  8. The cAMP Signaling Pathway and Direct Protein Kinase A Phosphorylation Regulate Polycystin-2 (TRPP2) Channel Function*

    PubMed Central

    Cantero, María del Rocío; Velázquez, Irina F.; Streets, Andrew J.; Ong, Albert C. M.; Cantiello, Horacio F.

    2015-01-01

    Polycystin-2 (PC2) is a TRP-type, Ca2+-permeable non-selective cation channel that plays an important role in Ca2+ signaling in renal and non-renal cells. The effect(s) of the cAMP pathway and kinase mediated phosphorylation of PC2 seem to be relevant to PC2 trafficking and its interaction with polycystin-1. However, the role of PC2 phosphorylation in channel function is still poorly defined. Here we reconstituted apical membranes of term human syncytiotrophoblast (hST), containing endogenous PC2 (PC2hst), and in vitro translated channel protein (PC2iv). Addition of the catalytic subunit of PKA increased by 566% the spontaneous PC2hst channel activity in the presence of ATP. Interestingly, 8-Br-cAMP also stimulated spontaneous PC2hst channel activity in the absence of the exogenous kinase. Either stimulation was inhibited by addition of alkaline phosphatase, which in turn, was reversed by the phosphatase inhibitor vanadate. Neither maneuver modified the single channel conductance but instead increased channel mean open time. PKA directly phosphorylated PC2, which increased the mean open time but not the single channel conductance of the channel. PKA phosphorylation did not modify either R742X truncated or S829A-mutant PC2iv channel function. The data indicate that the cAMP pathway regulates PC2-mediated cation transport in the hST. The relevant PKA site for PC2 channel regulation centers on a single residue serine 829, in the carboxyl terminus. PMID:26269590

  9. IκB Kinase 2 Regulates TPL-2 Activation of Extracellular Signal-Regulated Kinases 1 and 2 by Direct Phosphorylation of TPL-2 Serine 400

    PubMed Central

    Roget, Karine; Ben-Addi, Abduelhakem; Mambole-Dema, Agnes; Gantke, Thorsten; Yang, Huei-Ting; Janzen, Julia; Morrice, Nick; Abbott, Derek

    2012-01-01

    Tumor progression locus 2 (TPL-2) functions as a MEK-1/2 kinase, which is essential for Toll-like receptor 4 (TLR4) activation of extracellular signal-regulated kinase 1 and 2 (ERK-1/2) mitogen-activated protein (MAP) kinases in lipopolysaccharide (LPS)-stimulated macrophages and for inducing the production of the proinflammatory cytokines tumor necrosis factor and interleukin-1β. In unstimulated cells, association of TPL-2 with NF-κB1 p105 prevents TPL-2 phosphorylation of MEK-1/2. LPS stimulation of TPL-2 MEK-1/2 kinase activity requires TPL-2 release from p105. This is triggered by IκB kinase 2 (IKK-2) phosphorylation of the p105 PEST region, which promotes p105 ubiquitination and degradation by the proteasome. LPS activation of ERK-1/2 additionally requires transphosphorylation of TPL-2 on serine 400 in its C terminus, which controls TPL-2 signaling to ERK-1/2 independently of p105. However, the identity of the protein kinase responsible for TPL-2 serine 400 phosphorylation remained unknown. In the present study, we show that TPL-2 serine 400 phosphorylation is mediated by IKK2. The IKK complex therefore regulates two of the key regulatory steps required for TPL-2 activation of ERK-1/2, underlining the close linkage of ERK-1/2 MAP kinase activation to upregulation of NF-κB-dependent transcription. PMID:22988300

  10. Phosphorylation of tau is regulated by PKN.

    PubMed

    Taniguchi, T; Kawamata, T; Mukai, H; Hasegawa, H; Isagawa, T; Yasuda, M; Hashimoto, T; Terashima, A; Nakai, M; Mori, H; Ono, Y; Tanaka, C

    2001-03-30

    For the phosphorylation state of microtubule-associated protein, tau plays a pivotal role in regulating microtubule networks in neurons. Tau promotes the assembly and stabilization of microtubules. The potential for tau to bind to microtubules is down-regulated after local phosphorylation. When we investigated the effects of PKN activation on tau phosphorylation, we found that PKN triggers disruption of the microtubule array both in vitro and in vivo and predominantly phosphorylates tau in microtubule binding domains (MBDs). PKN has a catalytic domain highly homologous to protein kinase C (PKC), a kinase that phosphorylates Ser-313 (= Ser-324, the number used in this study) in MBDs. Thus, we identified the phosphorylation sites of PKN and PKC subtypes (PKC-alpha, -betaI, -betaII, -gamma, -delta, -epsilon, -zeta, and -lambda) in MBDs. PKN phosphorylates Ser-258, Ser-320, and Ser-352, although all PKC subtypes phosphorylate Ser-258, Ser-293, Ser-324, and Ser-352. There is a PKN-specific phosphorylation site, Ser-320, in MBDs. HIA3, a novel phosphorylation-dependent antibody recognizing phosphorylated tau at Ser-320, showed immunoreactivity in Chinese hamster ovary cells expressing tau and the active form of PKN, but not in Chinese hamster ovary cells expressing tau and the inactive form of PKN. The immunoreactivity for phosphorylated tau at Ser-320 increased in the presence of a phosphatase inhibitor, FK506 treatment, which means that calcineurin (protein phosphatase 2B) may be involved in dephosphorylating tau at Ser-320 site. We also noted that PKN reduces the phosphorylation recognized by the phosphorylation-dependent antibodies AT8, AT180, and AT270 in vivo. Thus PKN serves as a regulator of microtubules by specific phosphorylation of tau, which leads to disruption of tubulin assembly.

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

    SciTech Connect

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

    2015-04-17

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

  12. The regulation of STIM1 by phosphorylation

    PubMed Central

    Pozo-Guisado, Eulalia; Martin-Romero, Francisco Javier

    2013-01-01

    Calcium ion (Ca2+) concentration plays a key role in cell signaling in eukaryotic cells. At the cellular level, Ca2+ directly participates in such diverse cellular events as adhesion and migration, differentiation, contraction, secretion, synaptic transmission, fertilization, and cell death. As a consequence of these diverse actions, the cytosolic concentration of free Ca2+ is tightly regulated by the coordinated activity of Ca2+ channels, Ca2+ pumps, and Ca2+-binding proteins. Although many of these regulators have been studied in depth, other proteins have been described recently, and naturally far less is known about their contribution to cell physiology. Within this last group of proteins, STIM1 has emerged as a major contributor to Ca2+ signaling by means of its activity as Ca2+ channel regulator. STIM1 is a protein resident mainly, but not exclusively, in the endoplasmic reticulum (ER), and activates a set of plasma membrane Ca2+ channels termed store-operated calcium channels (SOCs) when the concentration of free Ca2+ within the ER drops transiently as a result of Ca2+ release from this compartment. Knowledge regarding the molecular architecture of STIM1 has grown considerably during the last years, and several structural domains within STIM1 have been reported to be required for the specific molecular interactions with other important players in Ca2+ signaling, such as Ca2+ channels and microtubules. Within the modulators of STIM1, phosphorylation has been shown to both activate and inactivate STIM1-dependent Ca2+ entry depending on the cell type, cell cycle phase, and the specific residue that becomes modified. Here we shall review current knowledge regarding the modulation of STIM1 by phosphorylation. PMID:24505502

  13. Palmitoylation gates phosphorylation-dependent regulation of BK potassium channels.

    PubMed

    Tian, Lijun; Jeffries, Owen; McClafferty, Heather; Molyvdas, Adam; Rowe, Iain C M; Saleem, Fozia; Chen, Lie; Greaves, Jennifer; Chamberlain, Luke H; Knaus, Hans-Guenther; Ruth, Peter; Shipston, Michael J

    2008-12-30

    Large conductance calcium- and voltage-gated potassium (BK) channels are important regulators of physiological homeostasis and their function is potently modulated by protein kinase A (PKA) phosphorylation. PKA regulates the channel through phosphorylation of residues within the intracellular C terminus of the pore-forming alpha-subunits. However, the molecular mechanism(s) by which phosphorylation of the alpha-subunit effects changes in channel activity are unknown. Inhibition of BK channels by PKA depends on phosphorylation of only a single alpha-subunit in the channel tetramer containing an alternatively spliced insert (STREX) suggesting that phosphorylation results in major conformational rearrangements of the C terminus. Here, we define the mechanism of PKA inhibition of BK channels and demonstrate that this regulation is conditional on the palmitoylation status of the channel. We show that the cytosolic C terminus of the STREX BK channel uniquely interacts with the plasma membrane via palmitoylation of evolutionarily conserved cysteine residues in the STREX insert. PKA phosphorylation of the serine residue immediately upstream of the conserved palmitoylated cysteine residues within STREX dissociates the C terminus from the plasma membrane, inhibiting STREX channel activity. Abolition of STREX palmitoylation by site-directed mutagenesis or pharmacological inhibition of palmitoyl transferases prevents PKA-mediated inhibition of BK channels. Thus, palmitoylation gates BK channel regulation by PKA phosphorylation. Palmitoylation and phosphorylation are both dynamically regulated; thus, cross-talk between these 2 major posttranslational signaling cascades provides a mechanism for conditional regulation of BK channels. Interplay of these distinct signaling cascades has important implications for the dynamic regulation of BK channels and physiological homeostasis.

  14. Regulation of renal fibrosis by Smad3 Thr388 phosphorylation.

    PubMed

    Qu, Xinli; Li, Xueling; Zheng, Yaowu; Ren, Yi; Puelles, Victor G; Caruana, Georgina; Nikolic-Paterson, David J; Li, Jinhua

    2014-04-01

    Transforming growth factor-β (TGF-β) promotes tissue fibrosis via receptor-mediated phosphorylation of the receptor-activated Smad2/3, together with Smad4. Of these, Smad3 plays a major profibrotic role in mouse models of tissue fibrosis. Transcriptional activity of the Smad3 protein is regulated by phosphorylation of residues in the C-terminal domain and the linker region. Herein, we examined the role of a novel phosphorylation site within the MH2 domain (T388) in the regulation of Smad3 activity. Confocal microscopy using an Smad3 phosphorylated T388-specific antibody identified phosphorylation of Smad3 T388 in myofibroblasts and tubular epithelial cells in human focal and segmental glomerulosclerosis and mouse models of unilateral ureteric obstruction and diabetic nephropathy, whereas phosphorylated T388 was largely absent in normal kidney. In vitro, TGF-β1 induced phosphorylation of Smad3 T388 in a biphasic pattern. A point mutation of T388/V in an Smad3 construct demonstrated that phosphorylation of T388 promotes Smad3 binding to Smad4 and CDK8, but was not necessary for nuclear translocation. Furthermore, T388 phosphorylation was required for TGF-β-induced collagen I gene promoter activity and extracellular matrix production in cultured fibroblasts. In conclusion, our study identifies phosphorylation of T388 in the Smad3 MH2 domain as an important mechanism that regulates the profibrotic TGF-β/Smad3 signaling pathway, which has direct relevance to human and experimental fibrotic kidney disease.

  15. Grb2 negatively regulates epidermal growth factor-induced phospholipase C-gamma1 activity through the direct interaction with tyrosine-phosphorylated phospholipase C-gamma1.

    PubMed

    Choi, Jang Hyun; Hong, Won-Pyo; Yun, Sanguk; Kim, Hyeon Soo; Lee, Jong-Ryul; Park, Jong Bae; Bae, Yun Soo; Ryu, Sung Ho; Suh, Pann-Ghill

    2005-10-01

    Phospholipase C-gamma1 (PLC-gamma1) plays pivotal roles in cellular growth and proliferation. Upon the stimulation of growth factors and hormones, PLC-gamma1 is rapidly phosphorylated at three known sites; Tyr771, Tyr783 and Tyr1254 and its enzymatic activity is up-regulated. In this study, we demonstrate for the first time that Grb2, an adaptor protein, specifically interacts with tyrosine-phosphorylated PLC-gamma1 at Tyr783. The association of Grb2 with PLC-gamma1 was induced by the treatment with epidermal growth factor (EGF). Replacement of Tyr783 with Phe completely blocked EGF-induced interaction of PLC-gamma1 with Grb2, indicating that tyrosine phosphorylation of PLC-gamma1 at Tyr783 is essential for the interaction with Grb2. Interestingly, the depletion of Grb2 from HEK-293 cells by RNA interference significantly enhanced increased EGF-induced PLC-gamma1 enzymatic activity and mobilization of the intracellular Ca2+, while it did not affect EGF-induced tyrosine phosphorylation of PLC-gamma1. Furthermore, overexpression of Grb2 inhibited PLC-gamma1 enzymatic activity. Taken together, these results suggest Grb2, in addition to its key function in signaling through Ras, may have a negatively regulatory role on EGF-induced PLC-gamma1 activation.

  16. Phosphorylation mechanisms in dopamine transporter regulation.

    PubMed

    Foster, James D; Vaughan, Roxanne A

    2016-11-09

    The dopamine transporter (DAT) is a plasma membrane phosphoprotein that actively translocates extracellular dopamine (DA) into presynaptic neurons. The transporter is the primary mechanism for control of DA levels and subsequent neurotransmission, and is the target for abused and therapeutic drugs that exert their effects by suppressing reuptake. The transport capacity of DAT is acutely regulated by signaling systems and drug exposure, providing neurons the ability to fine-tune DA clearance in response to specific conditions. Kinase pathways play major roles in these mechanisms, and this review summarizes the current status of DAT phosphorylation characteristics and the evidence linking transporter phosphorylation to control of reuptake and other functions. Greater understanding of these processes may aid in elucidation of their possible contributions to DA disease states and suggest specific phosphorylation sites as targets for therapeutic manipulation of reuptake.

  17. 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.

  18. ZDHHC3 Tyrosine Phosphorylation Regulates Neural Cell Adhesion Molecule Palmitoylation

    PubMed Central

    Lievens, Patricia Marie-Jeanne; Kuznetsova, Tatiana; Kochlamazashvili, Gaga; Cesca, Fabrizia; Gorinski, Natalya; Galil, Dalia Abdel; Cherkas, Volodimir; Ronkina, Natalia; Lafera, Juri; Gaestel, Matthias

    2016-01-01

    The neural cell adhesion molecule (NCAM) mediates cell-cell and cell-matrix adhesion. It is broadly expressed in the nervous system and regulates neurite outgrowth, synaptogenesis, and synaptic plasticity. Previous in vitro studies revealed that palmitoylation of NCAM is required for fibroblast growth factor 2 (FGF2)-stimulated neurite outgrowth and identified the zinc finger DHHC (Asp-His-His-Cys)-containing proteins ZDHHC3 and ZDHHC7 as specific NCAM-palmitoylating enzymes. Here, we verified that FGF2 controlled NCAM palmitoylation in vivo and investigated molecular mechanisms regulating NCAM palmitoylation by ZDHHC3. Experiments with overexpression and pharmacological inhibition of FGF receptor (FGFR) and Src revealed that these kinases control tyrosine phosphorylation of ZDHHC3 and that ZDHHC3 is phosphorylated by endogenously expressed FGFR and Src proteins. By site-directed mutagenesis, we found that Tyr18 is an FGFR1-specific ZDHHC3 phosphorylation site, while Tyr295 and Tyr297 are specifically phosphorylated by Src kinase in cell-based and cell-free assays. Abrogation of tyrosine phosphorylation increased ZDHHC3 autopalmitoylation, enhanced interaction with NCAM, and upregulated NCAM palmitoylation. Expression of ZDHHC3 with tyrosine mutated in cultured hippocampal neurons promoted neurite outgrowth. Our findings for the first time highlight that FGFR- and Src-mediated tyrosine phosphorylation of ZDHHC3 modulates ZDHHC3 enzymatic activity and plays a role in neuronal morphogenesis. PMID:27247265

  19. 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.

  20. The regulation of AMP-activated protein kinase by phosphorylation.

    PubMed Central

    Stein, S C; Woods, A; Jones, N A; Davison, M D; Carling, D

    2000-01-01

    The AMP-activated protein kinase (AMPK) cascade is activated by an increase in the AMP/ATP ratio within the cell. AMPK is regulated allosterically by AMP and by reversible phosphorylation. Threonine-172 within the catalytic subunit (alpha) of AMPK (Thr(172)) was identified as the major site phosphorylated by the AMP-activated protein kinase kinase (AMPKK) in vitro. We have used site-directed mutagenesis to study the role of phosphorylation of Thr(172) on AMPK activity. Mutation of Thr(172) to an aspartic acid residue (T172D) in either alpha1 or alpha2 resulted in a kinase complex with approx. 50% the activity of the corresponding wild-type complex. The activity of wild-type AMPK decreased by greater than 90% following treatment with protein phosphatases, whereas the activity of the T172D mutant complex fell by only 10-15%. Mutation of Thr(172) to an alanine residue (T172A) almost completely abolished kinase activity. These results indicate that phosphorylation of Thr(172) accounts for most of the activation by AMPKK, but that other sites are involved. In support of this we have shown that AMPKK phosphorylates at least two other sites on the alpha subunit and one site on the beta subunit. Furthermore, we provide evidence that phosphorylation of Thr(172) may be involved in the sensitivity of the AMPK complex to AMP. PMID:10642499

  1. Regulation of divalent metal transporter-1 by serine phosphorylation

    PubMed Central

    Seo, Young Ah; Kumara, Ruvin; Wetli, Herbert; Wessling-Resnick, Marianne

    2016-01-01

    Divalent metal transporter-1 (DMT1) mediates dietary iron uptake across the intestinal mucosa and facilitates peripheral delivery of iron released by transferrin in the endosome. Here, we report that classical cannabinoids (Δ9-tetrahydrocannabinol, Δ9-THC), nonclassical cannabinoids (CP 55,940), aminoalkylindoles (WIN 55,212-2) and endocannabinoids (anandamide) reduce 55Fe and 54Mn uptake by HEK293T(DMT1) cells stably expressing the transporter. siRNA knockdown of cannabinoid receptor type 2 (CB2) abrogated inhibition. CB2 is a G-protein (GTP-binding protein)-coupled receptor that negatively regulates signal transduction cascades involving serine/threonine kinases. Immunoprecipitation experiments showed that DMT1 is serine-phosphorylated under basal conditions, but that treatment with Δ9-THC reduced phosphorylation. Site-directed mutation of predicted DMT1 phosphosites further showed that substitution of serine with alanine at N-terminal position 43 (S43A) abolished basal phosphorylation. Concordantly, both the rate and extent of 55Fe uptake in cells expressing DMT1(S43A) was reduced compared with those expressing wild-type DMT1. Among kinase inhibitors that affected DMT1-mediated iron uptake, staurosporine also reduced DMT1 phosphorylation confirming a role for serine phosphorylation in iron transport regulation. These combined data indicate that phosphorylation at serine 43 of DMT1 promotes transport activity, whereas dephosphorylation is associated with loss of iron uptake. Since anti-inflammatory actions mediated through CB2 would be associated with reduced DMT1 phosphorylation, we postulate that this pathway provides a means to reduce oxidative stress by limiting iron uptake. PMID:27681840

  2. 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.

  3. In the Staphylococcus aureus two-component system sae, the response regulator SaeR binds to a direct repeat sequence and DNA binding requires phosphorylation by the sensor kinase SaeS.

    PubMed

    Sun, Fei; Li, Chunling; Jeong, Dowon; Sohn, Changmo; He, Chuan; Bae, Taeok

    2010-04-01

    Staphylococcus aureus uses the SaeRS two-component system to control the expression of many virulence factors such as alpha-hemolysin and coagulase; however, the molecular mechanism of this signaling has not yet been elucidated. Here, using the P1 promoter of the sae operon as a model target DNA, we demonstrated that the unphosphorylated response regulator SaeR does not bind to the P1 promoter DNA, while its C-terminal DNA binding domain alone does. The DNA binding activity of full-length SaeR could be restored by sensor kinase SaeS-induced phosphorylation. Phosphorylated SaeR is more resistant to digestion by trypsin, suggesting conformational changes. DNase I footprinting assays revealed that the SaeR protection region in the P1 promoter contains a direct repeat sequence (GTTAAN(6)GTTAA [where N is any nucleotide]). This sequence is critical to the binding of phosphorylated SaeR. Mutational changes in the repeat sequence greatly reduced both the in vitro binding of SaeR and the in vivo function of the P1 promoter. From these results, we concluded that SaeR recognizes the direct repeat sequence as a binding site and that binding requires phosphorylation by SaeS.

  4. A redox-regulated tyrosine phosphorylation cascade in rat spermatozoa.

    PubMed

    Lewis, B; Aitken, R J

    2001-01-01

    Rat spermatozoa from both the caput and cauda epididymidis were shown to generate superoxide anion (O2-.) both spontaneously and following stimulation with NAD(P)H. Caput spermatozoa gave a significantly greater O2- response to NADPH stimulation than caudal cells, whereas in both cell types the responses to exogenous NADPH and NADH were approximately equivalent. Analysis of H2O2 production revealed that this oxidant was generated only by caudal epididymal cells and only in these cells did the stimulation of reactive oxygen species (ROS) production with NADPH lead to an increase in tyrosine phosphorylation. Stimulation of ROS production with NADPH increased intracellular cyclic adenosine monophosphate (cAMP) levels in both caput and caudal epididymal cells, but only in caudal cells did cAMP stimulate tyrosine phosphorylation, in keeping with the NADPH results. On the basis of these findings we propose that tyrosine phosphorylation in rat spermatozoa is driven by ROS acting via 2 different but complementary mechanisms; O2-. stimulates tyrosine kinase activity indirectly through the elevation of intracellular cAMP while H2O2 acts directly on the kinase/phosphatase system, stimulating the former and inhibiting the latter. Zinc was examined as a potential regulator of this signal transduction cascade and was shown to suppress tyrosine phosphorylation in caput cells but to promote this activity in caudal spermatozoa, possibly through an inhibitory effect on tyrosine phosphatase activity. These results reveal the maturation of a redox-regulated, cAMP-mediated, signal transduction cascade during epididymal transit in the rat that is sensitive to zinc and plays a key role in the control of tyrosine phosphorylation events associated with capacitation.

  5. The Regulation of NF-κB Subunits by Phosphorylation

    PubMed Central

    Christian, Frank; Smith, Emma L.; Carmody, Ruaidhrí J.

    2016-01-01

    The NF-κB transcription factor is the master regulator of the inflammatory response and is essential for the homeostasis of the immune system. NF-κB regulates the transcription of genes that control inflammation, immune cell development, cell cycle, proliferation, and cell death. The fundamental role that NF-κB plays in key physiological processes makes it an important factor in determining health and disease. The importance of NF-κB in tissue homeostasis and immunity has frustrated therapeutic approaches aimed at inhibiting NF-κB activation. However, significant research efforts have revealed the crucial contribution of NF-κB phosphorylation to controlling NF-κB directed transactivation. Importantly, NF-κB phosphorylation controls transcription in a gene-specific manner, offering new opportunities to selectively target NF-κB for therapeutic benefit. This review will focus on the phosphorylation of the NF-κB subunits and the impact on NF-κB function. PMID:26999213

  6. Regulation of Endothelial Adherens Junctions by Tyrosine Phosphorylation

    PubMed Central

    Adam, Alejandro Pablo

    2015-01-01

    Endothelial cells form a semipermeable, regulated barrier that limits the passage of fluid, small molecules, and leukocytes between the bloodstream and the surrounding tissues. The adherens junction, a major mechanism of intercellular adhesion, is comprised of transmembrane cadherins forming homotypic interactions between adjacent cells and associated cytoplasmic catenins linking the cadherins to the cytoskeleton. Inflammatory conditions promote the disassembly of the adherens junction and a loss of intercellular adhesion, creating openings or gaps in the endothelium through which small molecules diffuse and leukocytes transmigrate. Tyrosine kinase signaling has emerged as a central regulator of the inflammatory response, partly through direct phosphorylation and dephosphorylation of the adherens junction components. This review discusses the findings that support and those that argue against a direct effect of cadherin and catenin phosphorylation in the disassembly of the adherens junction. Recent findings indicate a complex interaction between kinases, phosphatases, and the adherens junction components that allow a fine regulation of the endothelial permeability to small molecules, leukocyte migration, and barrier resealing. PMID:26556953

  7. A compartmentalized phosphorylation/dephosphorylation system that regulates U snRNA export from the nucleus.

    PubMed

    Kitao, Saori; Segref, Alexandra; Kast, Juergen; Wilm, Matthias; Mattaj, Iain W; Ohno, Mutsuhito

    2008-01-01

    PHAX (phosphorylated adaptor for RNA export) is the key regulator of U snRNA nuclear export in metazoa. Our previous work revealed that PHAX is phosphorylated in the nucleus and is exported as a component of the U snRNA export complex to the cytoplasm, where it is dephosphorylated (M. Ohno, A. Segref, A. Bachi, M. Wilm, and I. W. Mattaj, Cell 101:187-198, 2000). PHAX phosphorylation is essential for export complex assembly, whereas its dephosphorylation causes export complex disassembly. Thus, PHAX is subject to a compartmentalized phosphorylation/dephosphorylation cycle that contributes to transport directionality. However, neither essential PHAX phosphorylation sites nor the modifying enzymes that contribute to the compartmentalized system have been identified. Here, we identify PHAX phosphorylation sites that are necessary and sufficient for U snRNA export. Mutation of the phosphorylation sites inhibited U snRNA export in a dominant-negative way. We also show, by both biochemical and RNA interference knockdown experiments, that the nuclear kinase and the cytoplasmic phosphatase for PHAX are CK2 kinase and protein phosphatase 2A, respectively. Our results reveal the composition of the compartmentalized phosphorylation/dephosphorylation system that regulates U snRNA export. This finding was surprising in that such a specific system for U snRNA export regulation is composed of two such universal regulators, suggesting that this compartmentalized system is used more broadly for gene expression regulation.

  8. Regulation of Rho proteins by phosphorylation in the cardiovascular system.

    PubMed

    Loirand, Gervaise; Guilluy, Christophe; Pacaud, Pierre

    2006-08-01

    The small G protein Rho signaling pathways are recognized as major regulators of cardiovascular functions, and activation of Rho proteins appears to be a common component for the pathogenesis of hypertension and vascular proliferative disorders. Rho proteins are tightly regulated, and recent evidence suggests that modulation of Rho protein signaling by phosphorylation of Rho proteins provides an additional simple mechanism for coordinating Rho protein functions. This regulation by phosphorylation is particularly important in the arterial wall, where RhoA protein expressed in vascular smooth muscle cells is controlled by the endothelium through the nitric oxide/cGMP-dependent kinase pathway.

  9. 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

  10. Early growth response 1 (Egr-1) regulates phosphorylation of microtubule-associated protein tau in mammalian brain.

    PubMed

    Lu, Yifan; Li, Tong; Qureshi, Hamid Y; Han, Dong; Paudel, Hemant K

    2011-06-10

    In the normal brain, tau protein is phosphorylated at a number of proline- and non-proline directed sites, which reduce tau microtubule binding and thus regulate microtubule dynamics. In Alzheimer disease (AD), tau is abnormally hyperphosphorylated, leading to neurofibrillary tangle formation and microtubule disruption, suggesting a loss of regulatory mechanisms controlling tau phosphorylation. Early growth response 1 (Egr-1) is a transcription factor that is significantly up-regulated in AD brain. The pathological significance of this up-regulation is not known. In this study, we found that lentivirus-mediated overexpression of Egr-1 in rat brain hippocampus and primary neurons in culture activates proline-directed kinase Cdk5, inactivates PP1, promotes tau phosphorylation at both proline-directed Ser(396/404) and non-proline-directed Ser(262) sites, and destabilizes microtubules. Furthermore, in Egr-1(-/-) mouse brain, Cdk5 activity was decreased, PP1 activity was increased, and tau phosphorylation was reduced at both proline-directed and non-proline-directed sites. By using nerve growth factor-exposed PC12 cells, we determined that Egr-1 activates Cdk5 to promote phosphorylation of tau and inactivates PP1 via phosphorylation. When Cdk5 was inhibited, tau phosphorylation at both proline- and non-proline directed sites and PP1 phosphorylation were blocked, indicating that Egr-1 acts through Cdk5. By using an in vitro kinase assay and HEK-293 cells transfected with tau, PP1, and Cdk5, we found that Cdk5 phosphorylates Ser(396/404) directly. In addition, by phosphorylating and inactivating PP1, Cdk5 promotes tau phosphorylation at Ser(262) indirectly. Our results indicate that Egr-1 is an in vivo regulator of tau phosphorylation and suggest that in AD brain increased levels of Egr-1 aberrantly activate an Egr-1/Cdk5/PP1 pathway, leading to accumulation of hyperphosphorylated tau, thus destabilizing the microtubule cytoskeleton.

  11. Calcineurin regulates phosphorylation status of transcription factor osterix.

    PubMed

    Okamura, Hirohiko; Amorim, Bruna Rabelo; Wang, Jie; Yoshida, Kaya; Haneji, Tatsuji

    2009-02-06

    Osterix is an osteoblast-specific transcriptional factor that is essential for osteoblast differentiation and bone formation. Calcineurin regulates bone formation through modulating osteoblast differentiation. However, post-translational modification of osterix such as phosphorylation and interactions between osterix and calcineurin remains unclear. In the present study, we demonstrated that calcineurin interacted with osterix determined by immunoprecipitation assay and Western analysis. Immunocytochemical study also revealed that osterix and calcineurin were co-localized in nucleus. Deletion of calcineurin binding motif on osterix molecule disrupted osterix-calcineurin interaction. Phosphorylation status of osterix was augmented by treatment with phosphatase inhibitors, FK506 and calyculin A. In contrast, treatment of recombinant calcineurin reduced phosphorylation status of osterix. Our present study suggests that calcineurin has an important role in the function of osterix through its modification of phosphorylation.

  12. Identification of novel in vivo phosphorylation sites of the human proapoptotic protein BAD: pore-forming activity of BAD is regulated by phosphorylation.

    PubMed

    Polzien, Lisa; Baljuls, Angela; Rennefahrt, Ulrike E E; Fischer, Andreas; Schmitz, Werner; Zahedi, Rene P; Sickmann, Albert; Metz, Renate; Albert, Stefan; Benz, Roland; Hekman, Mirko; Rapp, Ulf R

    2009-10-09

    BAD is a proapoptotic member of the Bcl-2 protein family that is regulated by phosphorylation in response to survival factors. Although much attention has been devoted to the identification of phosphorylation sites in murine BAD, little data are available with respect to phosphorylation of human BAD protein. Using mass spectrometry, we identified here besides the established phosphorylation sites at serines 75, 99, and 118 several novel in vivo phosphorylation sites within human BAD (serines 25, 32/34, 97, and 124). Furthermore, we investigated the quantitative contribution of BAD targeting kinases in phosphorylating serine residues 75, 99, and 118. Our results indicate that RAF kinases represent, besides protein kinase A, PAK, and Akt/protein kinase B, in vivo BAD-phosphorylating kinases. RAF-induced phosphorylation of BAD was reduced to control levels using the RAF inhibitor BAY 43-9006. This phosphorylation was not prevented by MEK inhibitors. Consistently, expression of constitutively active RAF suppressed apoptosis induced by BAD and the inhibition of colony formation caused by BAD could be prevented by RAF. In addition, using the surface plasmon resonance technique, we analyzed the direct consequences of BAD phosphorylation by RAF with respect to association with 14-3-3 and Bcl-2/Bcl-X(L) proteins. Phosphorylation of BAD by active RAF promotes 14-3-3 protein association, in which the phosphoserine 99 represented the major binding site. Finally, we show here that BAD forms channels in planar bilayer membranes in vitro. This pore-forming capacity was dependent on phosphorylation status and interaction with 14-3-3 proteins. Collectively, our findings provide new insights into the regulation of BAD function by phosphorylation.

  13. Dynamic phosphorylation of Histone Deacetylase 1 by Aurora kinases during mitosis regulates zebrafish embryos development

    PubMed Central

    Loponte, Sara; Segré, Chiara V.; Senese, Silvia; Miccolo, Claudia; Santaguida, Stefano; Deflorian, Gianluca; Citro, Simona; Mattoscio, Domenico; Pisati, Federica; Moser, Mirjam A.; Visintin, Rosella; Seiser, Christian; Chiocca, Susanna

    2016-01-01

    Histone deacetylases (HDACs) catalyze the removal of acetyl molecules from histone and non-histone substrates playing important roles in chromatin remodeling and control of gene expression. Class I HDAC1 is a critical regulator of cell cycle progression, cellular proliferation and differentiation during development; it is also regulated by many post-translational modifications (PTMs). Herein we characterize a new mitosis-specific phosphorylation of HDAC1 driven by Aurora kinases A and B. We show that this phosphorylation affects HDAC1 enzymatic activity and it is critical for the maintenance of a proper proliferative and developmental plan in a complex organism. Notably, we find that Aurora-dependent phosphorylation of HDAC1 regulates histone acetylation by modulating the expression of genes directly involved in the developing zebrafish central nervous system. Our data represent a step towards the comprehension of HDAC1 regulation by its PTM code, with important implications in unravelling its roles both in physiology and pathology. PMID:27458029

  14. Akt phosphorylates and regulates Pdcd4 tumor suppressor protein.

    PubMed

    Palamarchuk, Alexey; Efanov, Alexey; Maximov, Vadim; Aqeilan, Rami I; Croce, Carlo M; Pekarsky, Yuri

    2005-12-15

    Programmed cell death 4 (Pdcd4) is a tumor suppressor protein that interacts with eukaryotic initiation factor 4A and inhibits protein synthesis. Pdcd4 also suppresses the transactivation of activator protein-1 (AP-1)-responsive promoters by c-Jun. The Akt (protein kinase B) serine/threonine kinase is a key mediator of phosphoinositide 3-kinase pathway involved in the regulation of cell proliferation, survival, and growth. Because Pdcd4 has two putative Akt phosphorylation sites at Ser(67) and Ser(457), we investigated whether Akt phosphorylates and regulates Pdcd4. Our results show that Akt specifically phosphorylates Ser(67) and Ser(457) residues of Pdcd4 in vitro and in vivo. We further show that phosphorylation of Pdcd4 by Akt causes nuclear translocation of Pdcd4. Using luciferase assay, we show that phosphorylation of Pdcd4 by Akt also causes a significant decrease of the ability of Pdcd4 to interfere with the transactivation of AP-1-responsive promoter by c-Jun.

  15. Protein Phosphorylation in Amyloplasts Regulates Starch Branching Enzyme Activity and Protein–Protein Interactions

    PubMed Central

    Tetlow, Ian J.; Wait, Robin; Lu, Zhenxiao; Akkasaeng, Rut; Bowsher, Caroline G.; Esposito, Sergio; Kosar-Hashemi, Behjat; Morell, Matthew K.; Emes, Michael J.

    2004-01-01

    Protein phosphorylation in amyloplasts and chloroplasts of Triticum aestivum (wheat) was investigated after the incubation of intact plastids with γ-32P-ATP. Among the soluble phosphoproteins detected in plastids, three forms of starch branching enzyme (SBE) were phosphorylated in amyloplasts (SBEI, SBEIIa, and SBEIIb), and both forms of SBE in chloroplasts (SBEI and SBEIIa) were shown to be phosphorylated after sequencing of the immunoprecipitated 32P-labeled phosphoproteins using quadrupole-orthogonal acceleration time of flight mass spectrometry. Phosphoamino acid analysis of the phosphorylated SBE forms indicated that the proteins are all phosphorylated on Ser residues. Analysis of starch granule–associated phosphoproteins after incubation of intact amyloplasts with γ-32P-ATP indicated that the granule-associated forms of SBEII and two granule-associated forms of starch synthase (SS) are phosphorylated, including SSIIa. Measurement of SBE activity in amyloplasts and chloroplasts showed that phosphorylation activated SBEIIa (and SBEIIb in amyloplasts), whereas dephosphorylation using alkaline phosphatase reduced the catalytic activity of both enzymes. Phosphorylation and dephosphorylation had no effect on the measurable activity of SBEI in amyloplasts and chloroplasts, and the activities of both granule-bound forms of SBEII in amyloplasts were unaffected by dephosphorylation. Immunoprecipitation experiments using peptide-specific anti-SBE antibodies showed that SBEIIb and starch phosphorylase each coimmunoprecipitated with SBEI in a phosphorylation-dependent manner, suggesting that these enzymes may form protein complexes within the amyloplast in vivo. Conversely, dephosphorylation of immunoprecipitated protein complex led to its disassembly. This article reports direct evidence that enzymes of starch metabolism (amylopectin synthesis) are regulated by protein phosphorylation and indicate a wider role for protein phosphorylation and protein

  16. Phosphorylation Regulates the Bound Structure of an Intrinsically Disordered Protein: The p53-TAZ2 Case.

    PubMed

    Ithuralde, Raúl Esteban; Turjanski, Adrián Gustavo

    2016-01-01

    Disordered regions and Intrinsically Disordered Proteins (IDPs) are involved in critical cellular processes and may acquire a stable three-dimensional structure only upon binding to their partners. IDPs may follow a folding-after-binding process, known as induced folding, or a folding-before-binding process, known as conformational selection. The transcription factor p53 is involved in the regulation of cellular events that arise upon stress or DNA damage. The p53 domain structure is composed of an N-terminal transactivation domain (p53TAD), a DNA Binding Domain and a tetramerization domain. The activity of TAD is tightly regulated by interactions with cofactors, inhibitors and phosphorylation. To initiate transcription, p53TAD binds to the TAZ2 domain of CBP, a co-transcription factor, and undergoes a folding and binding process, as revealed by the recent NMR structure of the complex. The activity of p53 is regulated by phosphorylation at multiple sites on the TAD domain and recent studies have shown that modifications at three residues affect the binding towards TAZ2. However, we still do not know how these phosphorylations affect the structure of the bound state and, therefore, how they regulate the p53 function. In this work, we have used computational simulations to understand how phosphorylation affects the structure of the p53TAD:TAZ2 complex and regulates the recognition mechanism. Phosphorylation has been proposed to enhance binding by direct interaction with the folded protein or by changing the unbound conformation of IDPs, for example by pre-folding the protein favoring the recognition mechanism. Here, we show an interesting turn in the p53 case: phosphorylation mainly affects the bound structure of p53TAD, highlighting the complexity of IDP protein-protein interactions. Our results are in agreement with previous experimental studies, allowing a clear picture of how p53 is regulated by phosphorylation and giving new insights into how post

  17. Phosphodiesterases Regulate BAY 41-2272-Induced VASP Phosphorylation in Vascular Smooth Muscle Cells

    PubMed Central

    Adderley, Shaquria P.; Joshi, Chintamani N.; Martin, Danielle N.; Tulis, David Anthony

    2012-01-01

    BAY 41-2272 (BAY), a stimulator of soluble guanylyl cyclase, increases cyclic nucleotides and inhibits proliferation of vascular smooth muscle cells (VSMCs). In this study, we elucidated mechanisms of action of BAY in its regulation of vasodilator-stimulated phosphoprotein (VASP) with an emphasis on VSMC phosphodiesterases (PDEs). BAY alone increased phosphorylation of VASPSer239 and VASPSer157, respective indicators of PKG and PKA signaling. IBMX, a non-selective inhibitor of PDEs, had no effect on BAY-induced phosphorylation at VASPSer239 but inhibited phosphorylation at VASPSer157. Selective inhibitors of PDE3 or PDE4 attenuated BAY-mediated increases at VASPSer239 and VASPSer157, whereas PDE5 inhibition potentiated BAY-mediated increases only at VASPSer157. In comparison, 8Br-cGMP increased phosphorylation at VASPSer239 and VASPSer157 which were not affected by selective PDE inhibitors. In the presence of 8Br-cAMP, inhibition of either PDE4 or PDE5 decreased VASPSer239 phosphorylation and inhibition of PDE3 increased phosphorylation at VASPSer239, while inhibition of PDE3 or PDE4 increased and PDE5 inhibition had no effect on VASPSer157 phosphorylation. These findings demonstrate that BAY operates via cAMP and cGMP along with regulation by PDEs to phosphorylate VASP in VSMCs and that the mechanism of action of BAY in VSMCs is different from that of direct cyclic nucleotide analogs with respect to VASP phosphorylation and the involvement of PDEs. Given a role for VASP as a critical cytoskeletal protein, these findings provide evidence for BAY as a regulator of VSMC growth and a potential therapeutic agent against vasculoproliferative disorders. PMID:22347188

  18. 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

  19. Phosphorylation of K+ channels at single residues regulates memory formation

    PubMed Central

    Vernon, Jeffrey; Irvine, Elaine E.; Peters, Marco; Jeyabalan, Jeshmi

    2016-01-01

    Phosphorylation is a ubiquitous post-translational modification of proteins, and a known physiological regulator of K+ channel function. Phosphorylation of K+ channels by kinases has long been presumed to regulate neuronal processing and behavior. Although circumstantial evidence has accumulated from behavioral studies of vertebrates and invertebrates, the contribution to memory of single phosphorylation sites on K+ channels has never been reported. We have used gene targeting in mice to inactivate protein kinase A substrate residues in the fast-inactivating subunit Kv4.2 (T38A mutants), and in the small-conductance Ca2+-activated subunit SK1 (S105A mutants). Both manipulations perturbed a specific form of memory, leaving others intact. T38A mutants had enhanced spatial memory for at least 4 wk after training, whereas performance in three tests of fear memory was unaffected. S105A mutants were impaired in passive avoidance memory, sparing fear, and spatial memory. Together with recent findings that excitability governs the participation of neurons in a memory circuit, this result suggests that the memory type supported by neurons may depend critically on the phosphorylation of specific K+ channels at single residues. PMID:26980786

  20. TARP phosphorylation regulates synaptic AMPA receptors through lipid bilayers.

    PubMed

    Sumioka, Akio; Yan, Dan; Tomita, Susumu

    2010-06-10

    Neurons use neurotransmitters to communicate across synapses, constructing neural circuits in the brain. AMPA-type glutamate receptors are the predominant excitatory neurotransmitter receptors mediating fast synaptic transmission. AMPA receptors localize at synapses by forming protein complexes with transmembrane AMPA receptor regulatory proteins (TARPs) and PSD-95-like membrane-associated guanylate kinases. Among the three classes of ionotropic glutamate receptors (AMPA, NMDA, and kainate type), AMPA receptor activity is most regulatable by neuronal activity to adjust synaptic strength. Here, we mutated the prototypical TARP, stargazin, and found that TARP phosphorylation regulates synaptic AMPA receptor activity in vivo. We also found that stargazin interacts with negatively charged lipid bilayers in a phosphorylation-dependent manner and that the lipid interaction inhibited stargazin binding to PSD-95. Cationic lipids dissociated stargazin from lipid bilayers and enhanced synaptic AMPA receptor activity in a stargazin phosphorylation-dependent manner. Thus, TARP phosphorylation plays a critical role in regulating AMPA receptor-mediated synaptic transmission via a lipid bilayer interaction.

  1. TARP phosphorylation regulates synaptic AMPA receptors through lipid bilayers

    PubMed Central

    Sumioka, Akio; Yan, Dan; Tomita, Susumu

    2010-01-01

    Summary Neurons use neurotransmitters to communicate across synapses, constructing neural circuits in the brain. AMPA-type glutamate receptors are the predominant excitatory neurotransmitter receptors mediating fast synaptic transmission. AMPA receptors localize at synapses by forming protein complexes with transmembrane AMPA receptor regulatory proteins (TARPs) and PSD-95-like MAGUKs. Among the three classes of ionotropic glutamate receptors (AMPA-, NMDA, kainate-type), AMPA receptor activity is most regulatable by neuronal activity to adjust synaptic strength. Here, we mutated the prototypical TARP, stargazin, and found that TARP phosphorylation regulates synaptic AMPA receptor activity in vivo. We also found that stargazin interacts with negatively-charged lipid bilayers in its phosphorylation dependent manner, and that the lipid interaction inhibited stargazin binding to PSD-95. Cationic lipids dissociated stargazin from lipid bilayers and enhanced synaptic AMPA receptor activity in a stargazin phosphorylation-dependent manner. Thus, TARP phosphorylation plays a critical role in regulating AMPA receptor-mediated synaptic transmission via a lipid bilayer interaction. PMID:20547132

  2. A unified molecular mechanism for the regulation of acetyl-CoA carboxylase by phosphorylation

    PubMed Central

    Wei, Jia; Zhang, Yixiao; Yu, Tai-Yuan; Sadre-Bazzaz, Kianoush; Rudolph, Michael J; Amodeo, Gabriele A; Symington, Lorraine S; Walz, Thomas; Tong, Liang

    2016-01-01

    Acetyl-CoA carboxylases (ACCs) are crucial metabolic enzymes and attractive targets for drug discovery. Eukaryotic acetyl-CoA carboxylases are 250 kDa single-chain, multi-domain enzymes and function as dimers and higher oligomers. Their catalytic activity is tightly regulated by phosphorylation and other means. Here we show that yeast ACC is directly phosphorylated by the protein kinase SNF1 at residue Ser1157, which potently inhibits the enzyme. Crystal structure of three ACC central domains (AC3–AC5) shows that the phosphorylated Ser1157 is recognized by Arg1173, Arg1260, Tyr1113 and Ser1159. The R1173A/R1260A double mutant is insensitive to SNF1, confirming that this binding site is crucial for regulation. Electron microscopic studies reveal dramatic conformational changes in the holoenzyme upon phosphorylation, likely owing to the dissociation of the biotin carboxylase domain dimer. The observations support a unified molecular mechanism for the regulation of ACC by phosphorylation as well as by the natural product soraphen A, a potent inhibitor of eukaryotic ACC. These molecular insights enhance our understanding of acetyl-CoA carboxylase regulation and provide a basis for drug discovery. PMID:27990296

  3. Coincident regulation of PKCdelta in human platelets by phosphorylation of Tyr311 and Tyr565 and phospholipase C signalling.

    PubMed

    Hall, Kellie J; Jones, Matthew L; Poole, Alastair W

    2007-09-15

    PKC (protein kinase C)d plays a complex role in platelets, having effects on both positive and negative signalling functions. It is phosphorylated on tyrosine residues in response to thrombin and collagen, and it has recently been shown that Tyr311 is phosphorylated in response to PAR (protease-activated receptor) 1 and PAR4 receptor activation. In the present study, we show that Tyr311 and Tyr565 are phosphorylated in response to thrombin, and have examined the interplay between phosphorylation and the classical lipid-mediated activation of PKCd. Phosphorylation of both Tyr311 and Tyr565 is dependent on Src kinase and PLC (phospholipase C) activity in response to thrombin. Importantly, direct allosteric activation of PKCd with PMA also induced phosphorylation of Tyr311 and Tyr565, and this was dependent on the activity of Src kinases, but not PLC. Membrane recruitment of PKCd is essential for phosphorylation of this tyrosine residue, but tyrosine phosphorylation is not required for membrane recruitment of PKCd. Both thrombin and PMA induce recruitment of PKCd to the membrane, and for thrombin, this recruitment is a PLC-dependent process. In order to address the functional role of tyrosine residue phosphorylation of PKCd, we demonstrate that phosphorylation can potentiate the activity of the kinase, although phosphorylation does not play a role in membrane recruitment of the kinase. PKCd is therefore regulated in a coincident fashion, PLC-dependent signals recruiting it to the plasma membrane and by phosphorylation on tyrosine residues, potentiating its activity.

  4. Phosphorylation by Nek1 regulates opening and closing of voltage dependent anion channel 1

    SciTech Connect

    Chen, Yumay; Gaczynska, Maria; Osmulski, Pawel; Polci, Rosaria; Riley, Daniel J.

    2010-04-09

    VDAC1 is a key component of the mitochondrial permeability transition pore. To initiate apoptosis and certain other forms of cell death, mitochondria become permeable such that cytochrome c and other pre-apoptotic molecules resident inside the mitochondria enter the cytosol and activate apoptotic cascades. We have shown recently that VDAC1 interacts directly with never-in-mitosis A related kinase 1 (Nek1), and that Nek1 phosphorylates VDAC1 on Ser193 to prevent excessive cell death after injury. How this phosphorylation regulates the activity of VDAC1, however, has not yet been reported. Here, we use atomic force microscopy (AFM) and cytochrome c conductance studies to examine the configuration of VDAC1 before and after phosphorylation by Nek1. Wild-type VDAC1 assumes an open configuration, but closes and prevents cytochrome c efflux when phosphorylated by Nek1. A VDAC1-Ser193Ala mutant, which cannot be phosphorylated by Nek1 under identical conditions, remains open and constitutively allows cytochrome c efflux. Conversely, a VDAC1-Ser193Glu mutant, which mimics constitutive phosphorylation by Nek1, remains closed by AFM and prevents cytochrome c leakage in the same liposome assays. Our data provide a mechanism to explain how Nek1 regulates cell death by affecting the opening and closing of VDAC1.

  5. Epidermal Growth Factor Receptor Fate Is Controlled by Hrs Tyrosine Phosphorylation Sites That Regulate Hrs Degradation▿

    PubMed Central

    Stern, Kathryn A.; Visser Smit, Gina D.; Place, Trenton L.; Winistorfer, Stanley; Piper, Robert C.; Lill, Nancy L.

    2007-01-01

    Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is an endosomal protein essential for the efficient sorting of activated growth factor receptors into the lysosomal degradation pathway. Hrs undergoes ligand-induced tyrosine phosphorylation on residues Y329 and Y334 downstream of epidermal growth factor receptor (EGFR) activation. It has been difficult to investigate the functional roles of phosphoHrs, as only a small proportion of the cellular Hrs pool is detectably phosphorylated. Using an HEK 293 model system, we found that ectopic expression of the protein Cbl enhances Hrs ubiquitination and increases Hrs phosphorylation following cell stimulation with EGF. We exploited Cbl's expansion of the phosphoHrs pool to determine whether Hrs tyrosine phosphorylation controls EGFR fate. In structure-function studies of Cbl and EGFR mutants, the level of Hrs phosphorylation and rapidity of apparent Hrs dephosphorylation correlated directly with EGFR degradation. Differential expression of wild-type versus Y329,334F mutant Hrs in Hrs-depleted cells revealed that one or both tyrosines regulate ligand-dependent Hrs degradation, as well as EGFR degradation. By modulating Hrs ubiquitination, phosphorylation, and protein levels, Cbl may control the composition of the endosomal sorting machinery and its ability to target EGFR for lysosomal degradation. PMID:17101784

  6. Phosphorylation and SCF-mediated degradation regulate CREB-H transcription of metabolic targets

    PubMed Central

    Barbosa, Sónia; Carreira, Suzanne; Bailey, Daniel; Abaitua, Fernando; O'Hare, Peter

    2015-01-01

    CREB‑H, an endoplasmic reticulum–anchored transcription factor, plays a key role in regulating secretion and in metabolic and inflammatory pathways, but how its activity is modulated remains unclear. We examined processing of the nuclear active form and identified a motif around S87–S90 with homology to DSG-type phosphodegrons. We show that this region is subject to multiple phosphorylations, which regulate CREB-H stability by targeting it to the SCFFbw1a E3 ubiquitin ligase. Data from phosphatase treatment, use of phosophospecific antibody, and substitution of serine residues demonstrate phosphorylation of candidate serines in the region, with the core S87/S90 motif representing a critical determinant promoting proteasome-mediated degradation. Candidate kinases CKII and GSK-3b phosphorylate CREB-H in vitro with specificities for different serines. Prior phosphorylation with GSK-3 at one or more of the adjacent serines substantially increases S87/S90-dependent phosphorylation by CKII. In vivo expression of a dominant-negative Cul1 enhances steady-state levels of CREB‑H, an effect augmented by Fbw1a. CREB-H directly interacts with Fbw1a in a phosphorylation-dependent manner. Finally, mutations within the phosphodegron, when incorporated into the full-length protein, result in increased levels of constitutively cleaved nuclear protein and increased transcription and secretion of a key endogenous target gene, apolipoprotein A IV. PMID:26108621

  7. Hedgehog-regulated atypical PKC promotes phosphorylation and activation of Smoothened and Cubitus interruptus in Drosophila

    PubMed Central

    Jiang, Kai; Liu, Yajuan; Fan, Junkai; Epperly, Garretson; Gao, Tianyan; Jiang, Jin; Jia, Jianhang

    2014-01-01

    Smoothened (Smo) is essential for transduction of the Hedgehog (Hh) signal in both insects and vertebrates. Cell surface/cilium accumulation of Smo is thought to play an important role in Hh signaling, but how the localization of Smo is controlled remains poorly understood. In this study, we demonstrate that atypical PKC (aPKC) regulates Smo phosphorylation and basolateral accumulation in Drosophila wings. Inactivation of aPKC by either RNAi or a mutation inhibits Smo basolateral accumulation and attenuates Hh target gene expression. In contrast, expression of constitutively active aPKC elevates basolateral accumulation of Smo and promotes Hh signaling. The aPKC-mediated phosphorylation of Smo at Ser680 promotes Ser683 phosphorylation by casein kinase 1 (CK1), and these phosphorylation events elevate Smo activity in vivo. Moreover, aPKC has an additional positive role in Hh signaling by regulating the activity of Cubitus interruptus (Ci) through phosphorylation of the Zn finger DNA-binding domain. Finally, the expression of aPKC is up-regulated by Hh signaling in a Ci-dependent manner. Our findings indicate a direct involvement of aPKC in Hh signaling beyond its role in cell polarity. PMID:25349414

  8. Regulation of microtubule dynamic instability in vitro by differentially phosphorylated stathmin.

    PubMed

    Manna, Tapas; Thrower, Douglas A; Honnappa, Srinivas; Steinmetz, Michel O; Wilson, Leslie

    2009-06-05

    Stathmin is an important regulator of microtubule polymerization and dynamics. When unphosphorylated it destabilizes microtubules in two ways, by reducing the microtubule polymer mass through sequestration of soluble tubulin into an assembly-incompetent T2S complex (two alpha:beta tubulin dimers per molecule of stathmin), and by increasing the switching frequency (catastrophe frequency) from growth to shortening at plus and minus ends by binding directly to the microtubules. Phosphorylation of stathmin on one or more of its four serine residues (Ser(16), Ser(25), Ser(38), and Ser(63)) reduces its microtubule-destabilizing activity. However, the effects of phosphorylation of the individual serine residues of stathmin on microtubule dynamic instability have not been investigated systematically. Here we analyzed the effects of stathmin singly phosphorylated at Ser(16) or Ser(63), and doubly phosphorylated at Ser(25) and Ser(38), on its ability to modulate microtubule dynamic instability at steady-state in vitro. Phosphorylation at either Ser(16) or Ser(63) strongly reduced or abolished the ability of stathmin to bind to and sequester soluble tubulin and its ability to act as a catastrophe factor by directly binding to the microtubules. In contrast, double phosphorylation of Ser(25) and Ser(38) did not affect the binding of stathmin to tubulin or microtubules or its catastrophe-promoting activity. Our results indicate that the effects of stathmin on dynamic instability are strongly but differently attenuated by phosphorylation at Ser(16) and Ser(63) and support the hypothesis that selective targeting by Ser(16)-specific or Ser(63)-specific kinases provides complimentary mechanisms for regulating microtubule function.

  9. A Compartmentalized Phosphorylation/Dephosphorylation System That Regulates U snRNA Export from the Nucleus▿ †

    PubMed Central

    Kitao, Saori; Segref, Alexandra; Kast, Juergen; Wilm, Matthias; Mattaj, Iain W.; Ohno, Mutsuhito

    2008-01-01

    PHAX (phosphorylated adaptor for RNA export) is the key regulator of U snRNA nuclear export in metazoa. Our previous work revealed that PHAX is phosphorylated in the nucleus and is exported as a component of the U snRNA export complex to the cytoplasm, where it is dephosphorylated (M. Ohno, A. Segref, A. Bachi, M. Wilm, and I. W. Mattaj, Cell 101:187-198, 2000). PHAX phosphorylation is essential for export complex assembly, whereas its dephosphorylation causes export complex disassembly. Thus, PHAX is subject to a compartmentalized phosphorylation/dephosphorylation cycle that contributes to transport directionality. However, neither essential PHAX phosphorylation sites nor the modifying enzymes that contribute to the compartmentalized system have been identified. Here, we identify PHAX phosphorylation sites that are necessary and sufficient for U snRNA export. Mutation of the phosphorylation sites inhibited U snRNA export in a dominant-negative way. We also show, by both biochemical and RNA interference knockdown experiments, that the nuclear kinase and the cytoplasmic phosphatase for PHAX are CK2 kinase and protein phosphatase 2A, respectively. Our results reveal the composition of the compartmentalized phosphorylation/dephosphorylation system that regulates U snRNA export. This finding was surprising in that such a specific system for U snRNA export regulation is composed of two such universal regulators, suggesting that this compartmentalized system is used more broadly for gene expression regulation. PMID:17967890

  10. Sec24 phosphorylation regulates autophagosome abundance during nutrient deprivation

    PubMed Central

    Davis, Saralin; Wang, Juan; Zhu, Ming; Stahmer, Kyle; Lakshminarayan, Ramya; Ghassemian, Majid; Jiang, Yu; Miller, Elizabeth A; Ferro-Novick, Susan

    2016-01-01

    Endoplasmic Reticulum (ER)-derived COPII coated vesicles constitutively transport secretory cargo to the Golgi. However, during starvation-induced stress, COPII vesicles have been implicated as a membrane source for autophagosomes, distinct organelles that engulf cellular components for degradation by macroautophagy (hereafter called autophagy). How cells regulate core trafficking machinery to fulfill dramatically different cellular roles in response to environmental cues is unknown. Here we show that phosphorylation of conserved amino acids on the membrane-distal surface of the Saccharomyces cerevisiae COPII cargo adaptor, Sec24, reprograms COPII vesicles for autophagy. We also show casein kinase 1 (Hrr25) is a key kinase that phosphorylates this regulatory surface. During autophagy, Sec24 phosphorylation regulates autophagosome number and its interaction with the C-terminus of Atg9, a component of the autophagy machinery required for autophagosome initiation. We propose that the acute need to produce autophagosomes during starvation drives the interaction of Sec24 with Atg9 to increase autophagosome abundance. DOI: http://dx.doi.org/10.7554/eLife.21167.001 PMID:27855785

  11. Phosphorylation and dephosphorylation regulate APC/CCdh1 substrate degradation

    PubMed Central

    Simpson-Lavy, Kobi J; Zenvirth, Drora; Brandeis, Michael

    2015-01-01

    The Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin ligase activated by its G1 specific adaptor protein Cdh1 is a major regulator of the cell cycle. The APC/CCdh1 mediates degradation of dozens of proteins, however, the kinetics and requirements for their degradation are largely unknown. We demonstrate that overexpression of the constitutive active CDH1m11 mutant that is not inhibited by phosphorylation results in mitotic exit in the absence of the FEAR and MEN pathways, and DNA re-replication in the absence of Cdc7 activity. This mode of mitotic exit also reveals additional requirements for APC/CCdh1 substrate degradation, which for some substrates such as Pds1 or Clb5 is dephosphorylation, but for others such as Cdc5 is phosphorylation. PMID:26252546

  12. PKM2 phosphorylates MLC2 and regulates cytokinesis of tumour cells.

    PubMed

    Jiang, Yuhui; Wang, Yugang; Wang, Ting; Hawke, David H; Zheng, Yanhua; Li, Xinjian; Zhou, Qin; Majumder, Sadhan; Bi, Erfei; Liu, David X; Huang, Suyun; Lu, Zhimin

    2014-11-21

    Pyruvate kinase M2 (PKM2) is expressed at high levels during embryonic development and tumour progression and is important for cell growth. However, it is not known whether it directly controls cell division. Here, we found that Aurora B phosphorylates PKM2, but not PKM1, at T45; this phosphorylation is required for PKM2's localization and interaction with myosin light chain 2 (MLC2) in the contractile ring region of mitotic cells during cytokinesis. PKM2 phosphorylates MLC2 at Y118, which primes the binding of ROCK2 to MLC2 and subsequent ROCK2-dependent MLC2 S15 phosphorylation. PKM2-regulated MLC2 phosphorylation, which is greatly enhanced by EGF stimulation or EGFRvIII, K-Ras G12V and B-Raf V600E mutant expression, plays a pivotal role in cytokinesis, cell proliferation and brain tumour development. These findings underscore the instrumental function of PKM2 in oncogenic EGFR-, K-Ras- and B-Raf-regulated cytokinesis and tumorigenesis.

  13. Phosphorylation Regulates the Bound Structure of an Intrinsically Disordered Protein: The p53-TAZ2 Case

    PubMed Central

    Ithuralde, Raúl Esteban; Turjanski, Adrián Gustavo

    2016-01-01

    Disordered regions and Intrinsically Disordered Proteins (IDPs) are involved in critical cellular processes and may acquire a stable three-dimensional structure only upon binding to their partners. IDPs may follow a folding-after-binding process, known as induced folding, or a folding-before-binding process, known as conformational selection. The transcription factor p53 is involved in the regulation of cellular events that arise upon stress or DNA damage. The p53 domain structure is composed of an N-terminal transactivation domain (p53TAD), a DNA Binding Domain and a tetramerization domain. The activity of TAD is tightly regulated by interactions with cofactors, inhibitors and phosphorylation. To initiate transcription, p53TAD binds to the TAZ2 domain of CBP, a co-transcription factor, and undergoes a folding and binding process, as revealed by the recent NMR structure of the complex. The activity of p53 is regulated by phosphorylation at multiple sites on the TAD domain and recent studies have shown that modifications at three residues affect the binding towards TAZ2. However, we still do not know how these phosphorylations affect the structure of the bound state and, therefore, how they regulate the p53 function. In this work, we have used computational simulations to understand how phosphorylation affects the structure of the p53TAD:TAZ2 complex and regulates the recognition mechanism. Phosphorylation has been proposed to enhance binding by direct interaction with the folded protein or by changing the unbound conformation of IDPs, for example by pre-folding the protein favoring the recognition mechanism. Here, we show an interesting turn in the p53 case: phosphorylation mainly affects the bound structure of p53TAD, highlighting the complexity of IDP protein-protein interactions. Our results are in agreement with previous experimental studies, allowing a clear picture of how p53 is regulated by phosphorylation and giving new insights into how post

  14. Subcellular distribution of cyclin-dependent kinase-like 5 (CDKL5) is regulated through phosphorylation by dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A).

    PubMed

    Oi, Ami; Katayama, Syouichi; Hatano, Naoya; Sugiyama, Yasunori; Kameshita, Isamu; Sueyoshi, Noriyuki

    2017-01-08

    Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase primarily expressed in the central nervous system and is known to cause X-linked neurodevelopmental disorders such as Rett syndrome. However, the mechanisms regulating CDKL5 have not yet been fully clarified. Therefore, in this study, we investigated the protein kinase that directly phosphorylates CDKL5, identifying it as dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), an enzyme binding to and phosphorylating CDKL5. We showed that subcellular distribution of CDKL5 was regulated by its phosphorylation by DYRK1A. In mouse neuroblastoma Neuro2a cells, CDKL5 was localized in both the cytosol and nucleus, whereas DYRK1A showed a typical nuclear localization. When CDKL5 and DYRK1A were co-expressed, the cytosolic localization of CDKL5 was significantly increased. Results of site-directed mutagenesis revealed that the phosphorylation site was Ser-308, in the vicinity of the nuclear localization signal. A mutation mimicking the phosphorylated serine residue by aspartate substitution (S308D) changed CDKL5 localization to the cytosol, whereas the corresponding alanine-substituted analog, CDKL5(S308A), was primarily localized to the nucleus. Taken together, these results strongly suggested that DYRK1A bound to CDKL5 and phosphorylated it on Ser-308, thus interfering with its nuclear localization.

  15. Akt phosphorylates and regulates the osteogenic activity of Osterix.

    PubMed

    Choi, You Hee; Jeong, Hyung Min; Jin, Yun-Hye; Li, Hongyan; Yeo, Chang-Yeol; Lee, Kwang-Youl

    2011-08-05

    Osterix (Osx), a zinc-finger transcription factor is required for osteoblast differentiation and new bone formation during embryonic development. Akt is a member of the serine/threonine-specific protein kinase and plays important roles in osteoblast differentiation. The function of Osterix can be also modulated by post-translational modification. But, the precise molecular signaling mechanisms between Osterix and Akt are not known. In this study, we investigated the potential regulation of Osterix function by Akt in osteoblast differentiation. We found that Akt phosphorylates Osterix and that Akt activation increases protein stability, osteogenic activity and transcriptional activity of Osterix. We also found that BMP-2 increases the protein level of Osterix in an Akt activity-dependent manner. These results suggest that Akt activity enhances the osteogenic function of Osterix, at least in part, through protein stabilization and that BMP-2 regulates the osteogenic function of Osterix, at least in part, through Akt.

  16. Regulation of the cystic fibrosis transmembrane conductance regulator anion channel by tyrosine phosphorylation

    PubMed Central

    Billet, Arnaud; Jia, Yanlin; Jensen, Tim; Riordan, John R.; Hanrahan, John W.

    2015-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) channel is activated by PKA phosphorylation of a regulatory domain that interacts dynamically with multiple CFTR domains and with other proteins. The large number of consensus sequences for phosphorylation by PKA has naturally focused most attention on regulation by this kinase. We report here that human CFTR is also phosphorylated by the tyrosine kinases p60c-Src (proto-oncogene tyrosine-protein kinase) and the proline-rich tyrosine kinase 2 (Pyk2), and they can also cause robust activation of quiescent CFTR channels. In excised patch-clamp experiments, CFTR activity during exposure to Src or Pyk2 reached ∼80% of that stimulated by PKA. Exposure to PKA after Src or Pyk2 caused a further increase to the level induced by PKA alone, implying a common limiting step. Channels became spontaneously active when v-Src or the catalytic domain of Pyk2 was coexpressed with CFTR and were further stimulated by the tyrosine phosphatase inhibitor dephostatin. Exogenous Src also activated 15SA-CFTR, a variant that lacks 15 potential PKA sites and has little response to PKA. PKA-independent activation by tyrosine phosphorylation has implications for the mechanism of regulation by the R domain and for the physiologic functions of CFTR.—Billet, A., Jia, Y., Jensen, T., Riordan, J. R., Hanrahan, J. W. Regulation of the cystic fibrosis transmembrane conductance regulator anion channel by tyrosine phosphorylation. PMID:26062600

  17. Sub1 Globally Regulates RNA Polymerase II C-Terminal Domain Phosphorylation

    PubMed Central

    García, Alicia; Rosonina, Emanuel; Manley, James L.; Calvo, Olga

    2010-01-01

    The transcriptional coactivator Sub1 has been implicated in several aspects of mRNA metabolism in yeast, such as activation of transcription, termination, and 3′-end formation. Here, we present evidence that Sub1 plays a significant role in controlling phosphorylation of the RNA polymerase II large subunit C-terminal domain (CTD). We show that SUB1 genetically interacts with the genes encoding all four known CTD kinases, SRB10, KIN28, BUR1, and CTK1, suggesting that Sub1 acts to influence CTD phosphorylation at more than one step of the transcription cycle. To address this directly, we first used in vitro kinase assays, and we show that, on the one hand, SUB1 deletion increased CTD phosphorylation by Kin28, Bur1, and Ctk1 but, on the other, it decreased CTD phosphorylation by Srb10. Second, chromatin immunoprecipitation assays revealed that SUB1 deletion decreased Srb10 chromatin association on the inducible GAL1 gene but increased Kin28 and Ctk1 chromatin association on actively transcribed genes. Taken together, our data point to multiple roles for Sub1 in the regulation of CTD phosphorylation throughout the transcription cycle. PMID:20823273

  18. Sub1 globally regulates RNA polymerase II C-terminal domain phosphorylation.

    PubMed

    García, Alicia; Rosonina, Emanuel; Manley, James L; Calvo, Olga

    2010-11-01

    The transcriptional coactivator Sub1 has been implicated in several aspects of mRNA metabolism in yeast, such as activation of transcription, termination, and 3'-end formation. Here, we present evidence that Sub1 plays a significant role in controlling phosphorylation of the RNA polymerase II large subunit C-terminal domain (CTD). We show that SUB1 genetically interacts with the genes encoding all four known CTD kinases, SRB10, KIN28, BUR1, and CTK1, suggesting that Sub1 acts to influence CTD phosphorylation at more than one step of the transcription cycle. To address this directly, we first used in vitro kinase assays, and we show that, on the one hand, SUB1 deletion increased CTD phosphorylation by Kin28, Bur1, and Ctk1 but, on the other, it decreased CTD phosphorylation by Srb10. Second, chromatin immunoprecipitation assays revealed that SUB1 deletion decreased Srb10 chromatin association on the inducible GAL1 gene but increased Kin28 and Ctk1 chromatin association on actively transcribed genes. Taken together, our data point to multiple roles for Sub1 in the regulation of CTD phosphorylation throughout the transcription cycle.

  19. Nutrient-regulated Phosphorylation of ATG13 Inhibits Starvation-induced Autophagy*

    PubMed Central

    Puente, Cindy; Hendrickson, Ronald C.; Jiang, Xuejun

    2016-01-01

    Autophagy is a conserved catabolic process that utilizes a defined series of membrane trafficking events to generate a de novo double-membrane vesicle termed the autophagosome, which matures by fusing to the lysosome. Subsequently, the lysosome facilitates the degradation and recycling of the cytoplasmic cargo. In yeast, the upstream signals that regulate the induction of starvation-induced autophagy are clearly defined. The nutrient-sensing kinase Tor inhibits the activation of autophagy by regulating the formation of the Atg1-Atg13-Atg17 complex, through hyperphosphorylation of Atg13. However, in mammals, the ortholog complex ULK1-ATG13-FIP200 is constitutively formed. As such, the molecular mechanism by which mTOR regulates mammalian autophagy is unknown. Here we report the identification and characterization of novel nutrient-regulated phosphorylation sites on ATG13: Ser-224 and Ser-258. mTOR directly phosphorylates ATG13 on Ser-258 while Ser-224 is modulated by the AMPK pathway. In ATG13 knock-out cells reconstituted with an unphosphorylatable mutant of ATG13, ULK1 kinase activity is more potent, and amino acid starvation induced more rapid ATG13 and ULK1 translocation. These events culminated in a more rapid starvation-induced autophagy response. Therefore, ATG13 phosphorylation plays a crucial role in autophagy regulation. PMID:26801615

  20. Nutrient-regulated Phosphorylation of ATG13 Inhibits Starvation-induced Autophagy.

    PubMed

    Puente, Cindy; Hendrickson, Ronald C; Jiang, Xuejun

    2016-03-11

    Autophagy is a conserved catabolic process that utilizes a defined series of membrane trafficking events to generate a de novo double-membrane vesicle termed the autophagosome, which matures by fusing to the lysosome. Subsequently, the lysosome facilitates the degradation and recycling of the cytoplasmic cargo. In yeast, the upstream signals that regulate the induction of starvation-induced autophagy are clearly defined. The nutrient-sensing kinase Tor inhibits the activation of autophagy by regulating the formation of the Atg1-Atg13-Atg17 complex, through hyperphosphorylation of Atg13. However, in mammals, the ortholog complex ULK1-ATG13-FIP200 is constitutively formed. As such, the molecular mechanism by which mTOR regulates mammalian autophagy is unknown. Here we report the identification and characterization of novel nutrient-regulated phosphorylation sites on ATG13: Ser-224 and Ser-258. mTOR directly phosphorylates ATG13 on Ser-258 while Ser-224 is modulated by the AMPK pathway. In ATG13 knock-out cells reconstituted with an unphosphorylatable mutant of ATG13, ULK1 kinase activity is more potent, and amino acid starvation induced more rapid ATG13 and ULK1 translocation. These events culminated in a more rapid starvation-induced autophagy response. Therefore, ATG13 phosphorylation plays a crucial role in autophagy regulation.

  1. Serine 249 phosphorylation by ATM protein kinase regulates hepatocyte nuclear factor-1α transactivation.

    PubMed

    Zhao, Long; Chen, Hui; Zhan, Yi-Qun; Li, Chang-Yan; Ge, Chang-Hui; Zhang, Jian-Hong; Wang, Xiao-Hui; Yu, Miao; Yang, Xiao-Ming

    2014-07-01

    Hepatocyte nuclear factor-1 alpha (HNF1α) exerts important effects on gene expression in multiple tissues. Several studies have directly or indirectly supported the role of phosphorylation processes in the activity of HNF1α. However, the molecular mechanism of this phosphorylation remains largely unknown. Using microcapillary liquid chromatography MS/MS and biochemical assays, we identified a novel phosphorylation site in HNF1α at Ser249. We also found that the ATM protein kinase phosphorylated HNF1α at Ser249 in vitro in an ATM-dependent manner and that ATM inhibitor KU55933 treatment inhibited phosphorylation of HNF1α at Ser249 in vivo. Coimmunoprecipitation assays confirmed the association between HNF1α and ATM. Moreover, ATM enhanced HNF1α transcriptional activity in a dose-dependent manner, whereas the ATM kinase-inactive mutant did not. The use of KU55933 confirmed our observation. Compared with wild-type HNF1α, a mutation in Ser249 resulted in a pronounced decrease in HNF1α transactivation, whereas no dominant-negative effect was observed. The HNF1αSer249 mutant also exhibited normal nuclear localization but decreased DNA-binding activity. Accordingly, the functional studies of HNF1αSer249 mutant revealed a defect in glucose metabolism. Our results suggested that ATM regulates the activity of HNF1α by phosphorylation of serine 249, particularly in glucose metabolism, which provides valuable insights into the undiscovered mechanisms of ATM in the regulation of glucose homeostasis.

  2. Regulation of DNA methylation patterns by CK2-mediated phosphorylation of Dnmt3a.

    PubMed

    Deplus, Rachel; Blanchon, Loïc; Rajavelu, Arumugam; Boukaba, Abdelhalim; Defrance, Matthieu; Luciani, Judith; Rothé, Françoise; Dedeurwaerder, Sarah; Denis, Hélène; Brinkman, Arie B; Simmer, Femke; Müller, Fabian; Bertin, Benjamin; Berdasco, Maria; Putmans, Pascale; Calonne, Emilie; Litchfield, David W; de Launoit, Yvan; Jurkowski, Tomasz P; Stunnenberg, Hendrik G; Bock, Christoph; Sotiriou, Christos; Fraga, Mario F; Esteller, Manel; Jeltsch, Albert; Fuks, François

    2014-08-07

    DNA methylation is a central epigenetic modification that is established by de novo DNA methyltransferases. The mechanisms underlying the generation of genomic methylation patterns are still poorly understood. Using mass spectrometry and a phosphospecific Dnmt3a antibody, we demonstrate that CK2 phosphorylates endogenous Dnmt3a at two key residues located near its PWWP domain, thereby downregulating the ability of Dnmt3a to methylate DNA. Genome-wide DNA methylation analysis shows that CK2 primarily modulates CpG methylation of several repeats, most notably of Alu SINEs. This modulation can be directly attributed to CK2-mediated phosphorylation of Dnmt3a. We also find that CK2-mediated phosphorylation is required for localization of Dnmt3a to heterochromatin. By revealing phosphorylation as a mode of regulation of de novo DNA methyltransferase function and by uncovering a mechanism for the regulation of methylation at repetitive elements, our results shed light on the origin of DNA methylation patterns.

  3. PGRMC1 regulation by phosphorylation: potential new insights in controlling biological activity!

    PubMed Central

    Cahill, Michael A.; Jazayeri, Jalal A.; Kovacevic, Zaklina; Richardson, Des R.

    2016-01-01

    Progesterone receptor membrane component 1 (PGRMC1) is a multifunctional protein implicated in multiple pathologies, including cancer and Alzheimer's disease. The recently published structure of PGRMC1 revealed heme-mediated dimerization that directed the PGRMC1-dependent cytochrome P450-mediated detoxification of doxorubicin. We describe here how the PGRMC1 structure also enables important new insights into the possible regulation of PGRMC1 function by phosphorylation. Predicted regulatory interaction sites for SH2- and SH3-domain proteins are in non-structured regions that could be available to cytoplasmic enzymes. Further to the published interpretation, we suggest that phosphorylation of PGRMC1 at position Y113 may promote the attested membrane trafficking function of PGRMC1. To stimulate further experimentation, we also discuss that heme-mediated dimerization of PGRMC1 and membrane trafficking may be mutually exclusive functions. These roles could potentially be reciprocally regulated by phosphorylation/dephosphorylation at Y113. It follows that the phosphorylation status of PGRMC1 should be further explored in order to better understand many of its proposed biological functions. PMID:27448967

  4. Regulation of Rnd3 Localization and Function By PKCα-Mediated Phosphorylation

    PubMed Central

    Madigan, James P.; Bodemann, Brian O.; Brady, Donita C.; Dewar, Brian J.; Keller, Patricia J.; Leitges, Michael; Philips, Mark R.; Ridley, Anne J.; Der, Channing J.; Cox, Adrienne D.

    2010-01-01

    The Rnd proteins (Rnd1, Rnd2 and Rnd3/RhoE) form a distinct branch of the Rho family of small GTPases. Altered Rnd3 expression causes changes in cytoskeletal organization and cell cycle progression. Rnd3 functions to decrease RhoA activity, but how Rnd3 itself is regulated to cause these changes is still under investigation. Unlike other Rho family proteins, Rnd3 is regulated not by GTP/GDP cycling, but at the level of expression and by posttranslational modifications such as prenylation and phosphorylation. We show here that, upon PKC agonist stimulation, Rnd3 undergoes an electrophoretic mobility shift and its subcellular localization becomes enriched at internal membranes. These changes are blocked by inhibition of conventional PKC isoforms and do not occur in PKCα-null cells or to a nonphosphorylatable mutant of Rnd3. We further show that PKCα directly phosphorylates Rnd3 in an in vitro kinase assay. Additionally, we provide evidence that the phosphorylation status of Rnd3 has a direct effect on its ability to block signaling from the Rho-ROCK pathway. These results identify an additional mechanism of regulation and provide clarification of how Rnd3 modulates Rho signaling to alter cytoskeletal organization. PMID:19723022

  5. Regulation of mitochondrial functions by protein phosphorylation and dephosphorylation.

    PubMed

    Lim, Sangbin; Smith, Kelly R; Lim, Ssang-Taek Steve; Tian, Rong; Lu, Jianrong; Tan, Ming

    2016-01-01

    The mitochondria are double membrane-bound organelles found in most eukaryotic cells. They generate most of the cell's energy supply of adenosine triphosphate (ATP). Protein phosphorylation and dephosphorylation are critical mechanisms in the regulation of cell signaling networks and are essential for almost all the cellular functions. For many decades, mitochondria were considered autonomous organelles merely functioning to generate energy for cells to survive and proliferate, and were thought to be independent of the cellular signaling networks. Consequently, phosphorylation and dephosphorylation processes of mitochondrial kinases and phosphatases were largely neglected. However, evidence accumulated in recent years on mitochondria-localized kinases/phosphatases has changed this longstanding view. Mitochondria are increasingly recognized as a hub for cell signaling, and many kinases and phosphatases have been reported to localize in mitochondria and play important functions. However, the strength of the evidence on mitochondrial localization and the activities of the reported kinases and phosphatases vary greatly, and the detailed mechanisms on how these kinases/phosphatases translocate to mitochondria, their subsequent function, and the physiological and pathological implications of their localization are still poorly understood. Here, we provide an updated perspective on the recent advancement in this area, with an emphasis on the implications of mitochondrial kinases/phosphatases in cancer and several other diseases.

  6. Brain phosphorylation of MeCP2 at serine 164 is developmentally regulated and globally alters its chromatin association

    PubMed Central

    Stefanelli, Gilda; Gandaglia, Anna; Costa, Mario; Cheema, Manjinder S.; Di Marino, Daniele; Barbiero, Isabella; Kilstrup-Nielsen, Charlotte; Ausió, Juan; Landsberger, Nicoletta

    2016-01-01

    MeCP2 is a transcriptional regulator whose functional alterations are responsible for several autism spectrum and mental disorders. Post-translational modifications (PTMs), and particularly differential phosphorylation, modulate MeCP2 function in response to diverse stimuli. Understanding the detailed role of MeCP2 phosphorylation is thus instrumental to ascertain how MeCP2 integrates the environmental signals and directs its adaptive transcriptional responses. The evolutionarily conserved serine 164 (S164) was found phosphorylated in rodent brain but its functional role has remained uncharacterized. We show here that phosphorylation of S164 in brain is dynamically regulated during neuronal maturation. S164 phosphorylation highly impairs MeCP2 binding to DNA in vitro and largely affects its nucleosome binding and chromatin affinity in vivo. Strikingly, the chromatin-binding properties of the global MeCP2 appear also extensively altered during the course of brain maturation. Functional assays reveal that proper temporal regulation of S164 phosphorylation controls the ability of MeCP2 to regulate neuronal morphology. Altogether, our results support the hypothesis of a complex PTM-mediated functional regulation of MeCP2 potentially involving a still poorly characterized epigenetic code. Furthermore, they demonstrate the relevance of the Intervening Domain of MeCP2 for binding to DNA. PMID:27323888

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

    PubMed Central

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

    2016-01-01

    Fibrous sheath CABYR binding protein (FSCB) is regulated by protein kinase A (PKA)-mediated tyrosine phosphorylation in the spermatozoa capacitation. Recently, we showed that FSCB phosphorylation activated spermatozoa motility. Nevertheless, the underlying mechanisms have not been completely elucidated. Here, we showed that FSCB phosphorylation inhibited SUMOylation of two crucial proteins ROPN1/ROPN1L that are associated with PKA/A kinase activity and spermatozoa motility. Suppression of SUMOylation of ROPN1/ROPN1L mimicked the effects of FSCB phosphorylation on spermatozoa motility. Immunoprecipitation assay showed that phosphorylated FSCB had a significantly higher affinity to ROPN1/ROPN1L than non-phosphorylated FSCB. Together, our data suggest that FSCB phosphorylation may regulate mouse spermatozoa capacitation through suppressing SUMOylation of ROPN1/ROPN1L, which sheds new light on creating a therapeutic strategy targeting FSCB phosphorylation in the study of infertility. PMID:27398160

  8. Mouse Sphingosine Kinase 1a Is Negatively Regulated through Conventional PKC-Dependent Phosphorylation at S373 Residue

    PubMed Central

    Oh, Yong-Seok; Bae, Sun Sik; Park, Jong Bae; Ha, Sang Hoon; Ryu, Sung Ho; Suh, Pann-Ghill

    2015-01-01

    Sphingosine kinase is a lipid kinase that converts sphingosine into sphingosine-1-phosphate, an important signaling molecule with intracellular and extracellular functions. Although diverse extracellular stimuli influence cellular sphingosine kinase activity, the molecular mechanisms underlying its regulation remain to be clarified. In this study, we investigated the phosphorylation-dependent regulation of mouse sphingosine kinase (mSK) isoforms 1 and 2. mSK1a was robustly phosphorylated in response to extracellular stimuli such as phorbol ester, whereas mSK2 exhibited a high basal level of phosphorylation in quiescent cells regardless of agonist stimulation. Interestingly, phorbol ester-induced phosphorylation of mSK1a correlated with suppression of its activity. Chemical inhibition of conventional PKCs (cPKCs) abolished mSK1a phosphorylation, while overexpression of PKCα, a cPKC isoform, potentiated the phosphorylation, in response to phorbol ester. Furthermore, an in vitro kinase assay showed that PKCα directly phosphorylated mSK1a. In addition, phosphopeptide mapping analysis determined that the S373 residue of mSK1a was the only site phosphorylated by cPKC. Interestingly, alanine substitution of S373 made mSK1a refractory to the inhibitory effect of phorbol esters, whereas glutamate substitution of the same residue resulted in a significant reduction in mSK1a activity, suggesting the significant role of this phosphorylation event. Taken together, we propose that mSK1a is negatively regulated through cPKC-dependent phosphorylation at S373 residue. PMID:26642194

  9. Mouse Sphingosine Kinase 1a Is Negatively Regulated through Conventional PKC-Dependent Phosphorylation at S373 Residue.

    PubMed

    Oh, Yong-Seok; Bae, Sun Sik; Park, Jong Bae; Ha, Sang Hoon; Ryu, Sung Ho; Suh, Pann-Ghill

    2015-01-01

    Sphingosine kinase is a lipid kinase that converts sphingosine into sphingosine-1-phosphate, an important signaling molecule with intracellular and extracellular functions. Although diverse extracellular stimuli influence cellular sphingosine kinase activity, the molecular mechanisms underlying its regulation remain to be clarified. In this study, we investigated the phosphorylation-dependent regulation of mouse sphingosine kinase (mSK) isoforms 1 and 2. mSK1a was robustly phosphorylated in response to extracellular stimuli such as phorbol ester, whereas mSK2 exhibited a high basal level of phosphorylation in quiescent cells regardless of agonist stimulation. Interestingly, phorbol ester-induced phosphorylation of mSK1a correlated with suppression of its activity. Chemical inhibition of conventional PKCs (cPKCs) abolished mSK1a phosphorylation, while overexpression of PKCα, a cPKC isoform, potentiated the phosphorylation, in response to phorbol ester. Furthermore, an in vitro kinase assay showed that PKCα directly phosphorylated mSK1a. In addition, phosphopeptide mapping analysis determined that the S373 residue of mSK1a was the only site phosphorylated by cPKC. Interestingly, alanine substitution of S373 made mSK1a refractory to the inhibitory effect of phorbol esters, whereas glutamate substitution of the same residue resulted in a significant reduction in mSK1a activity, suggesting the significant role of this phosphorylation event. Taken together, we propose that mSK1a is negatively regulated through cPKC-dependent phosphorylation at S373 residue.

  10. Auxin-regulated changes in protein phosphorylation in pea epicotyl segments

    SciTech Connect

    Reddy, A.S.N.; Chengappa, S.; Raghothama, K.G.; Poovaiah, B.W.

    1987-04-01

    Auxin-regulated changes in protein phosphorylation were studied by labeling pea epicotyl segments with (/sup 32/P) PO/sub 4//sup 3 -/ and analyzing the phosphoproteins by two dimensional (2-D) gel electrophoresis. Analysis of phosphoproteins revealed auxin-regulated changes in the phosphorylation of specific polypeptides. In the presence of auxin, phosphorylation of 23,000, 82,000, 105,000 and 110,000 molecular weight polypeptides was markedly decreased whereas phosphorylation of 19,000, 24,000, 28,000 molecular weight polypeptides was increased. Some of these changes are very rapid and could be observed within minutes. Furthermore, their studies with calmodulin antagonists indicate the possible involvement of calmodulin-dependent protein kinases and/or phosphatases in auxin-regulated changes in protein phosphorylation. In view of these results, they suggest that auxin-regulated protein phosphorylation could be the one of the earliest events in regulating diverse physiological processes by this hormone.

  11. How Phosphorylation and ATPase Activity Regulate Anion Flux though the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).

    PubMed

    Zwick, Matthias; Esposito, Cinzia; Hellstern, Manuel; Seelig, Anna

    2016-07-08

    The cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7), mutations of which cause cystic fibrosis, belongs to the ATP-binding cassette (ABC) transporter family and works as a channel for small anions, such as chloride and bicarbonate. Anion channel activity is known to depend on phosphorylation by cAMP-dependent protein kinase A (PKA) and CFTR-ATPase activity. Whereas anion channel activity has been extensively investigated, phosphorylation and CFTR-ATPase activity are still poorly understood. Here, we show that the two processes can be measured in a label-free and non-invasive manner in real time in live cells, stably transfected with CFTR. This study reveals three key findings. (i) The major contribution (≥90%) to the total CFTR-related ATP hydrolysis rate is due to phosphorylation by PKA and the minor contribution (≤10%) to CFTR-ATPase activity. (ii) The mutant CFTR-E1371S that is still conductive, but defective in ATP hydrolysis, is not phosphorylated, suggesting that phosphorylation requires a functional nucleotide binding domain and occurs in the post-hydrolysis transition state. (iii) CFTR-ATPase activity is inversely related to CFTR anion flux. The present data are consistent with a model in which CFTR is in a closed conformation with two ATPs bound. The open conformation is induced by ATP hydrolysis and corresponds to the post-hydrolysis transition state that is stabilized by phosphorylation and binding of chloride channel potentiators.

  12. LATS1/WARTS phosphorylates MYPT1 to counteract PLK1 and regulate mammalian mitotic progression

    PubMed Central

    Chiyoda, Tatsuyuki; Sugiyama, Naoyuki; Shimizu, Takatsune; Naoe, Hideaki; Kobayashi, Yusuke; Ishizawa, Jo; Arima, Yoshimi; Tsuda, Hiroshi; Ito, Masaaki; Kaibuchi, Kozo; Aoki, Daisuke; Ishihama, Yasushi

    2012-01-01

    In the mitotic exit network of budding yeast, Dbf2 kinase phosphorylates and regulates Cdc14 phosphatase. In contrast, no phosphatase substrates of LATS1/WARTS kinase, the mammalian equivalent of Dbf2, has been reported. To address this discrepancy, we performed phosphoproteomic screening using LATS1 kinase. Screening identified MYPT1 (myosin phosphatase–targeting subunit 1) as a new substrate for LATS1. LATS1 directly and preferentially phosphorylated serine 445 (S445) of MYPT1. An MYPT1 mutant (S445A) failed to dephosphorylate Thr 210 of PLK1 (pololike kinase 1), thereby activating PLK1. This suggests that LATS1 promotes MYPT1 to antagonize PLK1 activity. Consistent with this, LATS1-depleted HeLa cells or fibroblasts from LATS1 knockout mice showed increased PLK1 activity. We also found deoxyribonucleic acid (DNA) damage–induced LATS1 activation caused PLK1 suppression via the phosphorylation of MYPT1 S445. Furthermore, LATS1 knockdown cells showed reduced G2 checkpoint arrest after DNA damage. These results indicate that LATS1 phosphorylates a phosphatase as does the yeast Dbf2 and demonstrate a novel role of LATS1 in controlling PLK1 at the G2 DNA damage checkpoint. PMID:22641346

  13. Phosphorylation-dependent regulation cytosolic localization and oncogenic function of Skp2 by Akt/PKB

    PubMed Central

    Lin, Hui-Kuan; Wang, Guocan; Chen, Zhenbang; Teruya-Feldstein, Julie; Liu, Yan; Chan, Chia-Hsin; Yang, Wei-Lei; Erdjument-Bromage, Hediye; Nakayama, Keiichi I.; Nimer, Stephen; Tempst, Paul; Pandolfi, Pier Paolo

    2010-01-01

    Skp2 is an F-box protein that forms the SCF complex with Skp1 and Cullin-1 to constitute an E3 ligase for ubiquitylation. Ubiquitylation and degradation of the p27 is critical for Skp2-mediated cell cycle entry, and overexpression and cytosolic accumulation of Skp2 have been clearly associated with tumorigenesis although the functional significance of the latter has remained elusive. Here we show that the Akt/PKB interacts with and directly phosphorylates Skp2. We find that Skp2 phosphorylation by Akt triggers SCF complex formation and E3 ligase activity. Importantly, a phosphorylation-defective Skp2 mutant is drastically impaired in its ability to promote cell proliferation and tumorigenesis. Furthermore, we show that Akt-mediated phosphorylation triggers 14-3-3-β-dependent Skp2 relocalization to the cytosol, and we attribute a specific role to cytosolic Skp2 in the positive regulation of cell migration. Finally, we demonstrate that high levels of Akt activation correlate with Skp2 cytosolic accumulation in human cancer specimens. Our results therefore define a novel proto-oncogenic Akt/PKB-dependent signaling pathway. PMID:19270694

  14. Target gene specificity of USF-1 is directed via p38-mediated phosphorylation-dependent acetylation.

    PubMed

    Corre, Sébastien; Primot, Aline; Baron, Yorann; Le Seyec, Jacques; Goding, Colin; Galibert, Marie-Dominique

    2009-07-10

    How transcription factors interpret the output from signal transduction pathways to drive distinct programs of gene expression is a key issue that underpins development and disease. The ubiquitously expressed basic-helix-loop-helix leucine zipper upstream stimulating factor-1 binds E-box regulatory elements (CANNTG) to regulate a wide number of gene networks. In particular, USF-1 is a key component of the tanning process. Following UV irradiation, USF-1 is phosphorylated by the p38 stress-activated kinase on threonine 153 and directly up-regulates expression of the POMC, MC1R, TYR, TYRP-1 and DCT genes. However, how phosphorylation on Thr-153 might affect the activity of USF-1 is unclear. Here we show that, in response to DNA damage, oxidative stress and cellular infection USF-1 is acetylated in a phospho-Thr-153-dependent fashion. Phospho-acetylated USF-1 is nuclear and interacts with DNA but displays altered gene regulatory properties. Phospho-acetylated USF-1 is thus proposed to be associated with loss of transcriptional activation properties toward several target genes implicated in pigmentation process and cell cycle regulation. The identification of this critical stress-dependent USF-1 modification gives new insights into understanding USF-1 gene expression modulation associated with cancer development.

  15. Phosphorylation of K[superscript +] Channels at Single Residues Regulates Memory Formation

    ERIC Educational Resources Information Center

    Vernon, Jeffrey; Irvine, Elaine E.; Peters, Marco; Jeyabalan, Jeshmi; Giese, K. Peter

    2016-01-01

    Phosphorylation is a ubiquitous post-translational modification of proteins, and a known physiological regulator of K[superscript +] channel function. Phosphorylation of K[superscript +] channels by kinases has long been presumed to regulate neuronal processing and behavior. Although circumstantial evidence has accumulated from behavioral studies…

  16. Regulation of ceramide synthase by casein kinase 2-dependent phosphorylation in Saccharomyces cerevisiae.

    PubMed

    Fresques, Tara; Niles, Brad; Aronova, Sofia; Mogri, Huzefa; Rakhshandehroo, Taha; Powers, Ted

    2015-01-16

    Complex sphingolipids are important components of eukaryotic cell membranes and, together with their biosynthetic precursors, including sphingoid long chain bases and ceramides, have important signaling functions crucial for cell growth and survival. Ceramides are produced at the endoplasmic reticulum (ER) membrane by a multicomponent enzyme complex termed ceramide synthase (CerS). In budding yeast, this complex is composed of two catalytic subunits, Lac1 and Lag1, as well as an essential regulatory subunit, Lip1. Proper formation of ceramides by CerS has been shown previously to require the Cka2 subunit of casein kinase 2 (CK2), a ubiquitous enzyme with multiple cellular functions, but the precise mechanism involved has remained unidentified. Here we present evidence that Lac1 and Lag1 are direct targets for CK2 and that phosphorylation at conserved positions within the C-terminal cytoplasmic domain of each protein is required for optimal CerS activity. Our data suggest that phosphorylation of Lac1 and Lag1 is important for proper localization and distribution of CerS within the ER membrane and that phosphorylation of these sites is functionally linked to the COP I-dependent C-terminal dilysine ER retrieval pathway. Together, our data identify CK2 as an important regulator of sphingolipid metabolism, and additionally, because both ceramides and CK2 have been implicated in the regulation of cancer, our findings may lead to an enhanced understanding of their relationship in health and disease.

  17. Regulation of Ceramide Synthase by Casein Kinase 2-dependent Phosphorylation in Saccharomyces cerevisiae*

    PubMed Central

    Fresques, Tara; Niles, Brad; Aronova, Sofia; Mogri, Huzefa; Rakhshandehroo, Taha; Powers, Ted

    2015-01-01

    Complex sphingolipids are important components of eukaryotic cell membranes and, together with their biosynthetic precursors, including sphingoid long chain bases and ceramides, have important signaling functions crucial for cell growth and survival. Ceramides are produced at the endoplasmic reticulum (ER) membrane by a multicomponent enzyme complex termed ceramide synthase (CerS). In budding yeast, this complex is composed of two catalytic subunits, Lac1 and Lag1, as well as an essential regulatory subunit, Lip1. Proper formation of ceramides by CerS has been shown previously to require the Cka2 subunit of casein kinase 2 (CK2), a ubiquitous enzyme with multiple cellular functions, but the precise mechanism involved has remained unidentified. Here we present evidence that Lac1 and Lag1 are direct targets for CK2 and that phosphorylation at conserved positions within the C-terminal cytoplasmic domain of each protein is required for optimal CerS activity. Our data suggest that phosphorylation of Lac1 and Lag1 is important for proper localization and distribution of CerS within the ER membrane and that phosphorylation of these sites is functionally linked to the COP I-dependent C-terminal dilysine ER retrieval pathway. Together, our data identify CK2 as an important regulator of sphingolipid metabolism, and additionally, because both ceramides and CK2 have been implicated in the regulation of cancer, our findings may lead to an enhanced understanding of their relationship in health and disease. PMID:25429105

  18. Motile properties of the bi-directional kinesin-5 Cin8 are affected by phosphorylation in its motor domain

    PubMed Central

    Shapira, Ofer; Gheber, Larisa

    2016-01-01

    The Saccharomyces cerevisiae kinesin-5 Cin8 performs essential mitotic functions in spindle assembly and anaphase B spindle elongation. Recent work has shown that Cin8 is a bi-directional motor which moves towards the minus-end of microtubules (MTs) under high ionic strength (IS) conditions and changes directionality in low IS conditions and when bound between anti-parallel microtubules. Previous work from our laboratory has also indicated that Cin8 is differentially phosphorylated during late anaphase at cyclin-dependent kinase 1 (Cdk1)-specific sites located in its motor domain. In vivo, such phosphorylation causes Cin8 detachment from spindles and reduces the spindle elongation rate, while maintaining proper spindle morphology. To study the effect of phosphorylation on Cin8 motor function, we examined in vitro motile properties of wild type Cin8, as well as its phosphorylation using phospho-deficient and phospho-mimic variants, in a single molecule fluorescence motility assay. Analysis was performed on whole cell extracts and on purified Cin8 samples. We found that addition of negative charges in the phospho-mimic mutant weakened the MT-motor interaction, increased motor velocity and promoted minus-end-directed motility. These results indicate that phosphorylation in the catalytic domain of Cin8 regulates its motor function. PMID:27216310

  19. Phosphorylated Rad18 directs DNA Polymerase η to sites of stalled replication

    PubMed Central

    Day, Tovah A.; Palle, Komariah; Barkley, Laura R.; Kakusho, Naoko; Zou, Ying; Tateishi, Satoshi; Verreault, Alain; Masai, Hisao

    2010-01-01

    The E3 ubiquitin ligase Rad18 guides DNA Polymerase eta (Polη) to sites of replication fork stalling and mono-ubiquitinates proliferating cell nuclear antigen (PCNA) to facilitate binding of Y family trans-lesion synthesis (TLS) DNA polymerases during TLS. However, it is unclear exactly how Rad18 is regulated in response to DNA damage and how Rad18 activity is coordinated with progression through different phases of the cell cycle. Here we identify Rad18 as a novel substrate of the essential protein kinase Cdc7 (also termed Dbf4/Drf1-dependent Cdc7 kinase [DDK]). A serine cluster in the Polη-binding motif of Rad18 is phosphorylated by DDK. Efficient association of Rad18 with Polη is dependent on DDK and is necessary for redistribution of Polη to sites of replication fork stalling. This is the first demonstration of Rad18 regulation by direct phosphorylation and provides a novel mechanism for integration of S phase progression with postreplication DNA repair to maintain genome stability. PMID:21098111

  20. Mek1 Down Regulates Rad51 Activity during Yeast Meiosis by Phosphorylation of Hed1

    PubMed Central

    Callender, Tracy L.; Laljee, Saif; Zhou, Sai; Suhandynata, Ray T.; Gaines, William A.; Kwon, YoungHo; Börner, G. Valentin; Nicolas, Alain; Neiman, Aaron M.

    2016-01-01

    During meiosis, programmed double strand breaks (DSBs) are repaired preferentially between homologs to generate crossovers that promote proper chromosome segregation at Meiosis I. In many organisms, there are two strand exchange proteins, Rad51 and the meiosis-specific Dmc1, required for interhomolog (IH) bias. This bias requires the presence, but not the strand exchange activity of Rad51, while Dmc1 is responsible for the bulk of meiotic recombination. How these activities are regulated is less well established. In dmc1Δ mutants, Rad51 is actively inhibited, thereby resulting in prophase arrest due to unrepaired DSBs triggering the meiotic recombination checkpoint. This inhibition is dependent upon the meiosis-specific kinase Mek1 and occurs through two different mechanisms that prevent complex formation with the Rad51 accessory factor Rad54: (i) phosphorylation of Rad54 by Mek1 and (ii) binding of Rad51 by the meiosis-specific protein Hed1. An open question has been why inhibition of Mek1 affects Hed1 repression of Rad51. This work shows that Hed1 is a direct substrate of Mek1. Phosphorylation of Hed1 at threonine 40 helps suppress Rad51 activity in dmc1Δ mutants by promoting Hed1 protein stability. Rad51-mediated recombination occurring in the absence of Hed1 phosphorylation results in a significant increase in non-exchange chromosomes despite wild-type levels of crossovers, confirming previous results indicating a defect in crossover assurance. We propose that Rad51 function in meiosis is regulated in part by the coordinated phosphorylation of Rad54 and Hed1 by Mek1. PMID:27483004

  1. Protein phosphorylation and prevention of cytochrome oxidase inhibition by ATP: coupled mechanisms of energy metabolism regulation

    PubMed Central

    Acin-Perez, Rebeca; Gatti, Domenico L.; Bai, Yidong; Manfredi, Giovanni

    2011-01-01

    Summary Rapid regulation of oxidative phosphorylation is crucial for mitochondrial adaptation to swift changes in fuels availability and energy demands. An intra-mitochondrial signaling pathway regulates cytochrome oxidase (COX), the terminal enzyme of the respiratory chain, through reversible phosphorylation. We find that PKA-mediated phosphorylation of a COX subunit dictates mammalian mitochondrial energy fluxes, and identify the specific residue (S58) of COX subunit IV-1 (COXIV-1) that is involved in this mechanism of metabolic regulation. Using protein mutagenesis, molecular dynamics simulations, and induced fit docking, we show that mitochondrial energy metabolism regulation by phosphorylation of COXIV-1 is coupled with prevention of COX allosteric inhibition by ATP. This regulatory mechanism is essential for efficient oxidative metabolism and cell survival. We propose that S58 COXIV-1 phosphorylation has evolved as a metabolic switch that allows mammalian mitochondria to rapidly toggle between energy utilization and energy storage. PMID:21641552

  2. Serine 649 phosphorylation within the protein kinase C-regulated domain down-regulates CARMA1 activity in lymphocytes.

    PubMed

    Moreno-García, Miguel E; Sommer, Karen; Haftmann, Claudia; Sontheimer, Clayton; Andrews, Sarah F; Rawlings, David J

    2009-12-01

    Phosphorylation of CARMA1 is a crucial event initiating the assembly of IkappaB kinase and JNK signaling complexes downstream of activated Ag receptors. We previously mapped three protein kinase C (PKC) target sites in murine CARMA1 in vitro, and demonstrated that mutation of two of these serines (S564 and S657) resulted in reduced NF-kappaB activation, whereas mutation of the third serine (S649) had no clear effect. In this study, we report that when low concentrations of Ag receptor activators are used, loss of S649 (by mutation to alanine) promotes enhanced IkappaB kinase and JNK activation in both B and T cell lines. Reconstitution of CARMA1(-/-) DT40 B cells with CARMA1 S649A leads to increased cell death and reduced cell growth in comparison to wild-type CARMA1, likely a result of enhanced JNK activation. To directly determine whether S649 is modified in vivo, we generated phospho-specific Abs recognizing phospho-S649, and phospho-S657 as a positive control. Although phospho-S657 peaked and declined rapidly after Ag receptor stimulation, phospho-S649 occurred later and was maintained for a significantly longer period poststimulation in both B and T cells. Interestingly, phospho-S657 was completely abolished in PKCbeta-deficient B cells, whereas delayed phosphorylation at S649 was partially intact and depended, in part, upon novel PKC activity. Thus, distinct PKC-mediated CARMA1 phosphorylation events exert opposing effects on the activation status of CARMA1. We propose that early phosphorylation events at S657 and S564 promote the initial assembly of the CARMA1 signalosome, whereas later phosphorylation at S649 triggers CARMA1 down-regulation.

  3. HIF and reactive oxygen species regulate oxidative phosphorylation in cancer.

    PubMed

    Hervouet, Eric; Cízková, Alena; Demont, Jocelyne; Vojtísková, Alena; Pecina, Petr; Franssen-van Hal, Nicole L W; Keijer, Jaap; Simonnet, Hélène; Ivánek, Robert; Kmoch, Stanislav; Godinot, Catherine; Houstek, Josef

    2008-08-01

    A decrease in oxidative phosphorylation (OXPHOS) is characteristic of many cancer types and, in particular, of clear cell renal carcinoma (CCRC) deficient in von Hippel-Lindau (vhl) gene. In the absence of functional pVHL, hypoxia-inducible factor (HIF) 1-alpha and HIF2-alpha subunits are stabilized, which induces the transcription of many genes including those involved in glycolysis and reactive oxygen species (ROS) metabolism. Transfection of these cells with vhl is known to restore HIF-alpha subunit degradation and to reduce glycolytic genes transcription. We show that such transfection with vhl of 786-0 CCRC (which are devoid of HIF1-alpha) also increased the content of respiratory chain subunits. However, the levels of most transcripts encoding OXPHOS subunits were not modified. Inhibition of HIF2-alpha synthesis by RNA interference in pVHL-deficient 786-0 CCRC also restored respiratory chain subunit content and clearly demonstrated a key role of HIF in OXPHOS regulation. In agreement with these observations, stabilization of HIF-alpha subunit by CoCl(2) decreased respiratory chain subunit levels in CCRC cells expressing pVHL. In addition, HIF stimulated ROS production and mitochondrial manganese superoxide dismutase content. OXPHOS subunit content was also decreased by added H(2)O(2.) Interestingly, desferrioxamine (DFO) that also stabilized HIF did not decrease respiratory chain subunit level. While CoCl(2) significantly stimulates ROS production, DFO is known to prevent hydroxyl radical production by inhibiting Fenton reactions. This indicates that the HIF-induced decrease in OXPHOS is at least in part mediated by hydroxyl radical production.

  4. p38 regulates expression of osteoblast-specific genes by phosphorylation of osterix.

    PubMed

    Ortuño, María José; Ruiz-Gaspà, Silvia; Rodríguez-Carballo, Edgardo; Susperregui, Antonio R G; Bartrons, Ramon; Rosa, José Luis; Ventura, Francesc

    2010-10-15

    Osterix, a zinc finger transcription factor, is specifically expressed in osteoblasts and osteocytes of all developing bones. Because no bone formation occurs in Osx-null mice, Osterix is thought to be an essential regulator of osteoblast differentiation. We report that, in several mesenchymal and osteoblastic cell types, BMP-2 induces an increase in expression of the two isoforms of Osterix arising from two alternative promoters. We identified a consensus Sp1 sequence (GGGCGG) as Osterix binding regions in the fibromodulin and the bone sialoprotein promoters in vitro and in vivo. Furthermore, we show that Osterix is a novel substrate for p38 MAPK in vitro and in vivo and that Ser-73 and Ser-77 are the regulatory sites phosphorylated by p38. Our data also demonstrate that Osterix is able to increase recruitment of p300 and Brg1 to the promoters of its target genes fibromodulin and bone sialoprotein in vivo and that it directly associates with these cofactors through protein-protein interactions. Phosphorylation of Osterix at Ser-73/77 increased its ability to recruit p300 and SWI/SNF to either fibromodulin or bone sialoprotein promoters. We therefore propose that Osterix binds to Sp1 sequences on target gene promoters and that its phosphorylation by p38 enhances recruitment of coactivators to form transcriptionally active complexes.

  5. 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

  6. Regulation of photoreceptor gap junction phosphorylation by adenosine in zebrafish retina

    PubMed Central

    Li, Hongyan; Chuang, Alice Z.; O’Brien, John

    2014-01-01

    Electrical coupling of photoreceptors through gap junctions suppresses voltage noise, routes rod signals into cone pathways, expands the dynamic range of rod photoreceptors in high scotopic and mesopic illumination, and improves detection of contrast and small stimuli. In essentially all vertebrates, connexin 35/36 (gene homologues Cx36 in mammals, Cx35 in other vertebrates) is the major gap junction protein observed in photoreceptors, mediating rod-cone, cone-cone, and possibly rod-rod communication. Photoreceptor coupling is dynamically controlled by the day/night cycle and light/dark adaptation, and is directly correlated with phosphorylation of Cx35/36 at two sites, serine110 and serine 276/293 (homologous sites in teleost fish and mammals respectively). Activity of protein kinase A (PKA) plays a key role during this process. Previous studies have shown that activation of dopamine D4 receptors on photoreceptors inhibits adenylyl cyclase, down-regulates cAMP and PKA activity, and leads to photoreceptor uncoupling, imposing the daytime/light condition. In this study we explored the role of adenosine, a nighttime signal with a high extracellular concentration at night and a low concentration in the day, in regulating photoreceptor coupling by examining photoreceptor Cx35 phosphorylation in zebrafish retina. Adenosine enhanced photoreceptor Cx35 phosphorylation in daytime, but with a complex dose-response curve. Selective pharmacological manipulations revealed that adenosine A2a receptors provide a potent positive drive to phosphorylate photoreceptor Cx35 under the influence of endogenous adenosine at night. A2a receptors can be activated in the daytime as well by micromolar exogenous adenosine. However, the higher affinity adenosine A1 receptors are also present and have an antagonistic though less potent effect. Thus the nighttime/darkness signal adenosine provides a net positive drive on Cx35 phosphorylation at night, working in opposition to dopamine to

  7. Kin of IRRE-like Protein 2 Is a Phosphorylated Glycoprotein That Regulates Basal Insulin Secretion*

    PubMed Central

    Yesildag, Burcak; Bock, Thomas; Herrmanns, Karolin; Wollscheid, Bernd; Stoffel, Markus

    2015-01-01

    Direct interactions among pancreatic β-cells via cell surface proteins inhibit basal and enhance stimulated insulin secretion. Here, we functionally and biochemically characterized Kirrel2, an immunoglobulin superfamily protein with β-cell-specific expression in the pancreas. Our results show that Kirrel2 is a phosphorylated glycoprotein that co-localizes and interacts with the adherens junction proteins E-cadherin and β-catenin in MIN6 cells. We further demonstrate that the phosphosites Tyr595–596 are functionally relevant for the regulation of Kirrel2 stability and localization. Analysis of the extracellular and intracellular domains of Kirrel2 revealed that it is cleaved and shed from MIN6 cells and that the remaining membrane spanning cytoplasmic domain is processed by γ-secretase complex. Kirrel2 knockdown with RNA interference in MIN6 cells and ablation of Kirrel2 from mice with genetic deletion resulted in increased basal insulin secretion from β-cells, with no immediate influence on stimulated insulin secretion, total insulin content, or whole body glucose metabolism. Our results show that in pancreatic β-cells Kirrel2 localizes to adherens junctions, is regulated by multiple post-translational events, including glycosylation, extracellular cleavage, and phosphorylation, and engages in the regulation of basal insulin secretion. PMID:26324709

  8. How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria†

    PubMed Central

    Deutscher, Josef; Francke, Christof; Postma, Pieter W.

    2006-01-01

    The phosphoenolpyruvate(PEP):carbohydrate phosphotransferase system (PTS) is found only in bacteria, where it catalyzes the transport and phosphorylation of numerous monosaccharides, disaccharides, amino sugars, polyols, and other sugar derivatives. To carry out its catalytic function in sugar transport and phosphorylation, the PTS uses PEP as an energy source and phosphoryl donor. The phosphoryl group of PEP is usually transferred via four distinct proteins (domains) to the transported sugar bound to the respective membrane component(s) (EIIC and EIID) of the PTS. The organization of the PTS as a four-step phosphoryl transfer system, in which all P derivatives exhibit similar energy (phosphorylation occurs at histidyl or cysteyl residues), is surprising, as a single protein (or domain) coupling energy transfer and sugar phosphorylation would be sufficient for PTS function. A possible explanation for the complexity of the PTS was provided by the discovery that the PTS also carries out numerous regulatory functions. Depending on their phosphorylation state, the four proteins (domains) forming the PTS phosphorylation cascade (EI, HPr, EIIA, and EIIB) can phosphorylate or interact with numerous non-PTS proteins and thereby regulate their activity. In addition, in certain bacteria, one of the PTS components (HPr) is phosphorylated by ATP at a seryl residue, which increases the complexity of PTS-mediated regulation. In this review, we try to summarize the known protein phosphorylation-related regulatory functions of the PTS. As we shall see, the PTS regulation network not only controls carbohydrate uptake and metabolism but also interferes with the utilization of nitrogen and phosphorus and the virulence of certain pathogens. PMID:17158705

  9. HEY1 functions are regulated by its phosphorylation at Ser-68

    PubMed Central

    López-Mateo, Irene; Arruabarrena-Aristorena, Amaia; Artaza-Irigaray, Cristina; López, Juan A.; Calvo, Enrique; Belandia, Borja

    2016-01-01

    HEY1 (hairy/enhancer-of-split related with YRPW motif 1) is a member of the basic helix–loop–helix-orange (bHLH-O) family of transcription repressors that mediate Notch signalling. HEY1 acts as a positive regulator of the tumour suppressor p53 via still unknown mechanisms. A MALDI-TOF/TOF MS analysis has uncovered a novel HEY1 regulatory phosphorylation event at Ser-68. Strikingly, this single phosphorylation event controls HEY1 stability and function: simulation of HEY1 Ser-68 phosphorylation increases HEY1 protein stability but inhibits its ability to enhance p53 transcriptional activity. Unlike wild-type HEY1, expression of the phosphomimetic mutant HEY1-S68D failed to induce p53-dependent cell cycle arrest and it did not sensitize U2OS cells to p53-activating chemotherapeutic drugs. We have identified two related kinases, STK38 (serine/threonine kinase 38) and STK38L (serine/threonine kinase 38 like), which interact with and phosphorylate HEY1 at Ser-68. HEY1 is phosphorylated at Ser-68 during mitosis and it accumulates in the centrosomes of mitotic cells, suggesting a possible integration of HEY1-dependent signalling in centrosome function. Moreover, HEY1 interacts with a subset of p53-activating ribosomal proteins. Ribosomal stress causes HEY1 relocalization from the nucleoplasm to perinucleolar structures termed nucleolar caps. HEY1 interacts physically with at least one of the ribosomal proteins, RPL11, and both proteins cooperate in the inhibition of MDM2-mediated p53 degradation resulting in a synergistic positive effect on p53 transcriptional activity. HEY1 itself also interacts directly with MDM2 and it is subjected to MDM2-mediated degradation. Simulation of HEY1 Ser-68 phosphorylation prevents its interaction with p53, RPL11 and MDM2 and abolishes HEY1 migration to nucleolar caps upon ribosomal stress. Our findings uncover a novel mechanism for cross-talk between Notch signalling and nucleolar stress. PMID:27129302

  10. TCR-induced Akt serine 473 phosphorylation is regulated by protein kinase C-alpha

    SciTech Connect

    Yang, Lifen; Qiao, Guilin; Ying, Haiyan; Zhang, Jian; Yin, Fei

    2010-09-10

    Research highlights: {yields} Conventional PKC positively regulates TCR-induced phosphorylation of Akt. {yields} PKC-alpha is the PDK-2 responsible for phosphorylating Akt at Ser{sup 473} upon TCR stimulation. {yields} Knockdown of PKC-alpha decreases TCR-induced Akt phosphorylation. -- Abstract: Akt signaling plays a central role in T cell functions, such as proliferation, apoptosis, and regulatory T cell development. Phosphorylation at Ser{sup 473} in the hydrophobic motif, along with Thr{sup 308} in its activation loop, is considered necessary for Akt function. It is widely accepted that phosphoinositide-dependent kinase 1 (PDK-1) phosphorylates Akt at Thr{sup 308}, but the kinase(s) responsible for phosphorylating Akt at Ser{sup 473} (PDK-2) remains elusive. The existence of PDK-2 is considered to be specific to cell type and stimulus. PDK-2 in T cells in response to TCR stimulation has not been clearly defined. In this study, we found that conventional PKC positively regulated TCR-induced Akt Ser{sup 473} phosphorylation. PKC-alpha purified from T cells can phosphorylate Akt at Ser{sup 473} in vitro upon TCR stimulation. Knockdown of PKC-alpha in T-cell-line Jurkat cells reduced TCR-induced phosphorylation of Akt as well as its downstream targets. Thus our results suggest that PKC-alpha is a candidate for PDK-2 in T cells upon TCR stimulation.

  11. Phosphorylation Events in the Multiple Gene Regulator of Group A Streptococcus Significantly Influence Global Gene Expression and Virulence

    PubMed Central

    Sanson, Misu; Makthal, Nishanth; Gavagan, Maire; Cantu, Concepcion; Olsen, Randall J.; Musser, James M.

    2015-01-01

    Whole-genome sequencing analysis of ∼800 strains of group A Streptococcus (GAS) found that the gene encoding the multiple virulence gene regulator of GAS (mga) is highly polymorphic in serotype M59 strains but not in strains of other serotypes. To help understand the molecular mechanism of gene regulation by Mga and its contribution to GAS pathogenesis in serotype M59 GAS, we constructed an isogenic mga mutant strain. Transcriptome studies indicated a significant regulatory influence of Mga and altered metabolic capabilities conferred by Mga-regulated genes. We assessed the phosphorylation status of Mga in GAS cell lysates with Phos-tag gels. The results revealed that Mga is phosphorylated at histidines in vivo. Using phosphomimetic and nonphosphomimetic substitutions at conserved phosphoenolpyruvate:carbohydrate phosphotransferase regulation domain (PRD) histidines of Mga, we demonstrated that phosphorylation-mimicking aspartate replacements at H207 and H273 of PRD-1 and at H327 of PRD-2 are inhibitory to Mga-dependent gene expression. Conversely, non-phosphorylation-mimicking alanine substitutions at H273 and H327 relieved inhibition, and the mutant strains exhibited a wild-type phenotype. The opposing regulatory profiles observed for phosphorylation- and non-phosphorylation-mimicking substitutions at H273 extended to global gene regulation by Mga. Consistent with these observations, the H273D mutant strain attenuated GAS virulence, whereas the H273A strain exhibited a wild-type virulence phenotype in a mouse model of necrotizing fasciitis. Together, our results demonstrate phosphoregulation of Mga and its direct link to virulence in M59 GAS strains. These data also lay a foundation toward understanding how naturally occurring gain-of-function variations in mga, such as H201R, may confer an advantage to the pathogen and contribute to M59 GAS pathogenesis. PMID:25824840

  12. BRAF, KIT and NRAS mutations and expression of c-KIT, phosphorylated extracellular signal-regulated kinase and phosphorylated AKT in Japanese melanoma patients.

    PubMed

    Oyama, Satomi; Funasaka, Yoko; Watanabe, Atsushi; Takizawa, Toshihiro; Kawana, Seiji; Saeki, Hidehisa

    2015-05-01

    To clarify the status of gene mutation and activation of growth signal in melanoma of Japanese patients in vivo, we analyzed the mutation of BRAF exon 15, NRAS exon 2, and KIT exons 9, 11, 13, 17 and 18 in melanoma cells obtained by laser capture microdissection, and performed direct sequencing in 20 cases of acral lentiginous melanoma (ALM) and 17 cases of superficial spreading melanoma (SSM). In the study of the mutation of BRAF, pyrosequencing was also done. To examine the cell proliferation signaling, immunohistochemistry for phosphorylated extracellular signal-regulated kinase (pERK), phosphorylated AKT (phosphorylated AKT) and c-KIT was done. The mutation of BRAF p.V600E was detected in 13 cases of ALM (65.0%) and 12 cases of SSM (70.6%). No NRAS mutation was found in all cases. The mutation in exons 9, 11, and 18 of KIT was detected in nine cases. The mutation of BRAF and KIT showed no correlation with clinical stage, lymph node metastasis, tumor thickness, ulceration and histology. pERK and pAKT was observed in small population of melanoma cells and there was no correlation with gene mutation. Our results indicate that the mutations of BRAF and KIT exist in Japanese melanoma patients, however, the cell growth signaling may be regulated by not only these mutated genes, but by other unknown regulatory factors, which may affect the prognosis of melanoma.

  13. Regulation of VASP serine 157 phosphorylation in human neutrophils after stimulation by a chemoattractant.

    PubMed

    Eckert, Rachael E; Jones, Samuel L

    2007-11-01

    Vasodilator-stimulated phosphoprotein (VASP) is a cAMP-dependent protein kinase A (PKA) substrate, which links cellular signaling to cytoskeletal organization and cellular movement. VASP is phosphorylated by PKA on serine 157 (Ser 157), which is required for VASP function in platelet adhesion and fibroblast motility. Our hypothesis is that PKA regulates neutrophil migration through VASP Ser 157 phosphorylation. The objective of this study was to characterize VASP Ser 157 phosphorylation in chemoattractant-stimulated neutrophils. fMLF, IL-8, leukotriene B(4), or platelet-activating factor stimulation resulted in an initial increase in VASP Ser 157 phosphorylation, which was maximal by 30 s and was followed by a return to baseline Ser 157 phosphorylation by 10 min. In contrast, stimulation with the nonchemoattractant, proinflammatory cytokine TNF-alpha did not affect Ser 157 phosphorylation. The kinetics of fMLF-induced VASP Ser 157 phosphorylation levels closely matched the kinetics of the fold-change in F-actin levels in fMLF-stimulated neutrophils. fMLF-induced Ser 157 phosphorylation was abolished by pretreatment with the PKA inhibitor H89 and the adenylyl cyclase inhibitor SQ22536. In contrast, fMLF-induced Ser 157 phosphorylation was unaffected by the PKC inhibitors calphostin and staurosporine, the PKG inhibitors Rp-8-pCPT-cGMP and KT5823, and the calmodulin-dependent protein kinase II inhibitor KN-62. Inhibition of adhesion with EDTA or the anti-beta2-integrin antibody IB4 did not alter fMLF-induced VASP phosphorylation or dephosphorylation. These data show that chemoattractant stimulation of human neutrophils induces a rapid and transient PKA-dependent VASP Ser 157 phosphorylation. Adhesion does not appear to be an important regulator of the state of VASP Ser 157 phosphorylation in chemoattractant-stimulated neutrophils.

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

    PubMed Central

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

    2016-01-01

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

  15. Regulation of Hepatitis C Virion Production via Phosphorylation of the NS5A Protein

    PubMed Central

    Tellinghuisen, Timothy L.; Foss, Katie L.; Treadaway, Jason

    2008-01-01

    Hepatitis C virus (HCV) is a significant pathogen, infecting some 170 million people worldwide. Persistent virus infection often leads to cirrhosis and liver cancer. In the infected cell many RNA directed processes must occur to maintain and spread infection. Viral genomic RNA is constantly replicating, serving as template for translation, and being packaged into new virus particles; processes that cannot occur simultaneously. Little is known about the regulation of these events. The viral NS5A phosphoprotein has been proposed as a regulator of events in the HCV life cycle for years, but the details have remained enigmatic. NS5A is a three-domain protein and the requirement of domains I and II for RNA replication is well documented. NS5A domain III is not required for RNA replication, and the function of this region in the HCV lifecycle is unknown. We have identified a small deletion in domain III that disrupts the production of infectious virus particles without altering the efficiency of HCV RNA replication. This deletion disrupts virus production at an early stage of assembly, as no intracellular virus is generated and no viral RNA and nucleocapsid protein are released from cells. Genetic mapping has indicated a single serine residue within the deletion is responsible for the observed phenotype. This serine residue lies within a casein kinase II consensus motif, and mutations that mimic phosphorylation suggest that phosphorylation at this position regulates the production of infectious virus. We have shown by genetic silencing and chemical inhibition experiments that NS5A requires casein kinase II phosphorylation at this position for virion production. A mutation that mimics phosphorylation at this position is insensitive to these manipulations of casein kinase II activity. These data provide the first evidence for a function of the domain III of NS5A and implicate NS5A as an important regulator of the RNA replication and virion assembly of HCV. The ability to

  16. Mechanism of Ribonuclease III Catalytic Regulation by Serine Phosphorylation

    NASA Astrophysics Data System (ADS)

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

    2016-05-01

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

  17. Phosphorylation Affects DNA-Binding of the Senescence-Regulating bZIP Transcription Factor GBF1

    PubMed Central

    Smykowski, Anja; Fischer, Stefan M.; Zentgraf, Ulrike

    2015-01-01

    Massive changes in the transcriptome of Arabidopsis thaliana during onset and progression of leaf senescence imply a central role for transcription factors. While many transcription factors are themselves up- or down-regulated during senescence, the bZIP transcription factor G-box-binding factor 1 (GBF1/bZIP41) is constitutively expressed in Arabidopsis leaf tissue but at the same time triggers the onset of leaf senescence, suggesting posttranscriptional mechanisms for senescence-specific GBF1 activation. Here we show that GBF1 is phosphorylated by the threonine/serine CASEIN KINASE II (CKII) in vitro and that CKII phosphorylation had a negative effect on GBF1 DNA-binding to G-boxes of two direct target genes, CATALASE2 and RBSCS1a. Phosphorylation mimicry at three serine positions in the basic region of GBF1 also had a negative effect on DNA-binding. Kinase assays revealed that CKII phosphorylates at least one serine in the basic domain but has additional phosphorylation sites outside this domain. Two different ckII α subunit1 and one α subunit2 T-DNA insertion lines showed no visible senescence phenotype, but in all lines the expression of the senescence marker gene SAG12 was remarkably diminished. A model is presented suggesting that senescence-specific GBF1 activation might be achieved by lowering the phosphorylation of GBF1 by CKII. PMID:27135347

  18. CK2 phosphorylation of SAG at Thr10 regulates SAG stability, but not its E3 ligase activity.

    PubMed

    He, Hongbin; Tan, Mingjia; Pamarthy, Deepika; Wang, Guixia; Ahmed, Khalil; Sun, Yi

    2007-01-01

    Sensitive to Apoptosis Gene (SAG), a RING component of SCF E3 ubiquitin ligase, was shown to be phosphorylated by protein kinase CK2 at the Thr10 residue. It is, however, unknown whether this phosphorylation is stress-responsive or whether the phosphorylation changes its E3 ubiquitin ligase activity. To address these, we made a specific antibody against the phosphor-SAG(Thr10). Transient transfection experiment showed that SAG was phosphorylated at Thr10 which can be significantly inhibited by TBB, a relatively specific inhibitor of protein kinase CK2. To determine whether this SAG phosphorylation is stress-responsive, we defined a chemical-hypoxia condition in which SAG and CK2 were both induced. Under this condition, we failed to detect SAG phosphorylation at Thr10, which was readily detected, however, in the presence of MG132, a proteasome inhibitor, suggesting that the phosphorylated SAG has undergone a rapid degradation. To further define this, we made two SAG mutants, SAG-T10A which abolishes the SAG phosphorylation and SAG-T10E, which mimics the constitutive SAG phosphorylation. The half-life study revealed that indeed, SAG-T10E has a much shorter protein half-life (2 h), as compared to wild-type SAG (10 h). Again, rapid degradation of SAG-T10E in cells can be blocked by MG132. Thus, it appears that CK2-induced SAG phosphorylation at Thr10 regulates its stability through a proteasome-dependent pathway. Immunocytochemistry study showed that SAG as well as its phosphorylation mutants, was mainly localized in nucleus and lightly in cytoplasm. Hypoxia condition did not change their sub-cellular localization. Finally, an in vitro ubiqutination assay showed that SAG mutation at Thr10 did not change its E3 ligase activity when complexed with cullin-1. These studies suggested that CK2 might regulate SAG-SCF E3 ligase activity through modulating SAG's stability, rather than its enzymatic activity directly.

  19. Deoxycholic acid differentially regulates focal adhesion kinase phosphorylation: role of tyrosine phosphatase ShP2.

    PubMed

    Khare, Sharad; Holgren, Cory; Samarel, Allen M

    2006-12-01

    Environmental factors, including dietary fats, are implicated in colonic carcinogenesis. Dietary fats modulate secondary bile acids including deoxycholic acid (DCA) concentrations in the colon, which are thought to contribute to the nutritional-related component of colon cancer risk. Here we demonstrate, for the first time, that DCA differentially regulated the site-specific phosphorylation of focal adhesion kinase (FAK). DCA decreased adhesion of HCA-7 cells to the substratum and induced dephosphorylation of FAK at tyrosine-576/577 (Tyr-576/577) and Tyr-925. Tyrosine phosphorylation of FAK at Tyr-397 remained unaffected by DCA stimulation. Interestingly, we found that c-Src was constitutively associated with FAK and DCA actually activated Src, despite no change in FAK-397 and an inhibition of FAK-576 phosphorylation. DCA concomitantly and significantly increased association of tyrosine phosphatase ShP2 with FAK. Incubation of immunoprecipitated FAK, in vitro, with glutathione-S-transferase-ShP2 fusion protein resulted in tyrosine dephosphorylation of FAK in a concentration-dependent manner. Antisense oligodeoxynucleotides directed against ShP2 decreased ShP2 protein levels and attenuated DCA-induced FAK dephosphorylation. Inhibition of FAK by adenoviral-mediated overexpression of FAK-related nonkinase and gene silencing of Shp2 both abolished DCA's effect on cell adhesion, thus providing a possible mechanism for inside-out signaling by DCA in colon cancer cells. Our results suggest that DCA differentially regulates focal adhesion complexes and that tyrosine phosphatase ShP2 has a role in DCA signaling.

  20. Regulation of casein kinase 2 by phosphorylation/dephosphorylation.

    PubMed Central

    Agostinis, P; Goris, J; Pinna, L A; Merlevede, W

    1987-01-01

    The effects of various polycation-stimulated (PCS) phosphatases and of the active catalytic subunit of the ATPMg-dependent (AMDc) protein phosphatase on the activity of casein kinase 2 (CK-2) were investigated by using the synthetic peptide substrate Ser-Glu-Glu-Glu-Glu-Glu, whose phosphorylated derivative is entirely insensitive to these protein phosphatases. Previous dephosphorylation of native CK-2 enhances its specific activity 2-3-fold. Such an effect, accounted for by an increase in Vmax, is more readily promoted by the PCS phosphatases than by the AMDc phosphatase. The phosphate incorporated by autophosphorylation could not be removed by the protein phosphatases, suggesting the involvement of phosphorylation site(s) other than the one(s) affected by intramolecular autophosphorylation. The activation of CK-2 by the phosphatase pretreatment is neutralized during the kinase assay; the mechanism of this phenomenon, which is highly dependent on the kinase concentration, is discussed. Images Fig. 4. PMID:2829841

  1. Regulation of casein kinase 2 by phosphorylation/dephosphorylation.

    PubMed

    Agostinis, P; Goris, J; Pinna, L A; Merlevede, W

    1987-12-15

    The effects of various polycation-stimulated (PCS) phosphatases and of the active catalytic subunit of the ATPMg-dependent (AMDc) protein phosphatase on the activity of casein kinase 2 (CK-2) were investigated by using the synthetic peptide substrate Ser-Glu-Glu-Glu-Glu-Glu, whose phosphorylated derivative is entirely insensitive to these protein phosphatases. Previous dephosphorylation of native CK-2 enhances its specific activity 2-3-fold. Such an effect, accounted for by an increase in Vmax, is more readily promoted by the PCS phosphatases than by the AMDc phosphatase. The phosphate incorporated by autophosphorylation could not be removed by the protein phosphatases, suggesting the involvement of phosphorylation site(s) other than the one(s) affected by intramolecular autophosphorylation. The activation of CK-2 by the phosphatase pretreatment is neutralized during the kinase assay; the mechanism of this phenomenon, which is highly dependent on the kinase concentration, is discussed.

  2. Actin Polymerization: An Event Regulated by Tyrosine Phosphorylation During Buffalo Sperm Capacitation.

    PubMed

    Naresh, S; Atreja, S K

    2015-12-01

    In the female reproductive tract, the spermatozoa undergo a series of physiological and biochemical changes, prior to gaining the ability to fertilize, that result to capacitation. However, the actin polymerization and protein tyrosine phosphorylation are the two necessary steps for capacitation. In this study, we have demonstrated the actin polymerization and established the correlation between protein tyrosine phosphorylation and actin reorganization during in vitro capacitation in buffalo (Bubalus bubalis) spermatozoa. Indirect immunofluorescence and Western blot techniques were used to detect actin polymerization and tyrosine phosphorylation. The time-dependent fluorimetric studies revealed that the actin polymerization starts from the tail region and progressed towards the head region of spermatozoa during capacitation. The lysophosphatidyl choline (LPC)-induced acrosome reaction (AR) stimulated quick actin depolymerization. The inhibitor cytochalasin D (CD) blocked the in vitro capacitation by inhibiting the actin polymerization. In addition, we also performed different inhibitor (Genistein, H-89, PD9809 and GF-109) and enhancer (dbcAMP, H(2)O(2) and vanadate) studies on actin tyrosine phosphorylation and actin polymerization. The inhibitors of tyrosine phosphorylation inhibit actin tyrosine phosphorylation and polymerization, whereas enhancers of tyrosine phosphorylation stimulate F-actin formation and tyrosine phosphorylation. These observations suggest that the tyrosine phosphorylation regulates the actin polymerization, and both are coupled processes during capacitation of buffalo spermatozoa.

  3. Leptin down-regulates insulin action through phosphorylation of serine-318 in insulin receptor substrate 1.

    PubMed

    Hennige, Anita M; Stefan, Norbert; Kapp, Katja; Lehmann, Rainer; Weigert, Cora; Beck, Alexander; Moeschel, Klaus; Mushack, Joanne; Schleicher, Erwin; Häring, Hans-Ulrich

    2006-06-01

    Insulin resistance in skeletal muscle is found in obesity and type 2 diabetes. A mechanism for impaired insulin signaling in peripheral tissues is the inhibition of insulin action through serine phosphorylation of insulin receptor substrate (Irs) proteins that abolish the coupling of Irs proteins to the activated insulin receptor. Recently, we described serine-318 as a protein kinase C (PKC)-dependent phosphorylation site in Irs1 (Ser-318) activated by hyperinsulinemia. Here we show in various cell models that the adipose hormone leptin, a putative mediator in obesity-related insulin resistance, promotes phosphorylation of Ser-318 in Irs1 by a janus kinase 2, Irs2, and PKC-dependent pathway. Mutation of Ser-318 to alanine abrogates the inhibitory effect of leptin on insulin-induced Irs1 tyrosine phosphorylation and glucose uptake in L6 myoblasts. In C57Bl/6 mice, Ser-318 phosphorylation levels in muscle tissue were enhanced by leptin and insulin administration in lean animals while in diet-induced obesity Ser-318 phosphorylation levels were already up-regulated in the basal state, and further stimulation was diminished. In analogy, in lymphocytes of obese hyperleptinemic human subjects basal Ser-318 phosphorylation levels were increased compared to lean individuals. During a hyperinsulinemic euglycemic clamp, the increment in Ser-318 phosphorylation observed in lean individuals was absent in obese. In summary, these data suggest that phosphorylation of Ser-318 in Irs1 mediates the inhibitory signal of leptin on the insulin-signaling cascade in obese subjects.

  4. Cell Cycle Regulated Phosphorylation of the Telomere-Associated Protein TIN2

    PubMed Central

    Yang, Shuqun; Counter, Christopher M.

    2013-01-01

    The protein TIN2 is a member of telomere-binding protein complex that serves to cap and protect mammalian chromosome ends. As a number of proteins in this complex are phosphorylated in a cell cycle-dependent manner, we investigated whether TIN2 is modified by phosphorylation as well. We performed phospho-proteomic analysis of human TIN2, and identified two phosphorylated residues, serines 295 and 330. We demonstrated that both these sites were phosphorylated during mitosis in human cells, as detected by Phos-tag reagent and phosphorylation-specific antibodies. Phosphorylation of serines 295 and 330 appeared to be mediated, at least in part, by the mitotic kinase RSK2. Specifically, phosphorylation of TIN2 at both these residues was increased upon expression of RSK2 and reduced by an inhibitor of the RSK family of kinases. Moreover, RSK2 phosphorylated TIN2 in vitro. The identification of these specifically timed post-translational events during the cell cycle suggests a potential mitotic regulation of TIN2 by phosphorylation. PMID:23977114

  5. Regulation of nicotinic acetylcholine receptor phosphorylation in rat myotubes by forskolin and cAMP

    SciTech Connect

    Miles, K.; Anthony, D.T.; Rubin, L.L.; Greengard, P.; Huganir, R.L.

    1987-09-01

    The nicotinic acetylcholine receptor (Ac-ChoR) from rat myotubes prelabeled in culture with (/sup 32/P)orthophosphate was isolated by acetylcholine affinity chromatography followed by immunoaffinity chromatography. Under basal conditions, the nicotinic AcChoR was shown to be phosphorylated in situ on the ..beta.. and delta subunits. Regulation of AcChoR phosphorylation by cAMP-dependent protein kinase was explored by the addition of forskolin or cAMP analogues to prelabeled cell cultures. Forskolin, an activator of adenylate cyclase, stimulated the phosphorylation of the delta subunit 20-fold over basal phosphorylation and induced phosphorylation of the ..cap alpha.. subunit. The effect of forskolin was dose dependent with a half-maximal response at 8 ..mu..M in the presence of 35 ..mu..M Ro 20-1724, a phosphodiesterase inhibitor. Stimulation of delta subunit phosphorylation was almost maximal within 5 min, whereas stimulation of ..cap alpha.. subunit phosphorylation was not maximal until 45 min after forskolin treatment. Stimulation of AcChoR phosphorylation by 8-benzylthioadenosine 3',5'-cyclic monophosphate was identical to that obtained by forskolin. Two-dimensional thermolytic phosphopeptide maps of the delta subunit revealed a single major phosphopeptide. These results correlate closely with the observed effects of forskolin on AcChoR desensitization in muscle and suggest that cAMP-dependent phosphorylation of the delta subunit increases the rate of AcChoR desensitization in rat myotubes.

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

    SciTech Connect

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

    2010-08-13

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

  7. Shear stress regulates HUVEC hydraulic conductivity by occludin phosphorylation.

    PubMed

    Pang, Zhengyu; Antonetti, David A; Tarbell, John M

    2005-11-01

    Human umbilical vein endothelial cells (HUVECs) display hydraulic conductivity (L(P)) responses to shear stress that differ markedly from the responses of bovine aortic endothelial cells (BAECs). In HUVECs, 5, 10, and 20 dyn cm(-2) steady shear stress transiently increased L(P) with a return to preshear baseline after a 2-h exposure to shear stress. Pure oscillatory shear stress of 0 +/- 20 dyn cm(-2) (mean+/-amplitude) had no effect on L(P), whereas superposition of oscillatory shear stress on steady shear stress suppressed the effect induced by steady shear stress alone. Shear reversal (amplitude greater than mean) was not necessary for the inhibitory influence of oscillatory shear stress. The transient increase of L(P) by steady shear stress was not affected by incubation with BAPTA-AM (10 microM), suggesting calcium independence of the shear response. Decreasing nitric oxide (NO) concentration with L-NMMA (100 microM), a nitric oxide synthase (NOS) inhibitor, did not inhibit the HUVEC L(P) response to shear stress. At the protein level, 10 dyn cm(-2) shear stress did not affect the total content of occludin, but it did elevate the phosphorylation level transiently. The positive correlation between occludin phosphorylation and hydraulic conductivity parallels observations in BAECs and suggests that occludin phosphorylation may be a general mediator of shear-L(P) responses in diverse endothelial cell types.

  8. Focal Adhesion Targeting: The Critical Determinant of FAK Regulation and Substrate Phosphorylation

    PubMed Central

    Shen, Yu; Schaller, Michael D.

    1999-01-01

    The focal adhesion kinase (FAK) is discretely localized to focal adhesions via its C-terminal focal adhesion–targeting (FAT) sequence. FAK is regulated by integrin-dependent cell adhesion and can regulate tyrosine phosphorylation of downstream substrates, like paxillin. By the use of a mutational strategy, the regions of FAK that are required for cell adhesion–dependent regulation and for inducing tyrosine phosphorylation of paxillin were determined. The results show that the FAT sequence was the single region of FAK that was required for each function. Furthermore, the FAT sequence of FAK was replaced with a focal adhesion–targeting sequence from vinculin, and the resulting chimera exhibited cell adhesion–dependent tyrosine phosphorylation and could induce paxillin phosphorylation like wild-type FAK. These results suggest that subcellular localization is the major determinant of FAK function. PMID:10436008

  9. Cdk5/p35 phosphorylates lemur tyrosine kinase-2 to regulate protein phosphatase-1C phosphorylation and activity.

    PubMed

    Manser, Catherine; Vagnoni, Alessio; Guillot, Florence; Davies, Jennifer; Miller, Christopher C J

    2012-05-01

    Cyclin-dependent kinase-5 (cdk5)/p35 and protein phosphatase-1 (PP1) are two major enzymes that control a variety of physiological processes within the nervous system including neuronal differentiation, synaptic plasticity and axonal transport. Defective cdk5/p35 and PP1 function are also implicated in several major human neurodegenerative diseases. Cdk5/p35 and the catalytic subunit of PP1 (PP1C) both bind to the brain-enriched, serine-threonine kinase lemur tyrosine kinase-2 (LMTK2). Moreover, LMTK2 phosphorylates PP1C on threonine-320 (PP1Cthr³²⁰) to inhibit its activity. Here, we demonstrate that LMTK2 is phosphorylated on serine-1418 (LMTK2ser¹⁴¹⁸) by cdk5/p35 and present evidence that this regulates its ability to phosphorylate PP1Cthr³²⁰. We thus describe a new signalling pathway within the nervous system that links cdk5/p35 with PP1C and which has implications for a number of neuronal functions and neuronal dysfunction.

  10. Evidence for regulation of mitotic progression through temporal phosphorylation and dephosphorylation of CK2alpha.

    PubMed

    St-Denis, Nicole A; Derksen, D Richard; Litchfield, David W

    2009-04-01

    Proper mitotic progression is crucial for maintenance of genomic integrity in proliferating cells and is regulated through an intricate series of events, including protein phosphorylation governed by a complex network of protein kinases. One kinase family implicated in the regulation of mitotic progression is protein kinase CK2, a small family of enzymes that is overexpressed in cancer and induces transformation in mice and cultured fibroblasts. CK2alpha, one isoform of the catalytic subunits of CK2, is maximally phosphorylated at four sites in nocodazole-treated cells. To investigate the effects of CK2alpha phosphorylation on mitotic progression, we generated phosphospecific antibodies against its mitotic phosphorylation sites. In U2OS cells released from S-phase arrest, these antibodies reveal that CK2alpha is most highly phosphorylated in prophase and metaphase. Phosphorylation gradually decreases during anaphase and becomes undetectable during telophase and cytokinesis. Stable expression of phosphomimetic CK2alpha (CK2alpha-4D, CK2alpha-4E) results in aberrant centrosome amplification and chromosomal segregation defects and loss of mitotic cells through mitotic catastrophe. Conversely, cells expressing nonphosphorylatable CK2alpha (CK2alpha-4A) show a decreased ability to arrest in mitosis following nocodazole treatment, suggesting involvement in the spindle assembly checkpoint. Collectively, these studies indicate that reversible phosphorylation of CK2alpha requires precise regulation to allow proper mitotic progression.

  11. A novel mode of regulation of the Staphylococcus aureus Vancomycin-resistance-associated response regulator VraR mediated by Stk1 protein phosphorylation.

    PubMed

    Canova, Marc J; Baronian, Grégory; Brelle, Solène; Cohen-Gonsaud, Martin; Bischoff, Markus; Molle, Virginie

    2014-04-25

    The Staphylococcus aureus Vancomycin-resistance-associated response regulator VraR is known as an important response regulator, member of the VraTSR three-component signal transduction system that modulates the expression of the cell wall stress stimulon in response to a number of different cell wall active antibiotics. Given its crucial role in regulating gene expression in response to antibiotic challenges, VraR must be tightly regulated. We report here for the first time in S. aureus convergence of two major signal transduction systems, serine/threonine protein kinase and two (three)-component systems. We demonstrate that VraR can be phosphorylated by the staphylococcal Ser/Thr protein kinase Stk1 and that phosphorylation negatively affects its DNA-binding properties. Mass spectrometric analyses and site-directed mutagenesis identified Thr106, Thr119, Thr175 and Thr178 as phosphoacceptors. A S. aureus ΔvraR mutant expressing a VraR derivative that mimics constitutive phosphorylation, VraR_Asp, still exhibited markedly decreased antibiotic resistance against different cell wall active antibiotics, when compared to the wild-type, suggesting that VraR phosphorylation may represent a novel and presumably more general mechanism of regulation of the two (three)-component systems in staphylococci.

  12. H3S10 phosphorylation-mediated transcriptional regulation by Aurora kinase A.

    PubMed

    Kim, Se-Ryeon; Kim, Kee-Beom; Chae, Yun-Cheol; Park, Jin Woo; Seo, Sang-Beom

    2016-01-01

    Histone H3S10 phosphorylation has been known as a cell cycle-specific marker and has a role in transcriptional activation. Various kinases phosphorylate H3S10 in different species, however, the role of the mitotic serine/threonine protein kinase Aurora A (AURKA) is largely unknown. Here we present evidence that AURKA phosphorylates H3S10 and activates target gene transcription. We show that down-regulation of AURKA level during leukemia cell differentiation results in decreased H3S10 phosphorylation level. We further show that AURKA is recruited to target gene promoters and activates transcription via H3S10 phosphorylation. Furthermore, this recruitment can be disrupted by the AURKA inhibitor Alisertib and results in H3K9-me2 recruitment by G9a.

  13. Localized α4 Integrin Phosphorylation Directs Shear Stress-Induced Endothelial Cell Alignment

    PubMed Central

    Goldfinger, Lawrence E.; Tzima, Eleni; Stockton, Rebecca; Kiosses, William B.; Kinbara, Kayoko; Tkachenko, Eugene; Gutierrez, Edgar; Groisman, Alex; Nguyen, Phu; Chien, Shu; Ginsberg1, Mark H.

    2009-01-01

    Vascular endothelial cells respond to laminar shear stress by aligning in the direction of flow, a process which may contribute to athero-protection. Here we report that localized α4 integrin phosphorylation is a mechanism for establishing the directionality of shear stress-induced alignment in microvascular endothelial cells. Within 5 minutes of exposure to a physiological level of shear stress, endothelial α4 integrins became phosphorylated on Ser988. In wounded monolayers, phosphorylation was enhanced at the downstream edges of cells relative to the source of flow. The shear-induced α4 integrin phosphorylation was blocked by inhibitors of cAMP-dependent protein kinase A (PKA), an enzyme involved in the alignment of endothelial cells under prolonged shear. Moreover, shear-induced localized activation of the small GTPase Rac1, which specifies the directionality of endothelial alignment, was similarly blocked by PKA inhibitors. Furthermore, endothelial cells bearing a non-phosphorylatable α4(S988A) mutation failed to align in response to shear stress, thus establishing α4 as a relevant PKA substrate. We thereby show that shear-induced PKA-dependent α4 integrin phosphorylation at the downstream edge of endothelial cells promotes localized Rac1 activation, which in turn directs cytoskeletal alignment in response to shear stress. PMID:18583710

  14. Cortactin phosphorylation regulates cell invasion through a pH-dependent pathway

    PubMed Central

    Magalhaes, Marco A. O.; Larson, Daniel R.; Mader, Christopher C.; Bravo-Cordero, Jose Javier; Gil-Henn, Hava; Oser, Matthew; Chen, Xiaoming; Koleske, Anthony J.

    2011-01-01

    Invadopodia are invasive protrusions with proteolytic activity uniquely found in tumor cells. Cortactin phosphorylation is a key step during invadopodia maturation, regulating Nck1 binding and cofilin activity. The precise mechanism of cortactin-dependent cofilin regulation and the roles of this pathway in invadopodia maturation and cell invasion are not fully understood. We provide evidence that cortactin–cofilin binding is regulated by local pH changes at invadopodia that are mediated by the sodium–hydrogen exchanger NHE1. Furthermore, cortactin tyrosine phosphorylation mediates the recruitment of NHE1 to the invadopodium compartment, where it locally increases the pH to cause the release of cofilin from cortactin. We show that this mechanism involving cortactin phosphorylation, local pH increase, and cofilin activation regulates the dynamic cycles of invadopodium protrusion and retraction and is essential for cell invasion in 3D. Together, these findings identify a novel pH-dependent regulation of cell invasion. PMID:22105349

  15. Prostate Cell Specific Regulation of Androgen Receptor Phosphorylation in Vivo

    DTIC Science & Technology

    2009-11-01

    Gene Anal Tech 1988; 5: 22. 7. Schott S, Coustham V, Simonet T, Bedet C and Palladino F: Unique and redundant functions of C . elegans HP1 proteins in... C ., Hittelman, A., Rogatsky, I., Logan, S.K., and Garabedian, M.J. Glucocorticoid receptor phosphorylation differentially affects target gene ...Endocr Relat Cancer 2002;9:61–73. 5. Abate-Shen C , Shen MM. Molecular genetics of prostate cancer. Genes Dev 2000;14:2410–34. 6. Shang Y, Myers M

  16. Lens fiber connexin turnover and caspase-3-mediated cleavage are regulated alternately by phosphorylation.

    PubMed

    Yin, Xinye; Liu, Jialu; Jiang, Jean X

    2008-05-01

    Lens connexins are phosphorylated in vivo; however, the function and regulation of the phosphorylation remain largely unknown. We have previously identified an in vivo phosphorylation site, Ser(364), at the COOH terminus of lens connexin (Cx) Cx45.6 and phosphorylation appears to regulate connexin protein turnover. To assess the specific mechanism of Ser(364) phosphorylation in Cx45.6, exogenous wild type and Ser(364) mutant Cx45.6 were expressed in primary lens cultures through retroviral infection. Cx45.6 turnover was attenuated primarily by proteasomal inhibitors and to a lesser extent by lysosomal inhibitors. Furthermore, the level of Cx45.6 protein in ubiquitin co-expressed cells was significantly reduced as compared to the cells expressing Cx45.6 alone. Moreover, overexpression of ubiquitin led to a more significant decrease in wild type Cx45.6 than Cx45.6(S364A), a mutant deficient of phosphorylation site at Ser(364), although we did not detect any difference in the levels of ubiquitination between wild type and mutant Cx45.6. Interestingly, the mutant mimicking constitutive phosphorylation, Cx45.6(S364D), partially prevented the cleavage of Cx45.6 by caspase-3. Together, our data suggest that phosphorylation of Cx45.6 at Ser(364) appears to stimulate Cx45.6 turnover primarily through proteasome pathway and this phosphorylation inhibits the cleavage of Cx45.6 by caspase-3. These findings provide further insights into regulatory mechanism of the specific phosphorylation of connexins in the lens.

  17. Regulation of gap junction conductance by calcineurin through Cx43 phosphorylation: implications for action potential conduction.

    PubMed

    Jabr, Rita I; Hatch, Fiona S; Salvage, Samantha C; Orlowski, Alejandro; Lampe, Paul D; Fry, Christopher H

    2016-11-01

    Cardiac arrhythmias are associated with raised intracellular [Ca(2+)] and slowed action potential conduction caused by reduced gap junction (GJ) electrical conductance (Gj). Ventricular GJs are composed of connexin proteins (Cx43), with Gj determined by Cx43 phosphorylation status. Connexin phosphorylation is an interplay between protein kinases and phosphatases but the precise pathways are unknown. We aimed to identify key Ca(2+)-dependent phosphorylation sites on Cx43 that regulate cardiac gap junction conductance and action potential conduction velocity. We investigated the role of the Ca(2+)-dependent phosphatase, calcineurin. Intracellular [Ca(2+)] was raised in guinea-pig myocardium by a low-Na solution or increased stimulation. Conduction velocity and Gj were measured in multicellular strips. Phosphorylation of Cx43 serine residues (S365 and S368) and of the intermediary regulator I1 at threonine35 was measured by Western blot. Measurements were made in the presence and absence of inhibitors to calcineurin, I1 or protein phosphatase-1 and phosphatase-2.Raised [Ca(2)(+)]i decreased Gj, reduced Cx43 phosphorylation at S365 and increased it at S368; these changes were reversed by calcineurin inhibitors. Cx43-S368 phosphorylation was reversed by the protein kinase C inhibitor chelerythrine. Raised [Ca(2+)]i also decreased I1 phosphorylation, also prevented by calcineurin inhibitors, to increase activity of the Ca(2+)-independent phosphatase, PPI. The PP1 inhibitor, tautomycin, prevented Cx43-365 dephosphorylation, Cx43-S368 phosphorylation and Gj reduction in raised [Ca(2+)]i. PP2A had no role. Conduction velocity was reduced by raised [Ca(2+)]i and reversed by calcineurin inhibitors. Reduced action potential conduction and Gj in raised [Ca(2+)] are regulated by calcineurin-dependent Cx43-S365 phosphorylation, leading to Cx43-S368 dephosphorylation. The calcineurin action is indirect, via I1 dephosphorylation and subsequent activation of PP1.

  18. B-Cyclin/CDKs Regulate Mitotic Spindle Assembly by Phosphorylating Kinesins-5 in Budding Yeast

    PubMed Central

    Chee, Mark K.; Haase, Steven B.

    2010-01-01

    Although it has been known for many years that B-cyclin/CDK complexes regulate the assembly of the mitotic spindle and entry into mitosis, the full complement of relevant CDK targets has not been identified. It has previously been shown in a variety of model systems that B-type cyclin/CDK complexes, kinesin-5 motors, and the SCFCdc4 ubiquitin ligase are required for the separation of spindle poles and assembly of a bipolar spindle. It has been suggested that, in budding yeast, B-type cyclin/CDK (Clb/Cdc28) complexes promote spindle pole separation by inhibiting the degradation of the kinesins-5 Kip1 and Cin8 by the anaphase-promoting complex (APCCdh1). We have determined, however, that the Kip1 and Cin8 proteins are present at wild-type levels in the absence of Clb/Cdc28 kinase activity. Here, we show that Kip1 and Cin8 are in vitro targets of Clb2/Cdc28 and that the mutation of conserved CDK phosphorylation sites on Kip1 inhibits spindle pole separation without affecting the protein's in vivo localization or abundance. Mass spectrometry analysis confirms that two CDK sites in the tail domain of Kip1 are phosphorylated in vivo. In addition, we have determined that Sic1, a Clb/Cdc28-specific inhibitor, is the SCFCdc4 target that inhibits spindle pole separation in cells lacking functional Cdc4. Based on these findings, we propose that Clb/Cdc28 drives spindle pole separation by direct phosphorylation of kinesin-5 motors. PMID:20463882

  19. Nuclear import of the Drosophila Rel protein Dorsal is regulated by phosphorylation.

    PubMed

    Drier, E A; Huang, L H; Steward, R

    1999-03-01

    In Drosophila, dorsal-ventral polarity is determined by a maternally encoded signal transduction pathway that culminates in the graded nuclear localization of the Rel protein, Dorsal. Dorsal is retained in the cytoplasm by the IkappaB protein, Cactus. Signal-dependent phosphorylation of Cactus results in the degradation of Cactus and the nuclear targeting of Dorsal. We present an in-depth study of the functional importance of Dorsal phosphorylation. We find that Dorsal is phosphorylated by the ventral signal while associated with Cactus, and that Dorsal phosphorylation is essential for its nuclear import. In vivo phospholabeling of Dorsal is limited to serine residues in both ovaries and early embryos. A protein bearing mutations in six conserved serines abolishes Dorsal activity, is constitutively cytoplasmic, and appears to eliminate Dorsal phosphorylation, but still interacts with Cactus. Two individual serine-to-alanine mutations produce unexpected results. In a wild-type signaling background, a mutation in the highly conserved PKA site (S312) produces only a weak loss-of-function; however, it completely destabilizes the protein in a cactus mutant background. Significantly, the phosphorylation of another completely conserved serine (S317) regulates the high level of nuclear import found in ventral cells. We conclude that the formation of a wild-type Dorsal nuclear gradient requires the phosphorylation of both Cactus and Dorsal. The strong conservation of the serines suggests that phosphorylation of other Rel proteins is essential for their proper nuclear targeting.

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

    PubMed Central

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

    2008-01-01

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

  1. Phosphorylation of filamin A regulates chemokine receptor CCR2 recycling.

    PubMed

    Pons, Mònica; Izquierdo, Ismael; Andreu-Carbó, Mireia; Garrido, Georgina; Planagumà, Jesús; Muriel, Olivia; Del Pozo, Miguel A; Geli, M Isabel; Aragay, Anna M

    2017-01-15

    Proper endosomal trafficking of ligand-activated G-protein-coupled receptors (GPCRs) is essential to spatiotemporally tune their physiological responses. For the monocyte chemoattractant receptor 2 (CCR2B; one of two isoforms encoded by CCR2), endocytic recycling is important to sustain monocyte migration, whereas filamin A (FLNa) is essential for CCL2-induced monocyte migration. Here, we analyze the role of FLNa in the trafficking of CCR2B along the endocytic pathway. In FLNa-knockdown cells, activated CCR2B accumulated in enlarged EEA-1-positive endosomes, which exhibited slow movement and fast fluorescence recovery, suggesting an imbalance between receptor entry and exit rates. Utilizing super-resolution microscopy, we observed that FLNa-GFP, CCR2B and β2-adrenergic receptor (β2AR) were present in actin-enriched endosomal microdomains. Depletion of FLNa decreased CCR2B association with these microdomains and concomitantly delayed CCR2B endosomal traffic, without apparently affecting the number of microdomains. Interestingly, CCR2B and β2AR signaling induced phosphorylation of FLNa at residue S2152, and this phosphorylation event was contributes to sustain receptor recycling. Thus, our data strongly suggest that CCR2B and β2AR signals to FLNa to stimulate its endocytosis and recycling to the plasma membrane.

  2. Protein kinase D regulates positive selection of CD4+ thymocytes through phosphorylation of SHP-1

    PubMed Central

    Ishikawa, Eri; Kosako, Hidetaka; Yasuda, Tomoharu; Ohmuraya, Masaki; Araki, Kimi; Kurosaki, Tomohiro; Saito, Takashi; Yamasaki, Sho

    2016-01-01

    Thymic selection shapes an appropriate T cell antigen receptor (TCR) repertoire during T cell development. Here, we show that a serine/threonine kinase, protein kinase D (PKD), is crucial for thymocyte positive selection. In T cell-specific PKD-deficient (PKD2/PKD3 double-deficient) mice, the generation of CD4 single positive thymocytes is abrogated. This defect is likely caused by attenuated TCR signalling during positive selection and incomplete CD4 lineage specification in PKD-deficient thymocytes; however, TCR-proximal tyrosine phosphorylation is not affected. PKD is activated in CD4+CD8+ double positive (DP) thymocytes on stimulation with positively selecting peptides. By phosphoproteomic analysis, we identify SH2-containing protein tyrosine phosphatase-1 (SHP-1) as a direct substrate of PKD. Substitution of wild-type SHP-1 by phosphorylation-defective mutant (SHP-1S557A) impairs generation of CD4+ thymocytes. These results suggest that the PKD–SHP-1 axis positively regulates TCR signalling to promote CD4+ T cell development. PMID:27670070

  3. Cell cycle regulated phosphorylation of RPA-32 occurs within the replication initiation complex.

    PubMed Central

    Fotedar, R; Roberts, J M

    1992-01-01

    The transition from G1 to S phase of the cell cycle may be regulated by modification of proteins which are essential for initiating DNA replication. One of the first events during initiation is to unwind the origin DNA and this requires a single-stranded DNA binding protein. RPA, a highly conserved multi-subunit single-stranded DNA binding protein, was first identified as a cellular protein necessary for the initiation of SV40 DNA replication. The 32 kDa subunit of RPA has been shown to be phosphorylated at the start of S phase. Using SV40 replication as a model, we have reproduced in vitro the S phase-dependent phosphorylation of RPA-32 and show that it occurs specifically within the replication initiation complex. Phosphorylated RPA-32 is predominantly associated with DNA. Phosphorylation is not a pre-requisite for association with DNA, but occurs after RPA binds to single-stranded DNA formed at the origin during the initiation phase. The protein kinase(s) which phosphorylates RPA-32 is present at all stages of the cell cycle but RPA-32 does not bind to the SV40 origin or become phosphorylated in extracts from G1 cells. Therefore, the cell cycle-dependent phosphorylation of RPA-32 may be regulated by its binding to single-stranded origin DNA during replication initiation. Images PMID:1318194

  4. Ribosomal Protein S6 Phosphorylation in the Nervous System: From Regulation to Function

    PubMed Central

    Biever, Anne; Valjent, Emmanuel; Puighermanal, Emma

    2015-01-01

    Since the discovery of the phosphorylation of the 40S ribosomal protein S6 (rpS6) about four decades ago, much effort has been made to uncover the molecular mechanisms underlying the regulation of this post-translational modification. In the field of neuroscience, rpS6 phosphorylation is commonly used as a readout of the mammalian target of rapamycin complex 1 signaling activation or as a marker for neuronal activity. Nevertheless, its biological role in neurons still remains puzzling. Here we review the pharmacological and physiological stimuli regulating this modification in the nervous system as well as the pathways that transduce these signals into rpS6 phosphorylation. Altered rpS6 phosphorylation observed in various genetic and pathophysiological mouse models is also discussed. Finally, we examine the current state of knowledge on the physiological role of this post-translational modification and highlight the questions that remain to be addressed. PMID:26733799

  5. Regulation of RNA polymerase II activity by CTD phosphorylation and cell cycle control.

    PubMed

    Oelgeschläger, Thomas

    2002-02-01

    The carboxyl-terminal domain (CTD) of the largest subunit of mammalian RNA polymerase II (RNAP II) consists of 52 repeats of a consensus heptapeptide and is subject to phosphorylation and dephosphorylation events during each round of transcription. RNAP II activity is regulated during the cell cycle and cell cycle-dependend changes in RNAP II activity correlate well with CTD phosphorylation. In addition, global changes in the CTD phosphorylation status are observed in response to mitogenic or cytostatic signals such as growth factors, mitogens and DNA-damaging agents. Several CTD kinases are members of the cyclin-dependent kinase (CDK) superfamily and associate with transcription initiation complexes. Other CTD kinases implicated in cell cycle regulation include the mitogen-activated protein kinases ERK-1/2 and the c-Abl tyrosine kinase. These observations suggest that reversible RNAP II CTD phosphorylation may play a key role in linking cell cycle regulatory events to coordinated changes in transcription.

  6. P-TEFb Kinase Complex Phosphorylates Histone H1 to Regulate Expression of Cellular and HIV-1 Genes*

    PubMed Central

    O'Brien, Siobhan K.; Cao, Hong; Nathans, Robin; Ali, Akbar; Rana, Tariq M.

    2010-01-01

    Transcription of HIV-1 genes depends on the RNA polymerase II kinase and elongation factor positive transcription elongation factor b (P-TEFb), the complex of cyclin T1 and CDK9. Recent evidence suggests that regulation of transcription by P-TEFb involves chromatin binding and modifying factors. To determine how P-TEFb may connect chromatin remodeling to transcription, we investigated the relationship between P-TEFb and histone H1. We identify histone H1 as a substrate for P-TEFb involved in cellular and HIV-1 transcription. We show that P-TEFb interacts with H1 and that P-TEFb inhibition by RNAi, flavopiridol, or dominant negative CDK9 expression correlates with loss of phosphorylation and mobility of H1 in vivo. Importantly, P-TEFb directs H1 phosphorylation in response to wild-type HIV-1 infection, but not Tat-mutant HIV-1 infection. Our results show that P-TEFb phosphorylates histone H1 at a specific C-terminal phosphorylation site. Expression of a mutant H1.1 that cannot be phosphorylated by P-TEFb also disrupts Tat transactivation in an HIV reporter cell line as well as transcription of the c-fos and hsp70 genes in HeLa cells. We identify histone H1 as a novel P-TEFb substrate, and our results suggest new roles for P-TEFb in both cellular and HIV-1 transcription. PMID:20551309

  7. Negative regulation of RelA phosphorylation: emerging players and their roles in cancer.

    PubMed

    Lu, Xinyuan; Yarbrough, Wendell G

    2015-02-01

    NF-κB signaling contributes to human disease processes, notably inflammatory diseases and cancer. Many advances have been made in understanding mechanisms responsible for abnormal NF-κB activation with RelA post-translational modification, particularly phosphorylation, proven to be critical for RelA function. While the majority of studies have focused on identifying kinases responsible for NF-κB phosphorylation and pathway activation, recently progress has also been made in understanding the negative regulators important for restraining RelA activity. Here we summarize negative regulators of RelA phosphorylation, their targeting sites in RelA and biological functions through negative regulation of RelA activation. Finally, we emphasize the tumor suppressor-like roles that these negative regulators can assume in human cancers.

  8. Tyrosine phosphorylation of type Igamma phosphatidylinositol phosphate kinase by Src regulates an integrin-talin switch.

    PubMed

    Ling, Kun; Doughman, Renee L; Iyer, Vidhya V; Firestone, Ari J; Bairstow, Shawn F; Mosher, Deane F; Schaller, Michael D; Anderson, Richard A

    2003-12-22

    Engagement of integrin receptors with the extracellular matrix induces the formation of focal adhesions (FAs). Dynamic regulation of FAs is necessary for cells to polarize and migrate. Key interactions between FA scaffolding and signaling proteins are dependent on tyrosine phosphorylation. However, the precise role of tyrosine phosphorylation in FA development and maturation is poorly defined. Here, we show that phosphorylation of type Igamma phosphatidylinositol phosphate kinase (PIPKIgamma661) on tyrosine 644 (Y644) is critical for its interaction with talin, and consequently, localization to FAs. PIPKIgamma661 is specifically phosphorylated on Y644 by Src. Phosphorylation is regulated by focal adhesion kinase, which enhances the association between PIPKIgamma661 and Src. The phosphorylation of Y644 results in an approximately 15-fold increase in binding affinity to the talin head domain and blocks beta-integrin binding to talin. This defines a novel phosphotyrosine-binding site on the talin F3 domain and a "molecular switch" for talin binding between PIPKIgamma661 and beta-integrin that may regulate dynamic FA turnover.

  9. ERK/MAPK Regulates Hippocampal Histone Phosphorylation Following Contextual Fear Conditioning

    ERIC Educational Resources Information Center

    Levenson, Jonathan M.; Sweatt, J. David; Chwang, Wilson B.; O'Riordan, Kenneth J.

    2006-01-01

    Long-term memory formation is regulated by many distinct molecular mechanisms that control gene expression. An emerging model for effecting a stable, coordinated pattern of gene transcription involves epigenetic tagging through modifications of histones or DNA. In this study, we investigated the regulation of histone phosphorylation in the…

  10. Regulation of Auxin Transport by Phosphorylation and Flavonoids during Gravitropism in Arabidopsis

    NASA Technical Reports Server (NTRS)

    Muday, Gloria K.

    2005-01-01

    The focus of this research includes: 1) Regulation of Axin transport by flavonoids during gravitropism; 2) Phosphorylation control of auxin transport during gravity response; 3) Ethylene regulation of gravitropic curvature; 4) IBA transport and gravitropic response; and 5) Other collaborative projects.

  11. Akt-dependent Girdin phosphorylation regulates repair processes after acute myocardial infarction.

    PubMed

    Hayano, Shinji; Takefuji, Mikito; Maeda, Kengo; Noda, Tomonori; Ichimiya, Hitoshi; Kobayashi, Koichi; Enomoto, Atsushi; Asai, Naoya; Takahashi, Masahide; Murohara, Toyoaki

    2015-11-01

    Myocardial infarction is a leading cause of death, and cardiac rupture following myocardial infarction leads to extremely poor prognostic feature. A large body of evidence suggests that Akt is involved in several cardiac diseases. We previously reported that Akt-mediated Girdin phosphorylation is essential for angiogenesis and neointima formation. The role of Girdin expression and phosphorylation in myocardial infarction, however, is not understood. Therefore, we employed Girdin-deficient mice and Girdin S1416A knock-in (Girdin(SA/SA)) mice, replacing the Akt phosphorylation site with alanine, to address this question. We found that Girdin was expressed and phosphorylated in cardiac fibroblasts in vitro and that its phosphorylation was crucial for the proliferation and migration of cardiac fibroblasts. In vivo, Girdin was localized in non-cardiomyocyte interstitial cells and phosphorylated in α-smooth muscle actin-positive cells, which are likely to be cardiac myofibroblasts. In an acute myocardial infarction model, Girdin(SA/SA) suppressed the accumulation and proliferation of cardiac myofibroblasts in the infarcted area. Furthermore, lower collagen deposition in Girdin(SA/SA) mice impaired cardiac repair and resulted in increased mortality attributed to cardiac rupture. These findings suggest an important role of Girdin phosphorylation at serine 1416 in cardiac repair after acute myocardial infarction and provide insights into the complex mechanism of cardiac rupture through the Akt/Girdin-mediated regulation of cardiac myofibroblasts.

  12. 50 years of biological research--from oxidative phosphorylation to energy requiring transport regulation.

    PubMed

    Kalckar, H M

    1991-01-01

    In 1930 adenosine triphosphate appeared in the literature from W. A. Engelhardt's work on avian erythrocytes. This was an early example of oxidative phosphorylation in intact cells, and it required methylene blue and oxygen. Both Belitser and I realized that the use of Warburg manometers for aeration was critical in order to generate oxidative phosphorylation of glucose in tissue preparations. Test tube techniques did not work. In 1956 we were able to describe a human type of diabetes called "galactose diabetes," in which consumption of human or cows' milk provokes mental retardation. Replacement of human or cows' milk products with "vegetable milk" formula in early infancy can prevent retardation. We determined that the disease results from a defect of galactose-one-phosphate uridylyl-transferase, a hereditary enzyme. This type of enzyme defect, if discovered and treated in early infancy, is a benign molecular disease. Regulation of transport systems in mammalian cell cultures are frequently complex energized systems. Perhaps my greatest surprise in this regard was the mere fact that an all-cis "odd" hexose-D-allose turned out to be a highly intense down-regulator of the hexose transport system. Additions of inhibitors of oxidative phosphorylation (such as oligomycin or di-nitrophenol) arrested the allose-mediated down-regulation. We have reason to suspect that the strong down-regulator is a phosphorylated form of D-allose. Thus ends my story about oxidative energized biological phosphorylation systems.

  13. Transcriptional regulation by post-transcriptional modification--role of phosphorylation in Sp1 transcriptional activity.

    PubMed

    Chu, Shijian

    2012-10-15

    Sp1 is a ubiquitously expressed transcription factor involved in the regulation of a large number of genes including housekeeping genes as well as actively regulated genes. Although Sp1 was discovered nearly three decades ago, its functional diversity is still not completely understood. One of the ways that make Sp1 versatile in transcriptional regulation is its post-transcriptional modification, which alters Sp1 structure in different cells and at different times. Compared to other types of modifications of the Sp1 protein, phosphorylation has been studied far more extensively. This review focuses on the inducers, pathways, enzymes, and biological effects of Sp1 phosphorylation. Recent data are beginning to reveal the biological significance and universal presence of Sp1 phosphorylation-related cell/molecular responses. Studies in this field provide a quick glance at how a simple chemical modification of a transcription factor could produce significant functional diversity of the protein.

  14. Nik-related kinase regulates trophoblast proliferation and placental development by modulating AKT phosphorylation.

    PubMed

    Morioka, Yuka; Nam, Jin-Min; Ohashi, Takashi

    2017-01-01

    Nik-related kinase (Nrk) is a Ser/Thr kinase and was initially discovered as a molecule that was predominantly detected in skeletal muscles during development. A recent study using Nrk-null mice suggested the importance of Nrk in proper placental development; however, the molecular mechanism remains unknown. In this study, we demonstrated that differentiated trophoblasts from murine embryonic stem cells (ESCs) endogenously expressed Nrk and that Nrk disruption led to the enhanced proliferation of differentiated trophoblasts. This phenomenon may reflect the overproliferation of trophoblasts that has been reported in enlarged placentas of Nrk-null mice. Furthermore, we demonstrated that AKT phosphorylation at Ser473 was upregulated in Nrk-null trophoblasts and that inhibition of AKT phosphorylation cancelled the enhanced proliferation observed in differentiated Nrk-null trophoblasts. These results indicated that the upregulation of AKT phosphorylation was the possible cause of enhanced proliferation observed in Nrk-null trophoblasts. The upregulation of AKT phosphorylation was also confirmed in enlarged Nrk-null placentas in vivo, suggesting that proper regulation of AKT by Nrk was important for normal placental development. In addition, our detailed analysis on phosphorylation status of AKT isoforms in newly established trophoblast stem cells (TSCs) revealed that different levels of upregulation of AKT phosphorylation were occurred in Nrk-null TSCs depending on AKT isoforms. These results further support the importance of Nrk in proper development of trophoblast lineage cells and indicate the possible application of TSCs for the analysis of differently regulated activation mechanisms of AKT isoforms.

  15. Adenosine and dopamine receptors co-regulate photoreceptor coupling via gap junction phosphorylation in mouse retina

    PubMed Central

    Li, Hongyan; Zhang, Zhijing; Blackburn, Michael R.; Wang, Steven W.; Ribelayga, Christophe P.; O’Brien, John

    2013-01-01

    Gap junctions in retinal photoreceptors suppress voltage noise and facilitate input of rod signals into the cone pathway during mesopic vision. These synapses are highly plastic and regulated by light and circadian clocks. Recent studies have revealed an important role for connexin36 (Cx36) phosphorylation by protein kinase A (PKA) in regulating cell-cell coupling. Dopamine is a light-adaptive signal in the retina, causing uncoupling of photoreceptors via D4 receptors (D4R), which inhibits adenylyl cyclase (AC) and reduces PKA activity. We hypothesized that adenosine, with its extracellular levels increasing in darkness, may serve as a dark signal to co-regulate photoreceptor coupling through modulation of gap junction phosphorylation. Both D4R and A2a receptor (A2aR) mRNAs were present in photoreceptors, inner nuclear layer neurons, and ganglion cells in C57BL/6 mouse retina, and showed cyclic expression with partially overlapping rhythms. Pharmacologically activating A2aR or inhibiting D4R in light-adapted daytime retina increased photoreceptor coupling. Cx36 among photoreceptor terminals, representing predominantly rod-cone gap junctions but possibly including some rod-rod and cone-cone gap junctions, was phosphorylated in a PKA-dependent manner by the same treatments. Conversely, inhibiting A2aR or activating D4R in daytime dark-adapted retina decreased Cx36 phosphorylation with similar PKA dependence. A2a-deficient mouse retina showed defective regulation of photoreceptor gap junction phosphorylation, fairly regular dopamine release, and moderately down-regulated expression of D4R and AC type I mRNA. We conclude that adenosine and dopamine co-regulate photoreceptor coupling through opposite action on the PKA pathway and Cx36 phosphorylation. In addition, loss of the A2aR hampered D4R gene expression and function. PMID:23407968

  16. Binding of AP-2 adaptor complex to brain membrane is regulated by phosphorylation of proteins

    SciTech Connect

    Alberdi, A. . E-mail: aalberdi@fcm.uncu.edu.ar; Sartor, T.; Sosa, M.A.

    2005-05-13

    Phosphorylation of proteins appears as a key process in early steps of clathrin coated vesicle formation. Here, we report that treatment of post-nuclear fraction with alkaline phosphatase induced redistribution of {alpha} subunits of AP-2 adaptor complex to cytosol and this effect was higher in the {alpha}2 subunit. A high serine phosphorylation status of {alpha} subunits correlated with the higher affinity of AP-2 to membranes. Using a simple binding assay, where membranes were incubated with either purified adaptors or cytosols, we observed an inhibitory effect of tyrphostin, a tyrosine kinase inhibitor, on the binding of AP-2 to membranes, but also an unexpected decrease induced by the phosphatase inhibitor cyclosporine. We also show an inhibitory effect of ATP mediated by cytosolic proteins, although it could not be related to the phosphorylation of AP-2, suggesting an action upstream a cascade of phosphorylations that participate in the regulation of the assembly of AP-2 to membranes.

  17. Histone H3 phosphorylation in the rat pineal gland: adrenergic regulation and diurnal variation.

    PubMed

    Chik, C L; Arnason, T G; Dukewich, W G; Price, D M; Ranger, A; Ho, A K

    2007-04-01

    In this study, we investigated phosphorylation of Ser10 in histone H3 by norepinephrine (NE) in the rat pineal gland. In whole-animal studies, we demonstrated a marked increase in histone H3 phosphorylation in the rat pineal gland during the first half of the dark period. Exposure to light during this period caused a rapid decline in histone H3 phosphorylation with an estimated t1/2 of less than 15 min, indicating a high level of dephosphorylation activity. Corresponding studies in cultured pineal cells revealed that treatment with NE produced an increase in histone H3 phosphorylation that peaked between 2 and 3 h and declined rapidly by 4 h. The NE-induced histone H3 phosphorylation was blocked by cotreatment with propranolol or KT5720, a protein kinase A inhibitor, but not by prazosin or other kinase inhibitors. Moreover, only treatment with dibutyryl cAMP but not other kinase activators mimicked the effect of NE on histone H3 phosphorylation. The NE-stimulated H3 phosphorylation was markedly increased by cotreatment with a serine/threonine phosphatase inhibitor, tautomycin or okadaic acid, supporting a high level of ongoing histone H3 dephosphorylation activity. Together, our results indicate that histone H3 phosphorylation is a naturally occurring event at night in the rat pineal gland that is driven almost exclusively by a NE-->beta-adrenergic-->cAMP/protein kinase A signaling mechanism. This transient histone H3 phosphorylation probably reflects the nocturnal activation of multiple adrenergic-regulated genes in the rat pineal gland.

  18. Rictor regulates phosphorylation of the novel protein kinase C Apl II in Aplysia sensory neurons.

    PubMed

    Labban, Margaret; Dyer, John R; Sossin, Wayne S

    2012-09-01

    Rapamycin-insensitive companion of TOR (Rictor) is a conserved component of target of rapamycin complex 2 (TORC2), a complex implicated in phosphorylation of a number of signal transduction-related kinases, including protein kinase Cs (PKCs) at their 'hydrophobic' site in the carboxy-terminal extension domain. In the marine mollusk, Aplysia californica, an increase in phosphorylation of the novel PKC, Apl II, at the hydrophobic site is associated with a protein synthesis-dependent increase in synaptic strength seen after continuous application of serotonin. To determine if Rictor plays a role in this increase, we cloned the Aplysia ortholog of Rictor (ApRictor). An siRNA-mediated decrease in ApRictor levels in Aplysia sensory neurons led to a decrease in the phosphorylation of PKC Apl II at the hydrophobic site suggesting a role for ApRictor in hydrophobic site phosphorylation. However, over-expression of ApRictor was not sufficient to increase phosphorylation of PKC Apl II. Continuous application of serotonin increased phosphorylation of PKC Apl II at the hydrophobic site in cultured sensory neurons, and this was blocked by Torin, which inhibits both TORC1 and TORC2. Over-expression of ApRictor did not lead to change in the magnitude of serotonin-mediated phosphorylation, but did lead to a small increase in the membrane localization of phosphorylated PKC Apl II. In conclusion, these studies implicate Rictor in phosphorylation of a novel PKC during synaptic plasticity and suggest an additional role for Rictor in regulating the localization of PKCs.

  19. Regulation of NHE3 by lysophosphatidic acid is mediated by phosphorylation of NHE3 by RSK2

    PubMed Central

    No, Yi Ran; He, Peijian; Yoo, Byong Kwon

    2015-01-01

    Na+/H+ exchange by Na+/H+ exchanger 3 (NHE3) is a major route of sodium absorption in the intestine and kidney. We have shown previously that lysophosphatidic acid (LPA), a small phospholipid produced ubiquitously by all types of cells, stimulates NHE3 via LPA5 receptor. Stimulation of NHE3 activity by LPA involves LPA5 transactivating EGF receptor (EGFR) in the apical membrane. EGFR activates proline-rich tyrosine kinase 2 (Pyk2) and ERK, both of which are necessary for NHE3 regulation. However, Pyk2 and ERK are regulated by EGFR via independent pathways and appear to converge on an unidentified intermediate that ultimately targets NHE3. The p90 ribosomal S6 kinase (RSK) family of Ser/Thr protein kinases is a known effector of EGFR and ERK. Hence, we hypothesized that RSK may be the convergent effector of Pyk2 and ERK although it is not known whether Pyk2 regulates RSK. In this study, we show that Pyk2 is necessary for the maintenance of phosphoinositide-dependent kinase 1 (PDK1) autophosphorylation, and knockdown of Pyk2 or PDK1 mitigated LPA-induced phosphorylation of RSK and stimulation of NHE3 activity. Additionally, we show that RSK2, but not RSK1, is responsible for NHE3 regulation. RSK2 interacts with NHE3 at the apical membrane domain, where it phosphorylates NHE3. Alteration of S663 of NHE3 ablated LPA-induced phosphorylation of NHE3 and stimulation of the transport activity. Our study identifies RSK2 as a new kinase that regulates NHE3 activity by direct phosphorylation. PMID:25855080

  20. Characterization of four plasma membrane aquaporins in tulip petals: a putative homolog is regulated by phosphorylation.

    PubMed

    Azad, Abul Kalam; Katsuhara, Maki; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

    2008-08-01

    We suggested previously that temperature-dependent tulip (Tulipa gesneriana) petal movement that is concomitant with water transport is regulated by reversible phosphorylation of an unidentified plasma membrane intrinsic protein (PIP). In this study, four full-length cDNAs of PIPs from tulip petals were identified and cloned. Two PIPs, namely TgPIP1;1 and TgPIP1;2, are members of the PIP1 subfamily, and the remaining two PIPs, namely TgPIP2;1 and TgPIP2;2, belong to the PIP2 subfamily of aquaporins and were named according to the nomenclature of PIP genes in plants. Of these four homologs, only TgPIP2;2 displayed significant water channel activity in the heterologous expression assay using Xenopus laevis oocytes. The water channel activity of this functional isoform was abolished by mercury and was affected by inhibitors of protein kinase and protein phosphatase. Using a site-directed mutagenesis approach to substitute several serine residues with alanine, and assessing water channel activity using the methylotrophic yeast Pichia pastoris expression assay, we showed that Ser35, Ser116 and Ser274 are the putative phosphorylation sites of TgPIP2;2. Real-time reverse transcription-PCR analysis revealed that the transcript levels of TgPIP1;1 and TgPIP1;2 in tulip petals, stems, leaves, bulbs and roots are very low when compared with those of TgPIP2;1 and TgPIP2;2. The transcript level of TgPIP2;1 is negligible in roots, and TgPIP2;2 is ubiquitously expressed in all organs with significant transcript levels. From the data reported herein, we suggest that TgPIP2;2 might be modulated by phosphorylation and dephosphorylation for regulating water channel activity, and may play a role in transcellular water transport in all tulip organs.

  1. CaMKII-γ mediates phosphorylation of BAD at Ser170 to regulate cytokine-dependent survival and proliferation.

    PubMed

    Hojabrpour, Payman; Waissbluth, Ivan; Ghaffari, Mazyar; Cox, Michael E; Duronio, Vincent

    2012-02-15

    Phosphorylation of the BH3 (Bcl-2 homology domain 3)-only protein BAD (Bcl-2/Bcl-X(L)-antagonist, causing cell death) can either directly disrupt its association with the pro-survival proteins Bcl-X(L) and/or Bcl-2, or cause association of BAD with 14-3-3 proteins. In the present study, we further characterize phosphorylation of BAD at Ser170, a unique site with unclear function. We provide further evidence that mutation of Ser170 to a phospho-mimetic aspartic acid residue (S170D) can have a profound inhibitory effect on the pro-apoptosis function of BAD. Furthermore, mutated BAD with an alanine substitution inhibited cell proliferation, slowing progression specifically through S-phase. We identify the kinase responsible for phosphorylation at this site as CaMKII-γ (γ isoform of Ca2+/calmodulin-dependent kinase II), but not the other three isoforms of CaMKII, revealing an extraordinary specificity among these closely related kinases. Furthermore, cytokine treatment increased BAD-Ser170-directed CaMKII-γ activity and phosphorylation of CaMKII-γ at an activating site, and CaMKII activity directed to the BAD-Ser170 site was elevated during S-phase. Treating cells with a selective inhibitor of CaMKII caused apoptosis in cells expressing BAD, but not in cells expressing the BAD-S170D mutant. The present study provides support for BAD-Ser170 phosphorylation playing a key role not only in regulating BAD's pro-apoptotic activity, but also in cell proliferation.

  2. PPM1D controls nucleolar formation by up-regulating phosphorylation of nucleophosmin

    PubMed Central

    Kozakai, Yuuki; Kamada, Rui; Furuta, Junya; Kiyota, Yuhei; Chuman, Yoshiro; Sakaguchi, Kazuyasu

    2016-01-01

    An increase of nucleolar number and size has made nucleoli essential markers for cytology and tumour development. However, the underlying basis for their structural integrity and abundance remains unclear. Protein phosphatase PPM1D was found to be up-regulated in different carcinomas including breast cancers. Here, we demonstrate for the first time that PPM1D regulates nucleolar formation via inducing an increased phosphorylation of the nucleolar protein NPM. We show that PPM1D overexpression induces an increase in the nucleolar number regardless of p53 status. We also demonstrated that specific sequential phosphorylation of NPM is important for nucleolar formation and that PPM1D is a novel upstream regulator of this phosphorylation pathway. These results enhance our understanding of the molecular mechanisms that govern nucleoli formation by demonstrating that PPM1D regulates nucleolar formation by regulating NPM phosphorylation status through a novel signalling pathway, PPM1D-CDC25C-CDK1-PLK1. PMID:27619510

  3. Phosphorylation-dependent regulation of Notch1 signaling: the fulcrum of Notch1 signaling.

    PubMed

    Lee, Hye-Jin; Kim, Mi-Yeon; Park, Hee-Sae

    2015-08-01

    Notch signaling plays a pivotal role in cell fate determination, cellular development, cellular self-renewal, tumor progression, and has been linked to developmental disorders and carcinogenesis. Notch1 is activated through interactions with the ligands of neighboring cells, and acts as a transcriptional activator in the nucleus. The Notch1 intracellular domain (Notch1-IC) regulates the expression of target genes related to tumor development and progression. The Notch1 protein undergoes modification after translation by posttranslational modification enzymes. Phosphorylation modification is critical for enzymatic activation, complex formation, degradation, and subcellular localization. According to the nuclear cycle, Notch1-IC is degraded by E3 ligase, FBW7 in the nucleus via phosphorylation-dependent degradation. Here, we summarize the Notch signaling pathway, and resolve to understand the role of phosphorylation in the regulation of Notch signaling as well as to understand its relation to cancer.

  4. Regulation of lamin properties and functions: does phosphorylation do it all?

    PubMed Central

    Machowska, Magdalena; Piekarowicz, Katarzyna; Rzepecki, Ryszard

    2015-01-01

    The main functions of lamins are their mechanical and structural roles as major building blocks of the karyoskeleton. They are also involved in chromatin structure regulation, gene expression, intracellular signalling pathway modulation and development. All essential lamin functions seem to depend on their capacity for assembly or disassembly after the receipt of specific signals, and after specific, selective and precisely regulated interactions through their various domains. Reversible phosphorylation of lamins is crucial for their functions, so it is important to understand how lamin polymerization and interactions are modulated, and which sequences may undergo such modifications. This review combines experimental data with results of our in silico analyses focused on lamin phosphorylation in model organisms to show the presence of evolutionarily conserved sequences and to indicate specific in vivo phosphorylations that affect particular functions. PMID:26581574

  5. Phosphorylated STAT5 regulates p53 expression via BRCA1/BARD1-NPM1 and MDM2

    PubMed Central

    Ren, Zhuo; Aerts, Joeri L; Vandenplas, Hugo; Wang, Jiance A; Gorbenko, Olena; Chen, Jack P; Giron, Philippe; Heirman, Carlo; Goyvaerts, Cleo; Zacksenhaus, Eldad; Minden, Mark D; Stambolic, Vuk; Breckpot, Karine; De Grève, Jacques

    2016-01-01

    Signal transducer and activator of transcription 5 (STAT5) and nucleophosmin (NPM1) are critical regulators of multiple biological and pathological processes. Although a reciprocal regulatory relationship was established between STAT5A and a NPM–ALK fusion protein in T-cell lymphoma, no direct connection between STAT5 and wild-type NPM1 has been documented. Here we demonstrate a mutually regulatory relationship between STAT5 and NPM1. Induction of STAT5 phosphorylation at Y694 (P-STAT5) diminished NPM1 expression, whereas inhibition of STAT5 phosphorylation enhanced NPM1 expression. Conversely, NPM1 not only negatively regulated STAT5 phosphorylation but also preserved unphosphorylated STAT5 level. Mechanistically, we show that NPM1 downregulation by P-STAT5 is mediated by impairing the BRCA1-BARD1 ubiquitin ligase, which controls the stability of NPM1. In turn, decreased NPM1 levels led to suppression of p53 expression, resulting in enhanced cell survival. This study reveals a new STAT5 signaling pathway regulating p53 expression via NPM1 and uncovers new therapeutic targets for anticancer treatment in tumors driven by STAT5 signaling. PMID:28005077

  6. Phosphorylated STAT5 regulates p53 expression via BRCA1/BARD1-NPM1 and MDM2.

    PubMed

    Ren, Zhuo; Aerts, Joeri L; Vandenplas, Hugo; Wang, Jiance A; Gorbenko, Olena; Chen, Jack P; Giron, Philippe; Heirman, Carlo; Goyvaerts, Cleo; Zacksenhaus, Eldad; Minden, Mark D; Stambolic, Vuk; Breckpot, Karine; De Grève, Jacques

    2016-12-22

    Signal transducer and activator of transcription 5 (STAT5) and nucleophosmin (NPM1) are critical regulators of multiple biological and pathological processes. Although a reciprocal regulatory relationship was established between STAT5A and a NPM-ALK fusion protein in T-cell lymphoma, no direct connection between STAT5 and wild-type NPM1 has been documented. Here we demonstrate a mutually regulatory relationship between STAT5 and NPM1. Induction of STAT5 phosphorylation at Y694 (P-STAT5) diminished NPM1 expression, whereas inhibition of STAT5 phosphorylation enhanced NPM1 expression. Conversely, NPM1 not only negatively regulated STAT5 phosphorylation but also preserved unphosphorylated STAT5 level. Mechanistically, we show that NPM1 downregulation by P-STAT5 is mediated by impairing the BRCA1-BARD1 ubiquitin ligase, which controls the stability of NPM1. In turn, decreased NPM1 levels led to suppression of p53 expression, resulting in enhanced cell survival. This study reveals a new STAT5 signaling pathway regulating p53 expression via NPM1 and uncovers new therapeutic targets for anticancer treatment in tumors driven by STAT5 signaling.

  7. Regulation of 90-kilodalton ribosomal S6 kinase phosphorylation in the rat pineal gland.

    PubMed

    Ho, A K; Mackova, M; Cho, C; Chik, C L

    2003-08-01

    In this study we investigated diurnal changes in the activation state of the 90-kDa ribosomal S6 kinase (p90RSK) in the rat pineal gland. In animals housed under a lighting regimen with 12 h of light, we found an increase in phosphorylated p90RSK during the dark phase, and this increase was abolished by treatment with propranolol or continuous exposure to light. To determine the intracellular mechanism involved, rat pinealocytes were treated with norepinephrine. Norepinephrine caused a parallel increase in phosphorylated p42/44 MAPK (p42/44(MAPK)) and p90RSK that was reduced by prazosin or propranolol, indicating involvement of both alpha(1)- and beta-adrenergic receptors. Treatment with dibutyryl cGMP, 4beta-phorbol 12-myristate 13-acetate, or ionomycin mimicked norepinephrine-stimulated p90RSK phosphorylation, whereas dibutyryl cAMP caused a decrease in p90RSK phosphorylation. Inhibition of p42/44(MAPK) activation by UO126 was effective in reducing norepinephrine-stimulated p90RSK phosphorylation. Moreover, UO126 had an inhibitory effect on norepinephrine-stimulated arylalkyl-N-acetyltransferase activity. These results indicate that the adrenergically regulated nocturnal increase in p90RSK phosphorylation is mainly mediated through a cGMP-->p42/44(MAPK)-dependent mechanism.

  8. Regulation of GABA-modulin phosphorylation and GABA receptor binding by excitatory amino acids

    SciTech Connect

    Vaccarino, F.; Guidotti, A.

    1987-05-01

    Primary cultures of cerebellar granule cells phosphorylate numerous proteins including GABA-modulin (GM), which is a putative allosteric modulator of GABA receptors. Cell depolarization and treatment with dicarboxylic excitatory amino acids, which activate PI turnover, Ca/sup 2 +/ influx and guanylate cyclase in granule cells increase the phosphorylation of specific proteins. To determine GM phosphorylation by endogenous protein kinases in living granule cell cultures, GM was isolated by immunoprecipitation and reverse-phase HPLC. High K/sup +/, veratridine, glutamate and NMDA treatment stimulated GM phosphorylation over 2-fold. This increase was abolished by the absence of extracellular Ca/sup 2 +/ and was antagonized by Mg/sup 2 +/ ions and by AVP. The excitatory amino acid action was mimicked by phorbol esters but not by forskolin or by cGMP, and thus may be mediated by an activation of protein kinase C (PKC). Moreover, excitatory amino acids increase /sup 3/H-labelled phorbol ester binding sites in granule cell membrane. The same cultures, treated with glutamate or kainate, showed a 50-fold greater efficacy of muscimol for the stimulation of benzodiazepine (BZ) binding. These data-suggest that excitatory amino acid stimulation of neurons triggers PKC translocation and the activated enzyme phosphorylates GM. The extent of GM phosphorylation may regulate the coupling between GABA and BZ binding sites.

  9. Src regulates sequence-dependent beta-2 adrenergic receptor recycling via cortactin phosphorylation.

    PubMed

    Vistein, Rachel; Puthenveedu, Manojkumar A

    2014-11-01

    The recycling of internalized signaling receptors, which has direct functional consequences, is subject to multiple sequence and biochemical requirements. Why signaling receptors recycle via a specialized pathway, unlike many other proteins that recycle by bulk, is a fundamental unanswered question. Here, we show that these specialized pathways allow selective control of signaling receptor recycling by heterologous signaling. Using assays to visualize receptor recycling in living cells, we show that the recycling of the beta-2 adrenergic receptor (B2AR), a prototypic signaling receptor, is regulated by Src family kinases. The target of Src is cortactin, an essential factor for B2AR sorting into specialized recycling microdomains on the endosome. Phosphorylation of a single cortactin residue, Y466, regulates the rate of fission of B2AR recycling vesicles from these microdomains and, therefore, the rate of delivery of B2AR to the cell surface. Together, our results indicate that actin-stabilized microdomains that mediate signaling receptor recycling can serve as a functional point of convergence for crosstalk between signaling pathways.

  10. Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and trimeric complex formation

    PubMed Central

    Montagnoli, Alessia; Fiore, Francesca; Eytan, Esther; Carrano, Andrea C.; Draetta, Giulio F.; Hershko, Avram; Pagano, Michele

    1999-01-01

    The cellular abundance of the cyclin-dependent kinase (Cdk) inhibitor p27 is regulated by the ubiquitin–proteasome system. Activation of p27 degradation is seen in proliferating cells and in many types of aggressive human carcinomas. p27 can be phosphorylated on threonine 187 by Cdks, and cyclin E/Cdk2 overexpression can stimulate the degradation of wild-type p27, but not of a threonine 187-to-alanine p27 mutant [p27(T187A)]. However, whether threonine 187 phosphorylation stimulates p27 degradation through the ubiquitin–proteasome system or an alternative pathway is still not known. Here, we demonstrate that p27 ubiquitination (as assayed in vivo and in an in vitro reconstituted system) is cell-cycle regulated and that Cdk activity is required for the in vitro ubiquitination of p27. Furthermore, ubiquitination of wild-type p27, but not of p27(T187A), can occur in G1-enriched extracts only upon addition of cyclin E/Cdk2 or cyclin A/Cdk2. Using a phosphothreonine 187 site-specific antibody for p27, we show that threonine 187 phosphorylation of p27 is also cell-cycle dependent, being present in proliferating cells but undetectable in G1 cells. Finally, we show that in addition to threonine 187 phosphorylation, efficient p27 ubiquitination requires formation of a trimeric complex with the cyclin and Cdk subunits. In fact, cyclin B/Cdk1 which can phosphorylate p27 efficiently, but cannot form a stable complex with it, is unable to stimulate p27 ubiquitination by G1 extracts. Furthermore, another p27 mutant [p27(CK−)] that can be phosphorylated by cyclin E/Cdk2 but cannot bind this kinase complex, is refractory to ubiquitination. Thus throughout the cell cycle, both phosphorylation and trimeric complex formation act as signals for the ubiquitination of a Cdk inhibitor. PMID:10323868

  11. O-GlcNAcylation stabilizes β-catenin through direct competition with phosphorylation at threonine 41

    PubMed Central

    Olivier-Van Stichelen, Stéphanie; Dehennaut, Vanessa; Buzy, Armelle; Zachayus, Jean-Luc; Guinez, Céline; Mir, Anne-Marie; El Yazidi-Belkoura, Ikram; Copin, Marie-Christine; Boureme, Didier; Loyaux, Denis; Ferrara, Pascual; Lefebvre, Tony

    2014-01-01

    Dysfunctions in Wnt signaling increase β-catenin stability and are associated with cancers, including colorectal cancer. In addition, β-catenin degradation is decreased by nutrient-dependent O-GlcNAcylation. Human colon tumors and colons from mice fed high-carbohydrate diets exhibited higher amounts of β-catenin and O-GlcNAc relative to healthy tissues and mice fed a standard diet, respectively. Administration of the O-GlcNAcase inhibitor thiamet G to mice also increased colonic expression of β-catenin. By ETD-MS/MS, we identified 4 O-GlcNAcylation sites at the N terminus of β-catenin (S23/T40/T41/T112). Furthermore, mutation of serine and threonine residues within the D box of β-catenin reduced O-GlcNAcylation by 75%. Interestingly, elevating O-GlcNAcylation in human colon cell lines drastically reduced phosphorylation at T41, a key residue of the D box responsible for β-catenin stability. Analyses of β-catenin O-GlcNAcylation mutants reinforced T41 as the most crucial residue that controls the β-catenin degradation rate. Finally, inhibiting O-GlcNAcylation decreased the β-catenin/α-catenin interaction necessary for mucosa integrity, whereas O-GlcNAcase silencing improved this interaction. These results suggest that O-GlcNAcylation regulates not only the stability of β-catenin, but also affects its localization at the level of adherens junctions. Accordingly, we propose that O-GlcNAcylation of β-catenin is a missing link between the glucose metabolism deregulation observed in metabolic disorders and the development of cancer.—Olivier-Van Stichelen, S., Dehennaut, V., Buzy, A., Zachayus, J.-L., Guinez, C., Mir, A.-M., El Yazidi-Belkoura, I., Copin, M.-C., Boureme, D., Loyaux, D., Ferrara, P., Lefebvre, T. O-GlcNAcylation stabilizes β-catenin through direct competition with phosphorylation at threonine 41. PMID:24744147

  12. Cdk9 T-loop Phosphorylation is Regulated by the Calcium Signaling Pathway

    PubMed Central

    Ramakrishnan, Rajesh; Rice, Andrew P.

    2011-01-01

    Eukaryotic RNA polymerase II transcriptional elongation is a tightly regulated process and is dependent upon positive transcription elongation factor-b (P-TEFb). The core P-TEFb complex is composed of Cdk9 and Cyclin T and is essential for the expression of most protein coding genes. Cdk9 kinase function is dependent upon phosphorylation of Thr186 in its T-loop. In this study, we examined kinases and signaling pathways that influence Cdk9 T-loop phosphorylation. Using an RNAi screen in HeLa cells, we found that Cdk9 T-loop phosphorylation is regulated by Calcium/Calmodulin- dependent kinase 1D (CaMK1D). Using small molecules inhibitors in HeLa cells and primary CD4+ T lymphocytes, we found that the Ca2+ signaling pathway is required for Cdk9 T-loop phosphorylation. Inhibition of Ca2+ signaling led to dephosphorylation of Thr186 on Cdk9. In reporter plasmid assays, inhibition of the Ca2+ signaling pathway repressed the PCNA promoter and HIV-1 Tat transactivation of the HIV-1 LTR, but not HTLV-1 Tax transactivation of the HTLV-1 LTR, suggesting that perturbation of the Ca2+ pathway and reduction of Cdk9 T-loop phosphorylation inhibits transcription units that have a rigorous requirement for P-TEFb function. PMID:21448926

  13. Cdk9 T-loop phosphorylation is regulated by the calcium signaling pathway.

    PubMed

    Ramakrishnan, Rajesh; Rice, Andrew P

    2012-02-01

    Eukaryotic RNA polymerase II transcriptional elongation is a tightly regulated process and is dependent upon positive transcription elongation factor-b (P-TEFb). The core P-TEFb complex is composed of Cdk9 and Cyclin T and is essential for the expression of most protein coding genes. Cdk9 kinase function is dependent upon phosphorylation of Thr186 in its T-loop. In this study, we examined kinases and signaling pathways that influence Cdk9 T-loop phosphorylation. Using an RNAi screen in HeLa cells, we found that Cdk9 T-loop phosphorylation is regulated by Ca(2+)/calmodulin-dependent kinase 1D (CaMK1D). Using small molecules inhibitors in HeLa cells and primary CD4(+) T lymphocytes, we found that the Ca(2+) signaling pathway is required for Cdk9 T-loop phosphorylation. Inhibition of Ca(2+) signaling led to dephosphorylation of Thr186 on Cdk9. In reporter plasmid assays, inhibition of the Ca(2+) signaling pathway repressed the PCNA promoter and HIV-1 Tat transactivation of the HIV-1 LTR, but not HTLV-1 Tax transactivation of the HTLV-1 LTR, suggesting that perturbation of the Ca(2+) pathway and reduction of Cdk9 T-loop phosphorylation inhibits transcription units that have a rigorous requirement for P-TEFb function.

  14. VANADYL SULFATE INHIBITS NO PRODUCTION BY DIFFERENTIALLY REGULATING SERINE/THREONINE PHOSPHORYLATION OF ENOS

    EPA Science Inventory

    VANADYL SULFATE INHIBITS NO PRODUCTION BY DIFFERENTIALLY REGULATING SERINE/THREONINE PHOSPHORYLATION OF eNOS.

    Zhuowei Li, Jacqueline D. Carter, Lisa A. Dailey, Joleen Soukup, Yuh-Chin T. Huang. CEMALB, University of North Carolina and NHEERL, US EPA, Chapel Hill, North Ca...

  15. VANADL SULFATE INHIBITS NO PRODUCTION BY DIFFERENTIALLY REGULATING SERINE/THREONINE PHOSPHORYLATION OF ENOS

    EPA Science Inventory

    VANADYL SULFATE INHIBITS NO PRODUCTION BY DIFFERENTIALLY REGULATING SERINE/THREONINE PHOSPHORYLATION OF eNOS. Zhuowei Li, Jacqueline D. Carter, Lisa A. Dailey, Joleen Soukup, Yuh-Chin T. Huang. CEMALB, University of North Carolina and ORD, US EPA, Chapel Hill, North Carolina
    V...

  16. CDK2 Regulates HIV-1 Transcription by Phosphorylation of CDK9 on Serine 90

    PubMed Central

    2012-01-01

    Background HIV-1 transcription is activated by the viral Tat protein that recruits host positive transcription elongation factor-b (P-TEFb) containing CDK9/cyclin T1 to the HIV-1 promoter. P-TEFb in the cells exists as a lower molecular weight CDK9/cyclin T1 dimer and a high molecular weight complex of 7SK RNA, CDK9/cyclin T1, HEXIM1 dimer and several additional proteins. Our previous studies implicated CDK2 in HIV-1 transcription regulation. We also found that inhibition of CDK2 by iron chelators leads to the inhibition of CDK9 activity, suggesting a functional link between CDK2 and CDK9. Here, we investigate whether CDK2 phosphorylates CDK9 and regulates its activity. Results The siRNA-mediated knockdown of CDK2 inhibited CDK9 kinase activity and reduced CDK9 phosphorylation. Stable shRNA-mediated CDK2 knockdown inhibited HIV-1 transcription, but also increased the overall level of 7SK RNA. CDK9 contains a motif (90SPYNR94) that is consensus CDK2 phosphorylation site. CDK9 was phosphorylated on Ser90 by CDK2 in vitro. In cultured cells, CDK9 phosphorylation was reduced when Ser90 was mutated to an Ala. Phosphorylation of CDK9 on Ser90 was also detected with phospho-specific antibodies and it was reduced after the knockdown of CDK2. CDK9 expression decreased in the large complex for the CDK9-S90A mutant and was correlated with a reduced activity and an inhibition of HIV-1 transcription. In contrast, the CDK9-S90D mutant showed a slight decrease in CDK9 expression in both the large and small complexes but induced Tat-dependent HIV-1 transcription. Molecular modeling showed that Ser 90 of CDK9 is located on a flexible loop exposed to solvent, suggesting its availability for phosphorylation. Conclusion Our data indicate that CDK2 phosphorylates CDK9 on Ser 90 and thereby contributes to HIV-1 transcription. The phosphorylation of Ser90 by CDK2 represents a novel mechanism of HIV-1 regulated transcription and provides a new strategy for activation of latent HIV-1

  17. Casein kinase 2α regulates multidrug resistance-associated protein 1 function via phosphorylation of Thr249.

    PubMed

    Stolarczyk, Elzbieta I; Reiling, Cassandra J; Pickin, Kerry A; Coppage, Ryan; Knecht, Marc R; Paumi, Christian M

    2012-09-01

    We have shown previously that the function of Ycf1p, yeast ortholog of multidrug resistance-associated protein 1 (MRP1), is regulated by yeast casein kinase 2α (Cka1p) via phosphorylation at Ser251. In this study, we explored whether casein kinase 2α (CK2α), the human homolog of Cka1p, regulates MRP1 by phosphorylation at the semiconserved site Thr249. Knockdown of CK2α in MCF7-derived cells expressing MRP1 [MRP1 CK2α(-)] resulted in increased doxorubicin sensitivity. MRP1-dependent transport of leukotriene C(4) and estradiol-17β-d-glucuronide into vesicles derived from MRP1 CK2α(-) cells was decreased compared with MRP1 vesicles. Moreover, mutation of Thr249 to alanine (MRP1-T249A) also resulted in decreased MRP1-dependent transport, whereas a phosphomimicking mutation (MRP1-T249E) led to dramatic increase in MRP1-dependent transport. Studies in tissue culture confirmed these findings, showing increased intracellular doxorubicin accumulation in MRP1 CK2α(-) and MRP1-T249A cells compared with MRP1 cells. Inhibition of CK2 kinase by 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole resulted in increased doxorubicin accumulation in MRP1 cells, but not in MRP1 CK2α(-), MRP1-T249A, or MRP1-T249E cells, suggesting that CK2α regulates MRP1 function via phosphorylation of Thr249. Indeed, CK2α and MRP1 interact physically, and recombinant CK2 phosphorylates MRP1-derived peptide in vitro in a Thr249-dependent manner, whereas knockdown of CK2α results in decreased phosphorylation at MRP1-Thr249. The role of CK2 in regulating MRP1 was confirmed in other cancer cell lines where CK2 inhibition decreased MRP1-mediated efflux of doxorubicin and increased doxorubicin cytotoxicity. This study supports a model in which CK2α potentiates MRP1 function via direct phosphorylation of Thr249.

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

    PubMed Central

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

    2016-01-01

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

  19. Phosphorylation of human Jak3 at tyrosines 904 and 939 positively regulates its activity.

    PubMed

    Cheng, Hanyin; Ross, Jeremy A; Frost, Jeffrey A; Kirken, Robert A

    2008-04-01

    Janus tyrosine kinase 3 (Jak3) is essential for signaling by interleukin-2 (IL-2) family cytokines and proper immune function. Dysfunctional regulation of Jak3 may result in certain disease states. However, the molecular mechanisms governing Jak3 activation are not fully understood. In this study, we used a functional-proteomics approach to identify two novel tyrosine phosphorylation sites within Jak3, Y904 and Y939, which are conserved among Jak family proteins. By using phosphospecific antibodies, both residues were observed to be rapidly induced by stimulation of cells with IL-2 or other gammac cytokines. Mechanistic studies indicated that Y904 and Y939 regulate Jak3 activities. A phenylalanine substitution at either site greatly reduced Jak3 kinase activity in vitro and its ability to phosphorylate signal transducer and activator of transcription 5 (Stat5) in vivo, suggesting that phosphorylation of these previously unrecognized residues positively regulates Jak3 activity. Y904 and Y939 were required for optimal ATP usage by Jak3, while phosphorylation of Y939 preferentially promoted Stat5 activity in intact cells. Together, these findings demonstrate positive functional roles for two novel Jak3 phosphoregulatory sites which may be similarly important for other Jak family members. Identification of these sites also provides new therapeutic opportunities to modulate Jak3 function.

  20. Iron-independent phosphorylation of iron regulatory protein 2 regulates ferritin during the cell cycle.

    PubMed

    Wallander, Michelle L; Zumbrennen, Kimberly B; Rodansky, Eva S; Romney, S Joshua; Leibold, Elizabeth A

    2008-08-29

    Iron regulatory protein 2 (IRP2) is a key iron sensor that post-transcriptionally regulates mammalian iron homeostasis by binding to iron-responsive elements (IREs) in mRNAs that encode proteins involved in iron metabolism (e.g. ferritin and transferrin receptor 1). During iron deficiency, IRP2 binds IREs to regulate mRNA translation or stability, whereas during iron sufficiency IRP2 is degraded by the proteasome. Here, we identify an iron-independent IRP2 phosphorylation site that is regulated by the cell cycle. IRP2 Ser-157 is phosphorylated by Cdk1/cyclin B1 during G(2)/M and is dephosphorylated during mitotic exit by the phosphatase Cdc14A. Ser-157 phosphorylation during G(2)/M reduces IRP2 RNA-binding activity and increases ferritin synthesis, whereas Ser-157 dephosphorylation during mitotic exit restores IRP2 RNA-binding activity and represses ferritin synthesis. These data show that reversible phosphorylation of IRP2 during G(2)/M has a role in modulating the iron-independent expression of ferritin and other IRE-containing mRNAs during the cell cycle.

  1. AMP-activated protein kinase phosphorylates CtBP1 and down-regulates its activity

    SciTech Connect

    Kim, Jae-Hwan; Choi, Soo-Youn; Kang, Byung-Hee; Lee, Soon-Min; Cho, Eun-Jung; Youn, Hong-Duk

    2013-02-01

    Highlights: ► AMPK phosphorylates CtBP1 on serine 158. ► AMPK-mediated phosphorylation of CtBP1 causes the ubiquitination and nuclear export of CtBP1. ► AMPK downregulates the CtBP1-mediated repression of Bax transcription. -- Abstract: CtBP is a transcriptional repressor which plays a significant role in the regulation of cell proliferation and tumor progression. It was reported that glucose withdrawal causes induction of Bax due to the dissociation of CtBP from the Bax promoter. However, the precise mechanism involved in the regulation of CtBP still remains unclear. In this study, we found that an activated AMP-activated protein kinase (AMPK) phosphorylates CtBP1 on Ser-158 upon metabolic stresses. Moreover, AMPK-mediated phosphorylation of CtBP1 (S158) attenuates the repressive function of CtBP1. We also confirmed that triggering activation of AMPK by various factors resulted in an increase of Bax gene expression. These findings provide connections of AMPK with CtBP1-mediated regulation of Bax expression for cell death under metabolic stresses.

  2. RIC-3 phosphorylation enables dual regulation of excitation and inhibition of Caenorhabditis elegans muscle

    PubMed Central

    Safdie, Gracia; Liewald, Jana F.; Kagan, Sarah; Battat, Emil; Gottschalk, Alexander; Treinin, Millet

    2016-01-01

    Brain function depends on a delicate balance between excitation and inhibition. Similarly, Caenorhabditis elegans motor system function depends on a precise balance between excitation and inhibition, as C. elegans muscles receive both inhibitory, GABAergic and excitatory, cholinergic inputs from motor neurons. Here we show that phosphorylation of the ER-resident chaperone of nicotinic acetylcholine receptors, RIC-3, leads to increased muscle excitability. RIC-3 phosphorylation at Ser-164 depends on opposing functions of the phosphatase calcineurin (TAX-6), and of the casein kinase II homologue KIN-10. Effects of calcineurin down-regulation and of phosphorylated RIC-3 on muscle excitability are mediated by GABAA receptor inhibition. Thus RIC-3 phosphorylation enables effects of this chaperone on GABAA receptors in addition to nAChRs. This dual effect provides coordinated regulation of excitation and inhibition and enables fine-tuning of the excitation–inhibition balance. Moreover, regulation of inhibitory GABAA signaling by calcineurin, a calcium- and calmodulin-dependent phosphatase, enables homeostatic balancing of excitation and inhibition. PMID:27489343

  3. Activation of NF-kappa B in vivo is regulated by multiple phosphorylations.

    PubMed Central

    Naumann, M; Scheidereit, C

    1994-01-01

    The activation of nuclear factor kappa B (NF-kappa B) in intact cells is mechanistically not well understood. Therefore we investigated the modifications imposed on NF-kappa B/I kappa B components following stimulation and show that the final step of NF-kappa B induction in vivo involves phosphorylation of several members of the NF-kappa B/I kappa B protein families. In HeLa cells as well as in B cells, TNF-alpha rapidly induced nuclear translocation primarily of p50-p65, but not of c-rel. Both NF-kappa B precursors and I kappa B alpha became strongly phosphorylated with the same kinetics. In addition to the inducible phosphorylation after stimulation, B lymphocytes containing constitutive nuclear NF-kappa B revealed constitutively phosphorylated p65 and I kappa B alpha. Phosphorylation was accompanied by induced processing of the precursors p100 and p105 and by degradation of I kappa B alpha. As an in vitro model we show that phosphorylation of p105 impedes its ability to interact with NF-kappa B, as has been shown before for I kappa B alpha. Surprisingly, even p65, but not c-rel, was phosphorylated after induction in vivo, suggesting that TNF-alpha selectively activates only specific NF-kappa B heteromers and that modifications regulate not only I kappa B molecules but also NF-kappa B molecules. In fact, cellular NF-kappa B activity was phosphorylation-dependent and the DNA binding activity of p65-containing NF-kappa B was enhanced by phosphorylation in vitro. Furthermore, we found that the induction by hydrogen peroxide of NF-kappa B translocation to the nucleus, which is assumed to be triggered by reactive oxygen intermediates, also coincided with incorporation of phosphate into the same subunits that were modified after stimulation by TNF-alpha. Thus, phosphorylation appears to be a general mechanism for activation of NF-kappa B in vivo. Images PMID:7925300

  4. Synaptic Clustering of PSD-95 Is Regulated by c-Abl through Tyrosine Phosphorylation

    PubMed Central

    de Arce, Karen Perez; Varela-Nallar, Lorena; Farias, Olivia; Cifuentes, Alejandra; Bull, Paulina; Couch, Brian A.; Koleske, Anthony J.; Inestrosa, Nibaldo C.; Alvarez, Alejandra R.

    2010-01-01

    The c-Abl tyrosine kinase is present in mouse brain synapses, but its precise synaptic function is unknown. We found that c-Abl levels in the rat hippocampus increase postnatally, with expression peaking at the first postnatal week. In 14 d in vitro hippocampal neuron cultures, c-Abl localizes primarily to the postsynaptic compartment, in which it colocalizes with the postsynaptic scaffold protein postsynaptic density protein-95 (PSD-95) in apposition to presynaptic markers. c-Abl associates with PSD-95, and chemical or genetic inhibition of c-Abl kinase activity reduces PSD-95 tyrosine phosphorylation, leading to reduced PSD-95 clustering and reduced synapses in treated neurons. c-Abl can phosphorylate PSD-95 on tyrosine 533, and mutation of this residue reduces the ability of PSD-95 to cluster at postsynaptic sites. Our results indicate that c-Abl regulates synapse formation by mediating tyrosine phosphorylation and clustering of PSD-95. PMID:20220006

  5. Phosphorylation Provides a Negative Mode of Regulation for the Yeast Rab GTPase Sec4p

    PubMed Central

    Heger, Christopher D.; Wrann, Christiane D.; Collins, Ruth N.

    2011-01-01

    The Rab family of Ras-related GTPases are part of a complex signaling circuitry in eukaryotic cells, yet we understand little about the mechanisms that underlie Rab protein participation in such signal transduction networks, or how these networks are integrated at the physiological level. Reversible protein phosphorylation is widely used by cells as a signaling mechanism. Several phospho-Rabs have been identified, however the functional consequences of the modification appear to be diverse and need to be evaluated on an individual basis. In this study we demonstrate a role for phosphorylation as a negative regulatory event for the action of the yeast Rab GTPase Sec4p in regulating polarized growth. Our data suggest that the phosphorylation of the Rab Sec4p prevents interactions with its effector, the exocyst component Sec15p, and that the inhibition may be relieved by a PP2A phosphatase complex containing the regulatory subunit Cdc55p. PMID:21931684

  6. FLCN, a novel autophagy component, interacts with GABARAP and is regulated by ULK1 phosphorylation.

    PubMed

    Dunlop, Elaine A; Seifan, Sara; Claessens, Tijs; Behrends, Christian; Kamps, Miriam Af; Rozycka, Ewelina; Kemp, Alain J; Nookala, Ravi K; Blenis, John; Coull, Barry J; Murray, James T; van Steensel, Maurice Am; Wilkinson, Simon; Tee, Andrew R

    2014-10-01

    Birt-Hogg-Dubé (BHD) syndrome is a rare autosomal dominant condition caused by mutations in the FLCN gene and characterized by benign hair follicle tumors, pneumothorax, and renal cancer. Folliculin (FLCN), the protein product of the FLCN gene, is a poorly characterized tumor suppressor protein, currently linked to multiple cellular pathways. Autophagy maintains cellular homeostasis by removing damaged organelles and macromolecules. Although the autophagy kinase ULK1 drives autophagy, the underlying mechanisms are still being unraveled and few ULK1 substrates have been identified to date. Here, we identify that loss of FLCN moderately impairs basal autophagic flux, while re-expression of FLCN rescues autophagy. We reveal that the FLCN complex is regulated by ULK1 and elucidate 3 novel phosphorylation sites (Ser406, Ser537, and Ser542) within FLCN, which are induced by ULK1 overexpression. In addition, our findings demonstrate that FLCN interacts with a second integral component of the autophagy machinery, GABA(A) receptor-associated protein (GABARAP). The FLCN-GABARAP association is modulated by the presence of either folliculin-interacting protein (FNIP)-1 or FNIP2 and further regulated by ULK1. As observed by elevation of GABARAP, sequestome 1 (SQSTM1) and microtubule-associated protein 1 light chain 3 (MAP1LC3B) in chromophobe and clear cell tumors from a BHD patient, we found that autophagy is impaired in BHD-associated renal tumors. Consequently, this work reveals a novel facet of autophagy regulation by ULK1 and substantially contributes to our understanding of FLCN function by linking it directly to autophagy through GABARAP and ULK1.

  7. Extracellular signal-regulated kinase phosphorylation in forebrain neurones contributes to osmoregulatory mechanisms

    PubMed Central

    Dine, Julien; Ducourneau, Vincent R R; Fénelon, Valérie S; Fossat, Pascal; Amadio, Aurélie; Eder, Matthias; Israel, Jean-Marc; Oliet, Stéphane H R; Voisin, Daniel L

    2014-01-01

    Vasopressin secretion from the magnocellular neurosecretory cells (MNCs) is crucial for body fluid homeostasis. Osmotic regulation of MNC activity involves the concerted modulation of intrinsic mechanosensitive ion channels, taurine release from local astrocytes as well as excitatory inputs derived from osmosensitive forebrain regions. Extracellular signal-regulated protein kinases (ERK) are mitogen-activated protein kinases that transduce extracellular stimuli into intracellular post-translational and transcriptional responses, leading to changes in intrinsic neuronal properties and synaptic function. Here, we investigated whether ERK activation (i.e. phosphorylation) plays a role in the functioning of forebrain osmoregulatory networks. We found that within 10 min after intraperitoneal injections of hypertonic saline (3 m, 6 m) in rats, many phosphoERK-immunopositive neurones were observed in osmosensitive forebrain regions, including the MNC containing supraoptic nuclei. The intensity of ERK labelling was dose-dependent. Reciprocally, slow intragastric infusions of water that lower osmolality reduced basal ERK phosphorylation. In the supraoptic nucleus, ERK phosphorylation predominated in vasopressin neurones vs. oxytocin neurones and was absent from astrocytes. Western blot experiments confirmed that phosphoERK expression in the supraoptic nucleus was dose dependent. Intracerebroventricular administration of the ERK phosphorylation inhibitor U 0126 before a hyperosmotic challenge reduced the number of both phosphoERK-immunopositive neurones and Fos expressing neurones in osmosensitive forebrain regions. Blockade of ERK phosphorylation also reduced hypertonically induced depolarization and an increase in firing of the supraoptic MNCs recorded in vitro. It finally reduced hypertonically induced vasopressin release in the bloodstream. Altogether, these findings identify ERK phosphorylation as a new element contributing to the osmoregulatory mechanisms of

  8. Nik-related kinase regulates trophoblast proliferation and placental development by modulating AKT phosphorylation

    PubMed Central

    Morioka, Yuka; Nam, Jin-Min; Ohashi, Takashi

    2017-01-01

    Nik-related kinase (Nrk) is a Ser/Thr kinase and was initially discovered as a molecule that was predominantly detected in skeletal muscles during development. A recent study using Nrk-null mice suggested the importance of Nrk in proper placental development; however, the molecular mechanism remains unknown. In this study, we demonstrated that differentiated trophoblasts from murine embryonic stem cells (ESCs) endogenously expressed Nrk and that Nrk disruption led to the enhanced proliferation of differentiated trophoblasts. This phenomenon may reflect the overproliferation of trophoblasts that has been reported in enlarged placentas of Nrk-null mice. Furthermore, we demonstrated that AKT phosphorylation at Ser473 was upregulated in Nrk-null trophoblasts and that inhibition of AKT phosphorylation cancelled the enhanced proliferation observed in differentiated Nrk-null trophoblasts. These results indicated that the upregulation of AKT phosphorylation was the possible cause of enhanced proliferation observed in Nrk-null trophoblasts. The upregulation of AKT phosphorylation was also confirmed in enlarged Nrk-null placentas in vivo, suggesting that proper regulation of AKT by Nrk was important for normal placental development. In addition, our detailed analysis on phosphorylation status of AKT isoforms in newly established trophoblast stem cells (TSCs) revealed that different levels of upregulation of AKT phosphorylation were occurred in Nrk-null TSCs depending on AKT isoforms. These results further support the importance of Nrk in proper development of trophoblast lineage cells and indicate the possible application of TSCs for the analysis of differently regulated activation mechanisms of AKT isoforms. PMID:28152035

  9. Negative regulation of NF-κB p65 activity by serine 536 phosphorylation

    PubMed Central

    Pradère, Jean-Philippe; Hernandez, Céline; Koppe, Christiane; Friedman, Richard A; Luedde, Tom; Schwabe, Robert F.

    2016-01-01

    Nuclear factor κB (NF-κB) is a master regulator of inflammation and cell death. Whereas most of the activity of NF-κB is regulated through the inhibitor of kB (IκB) kinase (IKK)–dependent degradation of IκB, IKK also phosphorylates subunits of NF-κB. Here, we investigated the contribution of the phosphorylation of the NF-κ B subunit p65 at the IKK phosphorylation site serine 536 (Ser536) in humans, which is thought to be required for the activation and nuclear translocation of NF-κB. Through experiments with knock-in mice (S534A mice) expressing a mutant p65 with an alanine-to-serine substitution at position 534 (the murine homolog of human Ser536), we observed increased expression of NF-κ B–dependent genes after injection of mice with the inflammatory stimulus lipopolysaccharide (LPS) or exposure to gamma irradiation, and the enhanced gene expression was most pronounced at late time points. Compared to wild-type mice, S534A mice displayed increased mortality after injection with LPS. Increased NF-κ B signaling in the S534A mice was at least in part explained by the increased stability of the S534A p65 protein compared to that of the Ser534-phosphorylated wild-type protein. Together, our results suggest that Ser534 phosphorylation of p65 in mice (and, by extension, Ser536 phosphorylation of human p65) is not required for its nuclear translocation, but instead inhibits NF-κ B signaling to prevent deleterious inflammation. PMID:27555662

  10. Regulated phosphorylation and dephosphorylation of tau protein: effects on microtubule interaction, intracellular trafficking and neurodegeneration.

    PubMed Central

    Billingsley, M L; Kincaid, R L

    1997-01-01

    This review attempts to summarize what is known about tau phosphorylation in the context of both normal cellular function and dysfunction. However, conceptions of tau function continue to evolve, and it is likely that the regulation of tau distribution and metabolism is complex. The roles of microtubule-associated kinases and phosphatases have yet to be fully described, but may afford insight into how tau phosphorylation at the distal end of the axon regulates cytoskeletal-membrane interactions. Finally, lipid and glycosaminoglycan modification of tau structure affords yet more complexity for regulation and aggregation. Continued work will help to determine what is causal and what is coincidental in Alzheimer's disease, and may lead to identification of therapeutic targets for halting the progression of paired helical filament formation. PMID:9169588

  11. Fimbrin phosphorylation by metaphase Cdk1 regulates actin cable dynamics in budding yeast

    PubMed Central

    Miao, Yansong; Han, Xuemei; Zheng, Liangzhen; Xie, Ying; Mu, Yuguang; Yates, John R.; Drubin, David G.

    2016-01-01

    Actin cables, composed of actin filament bundles nucleated by formins, mediate intracellular transport for cell polarity establishment and maintenance. We previously observed that metaphase cells preferentially promote actin cable assembly through cyclin-dependent kinase 1 (Cdk1) activity. However, the relevant metaphase Cdk1 targets were not known. Here we show that the highly conserved actin filament crosslinking protein fimbrin is a critical Cdk1 target for actin cable assembly regulation in budding yeast. Fimbrin is specifically phosphorylated on threonine 103 by the metaphase cyclin–Cdk1 complex, in vivo and in vitro. On the basis of conformational simulations, we suggest that this phosphorylation stabilizes fimbrin's N-terminal domain, and modulates actin filament binding to regulate actin cable assembly and stability in cells. Overall, this work identifies fimbrin as a key target for cell cycle regulation of actin cable assembly in budding yeast, and suggests an underlying mechanism. PMID:27068241

  12. The Circadian Protein BMAL1 Regulates Translation in Response to S6K1-Mediated Phosphorylation.

    PubMed

    Lipton, Jonathan O; Yuan, Elizabeth D; Boyle, Lara M; Ebrahimi-Fakhari, Darius; Kwiatkowski, Erica; Nathan, Ashwin; Güttler, Thomas; Davis, Fred; Asara, John M; Sahin, Mustafa

    2015-05-21

    The circadian timing system synchronizes cellular function by coordinating rhythmic transcription via a transcription-translational feedback loop. How the circadian system regulates gene expression at the translational level remains a mystery. Here, we show that the key circadian transcription factor BMAL1 associates with the translational machinery in the cytosol and promotes protein synthesis. The mTOR-effector kinase, ribosomal S6 protein kinase 1 (S6K1), an important regulator of translation, rhythmically phosphorylates BMAL1 at an evolutionarily conserved site. S6K1-mediated phosphorylation is critical for BMAL1 to both associate with the translational machinery and stimulate protein synthesis. Protein synthesis rates demonstrate circadian oscillations dependent on BMAL1. Thus, in addition to its critical role in circadian transcription, BMAL1 is a translation factor that links circadian timing and the mTOR signaling pathway. More broadly, these results expand the role of the circadian clock to the regulation of protein synthesis.

  13. Tyrosine phosphorylation within the SH3 domain regulates CAS subcellular localization, cell migration, and invasiveness.

    PubMed

    Janoštiak, Radoslav; Tolde, Ondřej; Brůhová, Zuzana; Novotný, Marian; Hanks, Steven K; Rösel, Daniel; Brábek, Jan

    2011-11-01

    Crk-associated substrate (CAS) is a major tyrosine-phosphorylated protein in cells transformed by v-crk and v-src oncogenes and plays an important role in invasiveness of Src-transformed cells. A novel phosphorylation site on CAS, Tyr-12 (Y12) within the ligand-binding hydrophobic pocket of the CAS SH3 domain, was identified and found to be enriched in Src-transformed cells and invasive human carcinoma cells. To study the biological significance of CAS Y12 phosphorylation, phosphomimicking Y12E and nonphosphorylatable Y12F mutants of CAS were studied. The phosphomimicking mutation decreased interaction of the CAS SH3 domain with focal adhesion kinase (FAK) and PTP-PEST and reduced tyrosine phosphorylation of FAK. Live-cell imaging showed that green fluorescent protein-tagged CAS Y12E mutant is, in contrast to wild-type or Y12F CAS, excluded from focal adhesions but retains its localization to podosome-type adhesions. Expression of CAS-Y12F in cas-/- mouse embryonic fibroblasts resulted in hyperphosphorylation of the CAS substrate domain, and this was associated with slower turnover of focal adhesions and decreased cell migration. Moreover, expression of CAS Y12F in Src-transformed cells greatly decreased invasiveness when compared to wild-type CAS expression. These findings reveal an important role of CAS Y12 phosphorylation in the regulation of focal adhesion assembly, cell migration, and invasiveness of Src-transformed cells.

  14. Phosphorylation of the TAL1 oncoprotein by the extracellular-signal-regulated protein kinase ERK1.

    PubMed Central

    Cheng, J T; Cobb, M H; Baer, R

    1993-01-01

    Alteration of the TAL1 gene is the most common genetic lesion found in T-cell acute lymphoblastic leukemia. TAL1 encodes phosphoproteins, pp42TAL1 and pp22TAL1, that represent phosphorylated versions of the full-length (residues 1 to 331) and truncated (residues 176 to 331) TAL1 gene products, respectively. Both proteins contain the basic helix-loop-helix motif, a DNA-binding and protein dimerization motif common to several known transcriptional regulatory factors. We now report that serine residue 122 (S122) is a major phosphorylation site of pp42TAL1 in leukemic cell lines and transfected COS1 cells. In vivo phosphorylation of S122 is induced by epidermal growth factor with a rapid time course that parallels activation of the ERK/MAP2 protein kinases. Moreover, S122 is readily phosphorylated in vitro by the extracellular signal-regulated protein kinase ERK1. These data suggest that TAL1 residue S122 serves as an in vivo substrate for ERK/MAP2 kinases such as ERK1. Therefore, S122 phosphorylation may provide a mechanism whereby the properties of TAL1 polypeptides can be modulated by extracellular stimuli. Images PMID:8423803

  15. ELYS regulates the localization of LBR by modulating its phosphorylation state

    PubMed Central

    Mimura, Yasuhiro; Takagi, Masatoshi; Clever, Michaela

    2016-01-01

    ABSTRACT Lamin B receptor (LBR), an inner nuclear membrane (INM) protein, contributes to the functional integrity of the nucleus by tethering heterochromatin to the nuclear envelope. We have previously reported that the depletion of embryonic large molecule derived from yolk sac (ELYS; also known as AHCTF1), a component of the nuclear pore complex, from cells perturbs the localization of LBR to the INM, but little is known about the underlying molecular mechanism. In this study, we found that the depletion of ELYS promoted LBR phosphorylation at the residues known to be phosphorylated by cyclin-dependent kinase (CDK) and serine/arginine protein kinases 1 and 2 (SRPK1 and SRPK2, respectively). These phosphorylation events were most likely to be counter-balanced by protein phosphatase 1 (PP1), and the depletion of PP1 from cells consistently caused the mislocalization of LBR. These observations point to a new mechanism regulating the localization of LBR, which is governed by an ELYS-mediated phosphorylation network. This phosphorylation-dependent coordination between INM proteins and the nuclear pore complex might be important for the integrity of the nucleus. PMID:27802161

  16. Biphasic regulation of myosin light chain phosphorylation by p21-activated kinase modulates intestinal smooth muscle contractility.

    PubMed

    Chu, Ji; Pham, Ngoc T; Olate, Nicole; Kislitsyna, Karina; Day, Mary-Clare; LeTourneau, Phillip A; Kots, Alexander; Stewart, Randolph H; Laine, Glen A; Cox, Charles S; Uray, Karen

    2013-01-11

    Supraphysiological mechanical stretching in smooth muscle results in decreased contractile activity. However, the mechanism is unclear. Previous studies indicated that intestinal motility dysfunction after edema development is associated with increased smooth muscle stress and decreased myosin light chain (MLC) phosphorylation in vivo, providing an ideal model for studying mechanical stress-mediated decrease in smooth muscle contraction. Primary human intestinal smooth muscle cells (hISMCs) were subjected to either control cyclical stretch (CCS) or edema (increasing) cyclical stretch (ECS), mimicking the biophysical forces in non-edematous and edematous intestinal smooth muscle in vivo. ECS induced significant decreases in phosphorylation of MLC and MLC phosphatase targeting subunit (MYPT1) and a significant increase in p21-activated kinase (PAK) activity compared with CCS. PAK regulated MLC phosphorylation in an activity-dependent biphasic manner. PAK activation increased MLC and MYPT1 phosphorylation in CCS but decreased MLC and MYPT1 phosphorylation in hISMCs subjected to ECS. PAK inhibition had the opposite results. siRNA studies showed that PAK1 plays a critical role in regulating MLC phosphorylation in hISMCs. PAK1 enhanced MLC phosphorylation via phosphorylating MYPT1 on Thr-696, whereas PAK1 inhibited MLC phosphorylation via decreasing MYPT1 on both Thr-696 and Thr-853. Importantly, in vivo data indicated that PAK activity increased in edematous tissue, and inhibition of PAK in edematous intestine improved intestinal motility. We conclude that PAK1 positively regulates MLC phosphorylation in intestinal smooth muscle through increasing inhibitory phosphorylation of MYPT1 under physiologic conditions, whereas PAK1 negatively regulates MLC phosphorylation via inhibiting MYPT1 phosphorylation when PAK activity is increased under pathologic conditions.

  17. Regulation of CK2 by Phosphorylation and O-GlcNAcylation Revealed by Semisynthesis

    PubMed Central

    Tarrant, Mary Katherine; Rho, Hee-Sool; Xie, Zhi; Jiang, Yu Lin; Gross, Christopher; Culhane, Jeffrey C.; Yan, Gai; Qian, Jiang; Ichikawa, Yoshitaka; Matsuoka, Tatsuji; Zachara, Natasha; Etzkorn, Felicia A.; Hart, Gerald W.; Jeong, Jun Seop; Blackshaw, Seth; Zhu, Heng; Cole, Philip A.

    2011-01-01

    Protein Ser/Thr kinase CK2 (casein kinase II) is involved in a myriad of cellular processes including cell growth and proliferation by phosphorylating hundreds of substrates, yet the regulation process of CK2 function is poorly understood. Here we report that the CK2 catalytic subunit CK2α is modified by O-GlcNAc on Ser347, proximal to a cyclin-dependent kinase phosphorylation site (Thr344) on the same protein. We use protein semisynthesis to show that Thr344 phosphorylation increases CK2α cellular stability via Pin1 interaction whereas Ser347 glycosylation appears to be antagonistic to Thr344 phosphorylation and permissive to proteasomal degradation. By performing kinase assays with the site-specifically modified phospho- and glyco-modified CK2α in combination with CK2β and Pin1 binding partners on human protein microarrays, we show that CK2 kinase substrate selectivity is modulated by these specific posttranslational modifications. This study suggests how a promiscuous protein kinase can be regulated at multiple levels to achieve particular biological outputs. PMID:22267120

  18. Allosteric regulation of SERCA by phosphorylation-mediated conformational shift of phospholamban

    PubMed Central

    Gustavsson, Martin; Verardi, Raffaello; Mullen, Daniel G.; Mote, Kaustubh R.; Traaseth, Nathaniel J.; Gopinath, T.; Veglia, Gianluigi

    2013-01-01

    The membrane protein complex between the sarcoplasmic reticulum Ca2+-ATPase (SERCA) and phospholamban (PLN) controls Ca2+ transport in cardiomyocytes, thereby modulating cardiac contractility. β-Adrenergic-stimulated phosphorylation of PLN at Ser-16 enhances SERCA activity via an unknown mechanism. Using solid-state nuclear magnetic resonance spectroscopy, we mapped the physical interactions between SERCA and both unphosphorylated and phosphorylated PLN in membrane bilayers. We found that the allosteric regulation of SERCA depends on the conformational equilibrium of PLN, whose cytoplasmic regulatory domain interconverts between three different states: a ground T state (helical and membrane associated), an excited R state (unfolded and membrane detached), and a B state (extended and enzyme-bound), which is noninhibitory. Phosphorylation at Ser-16 of PLN shifts the populations toward the B state, increasing SERCA activity. We conclude that PLN’s conformational equilibrium is central to maintain SERCA’s apparent Ca2+ affinity within a physiological window. This model represents a paradigm shift in our understanding of SERCA regulation by posttranslational phosphorylation and suggests strategies for designing innovative therapeutic approaches to enhance cardiac muscle contractility. PMID:24101520

  19. Bub1 autophosphorylation feeds back to regulate kinetochore docking and promote localized substrate phosphorylation

    PubMed Central

    Asghar, Adeel; Lajeunesse, Audrey; Dulla, Kalyan; Combes, Guillaume; Thebault, Philippe; Nigg, Erich A.; Elowe, Sabine

    2015-01-01

    During mitosis, Bub1 kinase phosphorylates histone H2A-T120 to promote centromere sister chromatid cohesion through recruitment of shugoshin (Sgo) proteins. The regulation and dynamics of H2A-T120 phosphorylation are poorly understood. Using quantitative phosphoproteomics we show that Bub1 is autophosphorylated at numerous sites. We confirm mitosis-specific autophosphorylation of a several residues and show that Bub1 activation is primed in interphase but fully achieved only in mitosis. Mutation of a single autophosphorylation site T589 alters kinetochore turnover of Bub1 and results in uniform H2A-T120 phosphorylation and Sgo recruitment along chromosome arms. Consequently, improper sister chromatid resolution and chromosome segregation errors are observed. Kinetochore tethering of Bub1-T589A refocuses H2A-T120 phosphorylation and Sgo1 to centromeres. Recruitment of the Bub1-Bub3-BubR1 axis to kinetochores has recently been extensively studied. Our data provide novel insight into the regulation and kinetochore residency of Bub1 and indicate that its localization is dynamic and tightly controlled through feedback autophosphorylation. PMID:26399325

  20. Mitotic Phosphorylation of TREX1 C Terminus Disrupts TREX1 Regulation of the Oligosaccharyltransferase Complex.

    PubMed

    Kucej, Martin; Fermaintt, Charles S; Yang, Kun; Irizarry-Caro, Ricardo A; Yan, Nan

    2017-03-14

    TREX1 mutations are associated with several autoimmune and inflammatory diseases. The N-terminal DNase domain of TREX1 is important for preventing self-DNA from activating the interferon response. The C terminus of TREX1 is required for ER localization and regulation of oligosacchariyltransferase (OST) activity. Here, we show that during mitosis TREX1 is predominately phosphorylated at the C-terminal Serine-261 by Cyclin B/CDK1. TREX1 is dephosphorylated quickly at mitotic exit, likely by PP1/PP2-type serine/threonine phosphatase. Mitotic phosphorylation does not affect TREX1 DNase activity. Phosphomimetic mutations of mitotic phosphorylation sites in TREX1 disrupted the interaction with the OST subunit RPN1. RNA-seq analysis of Trex1(-/-) mouse embryonic fibroblasts expressing TREX1 wild-type or phosphor-mutants revealed a glycol-gene signature that is elevated when TREX1 mitotic phosphorylation sites are disrupted. Thus, the cell-cycle-dependent post-translation modification of TREX1 regulates its interaction with OST, which may have important implications for immune disease associated with the DNase-independent function of TREX1.

  1. Phosphorylation sites required for regulation of cardiac calcium channels in the fight-or-flight response.

    PubMed

    Fu, Ying; Westenbroek, Ruth E; Scheuer, Todd; Catterall, William A

    2013-11-26

    L-type Ca(2+) currents conducted by CaV1.2 channels initiate excitation-contraction coupling in the heart. Their activity is increased by β-adrenergic/cAMP signaling via phosphorylation by PKA in the fight-or-flight response, but the sites of regulation are unknown. We describe the functional role of phosphorylation of Ser1700 and Thr1704-sites of phosphorylation by PKA and casein kinase II at the interface between the proximal and distal C-terminal regulatory domains. Mutation of both residues to Ala in STAA mice reduced basal L-type Ca(2+) currents, due to a small decrease in expression and a substantial decrease in functional activity. The increase in L-type Ca(2+) current caused by isoproterenol was markedly reduced at physiological levels of stimulation (3-10 nM). Maximal increases in calcium current at nearly saturating concentrations of isoproterenol (100 nM) were also significantly reduced, but the mutation effects were smaller, suggesting that alternative regulatory mechanisms are engaged at maximal levels of stimulation. The β-adrenergic increase in cell contraction was also diminished. STAA ventricular myocytes exhibited arrhythmic contractions in response to isoproterenol, and up to 20% of STAA cells failed to sustain contractions when stimulated at 1 Hz. STAA mice have reduced exercise capacity, and cardiac hypertrophy is evident at 3 mo. We conclude that phosphorylation of Ser1700 and Thr1704 is essential for regulation of basal activity of CaV1.2 channels and for up-regulation by β-adrenergic signaling at physiological levels of stimulation. Disruption of phosphorylation at those sites leads to impaired cardiac function in vivo, as indicated by reduced exercise capacity and cardiac hypertrophy.

  2. The interaction between acetylation and serine-574 phosphorylation regulates the apoptotic function of FOXO3

    PubMed Central

    Li, Z; Bridges, B; Olson, J; Weinman, SA

    2017-01-01

    The multispecific transcription factor and tumor suppressor FOXO3 is an important mediator of apoptosis, but the mechanisms that control its proapoptotic function are poorly understood. There has long been evidence that acetylation promotes FOXO3-driven apoptosis and recently a specific JNK (c-Jun N-terminal kinase)-dependent S574 phosphorylated form (p-FOXO3) has been shown to be specifically apoptotic. This study examined whether acetylation and S574 phosphorylation act independently or in concert to regulate the apoptotic function of FOXO3. We observed that both sirtuins 1 and 7 (SIRT1 and SIRT7) are able to deacetylate FOXO3 in vitro and in vivo, and that lipopolysaccharide (LPS) treatment of THP-1 monocytes induced a rapid increase of FOXO3 acetylation, partly by suppression of SIRT1 and SIRT7. Acetylation was required for S574 phosphorylation and cellular apoptosis. Deacetylation of FOXO3 by SIRT activation or SIRT1 or SIRT7 overexpression prevented its S574 phosphorylation and blocked apoptosis in response to LPS. We also found that acetylated FOXO3 preferentially bound JNK1, and a mutant FOXO3 lacking four known acetylation sites (K242, 259, 290 and 569R) abolished JNK1 binding and failed to induce apoptosis. This interplay of acetylation and phosphorylation also regulated cell death in primary human peripheral blood monocytes (PBMs). PBMs isolated from alcoholic hepatitis patients had high expression of SIRT1 and SIRT7 and failed to induce p-FOXO3 and apoptosis in response to LPS. PBMs from healthy controls had lower SIRT1 and SIRT7 and readily formed p-FOXO3 and underwent apoptosis when similarly treated. These results reveal that acetylation is permissive for generation of the apoptotic form of FOXO3 and the activity of SIRT1 and particularly SIRT7 regulate this process in vivo, allowing control of monocyte apoptosis in response to LPS. PMID:27669435

  3. The interaction between acetylation and serine-574 phosphorylation regulates the apoptotic function of FOXO3.

    PubMed

    Li, Z; Bridges, B; Olson, J; Weinman, S A

    2017-03-30

    The multispecific transcription factor and tumor suppressor FOXO3 is an important mediator of apoptosis, but the mechanisms that control its proapoptotic function are poorly understood. There has long been evidence that acetylation promotes FOXO3-driven apoptosis and recently a specific JNK (c-Jun N-terminal kinase)-dependent S574 phosphorylated form (p-FOXO3) has been shown to be specifically apoptotic. This study examined whether acetylation and S574 phosphorylation act independently or in concert to regulate the apoptotic function of FOXO3. We observed that both sirtuins 1 and 7 (SIRT1 and SIRT7) are able to deacetylate FOXO3 in vitro and in vivo, and that lipopolysaccharide (LPS) treatment of THP-1 monocytes induced a rapid increase of FOXO3 acetylation, partly by suppression of SIRT1 and SIRT7. Acetylation was required for S574 phosphorylation and cellular apoptosis. Deacetylation of FOXO3 by SIRT activation or SIRT1 or SIRT7 overexpression prevented its S574 phosphorylation and blocked apoptosis in response to LPS. We also found that acetylated FOXO3 preferentially bound JNK1, and a mutant FOXO3 lacking four known acetylation sites (K242, 259, 290 and 569R) abolished JNK1 binding and failed to induce apoptosis. This interplay of acetylation and phosphorylation also regulated cell death in primary human peripheral blood monocytes (PBMs). PBMs isolated from alcoholic hepatitis patients had high expression of SIRT1 and SIRT7 and failed to induce p-FOXO3 and apoptosis in response to LPS. PBMs from healthy controls had lower SIRT1 and SIRT7 and readily formed p-FOXO3 and underwent apoptosis when similarly treated. These results reveal that acetylation is permissive for generation of the apoptotic form of FOXO3 and the activity of SIRT1 and particularly SIRT7 regulate this process in vivo, allowing control of monocyte apoptosis in response to LPS.

  4. Phosphorylation is the major mechanism regulating isocitrate lyase activity in Paracoccidioides brasiliensis yeast cells.

    PubMed

    Cruz, Aline H da Silva; Brock, Matthias; Zambuzzi-Carvalho, Patrícia F; Santos-Silva, Ludier K; Troian, Rogério F; Góes, Alfredo M; Soares, Célia M de Almeida; Pereira, Maristela

    2011-07-01

    The glyoxylate cycle plays an essential role for anaplerosis of oxaloacetate during growth of microorganisms on carbon sources such as acetate or fatty acids and has been shown to contribute to virulence of several pathogens. Here, we investigated the transcriptional and post-translational regulation of the glyoxylate cycle key enzyme isocitrate lyase (PbICL) in the human pathogenic fungus Paracoccidioides brasiliensis. Although sequence analyses on fungal isocitrate lyases revealed a high phylogenetic conservation, their regulation seems to differ significantly. Closely related Aspergillus species regulate the glyoxylate cycle at the transcriptional level, whereas Pbicl was constitutively expressed in yeast cells. However, only low PbICL activity was detected when cells were grown in the presence of glucose. Two-dimensional gel analyses with subsequent antibody hybridization revealed constitutive production of PbICL, but low PbICL activity on glucose coincided with extensive protein phosphorylation. Since an in vitro dephosphorylation of PbICL from glucose grown cells strongly increased ICL activity and resembled the phosphorylation pattern of highly active acetate grown cells, post-translational modification seems the main mechanism regulating PbICL activity in yeast cells. In agreement, a transfer of yeast cells from glucose to acetate medium increased PbICL activity without requirement of de novo protein synthesis. Thus, inactivation of PbICL by phosphorylation is reversible, denoting a new strategy for the rapid adaptation to changing environmental conditions.

  5. Coordinated ubiquitination and phosphorylation of RIP1 regulates necroptotic cell death

    PubMed Central

    de Almagro, M Cristina; Goncharov, Tatiana; Izrael-Tomasevic, Anita; Duttler, Stefanie; Kist, Matthias; Varfolomeev, Eugene; Wu, Xiumin; Lee, Wyne P; Murray, Jeremy; Webster, Joshua D; Yu, Kebing; Kirkpatrick, Donald S; Newton, Kim; Vucic, Domagoj

    2017-01-01

    Proper regulation of cell death signaling is crucial for the maintenance of homeostasis and prevention of disease. A caspase-independent regulated form of cell death called necroptosis is rapidly emerging as an important mediator of a number of human pathologies including inflammatory bowel disease and ischemia–reperfusion organ injury. Activation of necroptotic signaling through TNF signaling or organ injury leads to the activation of kinases receptor-interacting protein kinases 1 and 3 (RIP1 and RIP3) and culminates in inflammatory cell death. We found that, in addition to phosphorylation, necroptotic cell death is regulated by ubiquitination of RIP1 in the necrosome. Necroptotic RIP1 ubiquitination requires RIP1 kinase activity, but not necroptotic mediators RIP3 and MLKL (mixed lineage kinase-like). Using immunoaffinity enrichment and mass spectrometry, we profiled numerous ubiquitination events on RIP1 that are triggered during necroptotic signaling. Mutation of a necroptosis-related ubiquitination site on RIP1 reduced necroptotic cell death and RIP1 ubiquitination and phosphorylation, and disrupted the assembly of RIP1 and RIP3 in the necrosome, suggesting that necroptotic RIP1 ubiquitination is important for maintaining RIP1 kinase activity in the necrosome complex. We also observed RIP1 ubiquitination in injured kidneys consistent with a physiological role of RIP1 ubiquitination in ischemia–reperfusion disease. Taken together, these data reveal that coordinated and interdependent RIP1 phosphorylation and ubiquitination within the necroptotic complex regulate necroptotic signaling and cell death. PMID:27518435

  6. Phosphorylation of PP1 Regulator Sds22 by PLK1 Ensures Accurate Chromosome Segregation.

    PubMed

    Duan, Hequan; Wang, Chunli; Wang, Ming; Gao, Xinjiao; Yan, Maomao; Akram, Saima; Peng, Wei; Zou, Hanfa; Wang, Dong; Zhou, Jiajia; Chu, Youjun; Dou, Zhen; Barrett, Gregory; Green, Hadiyah-Nichole; Wang, Fangjun; Tian, Ruijun; He, Ping; Wang, Wenwen; Liu, Xing; Yao, Xuebiao

    2016-09-30

    During cell division, accurate chromosome segregation is tightly regulated by Polo-like kinase 1 (PLK1) and opposing activities of Aurora B kinase and protein phosphatase 1 (PP1). However, the regulatory mechanisms underlying the aforementioned hierarchical signaling cascade during mitotic chromosome segregation have remained elusive. Sds22 is a conserved regulator of PP1 activity, but how it regulates PP1 activity in space and time during mitosis remains elusive. Here we show that Sds22 is a novel and cognate substrate of PLK1 in mitosis, and the phosphorylation of Sds22 by PLK1 elicited an inhibition of PP1-mediated dephosphorylation of Aurora B at threonine 232 (Thr(232)) in a dose-dependent manner. Overexpression of a phosphomimetic mutant of Sds22 causes a dramatic increase in mitotic delay, whereas overexpression of a non-phosphorylatable mutant of Sds22 results in mitotic arrest. Mechanistically, the phosphorylation of Sds22 by PLK1 strengthens the binding of Sds22 to PP1 and inhibits the dephosphorylation of Thr(232) of Aurora B to ensure a robust, error-free metaphase-anaphase transition. These findings delineate a conserved signaling hierarchy that orchestrates dynamic protein phosphorylation and dephosphorylation of critical mitotic regulators during chromosome segregation to guard chromosome stability.

  7. TPX2 phosphorylation maintains metaphase spindle length by regulating microtubule flux

    PubMed Central

    Fu, Jingyan; Bian, Minglei; Xin, Guangwei; Deng, Zhaoxuan; Luo, Jia; Guo, Xiao; Chen, Hao; Wang, Yao; Jiang, Qing

    2015-01-01

    A steady-state metaphase spindle maintains constant length, although the microtubules undergo intensive dynamics. Tubulin dimers are incorporated at plus ends of spindle microtubules while they are removed from the minus ends, resulting in poleward movement. Such microtubule flux is regulated by the microtubule rescue factors CLASPs at kinetochores and depolymerizing protein Kif2a at the poles, along with other regulators of microtubule dynamics. How microtubule polymerization and depolymerization are coordinated remains unclear. Here we show that TPX2, a microtubule-bundling protein and activator of Aurora A, plays an important role. TPX2 was phosphorylated by Aurora A during mitosis. Its phospho-null mutant caused short metaphase spindles coupled with low microtubule flux rate. Interestingly, phosphorylation of TPX2 regulated its interaction with CLASP1 but not Kif2a. The effect of its mutant in shortening the spindle could be rescued by codepletion of CLASP1 and Kif2a that abolished microtubule flux. Together we propose that Aurora A–dependent TPX2 phosphorylation controls mitotic spindle length through regulating microtubule flux. PMID:26240182

  8. EGFR Signaling Regulates Maspin/SerpinB5 Phosphorylation and Nuclear Localization in Mammary Epithelial Cells

    PubMed Central

    Reina, Jeffrey; Morais Freitas, Vanessa

    2016-01-01

    Maspin (SerpinB5) is a non-inhibitory serpin (serine protease inhibitor) with very diverse biological activities including regulation of cell adhesion, migration, death, control of gene expression and oxidative stress response. Initially described as a tumor and metastasis suppressor, clinical data brought controversies to the field, as some studies reported no correlation between SerpinB5 expression and prognosis value. These data underscore the importance of understanding SerpinB5 function in a normal physiological context and the molecular mechanism involved. Several SerpinB5 phosphoforms have been detected in different cell lines, but the signaling pathways involved and the biological significance of this post-translational modification in vivo remains to be explored. In this study we investigated SerpinB5 expression, subcellular localization and phosphorylation in different stages of the mouse mammary gland development and the signaling pathway involved. Here we show that SerpinB5 is first detected in late pregnancy, reaches its highest levels in lactation and remains at constant levels during post-lactational regression (involution). Using high resolution isoelectric focusing followed but immunoblot, we found at least 8 different phosphoforms of SerpinB5 during lactation, which decreases steadily at the onset of involution. In order to investigate the signaling pathway involved in SerpinB5 phosphorylation, we took advantage of the non-transformed MCF-10A model system, as we have previously observed SerpinB5 phosphorylation in these cells. We detected basal levels of SerpinB5 phosphorylation in serum- and growth factor-starved cells, which is due to amphiregulin autocrine activity on MCF-10A cells. EGF and TGF alpha, two other EGFR ligands, promote important SerpinB5 phosphorylation. Interestingly, EGF treatment is followed by SerpinB5 nuclear accumulation. Altogether, these data indicate that SerpinB5 expression and phosphorylation are developmentally

  9. ATP-dependent phosphorylation of serine-46 in the phosphocarrier protein HPr regulates lactose/H+ symport in Lactobacillus brevis.

    PubMed Central

    Ye, J J; Reizer, J; Cui, X; Saier, M H

    1994-01-01

    Lactobacillus brevis takes up lactose and the nonmetabolizable lactose analogue thiomethyl beta-galactoside (TMG) by a permease-catalyzed lactose/H+ symport mechanism. Earlier studies have shown that TMG, previously accumulated in L. brevis cells, rapidly effluxes from the cells upon addition of glucose, and that glucose inhibits further uptake of TMG. We have developed a vesicular system to analyze this regulatory mechanism and have used electroporation to shock proteins and membrane-impermeant metabolites into the vesicles. Uptake of TMG was dependent on an energy source, effectively provided by intravesicular ATP or extravesicular arginine. TMG uptake into these vesicles was not inhibited, and preaccumulated TMG did not efflux from them upon addition of glucose. Intravesicular but not extravesicular wild-type phosphocarrier protein HPr of Bacillus subtilis restored regulation. Glucose could be replaced by intravesicular (but not extravesicular) fructose 1,6-bisphosphate, gluconate 6-phosphate, or 2-phosphoglycerate, but not by other phosphorylated metabolites, in agreement with the allosteric activating effects of these compounds on HPr(Ser) kinase measured in vitro. Intravesicular serine-46-->alanine mutant HPr cold not promote regulation of lactose permease activity when electroporated into the vesicles with or without glucose or the various phosphorylated metabolites, but the serine-46-->aspartate mutant HPr promoted regulation, even in the absence of glucose or a metabolite. HPr(Ser-P) appears to convert the lactose/H+ symporter into a sugar uniporter. These results establish that HPr serine phosphorylation by the ATP-dependent metabolite-activated HPr kinase regulates lactose permease activity in L. brevis. A direct allosteric mechanism is proposed. Images PMID:8159711

  10. Tyrosine Phosphorylation Based Homo-dimerization of Arabidopsis RACK1A Proteins Regulates Oxidative Stress Signaling Pathways in Yeast

    PubMed Central

    Sabila, Mercy; Kundu, Nabanita; Smalls, Deana; Ullah, Hemayet

    2016-01-01

    Scaffold proteins are known as important cellular regulators that can interact with multiple proteins to modulate diverse signal transduction pathways. RACK1 (Receptor for Activated C Kinase 1) is a WD-40 type scaffold protein, conserved in eukaryotes, from Chlamydymonas to plants and humans, plays regulatory roles in diverse signal transduction and stress response pathways. RACK1 in humans has been implicated in myriads of neuropathological diseases including Alzheimer and alcohol addictions. Model plant Arabidopsis thaliana genome maintains three different RACK1 genes termed RACK1A, RACK1B, and RACK1C with a very high (85–93%) sequence identity among them. Loss of function mutation in Arabidopsis indicates that RACK1 proteins regulate diverse environmental stress signaling pathways including drought and salt stress resistance pathway. Recently deduced crystal structure of Arabidopsis RACK1A- very first among all of the RACK1 proteins, indicates that it can potentially be regulated by post-translational modifications, like tyrosine phosphorylations and sumoylation at key residues. Here we show evidence that RACK1A proteins, depending on diverse environmental stresses, are tyrosine phosphorylated. Utilizing site-directed mutagenesis of key tyrosine residues, it is found that tyrosine phosphorylation can potentially dictate the homo-dimerization of RACK1A proteins. The homo-dimerized RACK1A proteins play a role in providing UV-B induced oxidative stress resistance. It is proposed that RACK1A proteins ability to function as scaffold protein may potentially be regulated by the homo-dimerized RACK1A proteins to mediate diverse stress signaling pathways. PMID:26941753

  11. Tyrosine Phosphorylation Based Homo-dimerization of Arabidopsis RACK1A Proteins Regulates Oxidative Stress Signaling Pathways in Yeast.

    PubMed

    Sabila, Mercy; Kundu, Nabanita; Smalls, Deana; Ullah, Hemayet

    2016-01-01

    Scaffold proteins are known as important cellular regulators that can interact with multiple proteins to modulate diverse signal transduction pathways. RACK1 (Receptor for Activated C Kinase 1) is a WD-40 type scaffold protein, conserved in eukaryotes, from Chlamydymonas to plants and humans, plays regulatory roles in diverse signal transduction and stress response pathways. RACK1 in humans has been implicated in myriads of neuropathological diseases including Alzheimer and alcohol addictions. Model plant Arabidopsis thaliana genome maintains three different RACK1 genes termed RACK1A, RACK1B, and RACK1C with a very high (85-93%) sequence identity among them. Loss of function mutation in Arabidopsis indicates that RACK1 proteins regulate diverse environmental stress signaling pathways including drought and salt stress resistance pathway. Recently deduced crystal structure of Arabidopsis RACK1A- very first among all of the RACK1 proteins, indicates that it can potentially be regulated by post-translational modifications, like tyrosine phosphorylations and sumoylation at key residues. Here we show evidence that RACK1A proteins, depending on diverse environmental stresses, are tyrosine phosphorylated. Utilizing site-directed mutagenesis of key tyrosine residues, it is found that tyrosine phosphorylation can potentially dictate the homo-dimerization of RACK1A proteins. The homo-dimerized RACK1A proteins play a role in providing UV-B induced oxidative stress resistance. It is proposed that RACK1A proteins ability to function as scaffold protein may potentially be regulated by the homo-dimerized RACK1A proteins to mediate diverse stress signaling pathways.

  12. Site-directed spectroscopy of cardiac myosin-binding protein C reveals effects of phosphorylation on protein structural dynamics

    PubMed Central

    Colson, Brett A.; Thompson, Andrew R.; Espinoza-Fonseca, L. Michel; Thomas, David D.

    2016-01-01

    We have used the site-directed spectroscopies of time-resolved fluorescence resonance energy transfer (TR-FRET) and double electron–electron resonance (DEER), combined with complementary molecular dynamics (MD) simulations, to resolve the structure and dynamics of cardiac myosin-binding protein C (cMyBP-C), focusing on the N-terminal region. The results have implications for the role of this protein in myocardial contraction, with particular relevance to β-adrenergic signaling, heart failure, and hypertrophic cardiomyopathy. N-terminal cMyBP-C domains C0–C2 (C0C2) contain binding regions for potential interactions with both thick and thin filaments. Phosphorylation by PKA in the MyBP-C motif regulates these binding interactions. Our spectroscopic assays detect distances between pairs of site-directed probes on cMyBP-C. We engineered intramolecular pairs of labeling sites within cMyBP-C to measure, with high resolution, the distance and disorder in the protein’s flexible regions using TR-FRET and DEER. Phosphorylation reduced the level of molecular disorder and the distribution of C0C2 intramolecular distances became more compact, with probes flanking either the motif between C1 and C2 or the Pro/Ala-rich linker (PAL) between C0 and C1. Further insight was obtained from microsecond MD simulations, which revealed a large structural change in the disordered motif region in which phosphorylation unmasks the surface of a series of residues on a stable α-helix within the motif with high potential as a protein–protein interaction site. These experimental and computational findings elucidate structural transitions in the flexible and dynamic portions of cMyBP-C, providing previously unidentified molecular insight into the modulatory role of this protein in cardiac muscle contractility. PMID:26908877

  13. Site-directed spectroscopy of cardiac myosin-binding protein C reveals effects of phosphorylation on protein structural dynamics.

    PubMed

    Colson, Brett A; Thompson, Andrew R; Espinoza-Fonseca, L Michel; Thomas, David D

    2016-03-22

    We have used the site-directed spectroscopies of time-resolved fluorescence resonance energy transfer (TR-FRET) and double electron-electron resonance (DEER), combined with complementary molecular dynamics (MD) simulations, to resolve the structure and dynamics of cardiac myosin-binding protein C (cMyBP-C), focusing on the N-terminal region. The results have implications for the role of this protein in myocardial contraction, with particular relevance to β-adrenergic signaling, heart failure, and hypertrophic cardiomyopathy. N-terminal cMyBP-C domains C0-C2 (C0C2) contain binding regions for potential interactions with both thick and thin filaments. Phosphorylation by PKA in the MyBP-C motif regulates these binding interactions. Our spectroscopic assays detect distances between pairs of site-directed probes on cMyBP-C. We engineered intramolecular pairs of labeling sites within cMyBP-C to measure, with high resolution, the distance and disorder in the protein's flexible regions using TR-FRET and DEER. Phosphorylation reduced the level of molecular disorder and the distribution of C0C2 intramolecular distances became more compact, with probes flanking either the motif between C1 and C2 or the Pro/Ala-rich linker (PAL) between C0 and C1. Further insight was obtained from microsecond MD simulations, which revealed a large structural change in the disordered motif region in which phosphorylation unmasks the surface of a series of residues on a stable α-helix within the motif with high potential as a protein-protein interaction site. These experimental and computational findings elucidate structural transitions in the flexible and dynamic portions of cMyBP-C, providing previously unidentified molecular insight into the modulatory role of this protein in cardiac muscle contractility.

  14. Phosphorylation of Complexin by PKA Regulates Activity-dependent Spontaneous Neurotransmitter Release and Structural Synaptic Plasticity

    PubMed Central

    Cho, Richard W.; Buhl, Lauren K.; Volfson, Dina; Tran, Adrienne; Li, Feng; Akbergenova, Yulia; Littleton, J. Troy

    2016-01-01

    Summary Synaptic plasticity is a fundamental feature of the nervous system that allows adaptation to changing behavioral environments. Most studies of synaptic plasticity have examined the regulated trafficking of postsynaptic glutamate receptors that generates alterations in synaptic transmission. Whether and how changes in the presynaptic release machinery contribute to neuronal plasticity is less clear. The SNARE complex mediates neurotransmitter release in response to presynaptic Ca++ entry. Here we show that the SNARE fusion clamp Complexin undergoes activity-dependent phosphorylation that alters the basic properties of neurotransmission in Drosophila. Retrograde signaling following stimulation activates PKA-dependent phosphorylation of the Complexin C-terminus that selectively and transiently enhances spontaneous release. Enhanced spontaneous release is required for activity-dependent synaptic growth. These data indicate that SNARE-dependent fusion mechanisms can be regulated in an activity-dependent manner and highlight the key role of spontaneous neurotransmitter release as a mediator of functional and structural plasticity. PMID:26590346

  15. Tyrosine phosphorylation on spleen tyrosine kinase (Syk) is differentially regulated in human and murine platelets by protein kinase C isoforms.

    PubMed

    Buitrago, Lorena; Bhavanasi, Dheeraj; Dangelmaier, Carol; Manne, Bhanu Kanth; Badolia, Rachit; Borgognone, Alessandra; Tsygankov, Alexander Y; McKenzie, Steven E; Kunapuli, Satya P

    2013-10-04

    Protein kinase C (PKC) isoforms differentially regulate platelet functional responses downstream of glycoprotein VI (GPVI) signaling, but the role of PKCs regulating upstream effectors such as Syk is not known. We investigated the role of PKC on Syk tyrosine phosphorylation using the pan-PKC inhibitor GF109203X (GFX). GPVI-mediated phosphorylation on Syk Tyr-323, Tyr-352, and Tyr-525/526 was rapidly dephosphorylated, but GFX treatment inhibited this dephosphorylation on Tyr-525/526 in human platelets but not in wild type murine platelets. GFX treatment did not affect tyrosine phosphorylation on FcRγ chain or Src family kinases. Phosphorylation of Lat Tyr-191 and PLCγ2 Tyr-759 was also increased upon treatment with GFX. We evaluated whether secreted ADP is required for such dephosphorylation. Exogenous addition of ADP to GFX-treated platelets did not affect tyrosine phosphorylation on Syk. FcγRIIA- or CLEC-2-mediated Syk tyrosine phosphorylation was also potentiated with GFX in human platelets. Because potentiation of Syk phosphorylation is not observed in murine platelets, PKC-deficient mice cannot be used to identify the PKC isoform regulating Syk phosphorylation. We therefore used selective inhibitors of PKC isoforms. Only PKCβ inhibition resulted in Syk hyperphosphorylation similar to that in platelets treated with GFX. This result indicates that PKCβ is the isoform responsible for Syk negative regulation in human platelets. In conclusion, we have elucidated a novel pathway of Syk regulation by PKCβ in human platelets.

  16. Mitotic protein kinase CDK1 phosphorylation of mRNA translation regulator 4E-BP1 Ser83 may contribute to cell transformation

    SciTech Connect

    Velásquez, Celestino; Cheng, Erdong; Shuda, Masahiro; Lee-Oesterreich, Paula J.; Pogge von Strandmann, Lisa; Gritsenko, Marina A.; Jacobs, Jon M.; Moore, Patrick S.; Chang, Yuan

    2016-07-11

    mTOR-directed 4E-BP1 phosphorylation promotes cap-dependent translation and tumorigen-esis. During mitosis, CDK1 substitutes for mTOR and fully phosphorylates 4E-BP1 at canoni-cal as well a non-canonical S83 site resulting in a mitosis-specific hyperphosphorylated δ isoform. Colocalization studies with a phospho-S83 specific antibody indicate that 4E-BP1 S83 phosphorylation accumulates at centrosomes during prophase, peaks at metaphase, and decreases through telophase. While S83 phosphorylation of 4E-BP1 does not affect in vitro cap-dependent translation, nor eIF4G/4E-BP1 cap-binding, expression of an alanine substitution mutant 4E-BP1.S83A partially reverses rodent cell transformation induced by Merkel cell polyomavirus (MCV) small T (sT) antigen viral oncoprotein. In contrast to inhibitory mTOR 4E-BP1 phosphorylation, these findings suggest that mitotic CDK1-directed phosphorylation of δ-4E-BP1 may yield a gain-of-function, distinct from translation regulation, that may be important in tumorigenesis and mitotic centrosome function.

  17. Mitotic protein kinase CDK1 phosphorylation of mRNA translation regulator 4E-BP1 Ser83 may contribute to cell transformation

    PubMed Central

    Velásquez, Celestino; Cheng, Erdong; Shuda, Masahiro; Lee-Oesterreich, Paula J.; Pogge von Strandmann, Lisa; Gritsenko, Marina A.; Jacobs, Jon M.; Moore, Patrick S.; Chang, Yuan

    2016-01-01

    Mammalian target of rapamycin (mTOR)-directed eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) phosphorylation promotes cap-dependent translation and tumorigenesis. During mitosis, cyclin-dependent kinase 1 (CDK1) substitutes for mTOR and fully phosphorylates 4E-BP1 at canonical sites (T37, T46, S65, and T70) and the noncanonical S83 site, resulting in a mitosis-specific hyperphosphorylated δ isoform. Colocalization studies with a phospho-S83 specific antibody indicate that 4E-BP1 S83 phosphorylation accumulates at centrosomes during prophase, peaks at metaphase, and decreases through telophase. Although S83 phosphorylation of 4E-BP1 does not affect general cap-dependent translation, expression of an alanine substitution mutant 4E-BP1.S83A partially reverses rodent cell transformation induced by Merkel cell polyomavirus small T antigen viral oncoprotein. In contrast to inhibitory mTOR 4E-BP1 phosphorylation, these findings suggest that mitotic CDK1-directed phosphorylation of δ-4E-BP1 may yield a gain of function, distinct from translation regulation, that may be important in tumorigenesis and mitotic centrosome function. PMID:27402756

  18. Cardiac myosin binding protein-C Ser302 phosphorylation regulates cardiac β-adrenergic reserve

    PubMed Central

    Mamidi, Ranganath; Gresham, Kenneth S.; Li, Jiayang; Stelzer, Julian E.

    2017-01-01

    Phosphorylation of cardiac myosin binding protein-C (MyBP-C) modulates cardiac contractile function; however, the specific roles of individual serines (Ser) within the M-domain that are targets for β-adrenergic signaling are not known. Recently, we demonstrated that significant accelerations in in vivo pressure development following β-agonist infusion can occur in transgenic (TG) mouse hearts expressing phospho-ablated Ser282 (that is, TGS282A) but not in hearts expressing phospho-ablation of all three serines [that is, Ser273, Ser282, and Ser302 (TG3SA)], suggesting an important modulatory role for other Ser residues. In this regard, there is evidence that Ser302 phosphorylation may be a key contributor to the β-agonist–induced positive inotropic responses in the myocardium, but its precise functional role has not been established. Thus, to determine the in vivo and in vitro functional roles of Ser302 phosphorylation, we generated TG mice expressing nonphosphorylatable Ser302 (that is, TGS302A). Left ventricular pressure-volume measurements revealed that TGS302A mice displayed no accelerations in the rate of systolic pressure rise and an inability to maintain systolic pressure following dobutamine infusion similar to TG3SA mice, implicating Ser302 phosphorylation as a critical regulator of enhanced systolic performance during β-adrenergic stress. Dynamic strain–induced cross-bridge (XB) measurements in skinned myocardium isolated from TGS302A hearts showed that the molecular basis for impaired β-adrenergic–mediated enhancements in systolic function is due to the absence of protein kinase A–mediated accelerations in the rate of cooperative XB recruitment. These results demonstrate that Ser302 phosphorylation regulates cardiac contractile reserve by enhancing contractile responses during β-adrenergic stress. PMID:28345052

  19. Regulation of BRCA1 Function by DNA Damage-Induced Site-Specific Phosphorylation

    DTIC Science & Technology

    2005-06-01

    Rad51p proteins, the lat - end-joining. BRCA-2 complexed with machinery. ter a member of the RAD-52 epi- RAD-51 is active in strand exchange BRCA- 1 has...AD Award Number: DAMD17-02- 1 -0584 TITLE: Regulation of BRCAl Function by DNA Damage-Induced Site- Specific Phosphorylation PRINCIPAL INVESTIGATOR...ApprovedREPORT DOCUMENTATION PAGE OMB No. 0704-0188 Public reporting burden for this collection of Information Is estimated to average 1 hour per

  20. Phosphorylation of the Kinase Domain Regulates Autophosphorylation of Myosin IIIA and Its Translocation in Microvilli

    PubMed Central

    2015-01-01

    Motor activity of myosin III is regulated by autophosphorylation. To investigate the role of the kinase activity on the transporter function of myosin IIIA (Myo3A), we identified the phosphorylation sites of kinase domain (KD), which is responsible for the regulation of kinase activity and thus motor function. Using mass spectrometry, we identified six phosphorylation sites in the KD, which are highly conserved among class III myosins and Ste20-related misshapen (Msn) kinases. Two predominant sites, Thr184 and Thr188, in KD are important for phosphorylation of the KD as well as the motor domain, which regulates the affinity for actin. In the Caco2 cells, the full-length human Myo3A (hMyo3AFull) markedly enlarged the microvilli, although it did not show discrete localization within the microvilli. On the other hand, hMyo3AFull(T184A) and hMyo3AFull(T188A) both showed clear localization at the microvilli tips. Our results suggest that Myo3A induces large actin bundle formation to form microvilli, and phosphorylation of KD at Thr184 and Thr188 is critical for the kinase activity of Myo3A, and regulation of Myo3A translocation to the tip of microvilli. Retinal extracts potently dephosphorylate both KD and motor domain without IQ motifs (MDIQo), which was inhibited by okadaic acid (OA) with nanomolar range and by tautomycetin (TMC) with micromolar range. The results suggest that Myo3A phosphatase is protein phosphatase type 2A (PP2A). Supporting this result, recombinant PP2Ac potently dephosphorylates both KD and MDIQo. We propose that the phosphorylation–dephosphorylation mechanism plays an essential role in mediating the transport and actin bundle formation and stability functions of hMyo3A. PMID:25402663

  1. Extracellular Signal-regulated Kinases (ERKs) Phosphorylate Lin28a Protein to Modulate P19 Cell Proliferation and Differentiation.

    PubMed

    Liu, Xiangyuan; Chen, Min; Li, Long; Gong, Liyan; Zhou, Hu; Gao, Daming

    2017-03-10

    Lin28a, originally discovered in the nematode Caenorhabditis elegans and highly conserved across species, is a well characterized regulator of let-7 microRNA (miRNA) and is implicated in cell proliferation and pluripotency control. However, little is known about how Lin28a function is modulated at the post-translational level and thereby responds to major signaling pathways. Here we show that Lin28a is directly phosphorylated by ERK1/2 kinases at Ser-200. By editing lin28a gene with the CRISPR/Cas9-based method, we generated P19 mouse embryonic carcinoma stem cells expressing Lin28a-S200A (phospho-deficient) and Lin28a-S200D (phospho-mimetic) mutants, respectively, to study the functional impact of Ser-200 phosphorylation. Lin28a-S200D-expressing cells, but not Lin28a-S200A-expressing or control P19 embryonic carcinoma cells, displayed impaired inhibition of let-7 miRNA and resulted in decreased cyclin D1, whereas Lin28a-S200A knock-in cells expressed less let-7 miRNA, proliferated faster, and exhibited differentiation defect upon retinoic acid induction. Therefore our results support that ERK kinase-mediated Lin28a phosphorylation may be an important mechanism for pluripotent cells to facilitate the escape from the self-renewal cycle and start the differentiation process.

  2. Chronic fluoxetine treatment directs energy metabolism towards the citric acid cycle and oxidative phosphorylation in rat hippocampal nonsynaptic mitochondria.

    PubMed

    Filipović, Dragana; Costina, Victor; Perić, Ivana; Stanisavljević, Andrijana; Findeisen, Peter

    2017-03-15

    Fluoxetine (Flx) is the principal treatment for depression; however, the precise mechanisms of its actions remain elusive. Our aim was to identify protein expression changes within rat hippocampus regulated by chronic Flx treatment versus vehicle-controls using proteomics. Fluoxetine-hydrohloride (15mg/kg) was administered daily to adult male Wistar rats for 3weeks, and cytosolic and nonsynaptic mitochondrial hippocampal proteomes were analyzed. All differentially expressed proteins were functionally annotated according to biological process and molecular function using Uniprot and Blast2GO. Our comparative study revealed that in cytosolic and nonsynaptic mitochondrial fractions, 60 and 3 proteins respectively, were down-regulated, and 23 and 60 proteins, respectively, were up-regulated. Proteins differentially regulated in cytosolic and nonsynaptic mitochondrial fractions were primarily related to cellular and metabolic processes. Of the identified proteins, the expressions of calretinin and parvalbumine were confirmed. The predominant molecular functions of differentially expressed proteins in both cell hippocampal fractions were binding and catalytic activity. Most differentially expressed proteins in nonsynaptic mitochondria were catalytic enzymes involved in the pyruvate metabolism, citric acid cycle, oxidative phosphorylation, ATP synthesis, ATP transduction and glutamate metabolism. Results indicate that chronic Flx treatment may influence proteins involved in calcium signaling, cytoskeletal structure, chaperone system and stimulates energy metabolism via the upregulation of GAPDH expression in cytoplasm, as well as directing energy metabolism toward the citric acid cycle and oxidative phosphorylation in nonsynaptic mitochondria. This approach provides new insight into the chronic effects of Flx treatment on protein expression in a key brain region associated with stress response and memory.

  3. NLK-mediated phosphorylation of HDAC1 negatively regulates Wnt signaling

    PubMed Central

    Masoumi, Katarzyna Chmielarska; Daams, Renée; Sime, Wondossen; Siino, Valentina; Ke, Hengning; Levander, Fredrik; Massoumi, Ramin

    2017-01-01

    The Wnt signaling pathway is essential in regulating various cellular processes. Different mechanisms of inhibition for Wnt signaling have been proposed. Besides β-catenin degradation through the proteasome, nemo-like kinase (NLK) is another molecule that is known to negatively regulate Wnt signaling. However, the mechanism by which NLK mediates the inhibition of Wnt signaling was not known. In the present study, we used primary embryonic fibroblast cells isolated from NLK-deficient mice and showed that these cells proliferate faster and have a shorter cell cycle than wild-type cells. In NLK-knockout cells, we observed sustained interaction between Lef1 and β-catenin, leading to elevated luciferase reporter of β-catenin/Lef1–mediated transcriptional activation. The mechanism for the reduced β-catenin/Lef1 promoter activation was explained by phosphorylation of HDAC1 at serine 421 via NLK. The phosphorylation of HDAC1 was achieved only in the presence of wild-type NLK because a catalytically inactive mutant of NLK was unable to phosphorylate HDAC1 and reduced the luciferase reporter of β-catenin/Lef1–mediated transcriptional activation. This result suggests that NLK and HDAC1 together negatively regulate Wnt signaling, which is vital in preventing aberrant proliferation of nontransformed primary fibroblast cells. PMID:27903773

  4. The heterocyst differentiation transcriptional regulator HetR of the filamentous cyanobacterium Anabaena forms tetramers and can be regulated by phosphorylation.

    PubMed

    Valladares, Ana; Flores, Enrique; Herrero, Antonia

    2016-02-01

    Many filamentous cyanobacteria respond to the external cue of nitrogen scarcity by the differentiation of heterocysts, cells specialized in the fixation of atmospheric nitrogen in oxic environments. Heterocysts follow a spatial pattern along the filament of two heterocysts separated by ca. 10-15 vegetative cells performing oxygenic photosynthesis. HetR is a transcriptional regulator that directs heterocyst differentiation. In the model strain Anabaena sp. PCC 7120, the HetR protein was observed in various oligomeric forms in vivo, including a tetramer that peaked with maximal hetR expression during differentiation. Tetramers were not detected in a hetR point mutant incapable of differentiation, but were conspicuous in an over-differentiating strain lacking the PatS inhibitor. In differentiated filaments the HetR tetramer was restricted to heterocysts, being undetectable in vegetative cells. HetR co-purified with RNA polymerase from Anabaena mainly as a tetramer. In vitro, purified recombinant HetR was distributed between monomers, dimers, trimers and tetramers, and it was phosphorylated when incubated with (γ-(32)P)ATP. Phosphorylation and PatS hampered the accumulation of HetR tetramers and impaired HetR binding to DNA. In summary, tetrameric HetR appears to represent a functionally relevant form of HetR, whose abundance in the Anabaena filament could be negatively regulated by phosphorylation and by PatS.

  5. Inhibition of CRMP2 phosphorylation repairs CNS by regulating neurotrophic and inhibitory responses.

    PubMed

    Nagai, Jun; Owada, Kazuki; Kitamura, Yoshiteru; Goshima, Yoshio; Ohshima, Toshio

    2016-03-01

    Central nervous system (CNS) regeneration is restricted by both the lack of neurotrophic responses and the presence of inhibitory factors. As of yet, a common mediator of these two pathways has not been identified. Microtubule dynamics is responsible for several key processes after CNS injuries: intracellular trafficking of receptors for neurotrophic factors, axonal retraction by inhibitory factors, and secondary tissue damages by inflammation and scarring. Kinases regulating microtubule organization, such as Cdk5 or GSK3β, may play pivotal roles during CNS recovery, but the molecular mechanisms remain to be elucidated. Collapsin response mediator protein 2 (CRMP2) stabilizes cytoskeletal polymerization, while CRMP2 phosphorylation by Cdk5 and GSK3β loses its affinity for cytoskeleton proteins, leading to the inhibition of axonal growth. Here, we characterized CRMP2 phosphorylation as the first crucial factor regulating neurotrophic and inhibitory responses after spinal cord injury (SCI). We found that pharmacological inhibition of GSK3β enhanced brain-derived neurotrophic factor (BDNF)-induced axonal growth response in cultured dorsal root ganglion (DRG) neurons. DRG neurons from CRMP2 knock-in (Crmp2KI/KI) mice, where CRMP2 phosphorylation was eliminated, showed elevated sensitivity to BDNF as well. Additionally, cultured Crmp2KI/KI neurons exhibited suppressed axonal growth inhibition by chondroitin sulfate proteoglycan (CSPG). These data suggest a couple of new molecular insights: the BDNF/GSK3β/CRMP2 and CSPG/GSK3β/CRMP2 pathways. Next, we tested the significance of CRMP2 phosphorylation after CNS injury in vivo. The phosphorylation level of CRMP2 was enhanced in the injured spinal cord. Crmp2KI/KI mice exhibited prominent recovery of locomotive and nociceptive functions after SCI, which correlated with the enhanced axonal growth of the motor and sensory neurons. Neuroprotective effects against SCI, such as microtubule stabilization, reduced inflammation

  6. Phosphorylation of Synaptojanin Differentially Regulates Endocytosis of Functionally Distinct Synaptic Vesicle Pools

    PubMed Central

    Geng, Junhua; Wang, Liping; Lee, Joo Yeun; Chen, Chun-Kan

    2016-01-01

    The rapid replenishment of synaptic vesicles through endocytosis is crucial for sustaining synaptic transmission during intense neuronal activity. Synaptojanin (Synj), a phosphoinositide phosphatase, is known to play an important role in vesicle recycling by promoting the uncoating of clathrin following synaptic vesicle uptake. Synj has been shown to be a substrate of the minibrain (Mnb) kinase, a fly homolog of the dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A); however, the functional impacts of Synj phosphorylation by Mnb are not well understood. Here we identify that Mnb phosphorylates Synj at S1029 in Drosophila. We find that phosphorylation of Synj at S1029 enhances Synj phosphatase activity, alters interaction between Synj and endophilin, and promotes efficient endocytosis of the active cycling vesicle pool (also referred to as exo-endo cycling pool) at the expense of reserve pool vesicle endocytosis. Dephosphorylated Synj, on the other hand, is deficient in the endocytosis of the active recycling pool vesicles but maintains reserve pool vesicle endocytosis to restore total vesicle pool size and sustain synaptic transmission. Together, our findings reveal a novel role for Synj in modulating reserve pool vesicle endocytosis and further indicate that dynamic phosphorylation and dephosphorylation of Synj differentially maintain endocytosis of distinct functional synaptic vesicle pools. SIGNIFICANCE STATEMENT Synaptic vesicle endocytosis sustains communication between neurons during a wide range of neuronal activities by recycling used vesicle membrane and protein components. Here we identify that Synaptojanin, a protein with a known role in synaptic vesicle endocytosis, is phosphorylated at S1029 in vivo by the Minibrain kinase. We further demonstrate that the phosphorylation status of Synaptojanin at S1029 differentially regulates its participation in the recycling of distinct synaptic vesicle pools. Our results reveal a new role for

  7. Serine 1179 Phosphorylation of Endothelial Nitric Oxide Synthase Increases Superoxide Generation and Alters Cofactor Regulation.

    PubMed

    Peng, Hu; Zhuang, Yugang; Harbeck, Mark C; He, Donghong; Xie, Lishi; Chen, Weiguo

    2015-01-01

    Endothelial nitric oxide synthase (eNOS) is responsible for maintaining systemic blood pressure, vascular remodeling and angiogenesis. In addition to producing NO, eNOS can also generate superoxide (O2-.) in the absence of the cofactor tetrahydrobiopterin (BH4). Previous studies have shown that bovine eNOS serine 1179 (Serine 1177/human) phosphorylation critically modulates NO synthesis. However, the effect of serine 1179 phosphorylation on eNOS superoxide generation is unknown. Here, we used the phosphomimetic form of eNOS (S1179D) to determine the effect of S1179 phosphorylation on superoxide generating activity, and its sensitivity to regulation by BH4, Ca2+, and calmodulin (CAM). S1179D eNOS exhibited significantly increased superoxide generating activity and NADPH consumption compared to wild-type eNOS (WT eNOS). The superoxide generating activities of S1179D eNOS and WT eNOS did not differ significantly in their sensitivity to regulation by either Ca2+ or CaM. The sensitivity of the superoxide generating activity of S1179D eNOS to inhibition by BH4 was significantly reduced compared to WT eNOS. In eNOS-overexpressing 293 cells, BH4 depletion with 10mM DAHP for 48 hours followed by 50ng/ml VEGF for 30 min to phosphorylate eNOS S1179 increased ROS accumulation compared to DAHP-only treated cells. Meanwhile, MTT assay indicated that overexpression of eNOS in HEK293 cells decreased cellular viability compared to control cells at BH4 depletion condition (P<0.01). VEGF-mediated Serine 1179 phosphorylation further decreased the cellular viability in eNOS-overexpressing 293 cells (P<0.01). Our data demonstrate that eNOS serine 1179 phosphorylation, in addition to enhancing NO production, also profoundly affects superoxide generation: S1179 phosphorylation increases superoxide production while decreasing sensitivity to the inhibitory effect of BH4 on this activity.

  8. Embryonic Stem Cell Growth Factors Regulate eIF2α Phosphorylation

    PubMed Central

    Friend, Kyle; Brooks, Hunter A.; Propson, Nicholas E.; Thomson, James A.; Kimble, Judith

    2015-01-01

    Growth factors and transcription factors are well known to regulate pluripotent stem cells, but less is known about translational control in stem cells. Here, we use embryonic stem cells (ESCs) to investigate a connection between ESC growth factors and eIF2α-mediated translational control (eIF2α phosphorylation promotes protein expression from mRNAs with upstream open-reading frames, or uORFs). We find abundant phosphorylated P-eIF2α (P-eIF2α) in both pluripotent mouse and human ESCs, but little P-eIF2α in ESCs triggered to differentiate. We show that the growth factors LIF (leukemia inhibitory factor) and BMP4 (bone morphogenic protein 4) both maintain P-eIF2α in mESCs, but use distinct mechanisms: LIF inhibits an eIF2α phosphatase whereas BMP4 activates an eIF2α kinase. The mRNAs encoding the pluripotency factors Nanog and c-Myc possess uORFs while Oct4 mRNA does not. We find that salubrinal, a chemical that increases eIF2α phosphorylation, promotes Nanog and c-Myc expression, but not Oct4 expression. These experiments connect ESC growth factors to eIF2α phosphorylation and suggest a chemical substitute for LIF to enhance Nanog and c-Myc expression. PMID:26406898

  9. Non-Canonical Role of IKKα in the Regulation of STAT1 Phosphorylation in Antiviral Signaling

    PubMed Central

    Xing, Fei; Matsumiya, Tomoh; Shiba, Yuko; Hayakari, Ryo; Yoshida, Hidemi; Imaizumi, Tadaatsu

    2016-01-01

    Non-self RNA is recognized by retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), inducing type I interferons (IFNs). Type I IFN promotes the expression of IFN-stimulated genes (ISGs), which requires the activation of signal transducer and activator of transcription-1 (STAT1). We previously reported that dsRNA induced STAT1 phosphorylation via a type I IFN-independent pathway in addition to the well-known type I IFN-dependent pathway. IκB kinase α (IKKα) is involved in antiviral signaling induced by dsRNA; however, its role is incompletely understood. Here, we explored the function of IKKα in RLR-mediated STAT1 phosphorylation. Silencing of IKKα markedly decreased the level of IFN-β and STAT1 phosphorylation inHeH response to dsRNA. However, the inhibition of IKKα did not alter the RLR signaling-mediated dimerization of interferon responsive factor 3 (IRF3) or the nuclear translocation of nuclear factor-κB (NFκB). These results suggest a non-canonical role of IKKα in RLR signaling. Furthermore, phosphorylation of STAT1 was suppressed by IKKα knockdown in cells treated with a specific neutralizing antibody for the type I IFN receptor (IFNAR) and in IFNAR-deficient cells. Collectively, the dual regulation of STAT1 by IKKα in antiviral signaling suggests a role for IKKα in the fine-tuning of antiviral signaling in response to non-self RNA. PMID:27992555

  10. The fission yeast meiotic checkpoint kinase Mek1 regulates nuclear localization of Cdc25 by phosphorylation.

    PubMed

    Pérez-Hidalgo, Livia; Moreno, Sergio; San-Segundo, Pedro A

    2008-12-01

    In eukaryotic cells, fidelity in transmission of genetic information during cell division is ensured by the action of cell cycle checkpoints. Checkpoints are surveillance mechanisms that arrest or delay cell cycle progression when critical cellular processes are defective or when the genome is damaged. During meiosis, the so-called meiotic recombination checkpoint blocks entry into meiosis I until recombination has been completed, thus avoiding aberrant chromosome segregation and the formation of aneuploid gametes. One of the key components of the meiotic recombination checkpoint is the meiosis-specific Mek1 kinase, which belongs to the family of Rad53/Cds1/Chk2 checkpoint kinases containing forkhead-associated domains. In fission yeast, several lines of evidence suggest that Mek1 targets the critical cell cycle regulator Cdc25 to delay meiotic cell cycle progression. Here, we investigate in more detail the molecular mechanism of action of the fission yeast Mek1 protein. We demonstrate that Mek1 acts independently of Cds1 to phosphorylate Cdc25, and this phosphorylation is required to trigger cell cycle arrest. Using ectopic overexpression of mek1(+) as a tool to induce in vivo activation of Mek1, we find that Mek1 promotes cytoplasmic accumulation of Cdc25 and results in prolonged phosphorylation of Cdc2 at tyrosine 15. We propose that at least one of the mechanisms contributing to the cell cycle delay when the meiotic recombination checkpoint is activated in fission yeast is the nuclear exclusion of the Cdc25 phosphatase by Mek1-dependent phosphorylation.

  11. SUMOylation of DNA topoisomerase IIα regulates histone H3 kinase Haspin and H3 phosphorylation in mitosis

    PubMed Central

    Yoshida, Makoto M.; Ting, Lily; Gygi, Steven P.

    2016-01-01

    DNA topoisomerase II (TOP2) plays a pivotal role in faithful chromosome separation through its strand-passaging activity that resolves tangled genomic DNA during mitosis. Additionally, TOP2 controls progression of mitosis by activating cell cycle checkpoints. Recent work showed that the enzymatically inert C-terminal domain (CTD) of TOP2 and its posttranslational modification are critical to this checkpoint regulation. However, the molecular mechanism has not yet been determined. By using Xenopus laevis egg extract, we found that SUMOylation of DNA topoisomerase IIα (TOP2A) CTD regulates the localization of the histone H3 kinase Haspin and phosphorylation of histone H3 at threonine 3 at the centromere, two steps known to be involved in the recruitment of the chromosomal passenger complex (CPC) to kinetochores in mitosis. Robust centromeric Haspin localization requires SUMOylated TOP2A CTD binding activity through SUMO-interaction motifs and the phosphorylation of Haspin. We propose a novel mechanism through which the TOP2 CTD regulates the CPC via direct interaction with Haspin at mitotic centromeres. PMID:27325792

  12. The Plastid Casein Kinase 2 Phosphorylates Rubisco Activase at the Thr-78 Site but Is Not Essential for Regulation of Rubisco Activation State

    PubMed Central

    Kim, Sang Y.; Bender, Kyle W.; Walker, Berkley J.; Zielinski, Raymond E.; Spalding, Martin H.; Ort, Donald R.; Huber, Steven C.

    2016-01-01

    Rubisco activase (RCA) is essential for the activation of Rubisco, the carboxylating enzyme of photosynthesis. In Arabidopsis, RCA is composed of a large RCAα and small RCAβ isoform that are formed by alternative splicing of a single gene (At2g39730). The activity of Rubisco is controlled in response to changes in irradiance by regulation of RCA activity, which is known to involve a redox-sensitive disulfide bond located in the carboxy-terminal extension of the RCAα subunit. Additionally, phosphorylation of RCA threonine-78 (Thr-78) has been reported to occur in the dark suggesting that phosphorylation may also be associated with dark-inactivation of RCA and deactivation of Rubisco. In the present study, we developed site-specific antibodies to monitor phosphorylation of RCA at the Thr-78 site and used non-reducing SDS-PAGE to monitor the redox status of the RCAα subunit. By immunoblotting, phosphorylation of both RCA isoforms occurred at low light and in the dark and feeding peroxide or DTT to leaf segments indicated that redox status of the chloroplast stroma was a critical factor controlling RCA phosphorylation. Use of a knockout mutant identified the plastid-targeted casein kinase 2 (cpCK2α) as the major protein kinase involved in RCA phosphorylation. Studies with recombinant cpCK2α and synthetic peptide substrates identified acidic residues at the –1, +2, and +3 positions surrounding Thr-78 as strong positive recognition elements. The cpck2 knockout mutant had strongly reduced phosphorylation at the Thr-78 site but was similar to wild type plants in terms of induction kinetics of photosynthesis following transfer from darkness or low light to high light, suggesting that if phosphorylation of RCA Thr-78 plays a direct role it would be redundant to redox regulation for control of Rubisco activation state under normal conditions. PMID:27064346

  13. G Protein-coupled Receptor Kinase-mediated Phosphorylation Regulates Post-endocytic Trafficking of the D2 Dopamine Receptor*S⃞

    PubMed Central

    Namkung, Yoon; Dipace, Concetta; Javitch, Jonathan A.; Sibley, David R.

    2009-01-01

    We investigated the role of G protein-coupled receptor kinase (GRK)-mediated phosphorylation in agonist-induced desensitization, arrestin association, endocytosis, and intracellular trafficking of the D2 dopamine receptor (DAR). Agonist activation of D2 DARs results in rapid and sustained receptor phosphorylation that is solely mediated by GRKs. A survey of GRKs revealed that only GRK2 or GRK3 promotes D2 DAR phosphorylation. Mutational analyses resulted in the identification of eight serine/threonine residues within the third cytoplasmic loop of the receptor that are phosphorylated by GRK2/3. Simultaneous mutation of these eight residues results in a receptor construct, GRK(-), that is completely devoid of agonist-promoted GRK-mediated receptor phosphorylation. We found that both wild-type (WT) and GRK(-) receptors underwent a similar degree of agonist-induced desensitization as assessed using [35S]GTPγS binding assays. Similarly, both receptor constructs internalized to the same extent in response to agonist treatment. Furthermore, using bioluminescence resonance energy transfer assays to directly assess receptor association with arrestin3, we found no differences between the WT and GRK(-) receptors. Thus, phosphorylation is not required for arrestin-receptor association or agonist-induced desensitization or internalization. In contrast, when we examined recycling of the D2 DARs to the cell surface, subsequent to agonist-induced endocytosis, the GRK(-) construct exhibited less recycling in comparison with the WT receptor. This impairment appears to be due to a greater propensity of the GRK(-) receptors to down-regulate once internalized. In contrast, if the receptor is highly phosphorylated, then receptor recycling is promoted. These results reveal a novel role for GRK-mediated phosphorylation in regulating the post-endocytic trafficking of a G protein-coupled receptor. PMID:19332542

  14. Phosphorylation of Rab11-FIP2 regulates polarity in MDCK cells

    PubMed Central

    Lapierre, Lynne A.; Avant, Kenya M.; Caldwell, Cathy M.; Oztan, Asli; Apodaca, Gerard; Knowles, Byron C.; Roland, Joseph T.; Ducharme, Nicole A.; Goldenring, James R.

    2012-01-01

    The Rab11 effector Rab11-family interacting protein 2 (Rab11-FIP2) regulates transcytosis through its interactions with Rab11a and myosin Vb. Previous studies implicated Rab11-FIP2 in the establishment of polarity in Madin–Darby canine kidney (MDCK) cells through phosphorylation of Ser-227 by MARK2. Here we examine the dynamic role of Rab11-FIP2 phosphorylation on MDCK cell polarity. Endogenous Rab11-FIP2 phosphorylated on Ser-227 coalesces on vesicular plaques during the reestablishment of polarity after either monolayer wounding or calcium switch. Whereas expression of the nonphosphorylatable Rab11-FIP2(S227A) elicits a loss in lumen formation in MDCK cell cysts grown in Matrigel, the putative pseudophosphorylated Rab11-FIP2(S227E) mutant induces the formation of cysts with multiple lumens. On permeable filters, Rab11-FIP2(S227E)–expressing cells exhibit alterations in the composition of both the adherens and tight junctions. At the adherens junction, p120 catenin and K-cadherin are retained, whereas the majority of the E-cadherin is lost. Although ZO-1 is retained at the tight junction, occludin is lost and the claudin composition is altered. Of interest, the effects of Rab11-FIP2 on cellular polarity did not involve myosin Vb or Rab11a. These results indicate that Ser-227 phosphorylation of Rab11-FIP2 regulates the composition of both adherens and tight junctions and is intimately involved in the regulation of polarity in epithelial cells. PMID:22553350

  15. Role of CBS and Bateman Domains in Phosphorylation-Dependent Regulation of a CLC Anion Channel.

    PubMed

    Yamada, Toshiki; Krzeminski, Mickael; Bozoky, Zoltan; Forman-Kay, Julie D; Strange, Kevin

    2016-11-01

    Eukaryotic CLC anion channels and transporters are homodimeric proteins composed of multiple α-helical membrane domains and large cytoplasmic C-termini containing two cystathionine-β-synthase domains (CBS1 and CBS2) that dimerize to form a Bateman domain. The Bateman domains of adjacent CLC subunits interact to form a Bateman domain dimer. The functions of CLC CBS and Bateman domains are poorly understood. We utilized the Caenorhabditis elegans CLC-1/2/Ka/Kb anion channel homolog CLH-3b to characterize the regulatory roles of CLC cytoplasmic domains. CLH-3b activity is reduced by phosphorylation or deletion of a 14-amino-acid activation domain (AD) located on the linker connecting CBS1 and CBS2. We demonstrate here that phosphorylation-dependent reductions in channel activity require an intact Bateman domain dimer and concomitant phosphorylation or deletion of both ADs. Regulation of a CLH-3b AD deletion mutant is reconstituted by intracellular perfusion with recombinant 14-amino-acid AD peptides. The sulfhydryl reactive reagent 2-(trimethylammonium)ethyl methanethiosulfonate bromide (MTSET) alters in a phosphorylation-dependent manner the activity of channels containing single cysteine residues that are engineered into the short intracellular loop connecting membrane α-helices H and I (H-I loop), the AD, CBS1, and CBS2. In contrast, MTSET has no effect on channels in which cysteine residues are engineered into intracellular regions that are dispensable for regulation. These studies together with our previous work suggest that binding and unbinding of the AD to the Bateman domain dimer induces conformational changes that are transduced to channel membrane domains via the H-I loop. Our findings provide new, to our knowledge, insights into the roles of CLC Bateman domains and the structure-function relationships that govern the regulation of CLC protein activity by diverse ligands and signaling pathways.

  16. Regulation of phenylalanine hydroxylase: conformational changes upon phosphorylation detected by H/D exchange and mass spectrometry.

    PubMed

    Li, Jun; Fitzpatrick, Paul F

    2013-07-15

    The enzyme phenylalanine hydroxylase catalyzes the hydroxylation of excess phenylalanine in the liver to tyrosine. The enzyme is regulated allosterically by phenylalanine and by phosphorylation of Ser16. Hydrogen/deuterium exchange monitored by mass spectrometry has been used to gain insight into any structural change upon phosphorylation. Peptides in both the catalytic and regulatory domains show increased deuterium incorporation into the phosphorylated protein. Deuterium is incorporated into fewer peptides than when the enzyme is activated by phenylalanine, and the incorporation is slower. This establishes that the conformational change upon phosphorylation of phenylalanine hydroxylase is different from and less extensive than that upon phenylalanine activation.

  17. KIF5C S176 Phosphorylation Regulates Microtubule Binding and Transport Efficiency in Mammalian Neurons

    PubMed Central

    Padzik, Artur; Deshpande, Prasannakumar; Hollos, Patrik; Franker, Mariella; Rannikko, Emmy H.; Cai, Dawen; Prus, Piotr; Mågård, Mats; Westerlund, Nina; Verhey, Kristen J.; James, Peter; Hoogenraad, Casper C.; Coffey, Eleanor T.

    2016-01-01

    Increased phosphorylation of the KIF5 anterograde motor is associated with impaired axonal transport and neurodegeneration, but paradoxically also with normal transport, though the details are not fully defined. JNK phosphorylates KIF5C on S176 in the motor domain; a site that we show is phosphorylated in brain. Microtubule pelleting assays demonstrate that phosphomimetic KIF5C(1-560)S176D associates weakly with microtubules compared to KIF5C(1-560)WT. Consistent with this, 50% of KIF5C(1-560)S176D shows diffuse movement in neurons. However, the remaining 50% remains microtubule bound and displays decreased pausing and increased bidirectional movement. The same directionality switching is observed with KIF5C(1-560)WT in the presence of an active JNK chimera, MKK7-JNK. Yet, in cargo trafficking assays where peroxisome cargo is bound, KIF5C(1-560)S176D-GFP-FRB transports normally to microtubule plus ends. We also find that JNK increases the ATP hydrolysis of KIF5C in vitro. These data suggest that phosphorylation of KIF5C-S176 primes the motor to either disengage entirely from microtubule tracks as previously observed in response to stress, or to display improved efficiency. The final outcome may depend on cargo load and motor ensembles. PMID:27013971

  18. A mutation of the fission yeast EB1 overcomes negative regulation by phosphorylation and stabilizes microtubules

    SciTech Connect

    Iimori, Makoto; Ozaki, Kanako; Chikashige, Yuji; Habu, Toshiyuki; Hiraoka, Yasushi; Maki, Takahisa; Hayashi, Ikuko; Obuse, Chikashi; Matsumoto, Tomohiro

    2012-02-01

    Mal3 is a fission yeast homolog of EB1, a plus-end tracking protein (+ TIP). We have generated a mutation (89R) replacing glutamine with arginine in the calponin homology (CH) domain of Mal3. Analysis of the 89R mutant in vitro has revealed that the mutation confers a higher affinity to microtubules and enhances the intrinsic activity to promote the microtubule-assembly. The mutant Mal3 is no longer a + TIP, but binds strongly the microtubule lattice. Live cell imaging has revealed that while the wild type Mal3 proteins dissociate from the tip of the growing microtubules before the onset of shrinkage, the mutant Mal3 proteins persist on microtubules and reduces a rate of shrinkage after a longer pausing period. Consequently, the mutant Mal3 proteins cause abnormal elongation of microtubules composing the spindle and aster. Mal3 is phosphorylated at a cluster of serine/threonine residues in the linker connecting the CH and EB1-like C-terminal motif domains. The phosphorylation occurs in a microtubule-dependent manner and reduces the affinity of Mal3 to microtubules. We propose that because the 89R mutation is resistant to the effect of phosphorylation, it can associate persistently with microtubules and confers a stronger stability of microtubules likely by reinforcing the cylindrical structure. -- Highlights: Black-Right-Pointing-Pointer We characterize a mutation (mal3-89R) in fission yeast homolog of EB1. Black-Right-Pointing-Pointer The mutation enhances the activity to assemble microtubules. Black-Right-Pointing-Pointer Mal3 is phosphorylated in a microtubule-dependent manner. Black-Right-Pointing-Pointer The phosphorylation negatively regulates the Mal3 activity.

  19. Regulation of Xenopus laevis DNA topoisomerase I activity by phosphorylation in vitro

    SciTech Connect

    Kaiserman, H.B.; Ingebritsen, T.S.; Benbow, R.M.

    1988-05-03

    DNA topoisomerase I has been purified to electrophoretic homogeneity from ovaries of the frog Xenopus laevis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most purified fraction revealed a single major band at 110 kDa and less abundant minor bands centered at 62 kDa. Incubation of the most purified fraction with immobilized calf intestinal alkaline phosphatase abolished all DNA topoisomerase enzymatic activity in a time-dependent reaction. Treatment of the dephosphorylated X. laevis DNA topoisomerase I with a X. laevis casein kinase type II activity and ATP restored DNA topoisomerase activity to a level higher than that observed in the most purified fraction. In vitro labeling experiments which employed the most purified DNA topoisomerase I fraction, (..gamma..-/sup 32/P)ATP, and the casein kinase type II enzyme showed that both the 110- and 62-kDa bands became phosphorylated in approximately molar proportions. Phosphoamino acid analysis showed that only serine residues became phosphorylated. Phosphorylation was accompanied by an increase in DNA topoisomerase activity in vitro. Dephosphorylation of DNA topoisomerase I appears to block formation of the initial enzyme-substrate complex on the basis of the failure of the dephosphorylated enzyme to nick DNA in the presence of camptothecin. The authors conclude that X. laevis DNA topoisomerase I is partially phosphorylated as isolated and that this phosphorylation is essential for expression of enzymatic activity in vitro. On the basis of the ability of the casein kinase type II activity to reactivate dephosphorylated DNA topoisomerase I, they speculate that this kinase may contribute to the physiological regulation of DNA topoisomerase I activity.

  20. Direct binding of MEK1 and MEK2 to AKT induces Foxo1 phosphorylation, cellular migration and metastasis

    PubMed Central

    Procaccia, Shiri; Ordan, Merav; Cohen, Izel; Bendetz-Nezer, Sarit; Seger, Rony

    2017-01-01

    Crosstalk between the ERK cascade and other signaling pathways is one of the means by which it acquires its signaling specificity. Here we identified a direct interaction of both MEK1 and MEK2 with AKT. The interaction is mediated by the proline rich domain of MEK1/2 and regulated by phosphorylation of Ser298 in MEK1, or Ser306 in MEK2, which we identified here as a novel regulatory site. We further developed a blocking peptide, which inhibits the interaction between MEK and AKT, and when applied to cells, affects migration and adhesion, but not proliferation. The specific mechanism of action of the MEK-AKT complex involves phosphorylation of the migration-related transcription factor FoxO1. Importantly, prevention of the interaction results in a decreased metastasis formation in a breast cancer mouse model. Thus, the identified interaction both sheds light on how signaling specificity is determined, and represents a possible new therapeutic target for metastatic cancer. PMID:28225038

  1. A Variable Active Site Residue Influences the Kinetics of Response Regulator Phosphorylation and Dephosphorylation.

    PubMed

    Immormino, Robert M; Silversmith, Ruth E; Bourret, Robert B

    2016-10-04

    Two-component regulatory systems, minimally composed of a sensor kinase and a response regulator protein, are common mediators of signal transduction in microorganisms. All response regulators contain a receiver domain with conserved active site residues that catalyze the signal activating and deactivating phosphorylation and dephosphorylation reactions. We explored the impact of variable active site position T+1 (one residue C-terminal to the conserved Thr/Ser) on reaction kinetics and signaling fidelity, using wild type and mutant Escherichia coli CheY, CheB, and NarL to represent the three major sequence classes observed across response regulators: Ala/Gly, Ser/Thr, and Val/Ile/Met, respectively, at T+1. Biochemical and structural data together suggested that different amino acids at T+1 impacted reaction kinetics by altering access to the active site while not perturbing overall protein structure. A given amino acid at position T+1 had similar effects on autodephosphorylation in each protein background tested, likely by modulating access of the attacking water molecule to the active site. Similarly, rate constants for CheY autophosphorylation with three different small molecule phosphodonors were consistent with the steric constraints on access to the phosphorylation site arising from combination of specific phosphodonors with particular amino acids at T+1. Because other variable active site residues also influence response regulator phosphorylation biochemistry, we began to explore how context (here, the amino acid at T+2) affected the influence of position T+1 on CheY autocatalytic reactions. Finally, position T+1 affected the fidelity and kinetics of phosphotransfer between sensor kinases and response regulators but was not a primary determinant of their interaction.

  2. Extracellular regulated kinase phosphorylates mitofusin 1 to control mitochondrial morphology and apoptosis.

    PubMed

    Pyakurel, Aswin; Savoia, Claudia; Hess, Daniel; Scorrano, Luca

    2015-04-16

    Controlled changes in mitochondrial morphology participate in cellular signaling cascades. However, the molecular mechanisms modifying mitochondrial shape are largely unknown. Here we show that the mitogen-activated protein (MAP) kinase cascade member extracellular-signal-regulated kinase (ERK) phosphorylates the pro-fusion protein mitofusin (MFN) 1, modulating its participation in apoptosis and mitochondrial fusion. Phosphoproteomic and biochemical analyses revealed that MFN1 is phosphorylated at an atypical ERK site in its heptad repeat (HR) 1 domain. This site proved essential to mediate MFN1-dependent mitochondrial elongation and apoptosis regulation by the MEK/ERK cascade. A mutant mimicking constitutive MFN1 phosphorylation was less efficient in oligomerizing and mitochondria tethering but bound more avidly to the proapoptotic BCL-2 family member BAK, facilitating its activation and cell death. Moreover, neuronal apoptosis following oxygen glucose deprivation and MEK/ERK activation required an intact MFN1(T562). Our data identify MFN1 as an ERK target to modulate mitochondrial shape and apoptosis.

  3. [Inhibition of NHE1 down-regulates IL-8 expression and enhances p38 phosphorylation].

    PubMed

    Gao, Wei; Zhang, Yu-Juan; Zhang, Hai-Rui; Jin, Wei-Na; Chang, Guo-Qiang; Zhang, Hong-Ju; Ma, Li; Lin, Ya-Ni; Li, Qing-Hua; Ru, Rong-Xin; Pang, Tian-Xiang

    2013-02-01

    This study was purposed to explore the changes of possible angiogenetic factors other than VEGF after inhibition of NHE1 and their related mechanisms. The K562 cells were treated by NHE1 specific inhibitor cariporide, the angiogenesis factors after inhibition of NHE1 were screened by using protein chip, the IL-8 expression level after cariporide treatment was detected by real-time quantitative PCR; the K562 cells with stable interference of NHE1 were constructed, the IL-8 expression level after interference of NHE1 was detected by real-time quantitative PCR; the p38 phosphorylation level in K562 cells treated with cariporide was detected by Western blot. After treatment of K562 cells with p38 inhibitor SB203580, the IL-8 expression level was decreased by real-time quantitative PCR. The results of protein chip showed that IL-8 expression decreased after cariporide treatment. Real-time quantitative PCR confirmed this inhibitory effect. The p38 phosphorylation level increased after cariporide treatment. The down-regulation of IL-8 expression induced by cariporide treatment was partially restored after K562 cells were treated with p38 inhibitor SB203580. It is concluded that the inhibition of NHE1 can inhibit IL-8 expression through up-regulation of p38 phosphorylation.

  4. mTORC1 phosphorylates UVRAG to negatively regulate autophagosome and endosome maturation.

    PubMed

    Kim, Young-Mi; Jung, Chang Hwa; Seo, Minchul; Kim, Eun Kyoung; Park, Ji-Man; Bae, Sun Sik; Kim, Do-Hyung

    2015-01-22

    mTORC1 plays a key role in autophagy as a negative regulator. The currently known targets of mTORC1 in the autophagy pathway mainly function at early stages of autophagosome formation. Here, we identify that mTORC1 inhibits later stages of autophagy by phosphorylating UVRAG. Under nutrient-enriched conditions, mTORC1 binds and phosphorylates UVRAG. The phosphorylation positively regulates the association of UVRAG with RUBICON, thereby enhancing the antagonizing effect of RUBICON on UVRAG-mediated autophagosome maturation. Upon dephosphorylation, UVRAG is released from RUBICON to interact with the HOPS complex, a component for the late endosome and lysosome fusion machinery, and enhances autophagosome and endosome maturation. Consequently, the dephosphorylation of UVRAG facilitates the lysosomal degradation of epidermal growth factor receptor (EGFR), reduces EGFR signaling, and suppresses cancer cell proliferation and tumor growth. These results demonstrate that mTORC1 engages in late stages of autophagy and endosome maturation, defining a broader range of mTORC1 functions in the membrane-associated processes.

  5. Casein kinase 1 α phosphorylates the Wnt regulator Jade-1 and modulates its activity.

    PubMed

    Borgal, Lori; Rinschen, Markus M; Dafinger, Claudia; Hoff, Sylvia; Reinert, Matthäus J; Lamkemeyer, Tobias; Lienkamp, Soeren S; Benzing, Thomas; Schermer, Bernhard

    2014-09-19

    Tight regulation of Wnt/β-catenin signaling is critical for vertebrate development and tissue maintenance, and deregulation can lead to a host of disease phenotypes, including developmental disorders and cancer. Proteins associated with primary cilia and centrosomes have been demonstrated to negatively regulate canonical Wnt signaling in interphase cells. The plant homeodomain zinc finger protein Jade-1 can act as an E3 ubiquitin ligase-targeting β-catenin for proteasomal degradation and concentrates at the centrosome and ciliary basal body in addition to the nucleus in interphase cells. We demonstrate that the destruction complex component casein kinase 1α (CK1α) phosphorylates Jade-1 at a conserved SLS motif and reduces the ability of Jade-1 to inhibit β-catenin signaling. Consistently, Jade-1 lacking the SLS motif is more effective than wild-type Jade-1 in reducing β-catenin-induced secondary axis formation in Xenopus laevis embryos in vivo. Interestingly, CK1α also phosphorylates β-catenin and the destruction complex component adenomatous polyposis coli at a similar SLS motif to the effect that β-catenin is targeted for degradation. The opposing effect of Jade-1 phosphorylation by CK1α suggests a novel example of the dual functions of CK1α activity to either oppose or promote canonical Wnt signaling in a context-dependent manner.

  6. Mad2 phosphorylation regulates its association with Mad1 and the APC/C

    PubMed Central

    Wassmann, Katja; Liberal, Vasco; Benezra, Robert

    2003-01-01

    Improper attachment of the mitotic spindle to the kinetochores of paired sister chromatids in mitosis is monitored by a checkpoint that leads to an arrest in early metaphase. This arrest requires the inhibitory association of Mad2 with the anaphase promoting complex/cyclosome (APC/C). It is not known how the association of Mad2 with the kinetochore and the APC/C is regulated in mitosis. Here, we demonstrate that human Mad2 is modified through phosphorylation on multiple serine residues in vivo in a cell cycle dependent manner and that only unphosphorylated Mad2 interacts with Mad1 or the APC/C in vivo. A Mad2 mutant containing serine to aspartic acid mutations mimicking the C-terminal phosphorylation events fails to interact with Mad1 or the APC/C and acts as a dominant-negative antagonist of wild-type Mad2. These data suggest that the phosphorylation state of Mad2 regulates its checkpoint activity by modulating its association with Mad1 and the APC/C. PMID:12574116

  7. Regulation of GABAergic synapse formation and plasticity by GSK3β-dependent phosphorylation of gephyrin

    PubMed Central

    Tyagarajan, Shiva K.; Ghosh, Himanish; Yévenes, Gonzalo E.; Nikonenko, Irina; Ebeling, Claire; Schwerdel, Cornelia; Sidler, Corinne; Zeilhofer, Hanns Ulrich; Gerrits, Bertran; Muller, Dominique; Fritschy, Jean-Marc

    2011-01-01

    Postsynaptic scaffolding proteins ensure efficient neurotransmission by anchoring receptors and signaling molecules in synapse-specific subcellular domains. In turn, posttranslational modifications of scaffolding proteins contribute to synaptic plasticity by remodeling the postsynaptic apparatus. Though these mechanisms are operant in glutamatergic synapses, little is known about regulation of GABAergic synapses, which mediate inhibitory transmission in the CNS. Here, we focused on gephyrin, the main scaffolding protein of GABAergic synapses. We identify a unique phosphorylation site in gephyrin, Ser270, targeted by glycogen synthase kinase 3β (GSK3β) to modulate GABAergic transmission. Abolishing Ser270 phosphorylation increased the density of gephyrin clusters and the frequency of miniature GABAergic postsynaptic currents in cultured hippocampal neurons. Enhanced, phosphorylation-dependent gephyrin clustering was also induced in vitro and in vivo with lithium chloride. Lithium is a GSK3β inhibitor used therapeutically as mood-stabilizing drug, which underscores the relevance of this posttranslational modification for synaptic plasticity. Conversely, we show that gephyrin availability for postsynaptic clustering is limited by Ca2+-dependent gephyrin cleavage by the cysteine protease calpain-1. Together, these findings identify gephyrin as synaptogenic molecule regulating GABAergic synaptic plasticity, likely contributing to the therapeutic action of lithium. PMID:21173228

  8. Phosphorylation of the Brome Mosaic Virus Capsid Regulates the Timing of Viral Infection

    PubMed Central

    Hoover, Haley S.; Wang, Joseph Che-Yen; Middleton, Stefani; Ni, Peng; Zlotnick, Adam

    2016-01-01

    ABSTRACT The four brome mosaic virus (BMV) RNAs (RNA1 to RNA4) are encapsidated in three distinct virions that have different disassembly rates in infection. The mechanism for the differential release of BMV RNAs from virions is unknown, since 180 copies of the same coat protein (CP) encapsidate each of the BMV genomic RNAs. Using mass spectrometry, we found that the BMV CP contains a complex pattern of posttranslational modifications. Treatment with phosphatase was found to not significantly affect the stability of the virions containing RNA1 but significantly impacted the stability of the virions that encapsidated BMV RNA2 and RNA3/4. Cryo-electron microscopy reconstruction revealed dramatic structural changes in the capsid and the encapsidated RNA. A phosphomimetic mutation in the flexible N-terminal arm of the CP increased BMV RNA replication and virion production. The degree of phosphorylation modulated the interaction of CP with the encapsidated RNA and the release of three of the BMV RNAs. UV cross-linking and immunoprecipitation methods coupled to high-throughput sequencing experiments showed that phosphorylation of the BMV CP can impact binding to RNAs in the virions, including sequences that contain regulatory motifs for BMV RNA gene expression and replication. Phosphatase-treated virions affected the timing of CP expression and viral RNA replication in plants. The degree of phosphorylation decreased when the plant hosts were grown at an elevated temperature. These results show that phosphorylation of the capsid modulates BMV infection. IMPORTANCE How icosahedral viruses regulate the release of viral RNA into the host is not well understood. The selective release of viral RNA can regulate the timing of replication and gene expression. Brome mosaic virus (BMV) is an RNA virus, and its three genomic RNAs are encapsidated in separate virions. Through proteomic, structural, and biochemical analyses, this work shows that posttranslational modifications

  9. Specific on-plate enrichment of phosphorylated peptides for direct MALDI-TOF MS analysis.

    PubMed

    Qiao, Liang; Roussel, Christophe; Wan, Jingjing; Yang, Pengyuan; Girault, Hubert H; Liu, Baohong

    2007-12-01

    An on-plate specific enrichment method is presented for the direct analysis of peptides phosphorylation. An array of sintered TiO 2 nanoparticle spots was prepared on a stainless steel plate to provide porous substrate with a very large specific surface and durable functions. These spots were used to selectively capture phosphorylated peptides from peptide mixtures, and the immobilized phosphopeptides could then be analyzed directly by MALDI MS after washing away the nonphosphorylated peptides. beta-Casein and protein mixtures were employed as model samples to investigate the selection efficiency. In this strategy, the steps of phosphopeptide capture, purification, and subsequent mass spectrometry analysis are all successfully accomplished on a single target plate, which greatly reduces sample loss and simplifies analytical procedures. The low detection limit, small sample size, and rapid selective entrapment show that this on-plate strategy is promising for online enrichment of phosphopeptides, which is essential for the analysis of minute amount of samples in high-throughput proteome research.

  10. ULK1 translocates to mitochondria and phosphorylates FUNDC1 to regulate mitophagy

    PubMed Central

    Wu, Wenxian; Tian, Weili; Hu, Zhe; Chen, Guo; Huang, Lei; Li, Wen; Zhang, Xingli; Xue, Peng; Zhou, Changqian; Liu, Lei; Zhu, Yushan; Zhang, Xingliang; Li, Longxuan; Zhang, Liangqing; Sui, Senfang; Zhao, Bin; Feng, Du

    2014-01-01

    Autophagy eliminates dysfunctional mitochondria in an intricate process known as mitophagy. ULK1 is critical for the induction of autophagy, but its substrate(s) and mechanism of action in mitophagy remain unclear. Here, we show that ULK1 is upregulated and translocates to fragmented mitochondria upon mitophagy induction by either hypoxia or mitochondrial uncouplers. At mitochondria, ULK1 interacts with FUNDC1, phosphorylating it at serine 17, which enhances FUNDC1 binding to LC3. A ULK1-binding-deficient mutant of FUNDC1 prevents ULK1 translocation to mitochondria and inhibits mitophagy. Finally, kinase-active ULK1 and a phospho-mimicking mutant of FUNDC1 rescue mitophagy in ULK1-null cells. Thus, we conclude that FUNDC1 regulates ULK1 recruitment to damaged mitochondria, where FUNDC1 phosphorylation by ULK1 is crucial for mitophagy. PMID:24671035

  11. ULK3 regulates cytokinetic abscission by phosphorylating ESCRT-III proteins.

    PubMed

    Caballe, Anna; Wenzel, Dawn M; Agromayor, Monica; Alam, Steven L; Skalicky, Jack J; Kloc, Magdalena; Carlton, Jeremy G; Labrador, Leticia; Sundquist, Wesley I; Martin-Serrano, Juan

    2015-05-26

    The endosomal sorting complexes required for transport (ESCRT) machinery mediates the physical separation between daughter cells during cytokinetic abscission. This process is regulated by the abscission checkpoint, a genome protection mechanism that relies on Aurora B and the ESCRT-III subunit CHMP4C to delay abscission in response to chromosome missegregation. In this study, we show that Unc-51-like kinase 3 (ULK3) phosphorylates and binds ESCRT-III subunits via tandem MIT domains, and thereby, delays abscission in response to lagging chromosomes, nuclear pore defects, and tension forces at the midbody. Our structural and biochemical studies reveal an unusually tight interaction between ULK3 and IST1, an ESCRT-III subunit required for abscission. We also demonstrate that IST1 phosphorylation by ULK3 is an essential signal required to sustain the abscission checkpoint and that ULK3 and CHMP4C are functionally linked components of the timer that controls abscission in multiple physiological situations.

  12. S-nitrosylation regulates VE-cadherin phosphorylation and internalization in microvascular permeability.

    PubMed

    Guequén, Anita; Carrasco, Rodrigo; Zamorano, Patricia; Rebolledo, Lorena; Burboa, Pia; Sarmiento, José; Boric, Mauricio P; Korayem, Adam; Durán, Walter N; Sánchez, Fabiola A

    2016-04-15

    The adherens junction complex, composed mainly of vascular endothelial (VE)-cadherin, β-catenin, p120, and γ-catenin, is the main element of the endothelial barrier in postcapillary venules.S-nitrosylation of β-catenin and p120 is an important step in proinflammatory agents-induced hyperpermeability. We investigated in vitro and in vivo whether or not VE-cadherin isS-nitrosylated using platelet-activating factor (PAF) as agonist. We report that PAF-stimulates S-nitrosylation of VE-cadherin, which disrupts its association with β-catenin. In addition, based on inhibition of nitric oxide production, our results strongly suggest that S-nitrosylation is required for VE-cadherin phosphorylation on tyrosine and for its internalization. Our results unveil an important mechanism to regulate phosphorylation of junctional proteins in association with S-nitrosylation.

  13. Mitosis-specific phosphorylation of PML at T409 regulates spindle checkpoint.

    PubMed

    Jin, J; Liu, J

    2016-08-31

    During mitosis, Promyelocytic leukemia nuclear bodies (PML NBs) change dramatically in morphology and composition, but little is known about function of PML in mitosis. Here, we show that PML is phosphorylated at T409 (PML p409) in a mitosis-specific manner. More importantly, PML p409 contributes to maintain the duration of pro-metaphase and regulates spindle checkpoint. Deficient PML p409 caused a shortening of pro-metaphase and challenged the nocodazole-triggered mitotic arrest. T409A mutation led to a higher frequency of misaligned chromosomes on metaphase plate, and subsequently death in late mitosis. In addition, inhibition of PML p409 repressed growth of tumor cells, suggesting that PML p409 is a potential target for cancer therapy. Collectively, our study demonstrated an important phosphorylated site of PML, which contributed to explore the role of PML in mitosis.

  14. Protein phosphorylation as a mechanism for regulation of spinach leaf sucrose-phosphate synthase activity

    SciTech Connect

    Huber, J.L.A.; Huber, S.C. )

    1989-04-01

    Protein phosphorylation has been identified as a mechanism for the light-dark regulation of spinach sucrose-phosphate synthase (SPS) activity, previously shown to involve some type of covalent modification of the enzyme. The 120 kD subunit of SPS in extracts of light-treated leaves was labeled with {sup 32}P in the presence of ({gamma}-{sup 32}P) ATP. In this in vitro system, {sup 32}P incorporation into light-activated SPS was dependent upon ATP and magnesium concentrations as well as time, and was closely paralleled by inactivation of the enzyme. The soluble protein kinase involved in the interconversion of SPS between activated and deactivated forms may be specific for SPS as it co-purifies with SPS during partial purification of the enzyme. The kinase appears not to be calcium activated and no evidence has been obtained for metabolite control of SPS phosphorylation/inactivation.

  15. Silica nanoparticles inhibit brown adipocyte differentiation via regulation of p38 phosphorylation

    NASA Astrophysics Data System (ADS)

    Son, Min Jeong; Kim, Won Kon; Kwak, Minjeong; Oh, Kyoung-Jin; Chang, Won Seok; Min, Jeong-Ki; Lee, Sang Chul; Song, Nam Woong; Bae, Kwang-Hee

    2015-10-01

    Nanoparticles are of great interest due to their wide variety of biomedical and bioengineering applications. However, they affect cellular differentiation and/or intracellular signaling when applied and exposed to target organisms or cells. The brown adipocyte is a cell type important in energy homeostasis and thus closely related to obesity. In this study, we assessed the effects of silica nanoparticles (SNPs) on brown adipocyte differentiation. The results clearly showed that brown adipocyte differentiation was significantly repressed by exposure to SNPs. The brown adipocyte-specific genes as well as mitochondrial content were also markedly reduced. Additionally, SNPs led to suppressed p38 phosphorylation during brown adipocyte differentiation. These effects depend on the size of SNPs. Taken together, these results lead us to suggest that SNP has anti-brown adipogenic effect in a size-dependent manner via regulation of p38 phosphorylation.

  16. Phosphorylation and dephosphorylation regulate APC/C(Cdh1) substrate degradation.

    PubMed

    Simpson-Lavy, Kobi J; Zenvirth, Drora; Brandeis, Michael

    2015-01-01

    The Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin ligase activated by its G1 specific adaptor protein Cdh1 is a major regulator of the cell cycle. The APC/C(Cdh1) mediates degradation of dozens of proteins, however, the kinetics and requirements for their degradation are largely unknown. We demonstrate that overexpression of the constitutive active CDH1(m11) mutant that is not inhibited by phosphorylation results in mitotic exit in the absence of the FEAR and MEN pathways, and DNA re-replication in the absence of Cdc7 activity. This mode of mitotic exit also reveals additional requirements for APC/C(Cdh1) substrate degradation, which for some substrates such as Pds1 or Clb5 is dephosphorylation, but for others such as Cdc5 is phosphorylation.

  17. Negative regulation of Jak2 by its auto-phosphorylation at tyrosine 913 via the Epo signaling pathway.

    PubMed

    Funakoshi-Tago, Megumi; Tago, Kenji; Kasahara, Tadashi; Parganas, Evan; Ihle, James N

    2008-11-01

    Janus kinase 2 (Jak2) has a pivotal role in erythropoietin (Epo) signaling pathway, including erythrocyte differentiation and Stat5 activation. In the course of screening for critical phosphorylation of tyrosine residues in Jak2, we identified tyrosine 913 (Y(913)) as a novel and functional phosphorylation site, which negatively regulates Jak2. Phosphorylation at Y(913) rapidly occurred and was sustained for at least 120 min after Epo stimulation, in contrast to the transient phosphorylation of Y(1007/1008) in the activation loop of Jak2. Interestingly, phosphorylation defective mutation of Y(913) (Y(913)F) results in a significant enhancement of Epo-induced Jak2 activation, whereas phosphorylation mimic mutation of Y(913) (Y(913)E) completely abrogated its activation. Furthermore, Jak2 deficient fetal liver cells expressing Y(913)F mutant generated many mature erythroid BFU-E and CFU-E colonies, while Y(913)E mutant failed to reconstitute Jak2 deficiency. We also demonstrate, in Jak1, phosphorylation of Y(939), a corresponding tyrosine residue with Y(913), negatively regulated Jak1 signaling pathway. Accordingly, our results suggest that this tyrosine phosphorylation in JH1 domain may be involved in common negative regulation mechanism for Jak family.

  18. Juvenile hormone prevents 20-hydroxyecdysone-induced metamorphosis by regulating the phosphorylation of a newly identified broad protein.

    PubMed

    Cai, Mei-Juan; Liu, Wen; Pei, Xu-Yang; Li, Xiang-Ru; He, Hong-Juan; Wang, Jin-Xing; Zhao, Xiao-Fan

    2014-09-19

    The steroid hormone 20-hydroxyecdysone (20E) initiates insect molting and metamorphosis. By contrast, juvenile hormone (JH) prevents metamorphosis. However, the mechanism by which JH inhibits metamorphosis remains unclear. In this study, we propose that JH induces the phosphorylation of Broad isoform Z7 (BrZ7), a newly identified protein, to inhibit 20E-mediated metamorphosis in the lepidopteran insect Helicoverpa armigera. The knockdown of BrZ7 in larvae inhibited metamorphosis by repressing the expression of the 20E response gene. BrZ7 was weakly expressed and phosphorylated during larval growth but highly expressed and non-phosphorylated during metamorphosis. JH regulated the rapid phosphorylation of BrZ7 via a G-protein-coupled receptor-, phospholipase C-, and protein kinase C-triggered pathway. The phosphorylated BrZ7 bound to the 5'-regulatory region of calponin to regulate its expression in the JH pathway. Exogenous JH induced BrZ7 phosphorylation to prevent metamorphosis by suppressing 20E-related gene transcription. JH promoted non-phosphorylated calponin interacting with ultraspiracle protein to activate the JH pathway and antagonize the 20E pathway. This study reveals one of the possible mechanisms by which JH counteracts 20E-regulated metamorphosis by inducing the phosphorylation of BrZ7.

  19. Regulation of pituitary tumor transforming gene (PTTG) expression and phosphorylation in thyroid cells.

    PubMed

    Lewy, Gregory D; Ryan, Gavin A; Read, Martin L; Fong, Jim C W; Poole, Vikki; Seed, Robert I; Sharma, Neil; Smith, Vicki E; Kwan, Perkin P K; Stewart, Sarah L; Bacon, Andrea; Warfield, Adrian; Franklyn, Jayne A; McCabe, Christopher J; Boelaert, Kristien

    2013-11-01

    Human pituitary tumor transforming gene (hPTTG) is a multifunctional proto-oncogene implicated in the initiation and progression of several tumors. Phosphorylation of hPTTG is mediated by cyclin-dependent kinase 2 (CDC2), whereas cellular expression is regulated by specificity protein 1 (SP1). The mechanisms underlying hPTTG propagation of aberrant thyroid cell growth have not been fully defined. We set out to investigate the interplay between hPTTG and growth factors, as well as the effects of phosphorylation and SP1 regulation on hPTTG expression and function. In our study, epidermal growth factor (EGF), TGFα, and IGF-1 induced hPTTG expression and phosphorylation in thyroid cells, which was associated with activation of MAPK and phosphoinositide 3-kinase. Growth factors induced hPTTG independently of CDC2 and SP1 in thyroid carcinoma cells. Strikingly, CDC2 depletion in TPC-1 cells resulted in enhanced expression and phosphorylation of hPTTG and reduced cellular proliferation. In reciprocal experiments, hPTTG overexpression induced EGF, IGF-1, and TGFα mRNAs in primary human thyrocytes. Treatment of primary human thyrocytes with conditioned media derived from hPTTG-transfected cells resulted in autocrine upregulation of hPTTG protein, which was ameliorated by growth factor depletion or growth factor receptor tyrosine kinase inhibitors. A transgenic murine model of thyroid targeted hPTTG overexpression (hPTTG-Tg) (FVB/N strain, both sexes) demonstrated smaller thyroids with reduced cellular proliferation and enhanced secretion of Egf. In contrast, Pttg(-/-) knockout mice (c57BL6 strain, both sexes) showed reduced thyroidal Egf mRNA expression. These results define hPTTG as having a central role in thyroid autocrine signaling mechanisms via growth factors, with profound implications for promotion of transformed cell growth.

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

    PubMed

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

    2003-11-01

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

  1. Tgf-beta induced Erk phosphorylation of smad linker region regulates smad signaling.

    PubMed

    Hough, Chris; Radu, Maria; Doré, Jules J E

    2012-01-01

    The Transforming Growth Factor-Beta (TGF-β) family is involved in regulating a variety of cellular processes such as apoptosis, differentiation, and proliferation. TGF-β binding to a Serine/Threonine kinase receptor complex causes the recruitment and subsequent activation of transcription factors known as smad2 and smad3. These proteins subsequently translocate into the nucleus to negatively or positively regulate gene expression. In this study, we define a second signaling pathway leading to TGF-β receptor activation of Extracellular Signal Regulated Kinase (Erk) in a cell-type dependent manner. TGF-β induced Erk activation was found in phenotypically normal mesenchymal cells, but not normal epithelial cells. By activating phosphotidylinositol 3-kinase (PI3K), TGF-β stimulates p21-activated kinase2 (Pak2) to phosphorylate c-Raf, ultimately resulting in Erk activation. Activation of Erk was necessary for TGF-β induced fibroblast replication. In addition, Erk phosphorylated the linker region of nuclear localized smads, resulting in increased half-life of C-terminal phospho-smad 2 and 3 and increased duration of smad target gene transcription. Together, these data show that in mesenchymal cell types the TGF-β/PI3K/Pak2/Raf/MEK/Erk pathway regulates smad signaling, is critical for TGF-β-induced growth and is part of an integrated signaling web containing multiple interacting pathways rather than discrete smad/non-smad pathways.

  2. The UL12 Protein of Herpes Simplex Virus 1 Is Regulated by Tyrosine Phosphorylation

    PubMed Central

    Fujii, Hikaru; Kato, Akihisa; Mugitani, Michio; Kashima, Yukie; Oyama, Masaaki; Kozuka-Hata, Hiroko; Arii, Jun

    2014-01-01

    ABSTRACT The herpes simplex virus 1 (HSV-1) UL12 protein (pUL12) is a nuclease that is critical for viral replication in vitro and neurovirulence in vivo. In this study, mass spectrometric analysis of pUL12 and phosphate-affinity SDS-polyacrylamide gel electrophoresis analysis identified tyrosine at pUL12 residue 371 (Tyr-371) as a pUL12 phosphorylation site: Tyr-371 is conserved in pUL12 homologs in herpesviruses in all Herpesviridae subfamilies. Replacement of Tyr-371 with phenylalanine (Y371F) in pUL12 (i) abolished its exonuclease activity in HSV-1-infected Vero, HEL, and A549 cells, (ii) reduced viral replication, cell-cell spread, and pUL12 expression in infected cells in a cell type-dependent manner, (iii) led to aberrant subcellular localization of pUL12 in infected cells in a cell type-dependent manner, and (iv) reduced HSV-1 neurovirulence in mice. The effects of the pUL12 Y371F mutation in cell cultures and mice were similar to those of a nuclease-dead double mutation in pUL12, although the Y371F mutation reduced viral replication severalfold more than the nuclease-dead double mutation in a cell type- and multiplicity-of-infection-dependent manner. Replacement of Tyr-371 with glutamic acid, which mimics constitutive phosphorylation, restored the wild-type phenotype in cell cultures and mice. These results suggested that phosphorylation of pUL12 Tyr-371 was essential for pUL12 to express its nuclease activity in HSV-1-infected cells and that this phosphorylation promoted viral replication and cell-cell spread in cell cultures and neurovirulence in mice mainly by upregulating pUL12 nuclease activity and, in part, by regulating the subcellular localization and expression of pUL12 in HSV-1-infected cells. IMPORTANCE Herpesviruses encode a considerable number of enzymes for their replication. Like cellular enzymes, the viral enzymes need to be properly regulated in infected cells. Although the functional aspects of herpesvirus enzymes have gradually been

  3. PLD1 regulates adipogenic differentiation through mTOR - IRS-1 phosphorylation at serine 636/639

    PubMed Central

    Song, Hae-In; Yoon, Mee-Sup

    2016-01-01

    Phospholipase D1 (PLD1) plays a known role in several differentiation processes, but its role in adipogenic differentiation remains unknown. In the present study, we identified PLD1 as a negative regulator of adipogenic differentiation. We showed that PLD activity was downregulated by both 3-Isobutyl-1-methylxanthine (IBMX) and insulin upon induction of differentiation in 3T3-L1 adipogenic cells. In line with this observation, PLD activity decreased in both high fat diet (HFD)-fed mice and ob/ob mice. We also found that differentiation of 3T3-L1 preadipocytes was enhanced by the depletion of PLD1 levels or inhibition of PLD1 activity by VU0155069, a PLD1-specific inhibitor. Conversely, treatment with phosphatidic acid (PA), a PLD product, and overexpression of PLD1 both caused a decrease in adipogenic differentiation. Moreover, the elevated differentiation in PLD1-knockdown 3T3-L1 cells was reduced by either PA treatment or PLD1 expression, confirming negative roles of PLD1 and PA in adipogenic differentiation. Further investigation revealed that PA displaces DEP domain-containing mTOR-interacting protein (DEPTOR) from mTORC1, which subsequently phosphorylates insulin receptor substrate-1 (IRS-1) at serine 636/639 in 3T3-L1 cells. Taken together, our findings provide convincing evidence for a direct role of PLD1 in adipogenic differentiation by regulating IRS-1 phosphorylation at serine 636/639 through DEPTOR displacement and mTOR activation. PMID:27872488

  4. Phosphorylation of Def Regulates Nucleolar p53 Turnover and Cell Cycle Progression through Def Recruitment of Calpain3

    PubMed Central

    Tao, Ting; Shi, Hui; Lo, Li Jan; Wang, Yingchun; Chen, Jun; Peng, Jinrong

    2016-01-01

    Digestive organ expansion factor (Def) is a nucleolar protein that plays dual functions: it serves as a component of the ribosomal small subunit processome for the biogenesis of ribosomes and also mediates p53 degradation through the cysteine proteinase calpain-3 (CAPN3). However, nothing is known about the exact relationship between Def and CAPN3 or the regulation of the Def function. In this report, we show that CAPN3 degrades p53 and its mutant proteins p53A138V, p53M237I, p53R248W, and p53R273P but not the p53R175H mutant protein. Importantly, we show that Def directly interacts with CAPN3 in the nucleoli and determines the nucleolar localisation of CAPN3, which is a prerequisite for the degradation of p53 in the nucleolus. Furthermore, we find that Def is modified by phosphorylation at five serine residues: S50, S58, S62, S87, and S92. We further show that simultaneous phosphorylations at S87 and S92 facilitate the nucleolar localisation of Capn3 that is not only essential for the degradation of p53 but is also important for regulating cell cycle progression. Hence, we propose that the Def-CAPN3 pathway serves as a nucleolar checkpoint for cell proliferation by selective inactivation of cell cycle-related substrates during organogenesis. PMID:27657329

  5. NF-κB-repressing factor phosphorylation regulates transcription elongation via its interactions with 5'→3' exoribonuclease 2 and negative elongation factor.

    PubMed

    Rother, Sascha; Bartels, Myriam; Schweda, Aike Torben; Resch, Klaus; Pallua, Norbert; Nourbakhsh, Mahtab

    2016-01-01

    NF-κB-repressing factor (NKRF) inhibits transcription elongation by binding to specific sequences in target promoters. Stimuli such as IL-1 have been shown to overcome this inhibitory action and enable the resumption of transcription elongation machinery by an unknown mechanism. Using mass spectrometry and in vitro phosphorylation analyses, we demonstrate that NKRF is phosphorylated within 3 different domains in unstimulated HeLa cells. Phosphoamino acid mapping and mutation analysis of NKRF further suggest that only Ser phosphorylation within aa 421-429 is regulated by IL-1 stimulation. In copurification studies, aa 421-429 is required for interactions between NKRF, 5'→3' exoribonuclease 2 (XRN2) and the negative elongation factor (NELF)-E in HeLa cells. Chromatin immunoprecipitation experiments further show that IL-1 stimulation leads to decrease in NKRF aa 421-429 phosphorylation and dissociation of NELF-E and XRN2 by concomitant resumption of transcription elongation of a synthetic reporter or the endogenous NKRF target gene, IL-8. Together, NKRF phosphorylation modulates promoter-proximal transcription elongation of NF-κB/NKRF-regulated genes via direct interactions with elongation complex in response to specific stimuli.

  6. Phosphorylation-regulated Binding of RNA Polymerase II to Fibrous Polymers of Low Complexity Domains

    PubMed Central

    Xiang, Siheng; Wu, Leeju; Theodoropoulos, Pano; Mirzaei, Hamid; Han, Tina; Xie, Shanhai; Corden, Jeffry L.; McKnight, Steven L.

    2014-01-01

    SUMMARY The low complexity (LC) domains of the products of the fused in sarcoma (FUS), Ewings sarcoma (EWS) and TAF15 genes are translocated onto a variety of different DNA-binding domains and thereby assist in driving the formation of cancerous cells. In the context of the translocated fusion proteins, these LC sequences function as transcriptional activation domains. Here we show that polymeric fibers formed from these LC domains directly bind the C-terminal domain (CTD) of RNA polymerase II in a manner reversible by phosphorylation of the iterated, heptad repeats of the CTD. Mutational analysis indicates that the degree of binding between the CTD and the LC domain polymers correlates with the strength of transcriptional activation. These studies offer a simple means of conceptualizing how RNA polymerase II is recruited to active genes in its unphosphorylated state, and released for elongation following phosphorylation of the CTD. PMID:24267890

  7. Cannabinoid WIN 55,212-2 regulates TRPV1 phosphorylation in sensory neurons.

    PubMed

    Jeske, Nathaniel A; Patwardhan, Amol M; Gamper, Nikita; Price, Theodore J; Akopian, Armen N; Hargreaves, Kenneth M

    2006-10-27

    Cannabinoids are known to have multiple sites of action in the nociceptive system, leading to reduced pain sensation. However, the peripheral mechanism(s) by which this phenomenon occurs remains an issue that has yet to be resolved. Because phosphorylation of TRPV1 (transient receptor potential subtype V1) plays a key role in the induction of thermal hyperalgesia in inflammatory pain models, we evaluated whether the cannabinoid agonist WIN 55,212-2 (WIN) regulates the phosphorylation state of TRPV1. Here, we show that treatment of primary rat trigeminal ganglion cultures with WIN led to dephosphorylation of TRPV1, specifically at threonine residues. Utilizing Chinese hamster ovary cell lines, we demonstrate that Thr(144) and Thr(370) were dephosphorylated, leading to desensitization of the TRPV1 receptor. This post-translational modification occurred through activation of the phosphatase calcineurin (protein phosphatase 2B) following WIN treatment. Furthermore, knockdown of TRPA1 (transient receptor potential subtype A1) expression in sensory neurons by specific small interfering RNA abolished the WIN effect on TRPV1 dephosphorylation, suggesting that WIN acts through TRPA1. We also confirm the importance of TRPA1 in WIN-induced dephosphorylation of TRPV1 in Chinese hamster ovary cells through targeted expression of one or both receptor channels. These results imply that the cannabinoid WIN modulates the sensitivity of sensory neurons to TRPV1 activation by altering receptor phosphorylation. In addition, our data could serve as a useful strategy in determining the potential use of certain cannabinoids as peripheral analgesics.

  8. Protein Kinase D Regulates RhoA Activity via Rhotekin Phosphorylation*

    PubMed Central

    Pusapati, Ganesh V.; Eiseler, Tim; Rykx, An; Vandoninck, Sandy; Derua, Rita; Waelkens, Etienne; Van Lint, Johan; von Wichert, Götz; Seufferlein, Thomas

    2012-01-01

    The members of the protein kinase D (PKD) family of serine/threonine kinases are major targets for tumor-promoting phorbol esters, G protein-coupled receptors, and activated protein kinase C isoforms (PKCs). The expanding list of cellular processes in which PKDs exert their function via phosphorylation of various substrates include proliferation, apoptosis, migration, angiogenesis, and vesicle trafficking. Therefore, identification of novel PKD substrates is necessary to understand the profound role of this kinase family in signal transduction. Here, we show that rhotekin, an effector of RhoA GTPase, is a novel substrate of PKD. We identified Ser-435 in rhotekin as the potential site targeted by PKD in vivo. Expression of a phosphomimetic S435E rhotekin mutant resulted in an increase of endogenous active RhoA GTPase levels. Phosphorylation of rhotekin by PKD2 modulates the anchoring of the RhoA in the plasma membrane. Consequently, the S435E rhotekin mutant displayed enhanced stress fiber formation when expressed in serum-starved fibroblasts. Our data thus identify a novel role of PKD as a regulator of RhoA activity and actin stress fiber formation through phosphorylation of rhotekin. PMID:22228765

  9. Phosphorylation of the Yeast γ-Tubulin Tub4 Regulates Microtubule Function

    PubMed Central

    Lin, Tien-chen; Gombos, Linda; Neuner, Annett; Sebastian, Dominik; Olsen, Jesper V.; Hrle, Ajla; Benda, Christian; Schiebel, Elmar

    2011-01-01

    The yeast γ-tubulin Tub4 is assembled with Spc97 and Spc98 into the small Tub4 complex. The Tub4 complex binds via the receptor proteins Spc72 and Spc110 to the spindle pole body (SPB), the functional equivalent of the mammalian centrosome, where the Tub4 complex organizes cytoplasmic and nuclear microtubules. Little is known about the regulation of the Tub4 complex. Here, we isolated the Tub4 complex with the bound receptors from yeast cells. Analysis of the purified Tub4 complex by mass spectrometry identified more than 50 phosphorylation sites in Spc72, Spc97, Spc98, Spc110 and Tub4. To examine the functional relevance of the phosphorylation sites, phospho-mimicking and non-phosphorylatable mutations in Tub4, Spc97 and Spc98 were analyzed. Three phosphorylation sites in Tub4 were found to be critical for Tub4 stability and microtubule organization. One of the sites is highly conserved in γ-tubulins from yeast to human. PMID:21573187

  10. Regulated phosphorylation of secretory granule membrane proteins of the rat parotid gland

    SciTech Connect

    Marino, C.R.; Castle, J.D.; Gorelick, F.S. )

    1990-07-01

    An antiserum raised against purified rat parotid secretory granule membrane proteins has been used to identify organelle-specific protein phosphorylation events following stimulation of intact cells from the rat parotid gland. After lobules were prelabeled with ({sup 32}P)orthophosphate and exposed to secretagogues, phosphoproteins were immunoprecipitated with the granule membrane protein antiserum, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized by autoradiography. Parallel studies of stimulated amylase release were performed. Isoproterenol treatment of parotid lobules resulted in an increase in the phosphate content of immunoprecipitable 60- and 72-kDa proteins that correlated with amylase release in a time-dependent manner. Forskolin addition mimicked these effects, but only the isoproterenol effects were reversed by propranolol treatment. To confirm the specificity of the antiserum to the secretory granule membrane fraction, subcellular isolation techniques were employed following in situ phosphorylation. The 60- and 72-kDa phosphoproteins were immunoprecipitated from both a particulate fraction and a purified secretory granule fraction. Furthermore, the extraction properties of both species suggest that they are integral membrane proteins. These findings support the possibility that stimulus-regulated secretion may involve phosphorylation of integral membrane proteins of the exocrine secretory granule.

  11. Aurora A phosphorylation of WD40-repeat protein 62 in mitotic spindle regulation

    PubMed Central

    Lim, Nicholas R.; Yeap, Yvonne Y. C.; Ang, Ching-Seng; Williamson, Nicholas A.; Bogoyevitch, Marie A.; Quinn, Leonie M.; Ng, Dominic C. H.

    2016-01-01

    ABSTRACT Mitotic spindle organization is regulated by centrosomal kinases that potentiate recruitment of spindle-associated proteins required for normal mitotic progress including the microcephaly protein WD40-repeat protein 62 (WDR62). WDR62 functions underlie normal brain development as autosomal recessive mutations and wdr62 loss cause microcephaly. Here we investigate the signaling interactions between WDR62 and the mitotic kinase Aurora A (AURKA) that has been recently shown to cooperate to control brain size in mice. The spindle recruitment of WDR62 is closely correlated with increased levels of AURKA following mitotic entry. We showed that depletion of TPX2 attenuated WDR62 localization at spindle poles indicating that TPX2 co-activation of AURKA is required to recruit WDR62 to the spindle. We demonstrated that AURKA activity contributed to the mitotic phosphorylation of WDR62 residues Ser49 and Thr50 and phosphorylation of WDR62 N-terminal residues was required for spindle organization and metaphase chromosome alignment. Our analysis of several MCPH-associated WDR62 mutants (V65M, R438H and V1314RfsX18) that are mislocalized in mitosis revealed that their interactions and phosphorylation by AURKA was substantially reduced consistent with the notion that AURKA is a key determinant of WDR62 spindle recruitment. Thus, our study highlights the role of AURKA signaling in the spatiotemporal control of WDR62 at spindle poles where it maintains spindle organization. PMID:26713495

  12. DYRK2 and GSK-3 phosphorylate and promote the timely degradation of OMA-1, a key regulator of the oocyte-to-embryo transition in C. elegans.

    PubMed

    Nishi, Yuichi; Lin, Rueyling

    2005-12-01

    Oocyte maturation and fertilization initiates a dynamic and tightly regulated process in which a non-dividing oocyte is transformed into a rapidly dividing embryo. We have shown previously that two C. elegans CCCH zinc finger proteins, OMA-1 and OMA-2, have an essential and redundant function in oocyte maturation. Both OMA-1 and OMA-2 are expressed only in oocytes and 1-cell embryos, and need to be degraded rapidly after the first mitotic division for embryogenesis to proceed normally. We report here a distinct redundant function for OMA-1 and OMA-2 in the 1-cell embryo. Depletion of both oma-1 and oma-2 in embryos leads to embryonic lethality. We also show that OMA-1 protein is directly phosphorylated at T239 by the DYRK kinase MBK-2, and that phosphorylation at T239 is required both for OMA-1 function in the 1-cell embryo and its degradation after the first mitosis. OMA-1 phosphorylated at T239 is only detected within a short developmental window of 1-cell embryos, beginning soon after the proposed activation of MBK-2. Phosphorylation at T239 facilitates subsequent phosphorylation of OMA-1 by another kinase, GSK-3, at T339 in vitro. Phosphorylation at both T239 and T339 are essential for correctly-timed OMA-1 degradation in vivo. We propose that a series of precisely-timed phosphorylation events regulates both the activity and the timing of degradation for OMA proteins, thereby allowing restricted and distinct functions of OMA-1 and OMA-2 in the maturing oocyte and 1-cell embryo, ensuring a normal oocyte-to-embryo transition in C. elegans.

  13. Protein kinase C phosphorylation disrupts Na+/H+ exchanger regulatory factor 1 autoinhibition and promotes cystic fibrosis transmembrane conductance regulator macromolecular assembly.

    PubMed

    Li, Jianquan; Poulikakos, Poulikos I; Dai, Zhongping; Testa, Joseph R; Callaway, David J E; Bu, Zimei

    2007-09-14

    An emerging theme in cell signaling is that membrane-bound channels and receptors are organized into supramolecular signaling complexes for optimum function and cross-talk. In this study, we determined how protein kinase C (PKC) phosphorylation influences the scaffolding protein Na(+)/H(+) exchanger regulatory factor 1 (NHERF) to assemble protein complexes of cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel that controls fluid and electrolyte transport across cell membranes. NHERF directs polarized expression of receptors and ion transport proteins in epithelial cells, as well as organizes the homo- and hetero-association of these cell surface proteins. NHERF contains two modular PDZ domains that are modular protein-protein interaction motifs, and a C-terminal domain. Previous studies have shown that NHERF is a phosphoprotein, but how phosphorylation affects NHERF to assemble macromolecular complexes is unknown. We show that PKC phosphorylates two amino acid residues Ser-339 and Ser-340 in the C-terminal domain of NHERF, but a serine 162 of PDZ2 is specifically protected from being phosphorylated by the intact C-terminal domain. PKC phosphorylation-mimicking mutant S339D/S340D of NHERF has increased affinity and stoichiometry when binding to C-CFTR. Moreover, solution small angle x-ray scattering indicates that the PDZ2 and C-terminal domains contact each other in NHERF, but such intramolecular domain-domain interactions are released in the PKC phosphorylation-mimicking mutant indicating that PKC phosphorylation disrupts the autoinhibition interactions in NHERF. The results demonstrate that the C-terminal domain of NHERF functions as an intramolecular switch that regulates the binding capability of PDZ2, and thus controls the stoichiometry of NHERF to assemble protein complexes.

  14. DNA-Dependent Protein Kinase Phosphorylation of IκBα and IκBβ Regulates NF-κB DNA Binding Properties

    PubMed Central

    Liu, Li; Kwak, Youn-Tae; Bex, Françoise; García-Martínez, León F.; Li, Xiao-Hua; Meek, Katheryn; Lane, William S.; Gaynor, Richard B.

    1998-01-01

    Regulation of the IκBα and IκBβ proteins is critical for modulating NF-κB-directed gene expression. Both IκBα and IκBβ are substrates for cellular kinases that phosphorylate the amino and carboxy termini of these proteins and regulate their function. In this study, we utilized a biochemical fractionation scheme to purify a kinase activity which phosphorylates residues in the amino and carboxy termini of both IκBα and IκBβ. Peptide microsequence analysis by capillary high-performance liquid chromatography ion trap mass spectroscopy revealed that this kinase was the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). DNA-PK phosphorylates serine residue 36 but not serine residue 32 in the amino terminus of IκBα and also phosphorylates threonine residue 273 in the carboxy terminus of this protein. To determine the biological relevance of DNA-PK phosphorylation of IκBα, murine severe combined immunodeficiency (SCID) cell lines which lack the DNA-PKcs gene were analyzed. Gel retardation analysis using extract prepared from these cells demonstrated constitutive nuclear NF-κB DNA binding activity, which was not detected in extracts prepared from SCID cells complemented with the human DNA-PKcs gene. Furthermore, IκBα that was phosphorylated by DNA-PK was a more potent inhibitor of NF-κB binding than nonphosphorylated IκBα. These results suggest that DNA-PK phosphorylation of IκBα increases its interaction with NF-κB to reduce NF-κB DNA binding properties. PMID:9632806

  15. LINGO-1 negatively regulates TrkB phosphorylation after ocular hypertension.

    PubMed

    Fu, Qing-Ling; Hu, Bing; Li, Xin; Shao, Zhaohui; Shi, Jian-Bo; Wu, Wutian; So, Kwok-Fai; Mi, Sha

    2010-03-01

    The antagonism of LINGO-1, a CNS-specific negative regulator of neuronal survival, was shown to promote short-term survival of retinal ganglion cell (RGC) in an ocular hypertension model. LINGO-1 antagonists, combined with brain-derived neurotrophic factor (BDNF), can increase the length of neuron survival through an unclear molecular mechanism. To determine the relationship between LINGO-1 and BDNF/TrkB receptor in neuronal protection, we show here that LINGO-1 forms a receptor complex with TrkB and negatively regulates its activation in the retina after ocular hypertension injury. LINGO-1 antagonist antibody 1A7 or soluble LINGO-1 (LINGO-1-Fc) treatment upregulates phospho-TrkB phosphorylation and leads to RGC survival after high intraocular pressure injury. This neuronal protective effect was blocked by anti-BDNF antibody. LINGO-1 antagonism therefore promotes RGC survival by regulating the BDNF and TrkB signaling pathway after ocular hypertension.

  16. Phosphorylation-dependent down-regulation of apolipoprotein A5 by insulin

    SciTech Connect

    Nowak, Maxine; Helleboid-Chapman, Audrey; Jakel, Heidelinde; Rommens, Corinne; Martin, Genevieve; Duran-Sandoval, Daniel; Staels, Bart; Rubin, Edward M.; Pennacchio, Len A.; Taskinen, Marja-Riitta; Fruchart-Najib, Jamila; Fruchart, Jean-Charles

    2004-02-15

    The apolipoprotein A5 (APOA5) gene has been shown to be important in lowering plasma triglyceride levels. Since several studies have shown that hyperinsulinemia is associated with hypertriglyceridemia, we sought to determine whether APOA5 gene is regulated by insulin. We show here that cell and mouse treatments with insulin down-regulated APOA5 expression in a dose-dependent manner. Furthermore, we determined that insulin decreases APOA5 promoter activity and subsequent deletion analyses revealed an E-box-containing fragment. We showed that Upstream Stimulatory Factors, USF1/USF2, bind to the identified E-box in the APOA5 promoter. Moreover, in cotransfection studies, USF1 stimulates APOA5 promoter activity. The treatment with insulin reduces the binding of USF1/USF2 to APOA5 promoter. The inhibition of PI3K pathway with wortmannin abolished the insulin s effect on APOA5 gene transcription. Using oligoprecipitation method of USF from nuclear extracts, we demonstrated that phosphorylated USF1 failed to bind to APOA5 promoter. This indicates that the APOA5 gene transrepression by insulin involves a phosphorylation of USF through PI3K, that modulate their binding to APOA5 promoter and results in APOA5 down-regulation. The effect of exogenous hyperinsulinemia in healthy men shows a decrease of the plasma ApoAV level. These data suggest a potential mechanism involving APOA5 gene in hypertriglyceridemia associated with hyperinsulinemia.

  17. Involvement of histone methylation and phosphorylation in regulation of transcription by thyroid hormone receptor.

    PubMed

    Li, Jiwen; Lin, Qiushi; Yoon, Ho-Geun; Huang, Zhi-Qing; Strahl, Brian D; Allis, C David; Wong, Jiemin

    2002-08-01

    Previous studies have established an important role of histone acetylation in transcriptional control by nuclear hormone receptors. With chromatin immunoprecipitation assays, we have now investigated whether histone methylation and phosphorylation are also involved in transcriptional regulation by thyroid hormone receptor (TR). We found that repression by unliganded TR is associated with a substantial increase in methylation of H3 lysine 9 (H3-K9) and a decrease in methylation of H3 lysine 4 (H3-K4), methylation of H3 arginine 17 (H3-R17), and a dual modification of phosphorylation of H3 serine 10 and acetylation of lysine 14 (pS10/acK14). On the other hand, transcriptional activation by liganded TR is coupled with a substantial decrease in both H3-K4 and H3-K9 methylation and a robust increase in H3-R17 methylation and the dual modification of pS10/acK14. Trichostatin A treatment results in not only histone hyperacetylation but also an increase in methylation of H3-K4, increase in dual modification of pS10/acK14, and reduction in methylation of H3-K9, revealing an extensive interplay between histone acetylation, methylation, and phosphorylation. In an effort to understand the underlying mechanism for an increase in H3-K9 methylation during repression by unliganded TR, we demonstrated that TR interacts in vitro with an H3-K9-specific histone methyltransferase (HMT), SUV39H1. Functional analysis indicates that SUV39H1 can facilitate repression by unliganded TR and in so doing requires its HMT activity. Together, our data uncover a novel role of H3-K9 methylation in repression by unliganded TR and provide strong evidence for the involvement of multiple distinct histone covalent modifications (acetylation, methylation, and phosphorylation) in transcriptional control by nuclear hormone receptors.

  18. Truncation and Activation of Dual Specificity Tyrosine Phosphorylation-regulated Kinase 1A by Calpain I

    PubMed Central

    Jin, Nana; Yin, Xiaomin; Gu, Jianlan; Zhang, Xinhua; Shi, Jianhua; Qian, Wei; Ji, Yuhua; Cao, Maohong; Gu, Xiaosong; Ding, Fei; Iqbal, Khalid; Gong, Cheng-Xin; Liu, Fei

    2015-01-01

    Hyperphosphorylation and dysregulation of exon 10 splicing of Tau are pivotally involved in pathogenesis of Alzheimer disease (AD) and/or other tauopathies. Alternative splicing of Tau exon 10, which encodes the second microtubule-binding repeat, generates Tau isoforms containing three and four microtubule-binding repeats, termed 3R-Taus and 4R-Taus, respectively. Dual specificity tyrosine-phosphorylation-regulated kinase 1A (Dyrk1A) lies at the Down syndrome critical region of chromosome 21. Overexpression of this kinase may contribute to the early Tau pathology in Down syndrome via phosphorylation of Tau and dysregulation of Tau exon 10. Here, we report that Dyrk1A was truncated at the C terminus and was associated with overactivation of calpain I in AD brain. Calpain I proteolyzed Dyrk1A in vitro first at the C terminus and further at the N terminus and enhanced its kinase activity toward Tau via increased Vmax but not Km. C-terminal truncation of Dyrk1A resulted in stronger activity than its full-length protein in promotion of exon 10 exclusion and phosphorylation of Tau. Dyrk1A was truncated in kainic acid-induced excitotoxic mouse brains and coincided with an increase in 3R-Tau expression and phosphorylation of Tau via calpain activation. Moreover, truncation of Dyrk1A was correlated with an increase in the ratio of 3R-Tau/4R-Tau and Tau hyperphosphorylation in AD brain. Collectively, these findings suggest that truncation/activation of Dyrk1A by Ca2+/calpain I might contribute to Tau pathology via promotion of exon 10 exclusion and hyperphosphorylation of Tau in AD brain. PMID:25918155

  19. Evidence for Regulation of Mitotic Progression through Temporal Phosphorylation and Dephosphorylation of CK2α▿ †

    PubMed Central

    St-Denis, Nicole A.; Derksen, D. Richard; Litchfield, David W.

    2009-01-01

    Proper mitotic progression is crucial for maintenance of genomic integrity in proliferating cells and is regulated through an intricate series of events, including protein phosphorylation governed by a complex network of protein kinases. One kinase family implicated in the regulation of mitotic progression is protein kinase CK2, a small family of enzymes that is overexpressed in cancer and induces transformation in mice and cultured fibroblasts. CK2α, one isoform of the catalytic subunits of CK2, is maximally phosphorylated at four sites in nocodazole-treated cells. To investigate the effects of CK2α phosphorylation on mitotic progression, we generated phosphospecific antibodies against its mitotic phosphorylation sites. In U2OS cells released from S-phase arrest, these antibodies reveal that CK2α is most highly phosphorylated in prophase and metaphase. Phosphorylation gradually decreases during anaphase and becomes undetectable during telophase and cytokinesis. Stable expression of phosphomimetic CK2α (CK2α-4D, CK2α-4E) results in aberrant centrosome amplification and chromosomal segregation defects and loss of mitotic cells through mitotic catastrophe. Conversely, cells expressing nonphosphorylatable CK2α (CK2α-4A) show a decreased ability to arrest in mitosis following nocodazole treatment, suggesting involvement in the spindle assembly checkpoint. Collectively, these studies indicate that reversible phosphorylation of CK2α requires precise regulation to allow proper mitotic progression. PMID:19188443

  20. TORC1 and TORC2 work together to regulate ribosomal protein S6 phosphorylation in Saccharomyces cerevisiae

    PubMed Central

    Yerlikaya, Seda; Meusburger, Madeleine; Kumari, Romika; Huber, Alexandre; Anrather, Dorothea; Costanzo, Michael; Boone, Charles; Ammerer, Gustav; Baranov, Pavel V.; Loewith, Robbie

    2016-01-01

    Nutrient-sensitive phosphorylation of the S6 protein of the 40S subunit of the eukaryote ribosome is highly conserved. However, despite four decades of research, the functional consequences of this modification remain unknown. Revisiting this enigma in Saccharomyces cerevisiae, we found that the regulation of Rps6 phosphorylation on Ser-232 and Ser-233 is mediated by both TOR complex 1 (TORC1) and TORC2. TORC1 regulates phosphorylation of both sites via the poorly characterized AGC-family kinase Ypk3 and the PP1 phosphatase Glc7, whereas TORC2 regulates phosphorylation of only the N-terminal phosphosite via Ypk1. Cells expressing a nonphosphorylatable variant of Rps6 display a reduced growth rate and a 40S biogenesis defect, but these phenotypes are not observed in cells in which Rps6 kinase activity is compromised. Furthermore, using polysome profiling and ribosome profiling, we failed to uncover a role of Rps6 phosphorylation in either global translation or translation of individual mRNAs. Taking the results together, this work depicts the signaling cascades orchestrating Rps6 phosphorylation in budding yeast, challenges the notion that Rps6 phosphorylation plays a role in translation, and demonstrates that observations made with Rps6 knock-ins must be interpreted cautiously. PMID:26582391

  1. New insights into the regulation of myosin light chain phosphorylation in retinal pigment epithelial cells.

    PubMed

    Ruiz-Loredo, Ariadna Yolanda; López-Colomé, Ana María

    2012-01-01

    The retinal pigment epithelium (RPE) plays an essential role in the function of the neural retina and the maintenance of vision. Most of the functions displayed by RPE require a dynamic organization of the acto-myosin cytoskeleton. Myosin II, a main cytoskeletal component in muscle and non-muscle cells, is directly involved in force generation required for organelle movement, selective molecule transport within cell compartments, exocytosis, endocytosis, phagocytosis, and cell division, among others. Contractile processes are triggered by the phosphorylation of myosin II light chains (MLCs), which promotes actin-myosin interaction and the assembly of contractile fibers. Considerable evidence indicates that non-muscle myosin II activation is critically involved in various pathological states, increasing the interest in studying the signaling pathways controlling MLC phosphorylation. Particularly, recent findings suggest a role for non-muscle myosin II-induced contraction in RPE cell transformation involved in the establishment of numerous retinal diseases. This review summarizes the current knowledge regarding myosin function in RPE cells, as well as the signaling networks leading to MLC phosphorylation under pathological conditions. Understanding the molecular mechanisms underlying RPE dysfunction would improve the development of new therapies for the treatment or prevention of different ocular disorders leading to blindness.

  2. Regulation of Ci-SCFSlimb binding, Ci proteolysis and Hedgehog pathway activity by Ci phosphorylation

    PubMed Central

    Smelkinson, Margery G.; Zhou, Qianhe; Kalderon, Daniel

    2007-01-01

    SUMMARY Hedgehog (Hh) proteins signal by inhibiting the proteolytic processing of Ci/Gli family transcription factors and by increasing Ci/Gli specific activity. In the absence of Hh, phosphorylation of Ci/Gli triggers binding to SCF ubiquitin ligase complexes and consequent proteolysis. Here we define the principal SCFSlimb binding site in Ci as an extended variant of a canonical Slimb/β-TRCP binding motif that can be created by PKA-priming of five successive CK1 sites. GSK3 enhances binding primarily through a nearby region of Ci, which may contact an SCF component other than Slimb. Studies of Ci variants with altered CK1 and GSK3 sites suggest that the large number of phosphorylation sites that direct SCFSlimb binding confers a Hh response that is both sensitive and graded, and that in the Drosophila wing disc, morphogenetic responses involve changes in both the level and specific activity of Ci. We also show that when Ci proteolysis is compromised, its specific activity is limited principally by Su(fu) and not by Cos2 cytoplasmic tethering or PKA phosphorylation. PMID:17925225

  3. Regulation of cytochrome c oxidase by adenylic nucleotides. Is oxidative phosphorylation feedback regulated by its end-products?

    PubMed

    Beauvoit, B; Rigoulet, M

    2001-01-01

    Cytochrome c oxidase, which catalyzes an irreversible step of the respiratory chain, is one of the rate-controlling steps of oxidative phosphorylation on isolated mitochondria. The rate of electron transfer through the complex is primarily controlled by the associated thermodynamic forces, i.e., the span in redox potential between oxygen and cytochrome c and the protonmotive force. However, the electron flux also depends on the various kinetic effectors, including adenylic nucleotides. Although the number of binding sites for ATP and ADP on cytochrome oxidase is still a matter of debate, experiments performed on the solubilized and reconstituted enzyme provide strong functional evidence that the mammalian cytochrome c oxidase binds adenylic nucleotides on both sides of the inner membrane. These effects include modification in cytochrome c affinity, allosteric inhibition and changes in proton pumping efficiency. Immunological studies have pointed out the role of subunit IV and that of an ATP-binding protein, subunit VIa, in these kinetic regulations. In yeast, the role of the nuclear-encoded subunits in assembly and regulation of the cytochrome c oxidase has been further substantiated by using gene-disruption analysis. Using a subunit VIa-null mutant, the consequences of the ATP regulation on oxidative phosphorylation have been further investigated on isolated mitochondria. Taken together, the data demonstrate that there are multiple regulating sites for ATP on the yeast cytochrome oxidase with respect to the location (matrix versus cytosolic side), kinetic effect (activation versus inhibition) and consequence on the flow-force relationships. The question is therefore raised as to the physiological meaning of such feedback regulation of the respiratory chain by ATP in the control and regulation of cellular energy metabolism.

  4. Site-directed spin labeling reveals a conformational switch in the phosphorylation domain of smooth muscle myosin.

    PubMed

    Nelson, Wendy D; Blakely, Sarah E; Nesmelov, Yuri E; Thomas, David D

    2005-03-15

    We have used site-directed spin labeling and EPR spectroscopy to detect structural changes within the regulatory light chain (RLC) of smooth muscle myosin upon phosphorylation. Smooth muscle contraction is activated by phosphorylation of S19 on RLC, but the structural basis of this process is unknown. There is no crystal structure containing a phosphorylated RLC, and there is no crystal structure for the N-terminal region of any RLC. Therefore, we have prepared single-Cys mutations throughout RLC, exchanged each mutant onto smooth muscle heavy meromyosin, verified normal regulatory function, and used EPR to determine dynamics and solvent accessibility at each site. A survey of spin-label sites throughout the RLC revealed that only the N-terminal region (first 24 aa) shows a significant change in dynamics upon phosphorylation, with most of the first 17 residues showing an increase in rotational amplitude. Therefore, we focused on this N-terminal region. Additional structural information was obtained from the pattern of oxygen accessibility along the sequence. In the absence of phosphorylation, little or no periodicity was observed, suggesting a lack of secondary structural order in this region. However, phosphorylation induced a strong helical pattern (3.6-residue periodicity) in the first 17 residues, while increasing accessibility throughout the first 24 residues. We have identified a domain within RLC, the N-terminal phosphorylation domain, in which phosphorylation increases helical order, internal dynamics, and accessibility. These results support a model in which this disorder-to-order transition within the phosphorylation domain results in decreased head-head interactions, activating myosin in smooth muscle.

  5. Protein tyrosine phosphatase PTPRB regulates Src phosphorylation and tumour progression in NSCLC.

    PubMed

    Qi, Yinliang; Dai, Yuanchang; Gui, Shuyu

    2016-10-01

    Protein tyrosine-phosphatases (PTPs) play important roles in various biological processes. Deregulation in PTP function has been implicated in carcinogenesis and tumour progression in many cancer types. However, the role of protein tyrosine phosphatase receptor type B (PTPRB) in non-small-cell lung cancer (NSCLC) tumorigenesis has not been investigated. Lentiviral vector expressing PTPRB cDNA or shRNA was infected into A549 and H1299 cell lines, followed by cell proliferation, colony formation, soft agar and invasion assays. A549 xenograft mouse model was used to evaluate in vivo function of PTPRB. Quantitative polymerase chain reaction (PCR) was used to measure PTPRB expression in NSCLC patient samples. Kaplan Meier analysis was performed to assess association between PTPRB expression and patient overall survival (OS). Multivariate analysis was performed to evaluate prognostic significance of PTPRB. Overexpression of PTPRB reduced cell proliferation rate, colony formation efficiency, soft agar growth and cell invasion in A549 and H1299 cells, as well as tumour growth rate in A549 xenograft. Knockdown of PTPRB increased Src phosphorylation and cell invasion, which was reversed by Src inhibitor PP2. Additionally, PTPRB was down-regulated in NSCLC patient and was associated with patient OS. PTPRB regulates Src phosphorylation and tumorigenesis in NSCLC. PTPRB may serve as an independent prognostic biomarker for NSCLC patients.

  6. Phosphorylation regulates binding of the human papillomavirus type 8 E2 protein to host chromosomes.

    PubMed

    Sekhar, Vandana; McBride, Alison A

    2012-09-01

    The papillomavirus E2 proteins are indispensable for the viral life cycle, and their functions are subject to tight regulation. The E2 proteins undergo posttranslational modifications that regulate their properties and roles in viral transcription, replication, and genome maintenance. During persistent infection, the E2 proteins from many papillomaviruses act as molecular bridges that tether the viral genomes to host chromosomes to retain them within the host nucleus and to partition them to daughter cells. The betapapillomavirus E2 proteins bind to pericentromeric regions of host mitotic chromosomes, including the ribosomal DNA loci. We recently reported that two residues (arginine 250 and serine 253) within the chromosome binding region of the human papillomavirus type 8 (HPV8) E2 protein are required for this binding. In this study, we show that serine 253 is phosphorylated, most likely by protein kinase A, and this modulates the interaction of the E2 protein with cellular chromatin. Furthermore, we show that this phosphorylation occurs in S phase, increases the half-life of the E2 protein, and promotes chromatin binding from S phase through mitosis.

  7. Regulation of the DNA Damage Response by DNA-PKcs Inhibitory Phosphorylation of ATM.

    PubMed

    Zhou, Yi; Lee, Ji-Hoon; Jiang, Wenxia; Crowe, Jennie L; Zha, Shan; Paull, Tanya T

    2017-01-05

    Ataxia-telangiectasia mutated (ATM) regulates the DNA damage response as well as DNA double-strand break repair through homologous recombination. Here we show that ATM is hyperactive when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is chemically inhibited or when the DNA-PKcs gene is deleted in human cells. Pre-incubation of ATM protein with active DNA-PKcs also significantly reduces ATM activity in vitro. We characterize several phosphorylation sites in ATM that are targets of DNA-PKcs and show that phospho-mimetic mutations at these residues significantly inhibit ATM activity and impair ATM signaling upon DNA damage. In contrast, phospho-blocking mutations at one cluster of sites increase the frequency of apoptosis during normal cell growth. DNA-PKcs, which is integral to the non-homologous end joining pathway, thus negatively regulates ATM activity through phosphorylation of ATM. These observations illuminate an important regulatory mechanism for ATM that also controls DNA repair pathway choice.

  8. C-terminal phosphorylation regulates the kinetics of a subset of melanopsin-mediated behaviors in mice.

    PubMed

    Somasundaram, Preethi; Wyrick, Glenn R; Fernandez, Diego Carlos; Ghahari, Alireza; Pinhal, Cindy M; Simmonds Richardson, Melissa; Rupp, Alan C; Cui, Lihong; Wu, Zhijian; Brown, R Lane; Badea, Tudor Constantin; Hattar, Samer; Robinson, Phyllis R

    2017-03-07

    Intrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin and mediate several non-image-forming visual functions, including circadian photoentrainment and the pupillary light reflex (PLR). ipRGCs act as autonomous photoreceptors via the intrinsic melanopsin-based phototransduction pathway and as a relay for rod/cone input via synaptically driven responses. Under low light intensities, where only synaptically driven rod/cone input activates ipRGCs, the duration of the ipRGC response will be determined by the termination kinetics of the rod/cone circuits. Little is known, however, about the termination kinetics of the intrinsic melanopsin-based phototransduction pathway and its contribution to several melanopsin-mediated behaviors. Here, we show that C-terminal phosphorylation of melanopsin determines the recovery kinetics of the intrinsic melanopsin-based photoresponse in ipRGCs, the duration of the PLR, and the speed of reentrainment. In contrast, circadian phase alignment and direct effects of light on activity (masking) are not influenced by C-terminal phosphorylation of melanopsin. Electrophysiological measurements demonstrate that expression of a virally encoded melanopsin lacking all C-terminal phosphorylation sites (C terminus phosphonull) leads to a prolonged intrinsic light response. In addition, mice expressing the C terminus phosphonull in ipRGCs reentrain faster to a delayed light/dark cycle compared with mice expressing virally encoded WT melanopsin; however, the phase angle of entrainment and masking were indistinguishable. Importantly, a sustained PLR in the phosphonull animals is only observed at brighter light intensities that activate melanopsin phototransduction, but not at dimmer light intensities that activate only the rod/cone pathway. Taken together, our results highlight how the kinetics of the melanopsin photoresponse differentially regulate distinct light-mediated behaviors.

  9. G protein-coupled receptor kinase GRK5 phosphorylates moesin and regulates metastasis in prostate cancer.

    PubMed

    Chakraborty, Prabir Kumar; Zhang, Yushan; Coomes, Alexandra S; Kim, Wan-Ju; Stupay, Rachel; Lynch, Lauren D; Atkinson, Tamieka; Kim, Jae I; Nie, Zhongzhen; Daaka, Yehia

    2014-07-01

    G protein-coupled receptor kinases (GRK) regulate diverse cellular functions ranging from metabolism to growth and locomotion. Here, we report an important contributory role for GRK5 in human prostate cancer. Inhibition of GRK5 kinase activity attenuated the migration and invasion of prostate cancer cells and, concordantly, increased cell attachment and focal adhesion formation. Mass spectrometric analysis of the phosphoproteome revealed the cytoskeletal-membrane attachment protein moesin as a putative GRK5 substrate. GRK5 regulated the subcellular distribution of moesin and colocalized with moesin at the cell periphery. We identified amino acid T66 of moesin as a principal GRK5 phosphorylation site and showed that enforcing the expression of a T66-mutated moesin reduced cell spreading. In a xenograft model of human prostate cancer, GRK5 silencing reduced tumor growth, invasion, and metastasis. Taken together, our results established GRK5 as a key contributor to the growth and metastasis of prostate cancer.

  10. Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility

    NASA Technical Reports Server (NTRS)

    Morfini, Gerardo; Szebenyi, Gyorgyi; Elluru, Ravindhra; Ratner, Nancy; Brady, Scott T.

    2002-01-01

    Membrane-bounded organelles (MBOs) are delivered to different domains in neurons by fast axonal transport. The importance of kinesin for fast antero grade transport is well established, but mechanisms for regulating kinesin-based motility are largely unknown. In this report, we provide biochemical and in vivo evidence that kinesin light chains (KLCs) interact with and are in vivo substrates for glycogen synthase kinase 3 (GSK3). Active GSK3 inhibited anterograde, but not retrograde, transport in squid axoplasm and reduced the amount of kinesin bound to MBOs. Kinesin microtubule binding and microtubule-stimulated ATPase activities were unaffected by GSK3 phosphorylation of KLCs. Active GSK3 was also localized preferentially to regions known to be sites of membrane delivery. These data suggest that GSK3 can regulate fast anterograde axonal transport and targeting of cargos to specific subcellular domains in neurons.

  11. Regulation of the mRNA-binding protein HuR by posttranslational modification: spotlight on phosphorylation.

    PubMed

    Eberhardt, Wolfgang; Doller, Anke; Pfeilschifter, Josef

    2012-06-01

    The ubiquitous mRNA-binding protein human antigen R (HuR) and its neuronal relatives (HuB, HuC, HuD) participate in the post-transcriptional regulation of many AU-rich element-bearing mRNAs. In addition to its originally described role in controlling mRNA decay, the binding of HuR to target mRNAs can affect many aspects of mRNA processing including splicing, polyadenylation, intracellular trafficking, translation and modulation of mRNA repression by miRNAs. In accordance to the growing list of signalling events which are involved in regulating these different HuR functions, recent data implicate that posttranslational modification, namely protein kinase-triggered phosphorylation of HuR plays a crucial role in connecting extracellular signal inputs to a specific post-transcriptional program by HuR. Notably, in addition to directly targeting HuR functions, posttranslational modifications of HuR have a major impact on the sequestration and binding to various HuR ligand proteins as has been demonstrated e.g. for the 14-3-3 chaperones. However, the detailed mechanisms of how a specific modification of HuR coordinates different aspects in HuR regulation are currently poorly understood. Due to the fact that most of the described HuR activities are closely related to its subcellular localization and the binding to cargo mRNA, this review will focus on these aspects of HuR functions and their control by posttranslational modification, particularly by HuR phosphorylations by different protein kinases.

  12. Differential phosphorylation of DNA-PKcs regulates the interplay between end-processing and end-ligation during non-homologous end-joining

    PubMed Central

    Jiang, Wenxia; Crowe, Jennifer L.; Liu, Xiangyu; Nakajima, Satoshi; Wang, Yunyue; Li, Chen; Lee, Brian J.; Dubois, Richard L.; Liu, Chao; Yu, Xiaochun; Lan, Li; Zha, Shan

    2015-01-01

    SUMMARY Non-homologous end-joining (NHEJ) is a major DNA double strand break repair pathway that is conserved in eukaryotes. In vertebrates, NHEJ further acquires end-processing capacities (e.g., hairpin opening) in addition to direct end-ligation. The catalytic subunit of DNA-PK (DNA-PKcs) is a vertebrate specific NHEJ factor that can be auto-phosphorylated or trans-phosphorylated by ATM kinase. Using a mouse model expressing a kinase-dead (KD) DNA-PKcs protein, we show that ATM-mediated trans-phosphorylation of DNA-PKcs regulates end-processing at the level of Artemis recruitment, while strictly auto-phosphorylation of DNA-PKcs is necessary to relieve the physical blockage on end-ligation imposed by the DNA-PKcs protein itself. Accordingly, DNA-PKcsKD/KD mice and cells show severe end-ligation defects and p53- and Ku-dependent embryonic lethality, but open hairpin-sealed ends normally in the presence of ATM kinase activity. Together, our findings identify DNA-PKcs as the molecular switch that coordinates end-processing and end-ligation at the DNA ends through differential phosphorylations. PMID:25818648

  13. The Role of TSC2 Phosphorylation in the Regulation of TSC2 Localization and mTOR Signaling

    DTIC Science & Technology

    2015-05-01

    mechanical stimuli. We then generated phosphodefective mutants of TSC2 and our preliminary results with these mutants indicate that these phosphorylation... mutants of TSC2. To date we have created a construct that contains mutations on all of the 6 phosphorylation sites that were found to be responsive...that are known to be regulated by insulin. Our original plan was to create a series of stable cell lines that express these mutant proteins (subtask 2b

  14. A Computational Model for the AMPA Receptor Phosphorylation Master Switch Regulating Cerebellar Long-Term Depression.

    PubMed

    Gallimore, Andrew R; Aricescu, A Radu; Yuzaki, Michisuke; Calinescu, Radu

    2016-01-01

    The expression of long-term depression (LTD) in cerebellar Purkinje cells results from the internalisation of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs) from the postsynaptic membrane. This process is regulated by a complex signalling pathway involving sustained protein kinase C (PKC) activation, inhibition of serine/threonine phosphatase, and an active protein tyrosine phosphatase, PTPMEG. In addition, two AMPAR-interacting proteins-glutamate receptor-interacting protein (GRIP) and protein interacting with C kinase 1 (PICK1)-regulate the availability of AMPARs for trafficking between the postsynaptic membrane and the endosome. Here we present a new computational model of these overlapping signalling pathways. The model reveals how PTPMEG cooperates with PKC to drive LTD expression by facilitating the effect of PKC on the dissociation of AMPARs from GRIP and thus their availability for trafficking. Model simulations show that LTD expression is increased by serine/threonine phosphatase inhibition, and negatively regulated by Src-family tyrosine kinase activity, which restricts the dissociation of AMPARs from GRIP under basal conditions. We use the model to expose the dynamic balance between AMPAR internalisation and reinsertion, and the phosphorylation switch responsible for the perturbation of this balance and for the rapid plasticity initiation and regulation. Our model advances the understanding of PF-PC LTD regulation and induction, and provides a validated extensible platform for more detailed studies of this fundamental synaptic process.

  15. A Computational Model for the AMPA Receptor Phosphorylation Master Switch Regulating Cerebellar Long-Term Depression

    PubMed Central

    Gallimore, Andrew R.; Aricescu, A. Radu; Yuzaki, Michisuke; Calinescu, Radu

    2016-01-01

    The expression of long-term depression (LTD) in cerebellar Purkinje cells results from the internalisation of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs) from the postsynaptic membrane. This process is regulated by a complex signalling pathway involving sustained protein kinase C (PKC) activation, inhibition of serine/threonine phosphatase, and an active protein tyrosine phosphatase, PTPMEG. In addition, two AMPAR-interacting proteins–glutamate receptor-interacting protein (GRIP) and protein interacting with C kinase 1 (PICK1)–regulate the availability of AMPARs for trafficking between the postsynaptic membrane and the endosome. Here we present a new computational model of these overlapping signalling pathways. The model reveals how PTPMEG cooperates with PKC to drive LTD expression by facilitating the effect of PKC on the dissociation of AMPARs from GRIP and thus their availability for trafficking. Model simulations show that LTD expression is increased by serine/threonine phosphatase inhibition, and negatively regulated by Src-family tyrosine kinase activity, which restricts the dissociation of AMPARs from GRIP under basal conditions. We use the model to expose the dynamic balance between AMPAR internalisation and reinsertion, and the phosphorylation switch responsible for the perturbation of this balance and for the rapid plasticity initiation and regulation. Our model advances the understanding of PF-PC LTD regulation and induction, and provides a validated extensible platform for more detailed studies of this fundamental synaptic process. PMID:26807999

  16. The M3-muscarinic receptor regulates learning and memory in a receptor phosphorylation/arrestin-dependent manner.

    PubMed

    Poulin, Benoit; Butcher, Adrian; McWilliams, Phillip; Bourgognon, Julie-Myrtille; Pawlak, Robert; Kong, Kok Choi; Bottrill, Andrew; Mistry, Sharad; Wess, Jürgen; Rosethorne, Elizabeth M; Charlton, Steven J; Tobin, Andrew B

    2010-05-18

    Degeneration of the cholinergic system is considered to be the underlying pathology that results in the cognitive deficit in Alzheimer's disease. This pathology is thought to be linked to a loss of signaling through the cholinergic M(1)-muscarinic receptor subtype. However, recent studies have cast doubt on whether this is the primary receptor mediating cholinergic-hippocampal learning and memory. The current study offers an alternative mechanism involving the M(3)-muscarinic receptor that is expressed in numerous brain regions including the hippocampus. We demonstrate here that M(3)-muscarinic receptor knockout mice show a deficit in fear conditioning learning and memory. The mechanism used by the M(3)-muscarinic receptor in this process involves receptor phosphorylation because a knockin mouse strain expressing a phosphorylation-deficient receptor mutant also shows a deficit in fear conditioning. Consistent with a role for receptor phosphorylation, we demonstrate that the M(3)-muscarinic receptor is phosphorylated in the hippocampus following agonist treatment and following fear conditioning training. Importantly, the phosphorylation-deficient M(3)-muscarinic receptor was coupled normally to G(q/11)-signaling but was uncoupled from phosphorylation-dependent processes such as receptor internalization and arrestin recruitment. It can, therefore, be concluded that M(3)-muscarinic receptor-dependent learning and memory depends, at least in part, on receptor phosphorylation/arrestin signaling. This study opens the potential for biased M(3)-muscarinic receptor ligands that direct phosphorylation/arrestin-dependent (non-G protein) signaling as being beneficial in cognitive disorders.

  17. Role of Protein Phosphorylation in the Regulation of Cell Cycle and DNA-Related Processes in Bacteria

    PubMed Central

    Garcia-Garcia, Transito; Poncet, Sandrine; Derouiche, Abderahmane; Shi, Lei; Mijakovic, Ivan; Noirot-Gros, Marie-Françoise

    2016-01-01

    In all living organisms, the phosphorylation of proteins modulates various aspects of their functionalities. In eukaryotes, protein phosphorylation plays a key role in cell signaling, gene expression, and differentiation. Protein phosphorylation is also involved in the global control of DNA replication during the cell cycle, as well as in the mechanisms that cope with stress-induced replication blocks. Similar to eukaryotes, bacteria use Hanks-type kinases and phosphatases for signal transduction, and protein phosphorylation is involved in numerous cellular processes. However, it remains unclear whether protein phosphorylation in bacteria can also regulate the activity of proteins involved in DNA-mediated processes such as DNA replication or repair. Accumulating evidence supported by functional and biochemical studies suggests that phospho-regulatory mechanisms also take place during the bacterial cell cycle. Recent phosphoproteomics and interactomics studies identified numerous phosphoproteins involved in various aspect of DNA metabolism strongly supporting the existence of such level of regulation in bacteria. Similar to eukaryotes, bacterial scaffolding-like proteins emerged as platforms for kinase activation and signaling. This review reports the current knowledge on the phosphorylation of proteins involved in the maintenance of genome integrity and the regulation of cell cycle in bacteria that reveals surprising similarities to eukaryotes. PMID:26909079

  18. Regulation of human polλ by ATM-mediated phosphorylation during non-homologous end joining.

    PubMed

    Sastre-Moreno, Guillermo; Pryor, John M; Moreno-Oñate, Marta; Herrero-Ruiz, Andrés M; Cortés-Ledesma, Felipe; Blanco, Luis; Ramsden, Dale A; Ruiz, Jose F

    2017-03-01

    DNA double strand breaks (DSBs) trigger a variety of cellular signaling processes, collectively termed the DNA-damage response (DDR), that are primarily regulated by protein kinase ataxia-telangiectasia mutated (ATM). Among DDR activated processes, the repair of DSBs by non-homologous end joining (NHEJ) is essential. The proper coordination of NHEJ factors is mainly achieved through phosphorylation by an ATM-related kinase, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), although the molecular basis for this regulation has yet to be fully elucidated. In this study we identify the major NHEJ DNA polymerase, DNA polymerase lambda (Polλ), as a target for both ATM and DNA-PKcs in human cells. We show that Polλ is efficiently phosphorylated by DNA-PKcs in vitro and predominantly by ATM after DSB induction with ionizing radiation (IR) in vivo. We identify threonine 204 (T204) as a main target for ATM/DNA-PKcs phosphorylation on human Polλ, and establish that its phosphorylation may facilitate the repair of a subset of IR-induced DSBs and the efficient Polλ-mediated gap-filling during NHEJ. Molecular evidence suggests that Polλ phosphorylation might favor Polλ interaction with the DNA-PK complex at DSBs. Altogether, our work provides the first demonstration of how Polλ is regulated by phosphorylation to connect with the NHEJ core machinery during DSB repair in human cells.

  19. Sleep deprivation impairs spatial memory and decreases extracellular signal-regulated kinase phosphorylation in the hippocampus.

    PubMed

    Guan, Zhiwei; Peng, Xuwen; Fang, Jidong

    2004-08-20

    Loss of sleep may result in memory impairment. However, little is known about the biochemical basis for memory deficits induced by sleep deprivation. Extracellular signal-regulated kinase (ERK) is involved in memory consolidation in different tasks. Phosphorylation of ERK is necessary for its activation and is an important step in mediating neuronal responses to synaptic activities. The aim of the present study was to determine the effects of total sleep deprivation (TSD) on memory and ERK phosphorylation in the brain. Rats were trained in Morris water maze to find a hidden platform (a spatial task) or a visible platform (a nonspatial task) after 6 h TSD or spontaneous sleep. TSD had no effect on spatial learning, but significantly impaired spatial memory tested 24 h after training. Nonspatial learning and memory were not impaired by TSD. Phospho-ERK levels in the hippocampus were significantly reduced after 6 h TSD compared to the controls and returned to the control levels after 2 h recovery sleep. Total ERK1 and ERK2 were slightly increased after 6 h TSD and returned to the control levels after 2 h recovery sleep. These alterations were not observed in the cortex after TSD. Protein phosphotase-1 and mitogen-activated protein kinase phosphatase-2, which dephosphorylates phospho-ERK, were also measured, but they were not altered by TSD. The impairments of both spatial memory and ERK phosphorylation indicate that the hippocampus is vulnerable to sleep loss. These results are consistent with the idea that decreased ERK activation in the hippocampus is involved in sleep deprivation-induced spatial memory impairment.

  20. Binding of E-MAP-115 to microtubules is regulated by cell cycle- dependent phosphorylation

    PubMed Central

    1995-01-01

    Expression levels of E-MAP-115, a microtubule-associated protein that stabilizes microtubules, increase with epithelial cell polarization and differentiation (Masson and Kreis, 1993). Although polarizing cells contain significant amounts of this protein, they can still divide and thus all stabilized microtubules must disassemble at the onset of mitosis to allow formation of the dynamic mitotic spindle. We show here that binding of E-MAP-115 to microtubules is regulated by phosphorylation during the cell cycle. Immunolabeling of HeLa cells for E-MAP-115 indicates that the protein is absent from microtubules during early prophase and progressively reassociates with microtubules after late prophase. A fraction of E-MAP-115 from HeLa cells released from a block at the G1/S boundary runs with higher apparent molecular weight on SDS-PAGE, with a peak correlating with the maximal number of cells in early stages of mitosis. E-MAP-115 from nocodazole-arrested mitotic cells, which can be obtained in larger amounts, displays identical modifications and was used for further biochemical characterization. The level of incorporation of 32P into mitotic E-MAP-115 is about 15- fold higher than into the interphase protein. Specific threonine phosphorylation occurs in mitosis, and the amount of phosphate associated with serine also increases. Hyperphosphorylated E-MAP-115 from mitotic cells cannot bind stably to microtubules in vitro. These results suggest that phosphorylation of E-MAP-115 is a prerequisite for increasing the dynamic properties of the interphase microtubules which leads to the assembly of the mitotic spindle at the onset of mitosis. Microtubule-associated proteins are thus most likely key targets for kinases which control changes in microtubule dynamic properties at the G2- to M-phase transition. PMID:7490279

  1. In vivo neuronal function of the fragile X mental retardation protein is regulated by phosphorylation.

    PubMed

    Coffee, R Lane; Williamson, Ashley J; Adkins, Christopher M; Gray, Marisa C; Page, Terry L; Broadie, Kendal

    2012-02-15

    Fragile X syndrome (FXS), caused by loss of the Fragile X Mental Retardation 1 (FMR1) gene product (FMRP), is the most common heritable cause of intellectual disability and autism spectrum disorders. It has been long hypothesized that the phosphorylation of serine 500 (S500) in human FMRP controls its function as an RNA-binding translational repressor. To test this hypothesis in vivo, we employed neuronally targeted expression of three human FMR1 transgenes, including wild-type (hFMR1), dephosphomimetic (S500A-hFMR1) and phosphomimetic (S500D-hFMR1), in the Drosophila FXS disease model to investigate phosphorylation requirements. At the molecular level, dfmr1 null mutants exhibit elevated brain protein levels due to loss of translational repressor activity. This defect is rescued for an individual target protein and across the population of brain proteins by the phosphomimetic, whereas the dephosphomimetic phenocopies the null condition. At the cellular level, dfmr1 null synapse architecture exhibits increased area, branching and bouton number. The phosphomimetic fully rescues these synaptogenesis defects, whereas the dephosphomimetic provides no rescue. The presence of Futsch-positive (microtubule-associated protein 1B) supernumerary microtubule loops is elevated in dfmr1 null synapses. The human phosphomimetic restores normal Futsch loops, whereas the dephosphomimetic provides no activity. At the behavioral level, dfmr1 null mutants exhibit strongly impaired olfactory associative learning. The human phosphomimetic targeted only to the brain-learning center restores normal learning ability, whereas the dephosphomimetic provides absolutely no rescue. We conclude that human FMRP S500 phosphorylation is necessary for its in vivo function as a neuronal translational repressor and regulator of synaptic architecture, and for the manifestation of FMRP-dependent learning behavior.

  2. β-Adrenergic stimulation activates protein kinase Cε and induces extracellular signal-regulated kinase phosphorylation and cardiomyocyte hypertrophy.

    PubMed

    Li, Lin; Cai, Hongyan; Liu, Hua; Guo, Tao

    2015-06-01

    The cardiac adrenergic signaling pathway is important in the induction of cardiac hypertrophy. The cardiac adrenergic pathway involves two main branches, phospholipase C (PLC)/protein kinase C (PKC) and the adenylate cyclase (cAMPase)/protein kinase A (PKA) signaling pathways. It is hypothesized that PLC/PKC and cAMPase/PKA are activated by the α‑adrenergic receptor (αAR) and the β‑adrenergic receptor (βAR), respectively. Previous studies have demonstrated that exchange protein directly activated by cAMP (Epac), a guanine exchange factor, activates phospholipase Cε. It is possible that there are βAR‑activated PKC pathways mediated by Epac and PLC. In the present study, the role of Epac and PLC in βAR activated PKC pathways in cardiomyocytes was investigated. It was found that PKCε activation and translocation were induced by the βAR agonist, isoproterenol (Iso). Epac agonist 8‑CPT‑2'OMe‑cAMP also enhanced PKCε activation. βAR stimulation activated PKCε in the cardiomyocytes and was regulated by Epac. Iso‑induced change in PKCε was not affected in the cardiomyocytes, which were infected with adenovirus coding rabbit muscle cAMP‑dependent protein kinase inhibitor. However, Iso‑induced PKCε activation was inhibited by the PLC inhibitor, U73122. The results suggested that Iso‑induced PKCε activation was independent of PKA, but was regulated by PLC. To further investigate the downstream signal target of PKCε activation, the expression of phosphorylated extracellular signal‑regulated kinase (pERK)1/2 and the levels of ERK phosphorylation was analyzed. The results revealed that Iso‑induced PKCε activation led to an increase in the expression of pERK1/2. ERK phosphorylation was inhibited by the PKCε inhibitor peptide. Taken together, these data demonstrated that the βAR is able to activate PKCε dependent on Epac and PLC, but independent of PKA.

  3. Featured Article: Differential regulation of endothelial nitric oxide synthase phosphorylation by protease-activated receptors in adult human endothelial cells.

    PubMed

    Tillery, Lakeisha C; Epperson, Tenille A; Eguchi, Satoru; Motley, Evangeline D

    2016-03-01

    Protease-activated receptors have been shown to regulate endothelial nitric oxide synthase through the phosphorylation of specific sites on the enzyme. It has been established that PAR-2 activation phosphorylates eNOS-Ser-1177 and leads to the production of the potent vasodilator nitric oxide, while PAR-1 activation phosphorylates eNOS-Thr-495 and decreases nitric oxide production in human umbilical vein endothelial cells. In this study, we hypothesize a differential coupling of protease-activated receptors to the signaling pathways that regulates endothelial nitric oxide synthase and nitric oxide production in primary adult human coronary artery endothelial cells. Using Western Blot analysis, we showed that thrombin and the PAR-1 activating peptide, TFLLR, lead to the phosphorylation of eNOS-Ser-1177 in human coronary artery endothelial cells, which was blocked by SCH-79797 (SCH), a PAR-1 inhibitor. Using the nitrate/nitrite assay, we also demonstrated that the thrombin- and TFLLR-induced production of nitric oxide was inhibited by SCH and L-NAME, a NOS inhibitor. In addition, we observed that TFLLR, unlike thrombin, significantly phosphorylated eNOS-Thr-495, which may explain the observed delay in nitric oxide production in comparison to that of thrombin. Activation of PAR-2 by SLIGRL, a PAR-2 specific ligand, leads to dual phosphorylation of both catalytic sites but primarily regulated eNOS-Thr-495 phosphorylation with no change in nitric oxide production in human coronary artery endothelial cells. PAR-3, known as the non-signaling receptor, was activated by TFRGAP, a PAR-3 mimicking peptide, and significantly induced the phosphorylation of eNOS-Thr-495 with minimal phosphorylation of eNOS-Ser-1177 with no change in nitric oxide production. In addition, we confirmed that PAR-mediated eNOS-Ser-1177 phosphorylation was Ca(2+)-dependent using the Ca(2+) chelator, BAPTA, while eNOS-Thr-495 phosphorylation was mediated via Rho kinase using the ROCK inhibitor, Y-27632

  4. Protein kinase CK2-mediated phosphorylation of HDAC2 regulates co-repressor formation, deacetylase activity and acetylation of HDAC2 by cigarette smoke and aldehydes.

    PubMed

    Adenuga, David; Rahman, Irfan

    2010-06-01

    Histone deacetylase 2 (HDAC2) mediates the repression of pro-inflammatory genes by deacetylating core histones, RelA/p65 and the glucocorticoid receptor. Reduced level of HDAC2 is associated with steroid resistant inflammation caused by cigarette smoke (CS)-derived oxidants and aldehydes. However, the molecular mechanisms regulating HDAC2 in response to CS and aldehydes is not known. Here, we report that CS extract, and aldehyde acrolein induced phosphorylation of HDAC2 which was abolished by mutations at serine sites S(394), S(411), S(422) and S(424). HDAC2 phosphorylation required direct interaction with serine-phosphorylated protein kinase CK2alpha and involved reduced HDAC2 deacetylase activity. Furthermore, HDAC2 phosphorylation was required for HDAC2 interaction with transcription factors, co-repressor complex formation, CBP recruitment, acetylation on lysine residues and modulates transrepression activity. Thus, phospho-acetylation of HDAC2 negatively regulates its deacetylase activity which has implications in steroid resistance in chronic inflammatory conditions.

  5. Histone H3 Threonine Phosphorylation Regulates Asymmetric Histone Inheritance in the Drosophila Male Germline

    PubMed Central

    Xie, Jing; Wooten, Matthew; Tran, Vuong; Chen, Bi-Chang; Pozmanter, Caitlin; Simbolon, Christine; Betzig, Eric; Chen, Xin

    2015-01-01

    SUMMARY A long-standing question concerns how stem cells maintain their identity through multiple divisions. Previously we reported that pre-existing and newly synthesized histone H3 are asymmetrically distributed during Drosophila male germline stem cell (GSC) asymmetric division. Here we show that phosphorylation at Threonine 3 of H3 (H3T3P) distinguishes preexisting versus newly synthesized H3. Converting T3 to the unphosphorylatable residue alanine (H3T3A) or to the phosphomimetic aspartate (H3T3D) disrupts asymmetric H3 inheritance. Expression of H3T3A or H3T3D specifically in early-stage germline also leads to cellular defects including GSC loss and germline tumors. Finally, compromising the activity of the H3T3 kinase Haspin enhances the H3T3A but suppresses the H3T3D phenotypes. Together these studies demonstrate that H3T3P distinguishes sister chromatids enriched with distinct pools of H3, coordinating asymmetric segregation of “old” H3 into GSCs, and that a tight regulation of H3T3 phosphorylation is required for male germline activity. PMID:26522592

  6. Histone H3 Threonine Phosphorylation Regulates Asymmetric Histone Inheritance in the Drosophila Male Germline.

    PubMed

    Xie, Jing; Wooten, Matthew; Tran, Vuong; Chen, Bi-Chang; Pozmanter, Caitlin; Simbolon, Christine; Betzig, Eric; Chen, Xin

    2015-11-05

    A long-standing question concerns how stem cells maintain their identity through multiple divisions. Previously, we reported that pre-existing and newly synthesized histone H3 are asymmetrically distributed during Drosophila male germline stem cell (GSC) asymmetric division. Here, we show that phosphorylation at threonine 3 of H3 (H3T3P) distinguishes pre-existing versus newly synthesized H3. Converting T3 to the unphosphorylatable residue alanine (H3T3A) or to the phosphomimetic aspartate (H3T3D) disrupts asymmetric H3 inheritance. Expression of H3T3A or H3T3D specifically in early-stage germline also leads to cellular defects, including GSC loss and germline tumors. Finally, compromising the activity of the H3T3 kinase Haspin enhances the H3T3A but suppresses the H3T3D phenotypes. These studies demonstrate that H3T3P distinguishes sister chromatids enriched with distinct pools of H3 in order to coordinate asymmetric segregation of "old" H3 into GSCs and that tight regulation of H3T3 phosphorylation is required for male germline activity.

  7. Polyamines regulate phosphorylation-dephosphorylation kinetics in a crustacean gill (Na(+), K(+))-ATPase.

    PubMed

    Lucena, Malson Neilson; Garçon, Daniela Pereira; Fontes, Carlos Frederico Leite; McNamara, John Campbell; Leone, Francisco Assis

    2017-05-01

    Aiming to clarify the mechanism of inhibition of (Na(+), K(+))-ATPase activity by polyamines, we examined the effects of exogenous putrescine, spermidine, and spermine on the kinetic behavior of phosphoenzyme-linked partial reactions using a microsomal gill (Na(+), K(+))-ATPase from juvenile and adult M. amazonicum, a freshwater palaemonid shrimp. The time course of phosphointermediate formation is greater (0.089 ± 0.006 s(-1)) in adults than in juveniles (0.053 ± 0.003 s(-1)) for spermidine, but similar to juveniles (0.059 ± 0.004 s(-1)) for putrescine. Maximum phosphointermediate formation for the (Na(+), K(+))-ATPase from juveniles decreased by 46% and 32% with spermidine and putrescine, respectively. In adults, maximum phosphointermediate levels decreased by 50% and 8%, respectively. For both spermidine and putrescine, dephosphorylation rates were higher for adults than for juveniles, and were higher than in controls without polyamines. Spermine had a negligible effect (<10%) on phosphorylation/dephosphorylation rates of both juvenile and adult enzymes. This is the first report on the effects of polyamines on phosphoenzyme-linked partial reactions in juvenile and adult M. amazonicum gill (Na(+), K(+))-ATPases. Our findings suggest that the phosphorylation/dephosphorylation steps of this gill enzyme may be regulated by polyamines during ontogenetic development.

  8. ULK3 regulates cytokinetic abscission by phosphorylating ESCRT-III proteins

    PubMed Central

    Caballe, Anna; Wenzel, Dawn M; Agromayor, Monica; Alam, Steven L; Skalicky, Jack J; Kloc, Magdalena; Carlton, Jeremy G; Labrador, Leticia; Sundquist, Wesley I; Martin-Serrano, Juan

    2015-01-01

    The endosomal sorting complexes required for transport (ESCRT) machinery mediates the physical separation between daughter cells during cytokinetic abscission. This process is regulated by the abscission checkpoint, a genome protection mechanism that relies on Aurora B and the ESCRT-III subunit CHMP4C to delay abscission in response to chromosome missegregation. In this study, we show that Unc-51-like kinase 3 (ULK3) phosphorylates and binds ESCRT-III subunits via tandem MIT domains, and thereby, delays abscission in response to lagging chromosomes, nuclear pore defects, and tension forces at the midbody. Our structural and biochemical studies reveal an unusually tight interaction between ULK3 and IST1, an ESCRT-III subunit required for abscission. We also demonstrate that IST1 phosphorylation by ULK3 is an essential signal required to sustain the abscission checkpoint and that ULK3 and CHMP4C are functionally linked components of the timer that controls abscission in multiple physiological situations. DOI: http://dx.doi.org/10.7554/eLife.06547.001 PMID:26011858

  9. Neuromedin B receptors regulate EGF receptor tyrosine phosphorylation in lung cancer cells

    PubMed Central

    Moody, Terry W.; Berna, Marc J.; Mantey, Samuel; Sancho, Veronica; Ridnour, Lisa; Wink, David A.; Chan, Daniel; Giaccone, Giuseppe; Jensen, Robert T.

    2014-01-01

    Neuromedin B (NMB), a member of the bombesin family of peptides, is an autocrine growth factor for many lung cancer cells. The present study investigated the ability of NMB to cause transactivation of the epidermal growth factor (EGF) receptor in lung cancer cells. By Western blot, addition of NMB or related peptides to NCI-H1299 human non-small cell lung cancer (NSCLC) cells, caused phosphorylation of Tyr1068 of the EGF receptor. The signal was amplified using NCI-H1299 cells stably transected with NMB receptors. The transactivation of the EGF receptor or the tyrosine phosphorylation of ERK caused by NMB-like peptides was inhibited by AG1478 or gefitinib (tyrosine kinase inhibitors) and NMB receptor antagonist PD168368 but not the GRP receptor antagonist, BW2258U89. The transactivation of the EGF receptor caused by NMB-like peptides was inhibited by GM6001 (matrix metalloprotease inhibitor), PP2 (Src inhibitor), or transforming growth factor (TGF)α antibody. The transactivation of the EGF receptor and the increase in reactive oxygen species caused by NMB-like peptides was inhibited by N-acetylcysteine (NAC) or Tiron. Gefitinib inhibited the proliferation of NCI-H1299 cells and its sensitivity was increased by the addition of PD168368. The results indicate that the NMB receptor regulates EGF receptor transactivation by a mechanism dependent on Src as well as metalloprotease activation and generation of reactive oxygen species. PMID:20388507

  10. Beta-adrenergic-regulated phosphorylation of the skeletal muscle Ca(V)1.1 channel in the fight-or-flight response.

    PubMed

    Emrick, Michelle A; Sadilek, Martin; Konoki, Keiichi; Catterall, William A

    2010-10-26

    Ca(V)1 channels initiate excitation-contraction coupling in skeletal and cardiac muscle. During the fight-or-flight response, epinephrine released by the adrenal medulla and norepinephrine released from sympathetic nerves increase muscle contractility by activation of the β-adrenergic receptor/cAMP-dependent protein kinase pathway and up-regulation of Ca(V)1 channels in skeletal and cardiac muscle. Although the physiological mechanism of this pathway is well defined, the molecular mechanism and the sites of protein phosphorylation required for Ca(V)1 channel regulation are unknown. To identify the regulatory sites of phosphorylation under physiologically relevant conditions, Ca(V)1.1 channels were purified from skeletal muscle and sites of phosphorylation on the α1 subunit were identified by mass spectrometry. Two phosphorylation sites were identified in the proximal C-terminal domain, serine 1575 (S1575) and threonine 1579 (T1579), which are conserved in cardiac Ca(V)1.2 channels (S1700 and T1704, respectively). In vitro phosphorylation revealed that Ca(V)1.1-S1575 is a substrate for both cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase II, whereas Ca(V)1.1-T1579 is a substrate for casein kinase 2. Treatment of rabbits with isoproterenol to activate β-adrenergic receptors increased phosphorylation of S1575 in skeletal muscle Ca(V)1.1 channels in vivo, and treatment with propranolol to inhibit β-adrenergic receptors reduced phosphorylation. As S1575 and T1579 in Ca(V)1.1 channels and their homologs in Ca(V)1.2 channels are located at a key regulatory interface between the distal and proximal C-terminal domains, it is likely that phosphorylation of these sites in skeletal and cardiac muscle is directly involved in calcium channel regulation in response to the sympathetic nervous system in the fight-or-flight response.

  11. β-Adrenergic–regulated phosphorylation of the skeletal muscle CaV1.1 channel in the fight-or-flight response

    PubMed Central

    Emrick, Michelle A.; Sadilek, Martin; Konoki, Keiichi; Catterall, William A.

    2010-01-01

    CaV1 channels initiate excitation–contraction coupling in skeletal and cardiac muscle. During the fight-or-flight response, epinephrine released by the adrenal medulla and norepinephrine released from sympathetic nerves increase muscle contractility by activation of the β-adrenergic receptor/cAMP-dependent protein kinase pathway and up-regulation of CaV1 channels in skeletal and cardiac muscle. Although the physiological mechanism of this pathway is well defined, the molecular mechanism and the sites of protein phosphorylation required for CaV1 channel regulation are unknown. To identify the regulatory sites of phosphorylation under physiologically relevant conditions, CaV1.1 channels were purified from skeletal muscle and sites of phosphorylation on the α1 subunit were identified by mass spectrometry. Two phosphorylation sites were identified in the proximal C-terminal domain, serine 1575 (S1575) and threonine 1579 (T1579), which are conserved in cardiac CaV1.2 channels (S1700 and T1704, respectively). In vitro phosphorylation revealed that CaV1.1-S1575 is a substrate for both cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase II, whereas CaV1.1-T1579 is a substrate for casein kinase 2. Treatment of rabbits with isoproterenol to activate β-adrenergic receptors increased phosphorylation of S1575 in skeletal muscle CaV1.1 channels in vivo, and treatment with propranolol to inhibit β-adrenergic receptors reduced phosphorylation. As S1575 and T1579 in CaV1.1 channels and their homologs in CaV1.2 channels are located at a key regulatory interface between the distal and proximal C-terminal domains, it is likely that phosphorylation of these sites in skeletal and cardiac muscle is directly involved in calcium channel regulation in response to the sympathetic nervous system in the fight-or-flight response. PMID:20937870

  12. IKKβ phosphorylation regulates RPS3 nuclear translocation and NF-κB function during Escherichia coli O157:H7 infection

    PubMed Central

    Wan, Fengyi; Weaver, Amanda; Gao, Xiaofei; Bern, Michael; Hardwidge, Philip R.; Lenardo, Michael J.

    2011-01-01

    NF-κB is a major gene regulator in immune responses and ribosomal protein S3 (RPS3) is an NF-κB subunit that directs specific gene transcription. However, it is unknown how RPS3 nuclear translocation is regulated. Here we report that IKKβ phosphorylation of serine 209 (S209) was crucial for RPS3 nuclear localization in response to activating stimuli. Moreover, the foodborne pathogen Escherichia coli O157:H7 virulence protein NleH1 specifically inhibited RPS3 S209 phosphorylation and blocked RPS3 function, thereby promoting bacterial colonization and diarrhea but decreasing mortality in a gnotobiotic piglet infection model. Thus, the IKKβ-dependent modification of a specific amino acid in RPS3 promotes specific NF-κB functions that underlie the molecular pathogenetic mechanisms of E. coli O157:H7. PMID:21399639

  13. [Phosphorylation of tau protein].

    PubMed

    Uchida, T; Ishiguro, K

    1990-05-01

    In aged human brain and particularly in Alzheimer's disease brain, paired helical filaments (PHFs) accumulate in the neuronal cell. Recently, it has been found that the highly phosphorylated tau protein, one of the microtubule-associated proteins (MAPs), is a component of PHF. The authors attempted to clarify the mechanism underlying the accumulation of PHF from the following two aspects; 1) What is the mechanism of phosphorylation of tau protein? 2) Is the highly phosphorylated tau protein capable of forming PHFs? From rat or bovine microtubule proteins we partially purified and characterized a novel protein kinase that specifically phosphorylated tau and MAP2 among many proteins in the brain extract, and which formed a PHF epitope on the phosphorylated human tau. This enzyme was one of the protein serine/threonine kinases and was independent of known second messengers. The phosphorylation of tau by this enzyme was stimulated by tubulin under the condition of microtubule formation, suggesting that the phosphorylation of tau could occur concomitantly with microtubule formation in the brain. Since this kinase was usually bound to tau but not directly to tubulin, the enzyme was associated with microtubules through tau. From these properties related to tau, this kinase is designated as tau protein kinase. The tau that been phosphorylated with this kinase using [gamma-32P]ATP as a phosphate donor, was digested by endoprotinase Lys-C to produce three labeled fragments, K1, K2 and K3. These three fragments were sequenced and the phosphorylation sites on tau by this kinase were identified. The K2 fragment overlapped with the tau-1 site known to be one of the phosphorylation site in PHF. This result strengthens the possibility that tau protein phosphorylated by tau protein kinase is incorporated into PHF. Tubulin binding sites on tau were located between K1 and K3 fragments, while K2 fragment was located in the neighboring to N-terminus of K1. No phosphorylated sites were

  14. TGF-β regulates phosphorylation and stabilization of Sox9 protein in chondrocytes through p38 and Smad dependent mechanisms

    PubMed Central

    Coricor, George; Serra, Rosa

    2016-01-01

    Members of the TGF-β superfamily are important regulators of chondrocyte function. Sox9, a key transcriptional regulator of chondrogenesis, is required for TGF-β-mediated regulation of specific cartilage genes. TGF-β can signal through a canonical, Smad-mediated pathway or non-conical pathways, including p38. Here we show that both pathways are activated in chondrocytes after treatment with TGF-β and that TGF-β stabilizes Sox9 protein and increases phosphorylation of Sox9. Mutagenesis of potential serine phosphorylation sites on Sox9 was used to demonstrate that serine 211 is required to maintain normal basal levels of Sox9 as well as mediate increased Sox9 levels in response to TGF-β. The serine 211 site is in a motif that is targeted by p38 kinase. We used siRNA and pharmacological agents to show that p38 and Smad3 independently regulate the phosphorylation and stability of Sox9. Previously, we demonstrated that Papss2 is a downstream transcriptional target of Sox9 and TGF-β. Here we show that p38 is required for TGF-β-mediated regulation of Papss2 mRNA. Together the results suggest a new mechanism for TGF-β-mediated gene regulation in chondrocytes via p38 and phosphorylation and stabilization of Sox9. Understanding how TGF-β regulates Sox9 may lead to identification of therapeutic targets for OA. PMID:27929080

  15. Regulation of µ-Opioid Receptors: Desensitization, Phosphorylation, Internalization, and Tolerance

    PubMed Central

    Williams, John T.; Ingram, Susan L.; Henderson, Graeme; Chavkin, Charles; von Zastrow, Mark; Schulz, Stefan; Koch, Thomas; Evans, Christopher J.

    2013-01-01

    Morphine and related µ-opioid receptor (MOR) agonists remain among the most effective drugs known for acute relief of severe pain. A major problem in treating painful conditions is that tolerance limits the long-term utility of opioid agonists. Considerable effort has been expended on developing an understanding of the molecular and cellular processes that underlie acute MOR signaling, short-term receptor regulation, and the progression of events that lead to tolerance for different MOR agonists. Although great progress has been made in the past decade, many points of contention and controversy cloud the realization of this progress. This review attempts to clarify some confusion by clearly defining terms, such as desensitization and tolerance, and addressing optimal pharmacological analyses for discerning relative importance of these cellular mechanisms. Cellular and molecular mechanisms regulating MOR function by phosphorylation relative to receptor desensitization and endocytosis are comprehensively reviewed, with an emphasis on agonist-biased regulation and areas where knowledge is lacking or controversial. The implications of these mechanisms for understanding the substantial contribution of MOR signaling to opioid tolerance are then considered in detail. While some functional MOR regulatory mechanisms contributing to tolerance are clearly understood, there are large gaps in understanding the molecular processes responsible for loss of MOR function after chronic exposure to opioids. Further elucidation of the cellular mechanisms that are regulated by opioids will be necessary for the successful development of MOR-based approaches to new pain therapeutics that limit the development of tolerance. PMID:23321159

  16. A Computational Screen for Regulators of Oxidative Phosphorylation Implicates SLIRP in Mitochondrial RNA Homeostasis

    PubMed Central

    Baughman, Joshua M.; Nilsson, Roland; Gohil, Vishal M.; Arlow, Daniel H.; Gauhar, Zareen; Mootha, Vamsi K.

    2009-01-01

    The human oxidative phosphorylation (OxPhos) system consists of approximately 90 proteins encoded by nuclear and mitochondrial genomes and serves as the primary cellular pathway for ATP biosynthesis. While the core protein machinery for OxPhos is well characterized, many of its assembly, maturation, and regulatory factors remain unknown. We exploited the tight transcriptional control of the genes encoding the core OxPhos machinery to identify novel regulators. We developed a computational procedure, which we call expression screening, which integrates information from thousands of microarray data sets in a principled manner to identify genes that are consistently co-expressed with a target pathway across biological contexts. We applied expression screening to predict dozens of novel regulators of OxPhos. For two candidate genes, CHCHD2 and SLIRP, we show that silencing with RNAi results in destabilization of OxPhos complexes and a marked loss of OxPhos enzymatic activity. Moreover, we show that SLIRP plays an essential role in maintaining mitochondrial-localized mRNA transcripts that encode OxPhos protein subunits. Our findings provide a catalogue of potential novel OxPhos regulators that advance our understanding of the coordination between nuclear and mitochondrial genomes for the regulation of cellular energy metabolism. PMID:19680543

  17. Aurora B Kinase Regulates the Postmitotic Endoreduplication Checkpoint via Phosphorylation of the Retinoblastoma Protein at Serine 780

    PubMed Central

    Nair, Jayasree S.; Ho, Alan L.; Tse, Archie N.; Coward, Jesse; Cheema, Haider; Ambrosini, Grazia; Keen, Nicholas

    2009-01-01

    The phenotypic change characteristic of Aurora B inhibition is the induction of polyploidy. Utilizing specific siRNA duplexes and a selective small molecule inhibitor (AZD1152) to inhibit Aurora B activity in tumor cells, we sought to elucidate the mechanism by which Aurora B inhibition results in polyploidy. Cells treated with AZD1152 progressed through mitosis with misaligned chromosomes and exited without cytokinesis and subsequently underwent endoreduplication of DNA despite activation of a p53-dependent pseudo G1 checkpoint. Concomitant with polyploid cell formation, we observed the appearance of Rb hypophosphorylation, an event that occurred independently of cyclin-dependent kinase inhibition. We went on to discover that Aurora B directly phosphorylates Rb at serine 780 both in vitro and in vivo. This novel interaction plays a critical role in regulating the postmitotic checkpoint to prevent endoreduplication after an aberrant mitosis. Thus, we propose for the first time that Aurora B determines cellular fate after an aberrant mitosis by directly regulating the Rb tumor suppressor protein. PMID:19225156

  18. Enzyme Activities of the Ceramide Synthases CERS2–6 Are Regulated by Phosphorylation in the C-terminal Region*

    PubMed Central

    Sassa, Takayuki; Hirayama, Taisuke; Kihara, Akio

    2016-01-01

    Ceramide and complex sphingolipids regulate important cellular functions including cell growth, apoptosis, and signaling. Dysregulation of sphingolipid metabolism leads to pathological consequences such as sphingolipidoses and insulin resistance. Ceramides in mammals vary greatly in their acyl-chain composition: six different ceramide synthase isozymes (CERS1–6) that exhibit distinct substrate specificity and tissue distribution account for this diversity. In the present study, we demonstrated that CERS2–6 were phosphorylated at the cytoplasmic C-terminal regions. Most of the phosphorylated residues conformed to a consensus motif for phosphorylation by casein kinase 2 (CK2), and treatment of cells with the CK2-specific inhibitor CX-4945 lowered the phosphorylation levels of CERS2, -4, -5, and -6. Phosphorylation of CERS2 was especially important for its catalytic activity, acting mainly by increasing its Vmax value. Phosphorylation modestly increased the catalytic activities of CERS4 and -5 and mildly increased those of CERS3 and -6. Dephosphorylation of endogenous ceramide synthases in the mouse brain led to severely reduced activity toward the Cers2 substrates C22:0/C24:0-CoAs and modestly reduced activity toward the Cers5/6 substrate C16:0-CoA. These results suggest that the phosphorylation of ceramide synthases may be a key regulatory point in the control of the distribution and levels of sphingolipids of various acyl-chain lengths. PMID:26887952

  19. Tyrosine phosphorylation/dephosphorylation of myosin II essential light chains of Entamoeba histolytica trophozoites regulates their motility.

    PubMed

    Bonilla-Moreno, Raúl; Pérez-Yépez, Eloy-Andrés; Villegas-Sepúlveda, Nicolás; Morales, Fernando O; Meza, Isaura

    2016-08-01

    Entamoeba histolytica trophozoites dwell in the human intestine as comensals although under still unclear circumstances become invasive and destroy the host tissues. For these activities, trophozoites relay on remarkable motility provided by the cytoskeleton organization. Amebic actin and some of its actin-associated proteins are well known, while components of the myosin II molecule, although predicted from the E. histolytica genome, need biochemical and functional characterization. Recently, an amebic essential light myosin II chain, named EhMLCI, was identified and reported to be phosphorylated in tyrosines. The phosphorylated form of the protein was associated with the soluble assembly incompetent conformation of the heavy myosin chains, while the non-phosphorylated protein was identified with filamentous heavy chains, organized in an assembly competent conformation. It was postulated that EhMLCI tyrosine phosphorylation could act as a negative regulator of myosin II activity by its phosphorylation/dephosphorylation cycles. To test this hypothesis, we constructed an expression vector containing an EhMLCI DNA sequence where two tyrosine residues, with strong probability of phosphorylation and fall within the single EF-hand domain that interacts with the N-terminus of myosin II heavy chains, were replaced by phenylalanines. Transfected trophozoites, expressing the mutant MutEhMLCI protein cannot process it, thereby not incorporated into the phosphorylation/dephosphorylation cycles required for myosin II activity, results in motility defective trophozoites.

  20. Interferon-γ-dependent tyrosine phosphorylation of MEKK4 via Pyk2 is regulated by annexin II and SHP2 in keratinocytes

    PubMed Central

    Halfter, Ursula M.; Derbyshire, Zachary E.; Vaillancourt, Richard R.

    2004-01-01

    IFNγ (interferon-γ) binding to its cognate receptor results, through JAK (Janus kinase), in direct activation of receptor-bound STAT1 (signal transducer and activator of transcription 1), although there is evidence for additional activation of a MAPK (mitogen-activated protein kinase) pathway. In the present paper, we report IFNγ-dependent activation of the MEKK4 (MAPK/extracellular-signal-regulated kinase kinase kinase 4) pathway in HaCaT human keratinocytes. MEKK4 is tyrosine­-phosphorylated and the IFNγ-dependent phosphorylation requires intracellular calcium. Calcium-dependent phosphorylation of MEKK4 is mediated by Pyk2. Moreover, MEKK4 and Pyk2 co-localize in an IFNγ-dependent manner in the perinuclear region. Furthermore, the calcium-binding protein, annexin II, and the calcium-regulated kinase, Pyk2, co-immunoprecipitate with MEKK4 after treatment with IFNγ. Immunofluorescence imaging of HaCaT cells shows an IFNγ-dependent co-localization of annexin II with Pyk2 in the perinuclear region, suggesting that annexin II mediates the calcium-dependent regulation of Pyk2. Tyrosine phosphorylation of MEKK4 correlates with its activity to phosphorylate MKK6 (MAPK kinase 6) in vitro and subsequent p38 MAPK activation in an IFNγ-dependent manner. Additional studies demonstrate that the SH2 (Src homology 2)-domain-containing tyrosine phosphatase SHP2 co-immunoprecipitates with MEKK4 in an IFNγ-dependent manner and co-localizes with MEKK4 after IFNγ stimulation in the perinuclear region in HaCaT cells. Furthermore, we provide evidence that SHP2 dephosphorylates MEKK4 and Pyk2, terminating the MEKK4-dependent branch of the IFNγ signalling pathway. PMID:15601262

  1. Fasting and Systemic Insulin Signaling Regulate Phosphorylation of Brain Proteins That Modulate Cell Morphology and Link to Neurological Disorders.

    PubMed

    Li, Min; Quan, Chao; Toth, Rachel; Campbell, David G; MacKintosh, Carol; Wang, Hong Yu; Chen, Shuai

    2015-12-11

    Diabetes is strongly associated with cognitive decline, but the molecular reasons are unknown. We found that fasting and peripheral insulin promote phosphorylation and dephosphorylation, respectively, of specific residues on brain proteins including cytoskeletal regulators such as slit-robo GTPase-activating protein 3 (srGAP3) and microtubule affinity-regulating protein kinases (MARKs), in which deficiency or dysregulation is linked to neurological disorders. Fasting activates protein kinase A (PKA) but not PKB/Akt signaling in the brain, and PKA can phosphorylate the purified srGAP3. The phosphorylation of srGAP3 and MARKs were increased when PKA signaling was activated in primary neurons. Knockdown of PKA decreased the phosphorylation of srGAP3. Furthermore, WAVE1, a protein kinase A-anchoring protein, formed a complex with srGAP3 and PKA in the brain of fasted mice to facilitate the phosphorylation of srGAP3 by PKA. Although brain cells have insulin receptors, our findings are inconsistent with the down-regulation of phosphorylation of target proteins being mediated by insulin signaling within the brain. Rather, our findings infer that systemic insulin, through a yet unknown mechanism, inhibits PKA or protein kinase(s) with similar specificity and/or activates an unknown phosphatase in the brain. Ser(858) of srGAP3 was identified as a key regulatory residue in which phosphorylation by PKA enhanced the GAP activity of srGAP3 toward its substrate, Rac1, in cells, thereby inhibiting the action of this GTPase in cytoskeletal regulation. Our findings reveal novel mechanisms linking peripheral insulin sensitivity with cytoskeletal remodeling in neurons, which may help to explain the association of diabetes with neurological disorders such as Alzheimer disease.

  2. Quantitative Phosphoproteomics Reveals Novel Phosphorylation Events in Insulin Signaling Regulated by Protein Phosphatase 1 Regulatory Subunit 12A

    PubMed Central

    Zhang, Xiangmin; Ma, Danjun; Caruso, Michael; Lewis, Monique; Qi, Yue; Yi, Zhengping

    2014-01-01

    Serine/threonine protein phosphatase 1 regulatory subunit 12A (PPP1R12A) modulates the activity and specificity of the catalytic subunit of protein phosphatase 1, regulating various cellular processes via dephosphorylation. Nonetheless, little is known about phosphorylation events controlled by PPP1R12A in skeletal muscle insulin signaling. Here, we used quantitative phosphoproteomics to generate a global picture of phosphorylation events regulated by PPP1R12A in a L6 skeletal muscle cell line, which were engineered for inducible PPP1R12A knockdown. Phosphoproteomics revealed 3876 phosphorylation sites (620 were novel) in these cells. Furthermore, PPP1R12A knockdown resulted in increased overall phosphorylation in L6 cells at the basal condition, and changed phosphorylation levels for 698 sites (assigned to 295 phosphoproteins) at the basal and/or insulin-stimulated conditions. Pathway analysis on the 295 phosphoproteins revealed multiple significantly enriched pathways related to insulin signaling, such as mTOR signaling and RhoA signaling. Moreover, phosphorylation levels for numerous regulatory sites in these pathways were significantly changed due to PPP1R12A knockdown. These results indicate that PPP1R12A indeed plays a role in skeletal muscle insulin signaling, providing novel insights into the biology of insulin action. This new information may facilitate the design of experiments to better understand mechanisms underlying skeletal muscle insulin resistance and type 2 diabetes. PMID:24972320

  3. The Rho-GTPase effector ROCK regulates meiotic maturation of the bovine oocyte via myosin light chain phosphorylation and cofilin phosphorylation.

    PubMed

    Lee, So-Rim; Xu, Yong-Nan; Jo, Yu-Jin; Namgoong, Suk; Kim, Nam-Hyung

    2015-11-01

    Oocyte meiosis involves a unique asymmetric division involving spindle movement from the central cytoplasm to the cortex, followed by polar body extrusion. ROCK is a Rho-GTPase effector involved in various cellular functions in somatic cells as well as oocyte meiosis. ROCK was previously shown to promote actin organization by phosphorylating several downstream targets, including LIM domain kinase (LIMK), phosphorylated cofilin (p-cofilin), and myosin light chain (MLC). In this study, we investigated the roles of ROCK and MLC during bovine oocyte meiosis. We found that ROCK was localized around the nucleus at the oocyte's germinal-vesicle (GV) stage, but spreads to the rest of the cytoplasm in later developmental stages. On the other hand, phosphorylated MLC (p-MLC) localized at the cortex, and its abundance decreased by the metaphase-II stage. Disrupting ROCK activity, via RNAi or the chemical inhibitor Y-27632, blocked both cell cycle progression and polar body extrusion. ROCK inhibition also resulted in decreased cortical actin, p-cofilin, and p-MLC levels. Similar to the phenotype associated with inhibition of ROCK activity, inhibition of MLC kinase by the chemical inhibitor ML-7 caused defects in polar body extrusion. Collectively, our results suggest that the ROCK/MLC/actomyosin as well as ROCK/LIMK/cofilin pathways regulate meiotic spindle migration and cytokinesis during bovine oocyte maturation.

  4. A Novel Alpha Kinase EhAK1 Phosphorylates Actin and Regulates Phagocytosis in Entamoeba histolytica

    PubMed Central

    Mansuri, M. Shahid; Bhattacharya, Sudha; Bhattacharya, Alok

    2014-01-01

    Phagocytosis plays a key role in nutrient uptake and virulence of the protist parasite Entamoeba histolytica. Phagosomes have been characterized by proteomics, and their maturation in the cells has been studied. However, there is so far not much understanding about initiation of phagocytosis and formation of phagosomes at the molecular level. Our group has been studying initiation of phagocytosis and formation of phagosomes in E. histolytica, and have described some of the molecules that play key roles in the process. Here we show the involvement of EhAK1, an alpha kinase and a SH3 domain containing protein in the pathway that leads to formation of phagosomes using red blood cell as ligand particle. A number of approaches, such as proteomics, biochemical, confocal imaging using specific antibodies or GFP tagged molecules, expression down regulation by antisense RNA, over expression of wild type and mutant proteins, were used to understand the role of EhAK1 in phagocytosis. EhAK1 was found in the phagocytic cups during the progression of cups, until closure of phagosomes, but not in the phagosomes themselves. It is recruited to the phagosomes through interaction with the calcium binding protein EhCaBP1. A reduction in phagocytosis was observed when EhAK1 was down regulated by antisense RNA, or by over expression of the kinase dead mutant. G-actin was identified as one of the major substrates of EhAK1. Phosphorylated actin preferentially accumulated at the phagocytic cups and over expression of a phosphorylation defective actin led to defects in phagocytosis. In conclusion, we describe an important component of the pathway that is initiated on attachment of red blood cells to E. histolytica cells. The main function of EhAK1 is to couple signalling events initiated after accumulation of EhC2PK to actin dynamics. PMID:25299184

  5. A novel alpha kinase EhAK1 phosphorylates actin and regulates phagocytosis in Entamoeba histolytica.

    PubMed

    Mansuri, M Shahid; Bhattacharya, Sudha; Bhattacharya, Alok

    2014-10-01

    Phagocytosis plays a key role in nutrient uptake and virulence of the protist parasite Entamoeba histolytica. Phagosomes have been characterized by proteomics, and their maturation in the cells has been studied. However, there is so far not much understanding about initiation of phagocytosis and formation of phagosomes at the molecular level. Our group has been studying initiation of phagocytosis and formation of phagosomes in E. histolytica, and have described some of the molecules that play key roles in the process. Here we show the involvement of EhAK1, an alpha kinase and a SH3 domain containing protein in the pathway that leads to formation of phagosomes using red blood cell as ligand particle. A number of approaches, such as proteomics, biochemical, confocal imaging using specific antibodies or GFP tagged molecules, expression down regulation by antisense RNA, over expression of wild type and mutant proteins, were used to understand the role of EhAK1 in phagocytosis. EhAK1 was found in the phagocytic cups during the progression of cups, until closure of phagosomes, but not in the phagosomes themselves. It is recruited to the phagosomes through interaction with the calcium binding protein EhCaBP1. A reduction in phagocytosis was observed when EhAK1 was down regulated by antisense RNA, or by over expression of the kinase dead mutant. G-actin was identified as one of the major substrates of EhAK1. Phosphorylated actin preferentially accumulated at the phagocytic cups and over expression of a phosphorylation defective actin led to defects in phagocytosis. In conclusion, we describe an important component of the pathway that is initiated on attachment of red blood cells to E. histolytica cells. The main function of EhAK1 is to couple signalling events initiated after accumulation of EhC2PK to actin dynamics.

  6. Roles of Phosphorylation of the Nucleocapsid Protein of Mumps Virus in Regulating Viral RNA Transcription and Replication

    PubMed Central

    Zengel, James; Pickar, Adrian; Xu, Pei; Lin, Alita

    2015-01-01

    ABSTRACT Mumps virus (MuV) is a paramyxovirus with a negative-sense nonsegmented RNA genome. The viral RNA genome is encapsidated by the nucleocapsid protein (NP) to form the ribonucleoprotein (RNP), which serves as a template for transcription and replication. In this study, we investigated the roles of phosphorylation sites of NP in MuV RNA synthesis. Using radioactive labeling, we first demonstrated that NP was phosphorylated in MuV-infected cells. Using both liquid chromatography-mass spectrometry (LC-MS) and in silico modeling, we identified nine putative phosphorylated residues within NP. We mutated these nine residues to alanine. Mutation of the serine residue at position 439 to alanine (S439A) was found to reduce the phosphorylation of NP in transfected cells by over 90%. The effects of these mutations on the MuV minigenome system were examined. The S439A mutant was found to have higher activity, four mutants had lower activity, and four mutants had similar activity compared to wild-type NP. MuV containing the S439A mutation had 90% reduced phosphorylation of NP and enhanced viral RNA synthesis and viral protein expression at early time points after infection, indicating that S439 is the major phosphorylation site of NP and its phosphorylation plays an important role in downregulating viral RNA synthesis. IMPORTANCE Mumps virus (MuV), a paramyxovirus, is an important human pathogen that is reemerging in human populations. Nucleocapsid protein (NP) of MuV is essential for viral RNA synthesis. We have identified the major phosphorylation site of NP. We have found that phosphorylation of NP plays a critical role in regulating viral RNA synthesis. The work will lead to a better understanding of viral RNA synthesis and possible novel targets for antiviral drug development. PMID:25948749

  7. Phosphorylation of Rac1 T108 by Extracellular Signal-Regulated Kinase in Response to Epidermal Growth Factor: a Novel Mechanism To Regulate Rac1 Function

    PubMed Central

    Tong, Junfeng; Li, Laiji; Ballermann, Barbara

    2013-01-01

    Accumulating evidence has implicated Rho GTPases, including Rac1, in many aspects of cancer development. Recent findings suggest that phosphorylation might further contribute to the tight regulation of Rho GTPases. Interestingly, sequence analysis of Rac1 shows that Rac1 T108 within the 106PNTP109 motif is likely an extracellular signal-regulated kinase (ERK) phosphorylation site and that Rac1 also has an ERK docking site, 183KKRKRKCLLL192 (D site), at the C terminus. Indeed, we show here that both transfected and endogenous Rac1 interacts with ERK and that this interaction is mediated by its D site. Green fluorescent protein (GFP)-Rac1 is threonine (T) phosphorylated in response to epidermal growth factor (EGF), and EGF-induced Rac1 threonine phosphorylation is dependent on the activation of ERK. Moreover, mutant Rac1 with the mutation of T108 to alanine (A) is not threonine phosphorylated in response to EGF. In vitro ERK kinase assay further shows that pure active ERK phosphorylates purified Rac1 but not mutant Rac1 T108A. We also show that Rac1 T108 phosphorylation decreases Rac1 activity, partially due to inhibiting its interaction with phospholipase C-γ1 (PLC-γ1). T108 phosphorylation targets Rac1 to the nucleus, which isolates Rac1 from other guanine nucleotide exchange factors (GEFs) and hinders Rac1's role in cell migration. We conclude that Rac1 T108 is phosphorylated by ERK in response to EGF, which plays an important role in regulating Rac1. PMID:24043306

  8. Adrenergic regulation and diurnal rhythm of p38 mitogen-activated protein kinase phosphorylation in the rat pineal gland.

    PubMed

    Chik, C L; Mackova, M; Price, D; Ho, A K

    2004-11-01

    In this study, we investigated adrenergic and photoneural regulation of p38MAPK phosphorylation in the rat pineal gland. Norepinephrine (NE), the endogenous neurotransmitter, dose-dependently increased the levels of phosphorylated MAPK kinase 3/6 (MKK3/6) and p38MAPK in rat pinealocytes. Time-course studies showed a gradual increase in MKK3/6 and p38MAPK phosphorylation that peaked between 1 and 2 h and persisted for 4 h post NE stimulation. In cells treated with NE for 2 and 4 h, the inclusion of prazosin or propranolol reduced NE-induced MKK3/6 and p38MAPK phosphorylation, indicating involvement of both alpha- and beta-adrenergic receptors for the sustained response. Whereas treatment with dibutyryl cAMP or ionomycin mimicked the NE-induced MKK3/6 and p38MAPK phosphorylation, neither dibutyryl cGMP nor 4beta-phorbol 12-myristate 13-acetate had an effect. The NE-induced increase in MKK3/6 and p38MAPK phosphorylation was blocked by KT5720 (a protein kinase A inhibitor) and KN93 (a Ca(2+)/calmodulin-dependent kinase inhibitor), but not by KT5823 (a protein kinase G inhibitor) or calphostin C (a protein kinase C inhibitor). In animals housed under a lighting regimen with 12 h of light, MKK3/6 and p38MAPK phosphorylation increased in the rat pineal gland at zeitgeber time 18. The nocturnal increase in p38MAPK phosphorylation was blocked by exposing the animal to constant light and reduced by treatment with propranolol, a beta-adrenergic blocker. Together, our results indicate that activation of p38MAPK is under photoneural control in the rat pineal gland and that protein kinase A and intracellular Ca(2+) signaling pathways are involved in NE regulation of p38MAPK.

  9. CaMKII regulates proteasome phosphorylation and activity and promotes memory destabilization following retrieval.

    PubMed

    Jarome, Timothy J; Ferrara, Nicole C; Kwapis, Janine L; Helmstetter, Fred J

    2016-02-01

    Numerous studies have suggested that memories "destabilize" and require de novo protein synthesis in order to reconsolidate following retrieval, but very little is known about how this destabilization process is regulated. Recently, ubiquitin-proteasome mediated protein degradation has been identified as a critical regulator of memory trace destabilization following retrieval, though the specific mechanisms controlling retrieval-induced changes in ubiquitin-proteasome activity remain equivocal. Here, we found that proteasome activity is increased in the amygdala in a CaMKII-dependent manner following the retrieval of a contextual fear memory. We show that in vitro inhibition of CaMKII reversed retrieval-induced increases in proteasome activity. Additionally, in vivo pharmacological blockade of CaMKII abolished increases in proteolytic activity and activity related regulatory phosphorylation in the amygdala following retrieval, suggesting that CaMKII was "upstream" of protein degradation during the memory reconsolidation process. Consistent with this, while inhibiting CaMKII in the amygdala did not impair memory following retrieval, it completely attenuated the memory impairments that resulted from post-retrieval protein synthesis blockade. Collectively, these results suggest that CaMKII controls the initiation of the memory reconsolidation process through regulation of the proteasome.

  10. DPPC regulates COX-2 expression in monocytes via phosphorylation of CREB

    SciTech Connect

    Morris, R.H.K. Tonks, A.J.; Jones, K.P.; Ahluwalia, M.K.; Thomas, A.W.; Tonks, A.; Jackson, S.K.

    2008-05-23

    The major phospholipid in pulmonary surfactant dipalmitoyl phosphatidylcholine (DPPC) has been shown to modulate inflammatory responses. Using human monocytes, this study demonstrates that DPPC significantly increased PGE{sub 2} (P < 0.05) production by 2.5-fold when compared to untreated monocyte controls. Mechanistically, this effect was concomitant with an increase in COX-2 expression which was abrogated in the presence of a COX-2 inhibitor. The regulation of COX-2 expression was independent of NF-{kappa}B activity. Further, DPPC increased the phosphorylation of the cyclic AMP response element binding protein (CREB; an important nuclear transcription factor important in regulating COX-2 expression). In addition, we also show that changing the fatty acid groups of PC (e.g. using L-{alpha}-phosphatidylcholine {beta}-arachidonoyl-{gamma}-palmitoyl (PAPC)) has a profound effect on the regulation of COX-2 expression and CREB activation. This study provides new evidence for the anti-inflammatory activity of DPPC and that this activity is at least in part mediated via CREB activation of COX-2.

  11. Imiquimod directly inhibits Hedgehog signalling by stimulating adenosine receptor/protein kinase A-mediated GLI phosphorylation

    PubMed Central

    Wolff, F; Loipetzberger, A; Gruber, W; Esterbauer, H; Aberger, F; Frischauf, A M

    2013-01-01

    Imiquimod (IMQ), a nucleoside analogue of the imidazoquinoline family, is used in the topical treatment of basal cell carcinoma (BCC) and other skin diseases. It is reported to be a TLR7 and TLR8 agonist and, as such, initiates a Th1 immune response by activating sentinel cells in the vicinity of the tumour. BCC is a hedgehog (HH)-driven malignancy with oncogenic glioma-associated oncogene (GLI) signalling activated in a ligand-independent manner. Here we show that IMQ can also directly repress HH signalling by negatively modulating GLI activity in BCC and medulloblastoma cells. Further, we provide evidence that the repressive effect of IMQ on HH signalling is not dependent on TLR/MYD88 signalling. Our results suggest a mechanism for IMQ engaging adenosine receptors (ADORAs) to control GLI signalling. Pharmacological activation of ADORA with either an ADORA agonist or IMQ resulted in a protein kinase A (PKA)-mediated GLI phosphorylation and reduction in GLI activator levels. The activation of PKA and HH pathway target gene downregulation in response to IMQ were abrogated by ADORA inhibition. Furthermore, activated Smoothened signalling, which positively signals to GLI transcription factors, could be effectively counteracted by IMQ. These results reveal a previously unknown mode of action of IMQ in the treatment of BCC and also suggest a role for ADORAs in the regulation of oncogenic HH signalling. PMID:23995793

  12. Raf kinase inhibitory protein function is regulated via a flexible pocket and novel phosphorylation-dependent mechanism.

    PubMed

    Granovsky, Alexey E; Clark, Matthew C; McElheny, Dan; Heil, Gary; Hong, Jia; Liu, Xuedong; Kim, Youngchang; Joachimiak, Grazyna; Joachimiak, Andrzej; Koide, Shohei; Rosner, Marsha Rich

    2009-03-01

    Raf kinase inhibitory protein (RKIP/PEBP1), a member of the phosphatidylethanolamine binding protein family that possesses a conserved ligand-binding pocket, negatively regulates the mammalian mitogen-activated protein kinase (MAPK) signaling cascade. Mutation of a conserved site (P74L) within the pocket leads to a loss or switch in the function of yeast or plant RKIP homologues. However, the mechanism by which the pocket influences RKIP function is unknown. Here we show that the pocket integrates two regulatory signals, phosphorylation and ligand binding, to control RKIP inhibition of Raf-1. RKIP association with Raf-1 is prevented by RKIP phosphorylation at S153. The P74L mutation increases kinase interaction and RKIP phosphorylation, enhancing Raf-1/MAPK signaling. Conversely, ligand binding to the RKIP pocket inhibits kinase interaction and RKIP phosphorylation by a noncompetitive mechanism. Additionally, ligand binding blocks RKIP association with Raf-1. Nuclear magnetic resonance studies reveal that the pocket is highly dynamic, rationalizing its capacity to interact with distinct partners and be involved in allosteric regulation. Our results show that RKIP uses a flexible pocket to integrate ligand binding- and phosphorylation-dependent interactions and to modulate the MAPK signaling pathway. This mechanism is an example of an emerging theme involving the regulation of signaling proteins and their interaction with effectors at the level of protein dynamics.

  13. Molecular dynamics simulation of phosphorylation-induced conformational transitions in the mycobacterium tuberculosis response regulator PrrA

    SciTech Connect

    Chen, Guo; Mcmahon, Benjamin H; Tung, Chang - Shung

    2008-01-01

    Phosphorylation-activated modulation of response regulators (RR) is predominantly used by bacteria as a strategy in regulating their two-component signaling (TCS) systems, the underlying molecular mechanisms are however far from fully understood. In this work we have conducted a molecular dynamics (MD) simulation of the phosphorylation-induced conformational transitions of RRs with the Mycobacterium Tuberculosis PrrA as a particular example. Starting from the full-length inactive structure of PrrA we introduced a local disturbance by phosphorylating the conserved aspartic acid residue, Asp-58, in the regulatory domain. A Go-model-type algorithm packaged with AMBER force fields was then applied to simulate the dynamics upon phosphorylation. The MD simulation shows that the phosphorylation of Asp-58 facilitates PrrA, whose inactive state has a compact conformation with a closed interdomain interface, to open up with its interdomain separation being increased by an average of about 1.5 {angstrom} for a simulation of 20 ns. The trans-activation loop, which is completely buried within the interdomain interface in the inactive PrrA, is found to become more exposed with the phosphorylated structure as well. These results provide more structural details of how the phosphorylation of a local aspartate activates PrrA to undergo a global conformational rearrangement toward its extended active state. This work also indicates that MD simulations can serve as a fast tool to unravel the regulation mechanisms of all RRs, which is especially valuable when the structures of full-length active RRs are currently unavailable.

  14. Phosphorylation of αS1-casein is regulated by different genes.

    PubMed

    Bijl, E; van Valenberg, H J F; Huppertz, T; van Hooijdonk, A C M; Bovenhuis, H

    2014-11-01

    Casein phosphorylation is a posttranslational modification catalyzed by kinase enzymes that attach phosphate groups to specific AA in the protein sequence. This modification is one of the key factors responsible for the stabilization of calcium phosphate nanoclusters in casein micelles and for the internal structure of the casein micelles. α(S1)-Casein (α(s1)-CN) is of special interest because it constitutes up to 40% of the total casein fraction in milk, and it has 2 common phosphorylation states, with 8 (α(S1)-CN-8P) and 9 (α(S1)-CN-9P) phosphorylated serine residues. Factors affecting this variation in the degree of phosphorylation are not currently known. The objective of this research was to determine the genetic background of α(S1)-CN-8P and α(S1)-CN-9P. The genetic and phenotypic correlation between α(S1)-CN-8P and α(S1)-CN-9P was low (0.18 and 0.19, respectively). This low genetic correlation suggests a different genetic background. These differences were further investigated by means of a genome-wide association study, which showed that both α(S1)-CN-8P and α(S1)-CN-9P were affected by a region on Bos taurus autosome (BTA) 6, but only α(S1)-CN-8P was affected by a region on BTA11 that contains the gene that encodes for β-lactoglobulin (β-LG), and only α(S1)-CN-9P was affected by a region on BTA14 that contains the diacylglycerol acyltransferase 1 (DGAT1) gene. Estimated effects of β-LG protein genotypes showed that only α(S1)-CN-8P was associated with the β-LG A/B polymorphism (g.1772G>A and g.3054C>T); the AA genotype of β-LG was associated with a lower concentration of α(S1)-CN-8P (-0.32% wt/wt) than the BB genotype (+0.41% wt/wt). Estimated effects of DGAT1 K232A genotypes showed that only α(S1)-CN-9P was associated with the DGAT1 gene polymorphism; DGAT1 AA genotype was associated with a higher α(S1)-CN-9P concentration (+0.53% wt/wt) than the DGAT1 KK genotype (-0.44% wt/wt). The results give insight in phosphorylation of α(S1

  15. Direct visualization of the phosphorylated epidermal growth factor receptor during its internalization in A-431 cells

    PubMed Central

    1987-01-01

    Epidermal growth factor (EGF) rapidly stimulates receptor autophosphorylation in A-431 cells. After 1 min the phosphorylated receptor can be identified at the plasma membrane using an anti- phosphotyrosine antibody. With further incubation at 37 degrees C, approximately 50% of the phosphorylated EGF receptor was internalized (t1/2 = 5 min) and associated with the tubulovesicular system and later with multivesicular bodies, but not the nucleus. During this period, there was no change in the extent or sites of phosphorylation. At all times the phosphotyrosine remained on the cytoplasmic side of the membrane, opposite to the EGF ligand identified by anti-EGF antibody. These data indicate that (a) the tyrosine-phosphorylated EGF receptor is internalized in its activated form providing a mechanism for translocation of the receptor kinase to substrates in the cell interior; (b) the internalized receptor remains intact for at least 60 min, does not associate with the nucleus, and does not generate any tyrosine-phosphorylated fragments; and (c) tyrosine phosphorylation alone is not the signal for receptor internalization. PMID:2447100

  16. Lck regulates the tyrosine phosphorylation of the T cell receptor subunits and ZAP-70 in murine thymocytes

    PubMed Central

    1996-01-01

    The Src-family and Syk/ZAP-70 family of protein tyrosine kinases (PTK) are required for T cell receptor (TCR) functions. We provide evidence that the Src-family PTK Lck is responsible for regulating the constitutive tyrosine phosphorylation of the TCR zeta subunit in murine thymocytes. Moreover, ligation of the TCR expressed on thymocytes from Lck-deficient mice largely failed to induce the phosphorylation of TCR- zeta, CD3 epsilon, or ZAP-70. In contrast, we find that the TCR-zeta subunit is weakly constitutively tyrosine phosphorylated in peripheral T cells isolated from Lck-null mice. These data suggest that Lck has a functional role in regulation of TCR signal transduction in thymocytes. In peripheral T cells, other Src-family PTKs such as Fyn may partially compensate for the absence of Lck. PMID:8642247

  17. Mitochondrial cytochrome c oxidase: mechanism of action and role in regulating oxidative phosphorylation.

    PubMed

    Wilson, David F; Vinogradov, Sergei A

    2014-12-15

    Mitochondrial oxidative phosphorylation has a central role in eukaryotic metabolism, providing the energy (ATP) required for survival. Regulation of this important pathway is, however, still not understood, largely due to limitations in the ability to measure the essential metabolites, including oxygen (pO2, oxygen pressure), ADP, and AMP. In addition, neither the mechanism of oxygen reduction by mitochondrial cytochrome c oxidase nor how its rate is controlled is understood, although this enzyme determines the rate of oxygen consumption and thereby the rate of ATP synthesis. Cytochrome c oxidase is responsible for reduction of molecular oxygen to water using reducing equivalents donated by cytochrome c and for site 3 energy coupling in oxidative phosphorylation. A mechanism-based model of the cytochrome c oxidase reaction is presented in which transfer of reducing equivalents from the lower- to the higher-potential region of the coupling site occurs against an opposing energy barrier, Q. The steady-state rate equation is fitted to data for the dependence of mitochondrial respiratory rate on cytochrome c reduction, oxygen pressure (pO2), and [ATP]/[ADP][Pi] at pH 6.5 to 8.35 (where Pi is inorganic phosphate). The fit of the rate expression to the experimental data is very good for all experimental conditions. Levels of the intermediates in oxygen reduction in the oxidase reaction site have been calculated. An intermediate in the reaction, tentatively identified as peroxide, bridged between the iron and copper atoms of the reaction site has a central role in coupling mitochondrial respiration to the [ATP]/[ADP][Pi].

  18. Density-enhanced Phosphatase 1 Regulates Phosphorylation of Tight Junction Proteins and Enhances Barrier Function of Epithelial Cells*

    PubMed Central

    Sallee, Jennifer L.; Burridge, Keith

    2009-01-01

    Cell-cell adhesion is a dynamic process that can activate multiple signaling pathways. These signaling pathways can be regulated through reversible tyrosine phosphorylation events. The level of tyrosine phosphorylation of junctional proteins reflects the balance between protein-tyrosine kinase and protein-tyrosine phosphatase activity. The receptor-tyrosine phosphatase DEP-1 (CD148/PTP-η) has been implicated in cell growth and differentiation as well as in regulating phosphorylation of junctional proteins. However, the role of DEP-1 in regulating tight junction phosphorylation and the integrity of cell-cell junctions is still under investigation. In this study, we used a catalytically dead substrate-trapping mutant of DEP-1 to identify potential substrates at cell-cell junctions. We have shown that in epithelial cells the trapping mutant of DEP-1 interacts with the tight junction proteins occludin and ZO-1 in a tyrosine phosphorylation-dependent manner. In contrast, PTP-PEST, Shp2, and PTPμ did not interact with these proteins, suggesting that the interaction of DEP-1 with occludin and ZO-1 is specific. In addition, occludin and ZO-1 were dephosphorylated by DEP-1 but not these other phosphatases in vitro. Overexpression of DEP-1 increased barrier function as measured by transepithelial electrical resistance and also reduced paracellular flux of fluorescein isothiocyanate-dextran following a calcium switch. Reduced DEP-1 expression by small interfering RNA had a small but significant increase in junction permeability. These data suggest that DEP-1 can modify the phosphorylation state of tight junction proteins and play a role in regulating permeability. PMID:19332538

  19. Tyrosine phosphorylation-independent regulation of lipopolysaccharide-mediated response by the transmembrane adaptor protein LAB.

    PubMed

    Zhu, Minghua; Fuller, Deirdre M; Ou-Yang, Chih-wen; Sullivan, Sarah A; Zhang, Weiguo

    2012-03-15

    Linker for activation of B cells (LAB)/non-T cell activation linker is a transmembrane adaptor protein that functions in immunoreceptor-mediated signaling. Published studies have shown that LAB has both positive and negative roles in regulating TCR and high-affinity Fc receptor-mediated signaling and cellular function. In this study, we showed that LAB was also expressed in dendritic cells and that LAB deficiency affected LPS-mediated signaling and cytokine production. LPS-mediated MAPK activation was enhanced in LAB(-/-) bone marrow-derived dendritic cells. These bone marrow-derived dendritic cells also produced more TNF-α, IL-6, and IL-10 than wild-type cells. Moreover, LAB(-/-) mice were hyperresponsive to LPS-induced septic shock. These data indicated that LAB has a negative role in LPS-mediated responses. By using LAB knockin mice, which harbor mutations at five membrane-distal tyrosines, we further showed that, in contrast to its role in immunoreceptor-mediated signaling, LAB function in LPS-mediated signaling pathway did not depend on its tyrosine phosphorylation. Our study suggested a novel mechanism by which LAB functions in the regulation of innate immunity.

  20. Rassf5 and Ndr kinases regulate neuronal polarity through Par3 phosphorylation in a novel pathway.

    PubMed

    Yang, Rui; Kong, Eryan; Jin, Jing; Hergovich, Alexander; Püschel, Andreas W

    2014-08-15

    The morphology and polarized growth of cells depend on pathways that control the asymmetric distribution of regulatory factors. The evolutionarily conserved Ndr kinases play important roles in cell polarity and morphogenesis in yeast and invertebrates but it is unclear whether they perform a similar function in mammalian cells. Here, we analyze the function of mammalian Ndr1 and Ndr2 (also known as STK38 or STK38L, respectively) in the establishment of polarity in neurons. We show that they act downstream of the tumor suppressor Rassf5 and upstream of the polarity protein Par3 (also known as PARD3). Rassf5 and Ndr1 or Ndr2 are required during the polarization of hippocampal neurons to prevent the formation of supernumerary axons. Mechanistically, the Ndr kinases act by phosphorylating Par3 at Ser383 to inhibit its interaction with dynein, thereby polarizing the distribution of Par3 and reinforcing axon specification. Our results identify a novel Rassf5-Ndr-Par3 signaling cascade that regulates the transport of Par3 during the establishment of neuronal polarity. Their role in neuronal polarity suggests that Ndr kinases perform a conserved function as regulators of cell polarity.

  1. Bordetella pertussis fim3 gene regulation by BvgA: phosphorylation controls the formation of inactive vs. active transcription complexes.

    PubMed

    Boulanger, Alice; Moon, Kyung; Decker, Kimberly B; Chen, Qing; Knipling, Leslie; Stibitz, Scott; Hinton, Deborah M

    2015-02-10

    Two-component systems [sensor kinase/response regulator (RR)] are major tools used by microorganisms to adapt to environmental conditions. RR phosphorylation is typically required for gene activation, but few studies have addressed how and if phosphorylation affects specific steps during transcription initiation. We characterized transcription complexes made with RNA polymerase and the Bordetella pertussis RR, BvgA, in its nonphosphorylated or phosphorylated (BvgA∼P) state at P(fim3), the promoter for the virulence gene fim3 (fimbrial subunit), using gel retardation, potassium permanganate and DNase I footprinting, cleavage reactions with protein conjugated with iron bromoacetamidobenzyl-EDTA, and in vitro transcription. Previous work has shown that the level of nonphosphorylated BvgA remains high in vivo under conditions in which BvgA is phosphorylated. Our results here indicate that surprisingly both BvgA and BvgA∼P form open and initiating complexes with RNA polymerase at P(fim3). However, phosphorylation of BvgA is needed to generate the correct conformation that can transition to competent elongation. Footprints obtained with the complexes made with nonphosphorylated BvgA are atypical; while the initiating complex with BvgA synthesizes short RNA, it does not generate full-length transcripts. Extended incubation of the BvgA/RNA polymerase initiated complex in the presence of heparin generates a stable, but defective species that depends on the initial transcribed sequence of fim3. We suggest that the presence of nonphosphorylated BvgA down-regulates P(fim3) activity when phosphorylated BvgA is present and may allow the bacterium to quickly adapt to the loss of inducing conditions by rapidly eliminating P(fim3) activation once the signal for BvgA phosphorylation is removed.

  2. Dietary salt regulates the phosphorylation of OSR1/SPAK kinases and the sodium chloride cotransporter through aldosterone.

    PubMed

    Chiga, Motoko; Rai, Tatemitsu; Yang, Sung-Sen; Ohta, Akihito; Takizawa, Toichiro; Sasaki, Sei; Uchida, Shinichi

    2008-12-01

    Pseudohypoaldosteronism type II (PHAII) is caused by mutations in the WNK1 and WNK4 genes (WNK with-no-lysine kinase). In a mouse model of this disease where a mutant of Wnk4 D561A was knocked in, increased phosphorylation of the sodium chloride cotransporter (NCC) was found and the transporter was concentrated on the apical membrane of the distal tubules. In addition, we recently found that other kinases, such as the oxidative stress response kinase-1/STE20/SPS1-related proline alanine-rich kinase (OSR1/SPAK), also showed increased phosphorylation in these mice. Here we determined whether this kinase cascade is regulated by dietary salt intake. We found that the phosphorylation states of NCC and OSR1/SPAK were increased by low-salt diets and decreased by high-salt diets; a regulation completely lost in the knock-in mice. Increased phosphorylation was reversed by spironolactone and this decreased phosphorylation was reversed by administration of exogenous aldosterone. These studies suggest that that the WNK-OSR1/SPAK-NCC cascade may be a novel effector system of aldosterone action in the kidney.

  3. Phosphorylation of syntaxin-3 at Thr 14 negatively regulates exocytosis in RBL-2H3 mast cells.

    PubMed

    Tadokoro, Satoshi; Shibata, Tetsuhiro; Inoh, Yoshikazu; Amano, Toshiro; Nakanishi, Mamoru; Hirashima, Naohide; Utsunomiya-Tate, Naoko

    2016-05-01

    Recent studies have revealed that soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins interact with each other, forming a SNARE complex that induces exocytosis in mast cells. Previously, we reported that syntaxin-3, a SNARE protein, regulates mast cell exocytosis and is constantly phosphorylated. In this study, we tried to identify the amino acid residue that is phosphorylated in mast cells, and to elucidate the regulatory mechanism of exocytosis by phosphorylation in syntaxin-3. We found that Thr 14 of syntaxin-3 was a phosphorylation site in mast cells. In addition, the overexpression of a constitutively dephosphorylated syntaxin-3 (T14A) mutant enhanced mast cell exocytosis. We also showed that the phosphomimetic mutation of syntaxin-3 at Thr 14 (T14E) induced structural changes in syntaxin-3, and this mutation inhibited binding of syntaxin-3 to Munc18-2. These results suggest that phosphorylated syntaxin-3 at Thr 14 negatively regulates mast cell exocytosis by impairing the interaction between syntaxin-3 and Munc18-2.

  4. Proto-Oncogenic Src Phosphorylates EB1 to Regulate the Microtubule-Focal Adhesion Crosstalk and Stimulate Cell Migration.

    PubMed

    Zhang, Yijun; Luo, Youguang; Lyu, Rui; Chen, Jie; Liu, Ruming; Li, Dengwen; Liu, Min; Zhou, Jun

    2016-01-01

    Cell migration, a complex process critical for tumor progression and metastasis, requires a dynamic crosstalk between microtubules (MTs) and focal adhesions (FAs). However, the molecular mechanisms underlying this event remain elusive. Herein we identify the proto-oncogenic protein Src as an important player in the regulation of the MT-FA crosstalk. Src interacts with and phosphorylates end-binding protein 1 (EB1), a member of MT plus end-tracking proteins (+TIPs), both in cells and in vitro. Systematic mutagenesis reveals that tyrosine-247 (Y247) is the primary residue of EB1 phosphorylated by Src. Interestingly, both constitutively activated Src and Y247-phosphorylated EB1 localize to the centrosome and FAs. Src-mediated EB1 phosphorylation diminishes its interactions with other +TIPs, including adenomatous polyposis coli (APC) and mitotic centromere associated kinesin (MCAK). In addition, EB1 phosphorylation at Y247 enhances the rate of MT catastrophe and significantly stimulates cell migration. These findings thus demonstrate that the Src-EB1 axis plays a crucial role in regulating the crosstalk between MTs and FAs to promote cell migration.

  5. Stress-evoked tyrosine phosphorylation of signal regulatory protein α regulates behavioral immobility in the forced swim test.

    PubMed

    Ohnishi, Hiroshi; Murata, Takaaki; Kusakari, Shinya; Hayashi, Yuriko; Takao, Keizo; Maruyama, Toshi; Ago, Yukio; Koda, Ken; Jin, Feng-Jie; Okawa, Katsuya; Oldenborg, Per-Arne; Okazawa, Hideki; Murata, Yoji; Furuya, Nobuhiko; Matsuda, Toshio; Miyakawa, Tsuyoshi; Matozaki, Takashi

    2010-08-04

    Severe stress induces changes in neuronal function that are implicated in stress-related disorders such as depression. The molecular mechanisms underlying the response of the brain to stress remain primarily unknown, however. Signal regulatory protein alpha (SIRPalpha) is an Ig-superfamily protein that undergoes tyrosine phosphorylation and binds the protein tyrosine phosphatase Shp2. Here we show that mice expressing a form of SIRPalpha that lacks most of the cytoplasmic region manifest prolonged immobility (depression-like behavior) in the forced swim (FS) test. FS stress induced marked tyrosine phosphorylation of SIRPalpha in the brain of wild-type mice through activation of Src family kinases. The SIRPalpha ligand CD47 was important for such SIRPalpha phosphorylation, and CD47-deficient mice also manifested prolonged immobility in the FS test. Moreover, FS stress-induced tyrosine phosphorylation of both the NR2B subunit of the NMDA subtype of glutamate receptor and the K+-channel subunit Kvbeta2 was regulated by SIRPalpha. Thus, tyrosine phosphorylation of SIRPalpha is important for regulation of depression-like behavior in the response of the brain to stress.

  6. The Late S-Phase Transcription Factor Hcm1 Is Regulated through Phosphorylation by the Cell Wall Integrity Checkpoint

    PubMed Central

    Negishi, Takahiro; Veis, Jiri; Hollenstein, David; Sekiya, Mizuho; Ammerer, Gustav

    2016-01-01

    The cell wall integrity (CWI) checkpoint in the budding yeast Saccharomyces cerevisiae coordinates cell wall construction and cell cycle progression. In this study, we showed that the regulation of Hcm1, a late-S-phase transcription factor, arrests the cell cycle via the cell wall integrity checkpoint. Although the HCM1 mRNA level remained unaffected when the cell wall integrity checkpoint was induced, the protein level decreased. The overproduction of Hcm1 resulted in the failure of the cell wall integrity checkpoint. We identified 39 Hcm1 phosphorylation sites, including 26 novel sites, by tandem mass spectrometry analysis. A mutational analysis revealed that phosphorylation of Hcm1 at S61, S65, and S66 is required for the proper onset of the cell wall integrity checkpoint by regulating the timely decrease in its protein level. Hyperactivation of the CWI mitogen-activated protein kinase (MAPK) signaling pathway significantly reduced the Hcm1 protein level, and the deletion of CWI MAPK Slt2 resulted in a failure to decrease Hcm1 protein levels in response to stress, suggesting that phosphorylation is regulated by CWI MAPK. In conclusion, we suggest that Hcm1 is regulated negatively by the cell wall integrity checkpoint through timely phosphorylation and degradation under stress to properly control budding yeast proliferation. PMID:26729465

  7. Structural insights into the recruitment of SMRT by the corepressor SHARP under phosphorylative regulation.

    PubMed

    Mikami, Suzuka; Kanaba, Teppei; Takizawa, Naoki; Kobayashi, Ayaho; Maesaki, Ryoko; Fujiwara, Toshinobu; Ito, Yutaka; Mishima, Masaki

    2014-01-07

    The transcriptional corepressors SMRT/NCoR, components of histone deacetylase complexes, interact with nuclear receptors and many other transcription factors. SMRT is a target for the ubiquitously expressed protein kinase CK2, which is known to phosphorylate a wide variety of substrates. Increasing evidence suggests that CK2 plays a regulatory role in many cellular events, particularly, in transcription. However, little is known about the precise mode of action involved. Here, we report the three-dimensional structure of a SMRT/HDAC1-associated repressor protein (SHARP) in complex with phosphorylated SMRT, as determined by solution NMR. Phosphorylation of the CK2 site on SMRT significantly increased affinity for SHARP. We also confirmed the significance of CK2 phosphorylation by reporter assay and propose a mechanism involving the process of phosphorylation acting as a molecular switch. Finally, we propose that the SPOC domain functions as a phosphorylation binding module.

  8. PKCdelta-mediated IRS-1 Ser24 phosphorylation negatively regulates IRS-1 function.

    PubMed

    Greene, Michael W; Ruhoff, Mary S; Roth, Richard A; Kim, Jeong-A; Quon, Michael J; Krause, Jean A

    2006-10-27

    The IRS-1 PH and PTB domains are essential for insulin-stimulated IRS-1 Tyr phosphorylation and insulin signaling, while Ser/Thr phosphorylation of IRS-1 disrupts these signaling events. To investigate consensus PKC phosphorylation sites in the PH-PTB domains of human IRS-1, we changed Ser24, Ser58, and Thr191 to Ala (3A) or Glu (3E), to block or mimic phosphorylation, respectively. The 3A mutant abrogated the inhibitory effect of PKCdelta on insulin-stimulated IRS-1 Tyr phosphorylation, while reductions in insulin-stimulated IRS-1 Tyr phosphorylation, cellular proliferation, and Akt activation were observed with the 3E mutant. When single Glu mutants were tested, the Ser24 to Glu mutant had the greatest inhibitory effect on insulin-stimulated IRS-1 Tyr phosphorylation. PKCdelta-mediated IRS-1 Ser24 phosphorylation was confirmed in cells with PKCdelta catalytic domain mutants and by an RNAi method. Mechanistic studies revealed that IRS-1 with Ala and Glu point mutations at Ser24 impaired phosphatidylinositol-4,5-bisphosphate binding. In summary, our data are consistent with the hypothesis that Ser24 is a negative regulatory phosphorylation site in IRS-1.

  9. PKC{delta}-mediated IRS-1 Ser24 phosphorylation negatively regulates IRS-1 function

    SciTech Connect

    Greene, Michael W. . E-mail: michael.greene@bassett.org; Ruhoff, Mary S.; Roth, Richard A.; Kim, Jeong-a; Quon, Michael J.; Krause, Jean A.

    2006-10-27

    The IRS-1 PH and PTB domains are essential for insulin-stimulated IRS-1 Tyr phosphorylation and insulin signaling, while Ser/Thr phosphorylation of IRS-1 disrupts these signaling events. To investigate consensus PKC phosphorylation sites in the PH-PTB domains of human IRS-1, we changed Ser24, Ser58, and Thr191 to Ala (3A) or Glu (3E), to block or mimic phosphorylation, respectively. The 3A mutant abrogated the inhibitory effect of PKC{delta} on insulin-stimulated IRS-1 Tyr phosphorylation, while reductions in insulin-stimulated IRS-1 Tyr phosphorylation, cellular proliferation, and Akt activation were observed with the 3E mutant. When single Glu mutants were tested, the Ser24 to Glu mutant had the greatest inhibitory effect on insulin-stimulated IRS-1 Tyr phosphorylation. PKC{delta}-mediated IRS-1 Ser24 phosphorylation was confirmed in cells with PKC{delta} catalytic domain mutants and by an RNAi method. Mechanistic studies revealed that IRS-1 with Ala and Glu point mutations at Ser24 impaired phosphatidylinositol-4,5-bisphosphate binding. In summary, our data are consistent with the hypothesis that Ser24 is a negative regulatory phosphorylation site in IRS-1.

  10. Bacillus subtilis Two-Component System Sensory Kinase DegS Is Regulated by Serine Phosphorylation in Its Input Domain

    PubMed Central

    Jers, Carsten; Kobir, Ahasanul; Søndergaard, Elsebeth Oline; Jensen, Peter Ruhdal; Mijakovic, Ivan

    2011-01-01

    Bacillus subtilis two-component system DegS/U is well known for the complexity of its regulation. The cytosolic sensory kinase DegS does not receive a single predominant input signal like most two-component kinases, instead it integrates a wide array of metabolic inputs that modulate its activity. The phosphorylation state of the response regulator DegU also does not confer a straightforward “on/off” response; it is fine-tuned and at different levels triggers different sub-regulons. Here we describe serine phosphorylation of the DegS sensing domain, which stimulates its kinase activity. We demonstrate that DegS phosphorylation can be carried out by at least two B. subtilis Hanks-type kinases in vitro, and this stimulates the phosphate transfer towards DegU. The consequences of this process were studied in vivo, using phosphomimetic (Ser76Asp) and non-phosphorylatable (Ser76Ala) mutants of DegS. In a number of physiological assays focused on different processes regulated by DegU, DegS S76D phosphomimetic mutant behaved like a strain with intermediate levels of DegU phosphorylation, whereas DegS S76A behaved like a strain with lower levels of DegU phophorylation. These findings suggest a link between DegS phosphorylation at serine 76 and the level of DegU phosphorylation, establishing this post-translational modification as an additional trigger for this two-component system. PMID:21304896

  11. Myosin 3A Kinase Activity Is Regulated by Phosphorylation of the Kinase Domain Activation Loop*

    PubMed Central

    Quintero, Omar A.; Unrath, William C.; Stevens, Stanley M.; Manor, Uri; Kachar, Bechara; Yengo, Christopher M.

    2013-01-01

    Class III myosins are unique members of the myosin superfamily in that they contain both a motor and kinase domain. We have found that motor activity is decreased by autophosphorylation, although little is known about the regulation of the kinase domain. We demonstrate by mass spectrometry that Thr-178 and Thr-184 in the kinase domain activation loop and two threonines in the loop 2 region of the motor domain are autophosphorylated (Thr-908 and Thr-919). The kinase activity of MYO3A 2IQ with the phosphomimic (T184E) or phosphoblock (T184A) mutations demonstrates that kinase activity is reduced 30-fold as a result of the T184A mutation, although the Thr-178 site only had a minor impact on kinase activity. Interestingly, the actin-activated ATPase activity of MYO3A 2IQ is slightly reduced as a result of the T178A and T184A mutations suggesting coupling between motor and kinase domains. Full-length GFP-tagged T184A and T184E MYO3A constructs transfected into COS7 cells do not disrupt the ability of MYO3A to localize to filopodia structures. In addition, we demonstrate that T184E MYO3A reduces filopodia elongation in the presence of espin-1, whereas T184A enhances filopodia elongation in a similar fashion to kinase-dead MYO3A. Our results suggest that as MYO3A accumulates at the tips of actin protrusions, autophosphorylation of Thr-184 enhances kinase activity resulting in phosphorylation of the MYO3A motor and reducing motor activity. The differential regulation of the kinase and motor activities allows for MYO3A to precisely self-regulate its concentration in the actin bundle-based structures of cells. PMID:24214986

  12. Nuclear phosphoproteome analysis of 3T3-L1 preadipocyte differentiation reveals system-wide phosphorylation of transcriptional regulators.

    PubMed

    Rabiee, Atefeh; Schwämmle, Veit; Sidoli, Simone; Dai, Jie; Rogowska-Wrzesinska, Adelina; Mandrup, Susanne; Jensen, Ole N

    2017-03-01

    Adipocytes (fat cells) are important endocrine and metabolic cells critical for systemic insulin sensitivity. Both adipose excess and insufficiency are associated with adverse metabolic function. Adipogenesis is the process whereby preadipocyte precursor cells differentiate into lipid-laden mature adipocytes. This process is driven by a network of transcriptional regulators (TRs). We hypothesized that protein PTMs, in particular phosphorylation, play a major role in activating and propagating signals within TR networks upon induction of adipogenesis by extracellular stimulus. We applied MS-based quantitative proteomics and phosphoproteomics to monitor the alteration of nuclear proteins during the early stages (4 h) of preadipocyte differentiation. We identified a total of 4072 proteins including 2434 phosphorylated proteins, a majority of which were assigned as regulators of gene expression. Our results demonstrate that adipogenic stimuli increase the nuclear abundance and/or the phosphorylation levels of proteins involved in gene expression, cell organization, and oxidation-reduction pathways. Furthermore, proteins acting as negative modulators involved in negative regulation of gene expression, insulin stimulated glucose uptake, and cytoskeletal organization showed a decrease in their nuclear abundance and/or phosphorylation levels during the first 4 h of adipogenesis. Among 288 identified TRs, 49 were regulated within 4 h of adipogenic stimulation including several known and many novel potential adipogenic regulators. We created a kinase-substrate database for 3T3-L1 preadipocytes by investigating the relationship between protein kinases and protein phosphorylation sites identified in our dataset. A majority of the putative protein kinases belong to the cyclin-dependent kinase family and the mitogen-activated protein kinase family including P38 and c-Jun N-terminal kinases, suggesting that these kinases act as orchestrators of early adipogenesis.

  13. Tissue Strain Reorganizes Collagen With a Switchlike Response That Regulates Neuronal Extracellular Signal-Regulated Kinase Phosphorylation In Vitro: Implications for Ligamentous Injury and Mechanotransduction

    PubMed Central

    Zhang, Sijia; Cao, Xuan; Stablow, Alec M.; Shenoy, Vivek B.; Winkelstein, Beth A.

    2016-01-01

    Excessive loading of ligaments can activate the neural afferents that innervate the collagenous tissue, leading to a host of pathologies including pain. An integrated experimental and modeling approach was used to define the responses of neurons and the surrounding collagen fibers to the ligamentous matrix loading and to begin to understand how macroscopic deformation is translated to neuronal loading and signaling. A neuron-collagen construct (NCC) developed to mimic innervation of collagenous tissue underwent tension to strains simulating nonpainful (8%) or painful ligament loading (16%). Both neuronal phosphorylation of extracellular signal-regulated kinase (ERK), which is related to neuroplasticity (R2 ≥ 0.041; p ≤ 0.0171) and neuronal aspect ratio (AR) (R2 ≥ 0.250; p < 0.0001), were significantly correlated with tissue-level strains. As NCC strains increased during a slowly applied loading (1%/s), a “switchlike” fiber realignment response was detected with collagen reorganization occurring only above a transition point of 11.3% strain. A finite-element based discrete fiber network (DFN) model predicted that at bulk strains above the transition point, heterogeneous fiber strains were both tensile and compressive and increased, with strains in some fibers along the loading direction exceeding the applied bulk strain. The transition point identified for changes in collagen fiber realignment was consistent with the measured strain threshold (11.7% with a 95% confidence interval of 10.2–13.4%) for elevating ERK phosphorylation after loading. As with collagen fiber realignment, the greatest degree of neuronal reorientation toward the loading direction was observed at the NCC distraction corresponding to painful loading. Because activation of neuronal ERK occurred only at strains that produced evident collagen fiber realignment, findings suggest that tissue strain-induced changes in the micromechanical environment, especially altered local

  14. Interaction of Nck1 and PERK phosphorylated at Y⁵⁶¹ negatively modulates PERK activity and PERK regulation of pancreatic β-cell proinsulin content.

    PubMed

    Yamani, Lama; Latreille, Mathieu; Larose, Louise

    2014-03-01

    PERK, the PKR-like endoplasmic reticulum (ER) kinase, is an ER transmembrane serine/threonine protein kinase activated during ER stress. In this study, we provide evidence that the Src-homology domain-containing adaptor Nck1 negatively regulates PERK. We show that Nck directly binds to phosphorylated Y(561) in the PERK juxtamembrane domain through its SH2 domain. We demonstrate that mutation of Y(561) to a nonphosphorylatable residue (Y561F) promotes PERK activity, suggesting that PERK phosphorylation at Y(561) (pY(561)PERK) negatively regulates PERK. In agreement, we show that pY(561)PERK delays PERK activation and signaling during ER stress. Compatible with a role for PERK in pancreatic β-cells, we provide strong evidence that Nck1 contributes to PERK regulation of pancreatic β-cell proteostasis. In fact, we demonstrated that down-regulation of Nck1 in mouse insulinoma MIN6 cells results in faster dephosphorylation of pY(561)PERK, which correlates with enhanced PERK activation, increased insulin biosynthesis, and PERK-dependent increase in proinsulin content. Furthermore, we report that pancreatic islets in whole-body Nck1-knockout mice contain more insulin than control littermates. Together our data strongly suggest that Nck1 negatively regulates PERK by interacting with PERK and protecting PERK from being dephosphorylated at its inhibitory site pY(561) and in this way affects pancreatic β-cell proinsulin biogenesis.

  15. The mitosis-regulating and protein-protein interaction activities of astrin are controlled by aurora-A-induced phosphorylation.

    PubMed

    Chiu, Shao-Chih; Chen, Jo-Mei Maureen; Wei, Tong-You Wade; Cheng, Tai-Shan; Wang, Ya-Hui Candice; Ku, Chia-Feng; Lian, Chiao-Hsuan; Liu, Chun-Chih Jared; Kuo, Yi-Chun; Yu, Chang-Tze Ricky

    2014-09-01

    Cells display dramatic morphological changes in mitosis, where numerous factors form regulatory networks to orchestrate the complicated process, resulting in extreme fidelity of the segregation of duplicated chromosomes into two daughter cells. Astrin regulates several aspects of mitosis, such as maintaining the cohesion of sister chromatids by inactivating Separase and stabilizing spindle, aligning and segregating chromosomes, and silencing spindle assembly checkpoint by interacting with Src kinase-associated phosphoprotein (SKAP) and cytoplasmic linker-associated protein-1α (CLASP-1α). To understand how Astrin is regulated in mitosis, we report here that Astrin acts as a mitotic phosphoprotein, and Aurora-A phosphorylates Astrin at Ser(115). The phosphorylation-deficient mutant Astrin S115A abnormally activates spindle assembly checkpoint and delays mitosis progression, decreases spindle stability, and induces chromosome misalignment. Mechanistic analyses reveal that Astrin phosphorylation mimicking mutant S115D, instead of S115A, binds and induces ubiquitination and degradation of securin, which sequentially activates Separase, an enzyme required for the separation of sister chromatids. Moreover, S115A fails to bind mitosis regulators, including SKAP and CLASP-1α, which results in the mitotic defects observed in Astrin S115A-transfected cells. In conclusion, Aurora-A phosphorylates Astrin and guides the binding of Astrin to its cellular partners, which ensures proper progression of mitosis.

  16. Prostate Cell-Specific Regulation of Androgen Receptor Phosphorylation In Vivo

    DTIC Science & Technology

    2007-11-01

    phosphorylation of S650 is enhanced by treatment with forskolin (FSK), Epidermal Growth Factor (EGF) and phorbol-12-myristate-13-acetate (PMA)[Gioeli, D., J. Biol...phosphorylation. - 7 - Figure 2: A PMA but not R1881 or Forskolin induces S650 phosporylation. LNCaP cells were steroid starved and

  17. Calcium-regulated in vivo protein phosphorylation in Zea mays L. root tips

    NASA Technical Reports Server (NTRS)

    Raghothama, K. G.; Reddy, A. S.; Friedmann, M.; Poovaiah, B. W.

    1987-01-01

    Calcium dependent protein phosphorylation was studied in corn (Zea mays L.) root tips. Prior to in vivo protein phosphorylation experiments, the effect of calcium, ethyleneglycol-bis-(beta-aminoethyl ether)-N-N' -tetraacetic acid (EGTA) and calcium ionophore (A-23187) on phosphorus uptake was studied. Calcium increased phosphorus uptake, whereas EGTA and A-23187 decreased it. Consequently, phosphorus concentration in the media was adjusted so as to attain similar uptake in different treatments. Phosphoproteins were analyzed by two-dimensional gel electrophoresis. Distinct changes in phosphorylation were observed following altered calcium levels. Calcium depletion in root tips with EGTA and A-23187 decreased protein phosphorylation. However, replenishment of calcium following EGTA and ionophore pretreatment enhanced phosphorylation of proteins. Preloading of the root tips with 32P in the presence of EGTA and A-23187 followed by a ten minute calcium treatment, resulted in increased phosphorylation indicating the involvement of calcium, calcium and calmodulin-dependent kinases. Calmodulin antagonist W-7 was effective in inhibiting calcium-promoted phosphorylation. These studies suggest a physiological role for calcium-dependent phosphorylation in calcium-mediated processes in plants.

  18. Papaverine Prevents Vasospasm by Regulation of Myosin Light Chain Phosphorylation and Actin Polymerization in Human Saphenous Vein

    PubMed Central

    Hocking, Kyle M.; Putumbaka, Gowthami; Wise, Eric S.; Cheung-Flynn, Joyce; Brophy, Colleen M.; Komalavilas, Padmini

    2016-01-01

    Objective Papaverine is used to prevent vasospasm in human saphenous veins (HSV) during vein graft preparation prior to implantation as a bypass conduit. Papaverine is a nonspecific inhibitor of phosphodiesterases, leading to increases in both intracellular cGMP and cAMP. We hypothesized that papaverine reduces force by decreasing intracellular calcium concentrations ([Ca2+]i) and myosin light chain phosphorylation, and increasing actin depolymerization via regulation of actin regulatory protein phosphorylation. Approach and Results HSV was equilibrated in a muscle bath, pre-treated with 1 mM papaverine followed by 5 μM norepinephrine, and force along with [Ca2+]i levels were concurrently measured. Filamentous actin (F-actin) level was measured by an in vitro actin assay. Tissue was snap frozen to measure myosin light chain and actin regulatory protein phosphorylation. Pre-treatment with papaverine completely inhibited norepinephrine-induced force generation, blocked increases in [Ca2+]i and led to a decrease in the phosphorylation of myosin light chain. Papaverine pre-treatment also led to increased phosphorylation of the heat shock-related protein 20 (HSPB6) and the vasodilator stimulated phosphoprotein (VASP), as well as decreased filamentous actin (F-actin) levels suggesting depolymerization of actin. Conclusions These results suggest that papaverine-induced force inhibition of HSV involves [Ca2+]i-mediated inhibition of myosin light chain phosphorylation and actin regulatory protein phosphorylation-mediated actin depolymerization. Thus, papaverine induces sustained inhibition of contraction of HSV by the modulation of both myosin cross-bridge formation and actin cytoskeletal dynamics and is a pharmacological alternative to high pressure distention to prevent vasospasm. PMID:27136356

  19. Cardiac β2-Adrenergic Receptor Phosphorylation at Ser355/356 Regulates Receptor Internalization and Functional Resensitization.

    PubMed

    Fan, Xiaofang; Gu, Xuejiang; Zhao, Ru; Zheng, Qingqing; Li, Lan; Yang, Wenbing; Ding, Lu; Xue, Feng; Fan, Junming; Gong, Yongsheng; Wang, Yongyu

    2016-01-01

    Previous studies have demonstrated that β2-adrenergic receptors (β2ARs) can be phosphorylated by G protein-coupled receptor kinases (GRKs) and protein kinase A (PKA), affecting β2AR internalization and desensitization. However, the exact physiological function of β2ARs in cardiomyocytes is unknown. In this study, we showed that neonatal mouse cardiomyocytes had different contraction and internalization responses to sustained or repeated, transient agonist stimulation. Specifically, short-time stimulation (10 min) with epinephrine or norepinephrine increased the cardiomyocyte contraction rate, reaching a maximum at 5 min, followed by a slow decline. When the agonist was re-added after a 60-min wash-out period, the increase in the cardiomyocyte contraction rate was similar to the initial response. In contrast, when cardiomyocytes were exposed continuously to epinephrine or norepinephrine for 60 min, the second agonist stimulation did not increase the contraction response. These results indicated that continuous β2AR stimulation caused functional desensitization. Phosphorylation of β2ARs at serine (Ser)355/356 GRK phosphorylation sites, but not at Ser345/346 PKA phosphorylation sites increased with continuous epinephrine stimulation for 60 min. Accordingly, β2AR internalization increased. Interestingly, β2AR internalization was blocked by mutations at the GRK phosphorylation sites, but not by mutations at the PKA phosphorylation sites. Furthermore, inhibition of β2AR dephosphorylation by okadaic acid, a phosphatase 2A inhibitor, impaired the recovery of internalized β2ARs and reduced the cardiomyocyte contraction rate in response to epinephrine. Finally, epinephrine treatment induced the physical interaction of β-arrestin with internalized β2ARs in cardiomyocytes. Together, these data revealed the essential role of the Ser355/356 phosphorylation status of β2ARs in regulating receptor internalization and physiological resensitization in neonatal

  20. A model of yeast cell-cycle regulation based on multisite phosphorylation

    PubMed Central

    Barik, Debashis; Baumann, William T; Paul, Mark R; Novak, Bela; Tyson, John J

    2010-01-01

    In order for the cell's genome to be passed intact from one generation to the next, the events of the cell cycle (DNA replication, mitosis, cell division) must be executed in the correct order, despite the considerable molecular noise inherent in any protein-based regulatory system residing in the small confines of a eukaryotic cell. To assess the effects of molecular fluctuations on cell-cycle progression in budding yeast cells, we have constructed a new model of the regulation of Cln- and Clb-dependent kinases, based on multisite phosphorylation of their target proteins and on positive and negative feedback loops involving the kinases themselves. To account for the significant role of noise in the transcription and translation steps of gene expression, the model includes mRNAs as well as proteins. The model equations are simulated deterministically and stochastically to reveal the bistable switching behavior on which proper cell-cycle progression depends and to show that this behavior is robust to the level of molecular noise expected in yeast-sized cells (∼50 fL volume). The model gives a quantitatively accurate account of the variability observed in the G1-S transition in budding yeast, which is governed by an underlying sizer+timer control system. PMID:20739927

  1. ATR-mediated phosphorylation of FANCI regulates dormant origin firing in response to replication stress.

    PubMed

    Chen, Yu-Hung; Jones, Mathew J K; Yin, Yandong; Crist, Sarah B; Colnaghi, Luca; Sims, Robert J; Rothenberg, Eli; Jallepalli, Prasad V; Huang, Tony T

    2015-04-16

    Excess dormant origins bound by the minichromosome maintenance (MCM) replicative helicase complex play a critical role in preventing replication stress, chromosome instability, and tumorigenesis. In response to DNA damage, replicating cells must coordinate DNA repair and dormant origin firing to ensure complete and timely replication of the genome; how cells regulate this process remains elusive. Herein, we identify a member of the Fanconi anemia (FA) DNA repair pathway, FANCI, as a key effector of dormant origin firing in response to replication stress. Cells lacking FANCI have reduced number of origins, increased inter-origin distances, and slowed proliferation rates. Intriguingly, ATR-mediated FANCI phosphorylation inhibits dormant origin firing while promoting replication fork restart/DNA repair. Using super-resolution microscopy, we show that FANCI co-localizes with MCM-bound chromatin in response to replication stress. These data reveal a unique role for FANCI as a modulator of dormant origin firing and link timely genome replication to DNA repair.

  2. Cell-cycle dependent phosphorylation of yeast pericentrin regulates γ-TuSC-mediated microtubule nucleation

    PubMed Central

    Lin, Tien-chen; Neuner, Annett; Schlosser, Yvonne T; Scharf, Annette ND; Weber, Lisa; Schiebel, Elmar

    2014-01-01

    Budding yeast Spc110, a member of γ-tubulin complex receptor family (γ-TuCR), recruits γ-tubulin complexes to microtubule (MT) organizing centers (MTOCs). Biochemical studies suggest that Spc110 facilitates higher-order γ-tubulin complex assembly (Kollman et al., 2010). Nevertheless the molecular basis for this activity and the regulation are unclear. Here we show that Spc110 phosphorylated by Mps1 and Cdk1 activates γ-TuSC oligomerization and MT nucleation in a cell cycle dependent manner. Interaction between the N-terminus of the γ-TuSC subunit Spc98 and Spc110 is important for this activity. Besides the conserved CM1 motif in γ-TuCRs (Sawin et al., 2004), a second motif that we named Spc110/Pcp1 motif (SPM) is also important for MT nucleation. The activating Mps1 and Cdk1 sites lie between SPM and CM1 motifs. Most organisms have both SPM-CM1 (Spc110/Pcp1/PCNT) and CM1-only (Spc72/Mto1/Cnn/CDK5RAP2/myomegalin) types of γ-TuCRs. The two types of γ-TuCRs contain distinct but conserved C-terminal MTOC targeting domains. DOI: http://dx.doi.org/10.7554/eLife.02208.001 PMID:24842996

  3. c-Jun-N-terminal phosphorylation regulates DNMT1 expression and genome wide methylation in gliomas

    PubMed Central

    Heiland, Dieter H; Ferrarese, Roberto; Claus, Rainer; Dai, Fangping; Masilamani, Anie P; Kling, Eva; Weyerbrock, Astrid; Kling, Teresia; Nelander, Sven; Carro, Maria S

    2017-01-01

    High-grade gliomas (HGG) are the most common brain tumors, with an average survival time of 14 months. A glioma-CpG island methylator phenotype (G-CIMP), associated with better clinical outcome, has been described in low and high-grade gliomas. Mutation of IDH1 is known to drive the G-CIMP status. In some cases, however, the hypermethylation phenotype is independent of IDH1 mutation, suggesting the involvement of other mechanisms. Here, we demonstrate that DNMT1 expression is higher in low-grade gliomas compared to glioblastomas and correlates with phosphorylated c-Jun. We show that phospho-c-Jun binds to the DNMT1 promoter and causes DNA hypermethylation. Phospho-c-Jun activation by Anisomycin treatment in primary glioblastoma-derived cells attenuates the aggressive features of mesenchymal glioblastomas and leads to promoter methylation and downregulation of key mesenchymal genes (CD44, MMP9 and CHI3L1). Our findings suggest that phospho-c-Jun activates an important regulatory mechanism to control DNMT1 expression and regulate global DNA methylation in Glioblastoma. PMID:28036297

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

    PubMed Central

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

    2016-01-01

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

  5. ATM and GLUT1-S490 Phosphorylation Regulate GLUT1 Mediated Transport in Skeletal Muscle

    PubMed Central

    Andrisse, Stanley; Patel, Gaytri D.; Chen, Joseph E.; Webber, Andrea M.; Spears, Larry D.; Koehler, Rikki M.; Robinson-Hill, Rona M.; Ching, James K.; Jeong, Imju; Fisher, Jonathan S.

    2013-01-01

    Objective The glucose and dehydroascorbic acid (DHA) transporter GLUT1 contains a phosphorylation site, S490, for ataxia telangiectasia mutated (ATM). The objective of this study was to determine whether ATM and GLUT1-S490 regulate GLUT1. Research Design and Methods L6 myoblasts and mouse skeletal muscles were used to study the effects of ATM inhibition, ATM activation, and S490 mutation on GLUT1 localization, trafficking, and transport activity. Results In myoblasts, inhibition of ATM significantly diminished cell surface GLUT1, glucose and DHA transport, GLUT1 externalization, and association of GLUT1 with Gα-interacting protein-interacting protein, C-terminus (GIPC1), which has been implicated in recycling of endosomal proteins. In contrast, ATM activation by doxorubicin (DXR) increased DHA transport, cell surface GLUT1, and the GLUT1/GIPC1 association. S490A mutation decreased glucose and DHA transport, cell surface GLUT1, and interaction of GLUT1 with GIPC1, while S490D mutation increased transport, cell surface GLUT1, and the GLUT1/GIPC1 interaction. ATM dysfunction or ATM inhibition reduced DHA transport in extensor digitorum longus (EDL) muscles and decreased glucose transport in EDL and soleus. In contrast, DXR increased DHA transport in EDL. Conclusions These results provide evidence that ATM and GLUT1-S490 promote cell surface GLUT1 and GLUT1-mediated transport in skeletal muscle associated with upregulation of the GLUT1/GIPC1 interaction. PMID:23776597

  6. Phosphorylation of the Synaptonemal Complex Protein Zip1 Regulates the Crossover/Noncrossover Decision during Yeast Meiosis.

    PubMed

    Chen, Xiangyu; Suhandynata, Ray T; Sandhu, Rima; Rockmill, Beth; Mohibullah, Neeman; Niu, Hengyao; Liang, Jason; Lo, Hsiao-Chi; Miller, Danny E; Zhou, Huilin; Börner, G Valentin; Hollingsworth, Nancy M

    2015-12-01

    Interhomolog crossovers promote proper chromosome segregation during meiosis and are formed by the regulated repair of programmed double-strand breaks. This regulation requires components of the synaptonemal complex (SC), a proteinaceous structure formed between homologous chromosomes. In yeast, SC formation requires the "ZMM" genes, which encode a functionally diverse set of proteins, including the transverse filament protein, Zip1. In wild-type meiosis, Zmm proteins promote the biased resolution of recombination intermediates into crossovers that are distributed throughout the genome by interference. In contrast, noncrossovers are formed primarily through synthesis-dependent strand annealing mediated by the Sgs1 helicase. This work identifies a conserved region on the C terminus of Zip1 (called Zip1 4S), whose phosphorylation is required for the ZMM pathway of crossover formation. Zip1 4S phosphorylation is promoted both by double-strand breaks (DSBs) and the meiosis-specific kinase, MEK1/MRE4, demonstrating a role for MEK1 in the regulation of interhomolog crossover formation, as well as interhomolog bias. Failure to phosphorylate Zip1 4S results in meiotic prophase arrest, specifically in the absence of SGS1. This gain of function meiotic arrest phenotype is suppressed by spo11Δ, suggesting that it is due to unrepaired breaks triggering the meiotic recombination checkpoint. Epistasis experiments combining deletions of individual ZMM genes with sgs1-md zip1-4A indicate that Zip1 4S phosphorylation functions prior to the other ZMMs. These results suggest that phosphorylation of Zip1 at DSBs commits those breaks to repair via the ZMM pathway and provides a mechanism by which the crossover/noncrossover decision can be dynamically regulated during yeast meiosis.

  7. Phosphorylation of the Synaptonemal Complex Protein Zip1 Regulates the Crossover/Noncrossover Decision during Yeast Meiosis

    PubMed Central

    Chen, Xiangyu; Suhandynata, Ray T.; Sandhu, Rima; Rockmill, Beth; Mohibullah, Neeman; Niu, Hengyao; Liang, Jason; Lo, Hsiao-Chi; Miller, Danny E.; Zhou, Huilin; Börner, G. Valentin; Hollingsworth, Nancy M.

    2015-01-01

    Interhomolog crossovers promote proper chromosome segregation during meiosis and are formed by the regulated repair of programmed double-strand breaks. This regulation requires components of the synaptonemal complex (SC), a proteinaceous structure formed between homologous chromosomes. In yeast, SC formation requires the “ZMM” genes, which encode a functionally diverse set of proteins, including the transverse filament protein, Zip1. In wild-type meiosis, Zmm proteins promote the biased resolution of recombination intermediates into crossovers that are distributed throughout the genome by interference. In contrast, noncrossovers are formed primarily through synthesis-dependent strand annealing mediated by the Sgs1 helicase. This work identifies a conserved region on the C terminus of Zip1 (called Zip1 4S), whose phosphorylation is required for the ZMM pathway of crossover formation. Zip1 4S phosphorylation is promoted both by double-strand breaks (DSBs) and the meiosis-specific kinase, MEK1/MRE4, demonstrating a role for MEK1 in the regulation of interhomolog crossover formation, as well as interhomolog bias. Failure to phosphorylate Zip1 4S results in meiotic prophase arrest, specifically in the absence of SGS1. This gain of function meiotic arrest phenotype is suppressed by spo11Δ, suggesting that it is due to unrepaired breaks triggering the meiotic recombination checkpoint. Epistasis experiments combining deletions of individual ZMM genes with sgs1-md zip1-4A indicate that Zip1 4S phosphorylation functions prior to the other ZMMs. These results suggest that phosphorylation of Zip1 at DSBs commits those breaks to repair via the ZMM pathway and provides a mechanism by which the crossover/noncrossover decision can be dynamically regulated during yeast meiosis. PMID:26682552

  8. CDC28 phosphorylates Cac1p and regulates the association of chromatin assembly factor I with chromatin.

    PubMed

    Jeffery, Daniel C B; Kakusho, Naoko; You, Zhiying; Gharib, Marlene; Wyse, Brandon; Drury, Erin; Weinreich, Michael; Thibault, Pierre; Verreault, Alain; Masai, Hisao; Yankulov, Krassimir

    2015-01-01

    Chromatin Assembly Factor I (CAF-I) plays a key role in the replication-coupled assembly of nucleosomes. It is expected that its function is linked to the regulation of the cell cycle, but little detail is available. Current models suggest that CAF-I is recruited to replication forks and to chromatin via an interaction between its Cac1p subunit and the replication sliding clamp, PCNA, and that this interaction is stimulated by the kinase CDC7. Here we show that another kinase, CDC28, phosphorylates Cac1p on serines 94 and 515 in early S phase and regulates its association with chromatin, but not its association with PCNA. Mutations in the Cac1p-phosphorylation sites of CDC28 but not of CDC7 substantially reduce the in vivo phosphorylation of Cac1p. However, mutations in the putative CDC7 target sites on Cac1p reduce its stability. The association of CAF-I with chromatin is impaired in a cdc28-1 mutant and to a lesser extent in a cdc7-1 mutant. In addition, mutations in the Cac1p-phosphorylation sites by both CDC28 and CDC7 reduce gene silencing at the telomeres. We propose that this phosphorylation represents a regulatory step in the recruitment of CAF-I to chromatin in early S phase that is distinct from the association of CAF-I with PCNA. Hence, we implicate CDC28 in the regulation of chromatin reassembly during DNA replication. These findings provide novel mechanistic insights on the links between cell-cycle regulation, DNA replication and chromatin reassembly.

  9. CDC28 phosphorylates Cac1p and regulates the association of chromatin assembly factor i with chromatin

    PubMed Central

    Jeffery, Daniel CB; Kakusho, Naoko; You, Zhiying; Gharib, Marlene; Wyse, Brandon; Drury, Erin; Weinreich, Michael; Thibault, Pierre; Verreault, Alain; Masai, Hisao; Yankulov, Krassimir

    2015-01-01

    Chromatin Assembly Factor I (CAF-I) plays a key role in the replication-coupled assembly of nucleosomes. It is expected that its function is linked to the regulation of the cell cycle, but little detail is available. Current models suggest that CAF-I is recruited to replication forks and to chromatin via an interaction between its Cac1p subunit and the replication sliding clamp, PCNA, and that this interaction is stimulated by the kinase CDC7. Here we show that another kinase, CDC28, phosphorylates Cac1p on serines 94 and 515 in early S phase and regulates its association with chromatin, but not its association with PCNA. Mutations in the Cac1p-phosphorylation sites of CDC28 but not of CDC7 substantially reduce the in vivo phosphorylation of Cac1p. However, mutations in the putative CDC7 target sites on Cac1p reduce its stability. The association of CAF-I with chromatin is impaired in a cdc28–1 mutant and to a lesser extent in a cdc7–1 mutant. In addition, mutations in the Cac1p-phosphorylation sites by both CDC28 and CDC7 reduce gene silencing at the telomeres. We propose that this phosphorylation represents a regulatory step in the recruitment of CAF-I to chromatin in early S phase that is distinct from the association of CAF-I with PCNA. Hence, we implicate CDC28 in the regulation of chromatin reassembly during DNA replication. These findings provide novel mechanistic insights on the links between cell-cycle regulation, DNA replication and chromatin reassembly. PMID:25602519

  10. Activation of Cdk5/p25 and tau phosphorylation following chronic brain hypoperfusion in rats involves microRNA-195 down-regulation.

    PubMed

    Sun, Li-Hua; Ban, Tao; Liu, Cheng-Di; Chen, Qing-Xin; Wang, Xu; Yan, Mei-Ling; Hu, Xue-Ling; Su, Xiao-Lin; Bao, Ya-Nan; Sun, Lin-Lin; Zhao, Lin-Jing; Pei, Shuang-Chao; Jiang, Xue-Mei; Zong, De-Kang; Ai, Jing

    2015-09-01

    . Schematic diagram of miR-195 mediated Aβ aggregation and tau hyperphosphorylation in chronic brain hypoperfusion (CBH). First, CBH results in the elevation of nuclear factor-κB (NF-κB), which binds with the promoter sequences of miR-195 and negatively regulates the expression of miR-195. Second, down-regulated miR-195 induces up-regulation of APP and BACE1 and leads to an increase in Aβ levels. Third, some of the elevated Aβ then enter the intracellular space and activate calpain, which promotes the conversion of Cdk5/p35 to Cdk5/p25 and catalyzes the degradation of IκB; IκB is an inhibitor of NF-κB, which activates NF-κB. Cdk5/p25 directly phosphorylates Tau. Fourth, down-regulated miR-195 induces an up-regulation of p35, which provides the active substrates of p25. Our findings demonstrated that the down-regulation of miR-195 plays a key role in the increased vulnerability to dementia via the regulation of multiple targets following CBH.

  11. Regulation of NuA4 histone acetyltransferase activity in transcription and DNA repair by phosphorylation of histone H4.

    PubMed

    Utley, Rhea T; Lacoste, Nicolas; Jobin-Robitaille, Olivier; Allard, Stéphane; Côté, Jacques

    2005-09-01

    The NuA4 complex is a histone H4/H2A acetyltransferase involved in transcription and DNA repair. While histone acetylation is important in many processes, it has become increasingly clear that additional histone modifications also play a crucial interrelated role. To understand how NuA4 action is regulated, we tested various H4 tail peptides harboring known modifications in HAT assays. While dimethylation at arginine 3 (R3M) had little effect on NuA4 activity, phosphorylation of serine 1 (S1P) strongly decreased the ability of the complex to acetylate H4 peptides. However, R3M in combination with S1P alleviates the repression of NuA4 activity. Chromatin from cells treated with DNA damage-inducing agents shows an increase in phosphorylation of serine 1 and a concomitant decrease in H4 acetylation. We found that casein kinase 2 phosphorylates histone H4 and associates with the Rpd3 deacetylase complex, demonstrating a physical connection between phosphorylation of serine 1 and unacetylated H4 tails. Chromatin immunoprecipitation experiments also link local phosphorylation of H4 with its deacetylation, during both transcription and DNA repair. Time course chromatin immunoprecipitation data support a model in which histone H4 phosphorylation occurs after NuA4 action during double-strand break repair at the step of chromatin restoration and deacetylation. These findings demonstrate that H4 phospho-serine 1 regulates chromatin acetylation by the NuA4 complex and that this process is important for normal gene expression and DNA repair.

  12. A cytoplasmic negative regulator isoform of ATF7 impairs ATF7 and ATF2 phosphorylation and transcriptional activity.

    PubMed

    Diring, Jessica; Camuzeaux, Barbara; Donzeau, Mariel; Vigneron, Marc; Rosa-Calatrava, Manuel; Kedinger, Claude; Chatton, Bruno

    2011-01-01

    Alternative splicing and post-translational modifications are processes that give rise to the complexity of the proteome. The nuclear ATF7 and ATF2 (activating transcription factor) are structurally homologous leucine zipper transcription factors encoded by distinct genes. Stress and growth factors activate ATF2 and ATF7 mainly via sequential phosphorylation of two conserved threonine residues in their activation domain. Distinct protein kinases, among which mitogen-activated protein kinases (MAPK), phosphorylate ATF2 and ATF7 first on Thr71/Thr53 and next on Thr69/Thr51 residues respectively, resulting in transcriptional activation. Here, we identify and characterize a cytoplasmic alternatively spliced isoform of ATF7. This variant, named ATF7-4, inhibits both ATF2 and ATF7 transcriptional activities by impairing the first phosphorylation event on Thr71/Thr53 residues. ATF7-4 indeed sequesters the Thr53-phosphorylating kinase in the cytoplasm. Upon stimulus-induced phosphorylation, ATF7-4 is poly-ubiquitinated and degraded, enabling the release of the kinase and ATF7/ATF2 activation. Our data therefore conclusively establish that ATF7-4 is an important cytoplasmic negative regulator of ATF7 and ATF2 transcription factors.

  13. Diverse roles for auxiliary subunits in phosphorylation-dependent regulation of mammalian brain voltage-gated potassium channels.

    PubMed

    Vacher, Helene; Trimmer, James S

    2011-11-01

    Voltage-gated ion channels are a diverse family of signaling proteins that mediate rapid electrical signaling events. Among these, voltage-gated potassium or Kv channels are the most diverse partly due to the large number of principal (or α) subunits and auxiliary subunits that can assemble in different combinations to generate Kv channel complexes with distinct structures and functions. The diversity of Kv channels underlies much of the variability in the active properties between different mammalian central neurons and the dynamic changes that lead to experience-dependent plasticity in intrinsic excitability. Recent studies have revealed that Kv channel α subunits and auxiliary subunits are extensively phosphorylated, contributing to additional structural and functional diversity. Here, we highlight recent studies that show that auxiliary subunits exert some of their profound effects on dendritic Kv4 and axonal Kv1 channels through phosphorylation-dependent mechanisms, either due to phosphorylation on the auxiliary subunit itself or by influencing the extent and/or impact of α subunit phosphorylation. The complex effects of auxiliary subunits and phosphorylation provide a potent mechanism to generate additional diversity in the structure and function of Kv4 and Kv1 channels, as well as allowing for dynamic reversible regulation of these important ion channels.

  14. A Cytoplasmic Negative Regulator Isoform of ATF7 Impairs ATF7 and ATF2 Phosphorylation and Transcriptional Activity

    PubMed Central

    Diring, Jessica; Camuzeaux, Barbara; Donzeau, Mariel; Vigneron, Marc; Rosa-Calatrava, Manuel; Kedinger, Claude; Chatton, Bruno

    2011-01-01

    Alternative splicing and post-translational modifications are processes that give rise to the complexity of the proteome. The nuclear ATF7 and ATF2 (activating transcription factor) are structurally homologous leucine zipper transcription factors encoded by distinct genes. Stress and growth factors activate ATF2 and ATF7 mainly via sequential phosphorylation of two conserved threonine residues in their activation domain. Distinct protein kinases, among which mitogen-activated protein kinases (MAPK), phosphorylate ATF2 and ATF7 first on Thr71/Thr53 and next on Thr69/Thr51 residues respectively, resulting in transcriptional activation. Here, we identify and characterize a cytoplasmic alternatively spliced isoform of ATF7. This variant, named ATF7-4, inhibits both ATF2 and ATF7 transcriptional activities by impairing the first phosphorylation event on Thr71/Thr53 residues. ATF7-4 indeed sequesters the Thr53-phosphorylating kinase in the cytoplasm. Upon stimulus-induced phosphorylation, ATF7-4 is poly-ubiquitinated and degraded, enabling the release of the kinase and ATF7/ATF2 activation. Our data therefore conclusively establish that ATF7-4 is an important cytoplasmic negative regulator of ATF7 and ATF2 transcription factors. PMID:21858082

  15. The cAMP pathway regulates mRNA decay through phosphorylation of the RNA-binding protein TIS11b/BRF1

    PubMed Central

    Rataj, Felicitas; Planel, Séverine; Desroches-Castan, Agnès; Le Douce, Juliette; Lamribet, Khadija; Denis, Josiane; Feige, Jean-Jacques; Cherradi, Nadia

    2016-01-01

    TPA-inducible sequence 11b/butyrate response factor 1 (TIS11b/BRF1) belongs to the tristetraprolin (TTP) family of zinc-finger proteins, which bind to mRNAs containing AU-rich elements in their 3′-untranslated region and target them for degradation. Regulation of TTP family function through phosphorylation by p38 MAP kinase and Akt/protein kinase B signaling pathways has been extensively studied. In contrast, the role of cAMP-dependent protein kinase (PKA) in the control of TTP family activity in mRNA decay remains largely unknown. Here we show that PKA activation induces TIS11b gene expression and protein phosphorylation. Site-directed mutagenesis combined with kinase assays and specific phosphosite immunodetection identified Ser-54 (S54) and Ser-334 (S334) as PKA target amino acids in vitro and in vivo. Phosphomimetic mutation of the C-terminal S334 markedly increased TIS11b half-life and, unexpectedly, enhanced TIS11b activity on mRNA decay. Examination of protein–protein interactions between TIS11b and components of the mRNA decay machinery revealed that mimicking phosphorylation at S334 enhances TIS11b interaction with the decapping coactivator Dcp1a, while preventing phosphorylation at S334 potentiates its interaction with the Ccr4-Not deadenylase complex subunit Cnot1. Collectively our findings establish for the first time that cAMP-elicited phosphorylation of TIS11b plays a key regulatory role in its mRNA decay-promoting function. PMID:27708140

  16. Nuclear phosphorylated Y142 β-catenin accumulates in astrocytomas and glioblastomas and regulates cell invasion

    PubMed Central

    Náger, Mireia; Santacana, Maria; Bhardwaj, Deepshikha; Valls, Joan; Ferrer, Isidre; Nogués, Pere; Cantí, Carles; Herreros, Judit

    2015-01-01

    Abstract Glioblastoma multiforme (GBM) is a fast growing brain tumor characterized by extensive infiltration into the surrounding tissue and one of the most aggressive cancers. GBM is the most common glioma (originating from glial-derived cells) that either evolves from a low grade astrocytoma or appears de novo. Wnt/β-catenin and Hepatocyte Growth Factor (HGF)/c-Met signaling are hyperactive in human gliomas, where they regulate cell proliferation, migration and stem cell behavior. We previously demonstrated that β-catenin is phosphorylated at Y142 by recombinant c-Met kinase and downstream of HGF signaling in neurons. Here we studied phosphoY142 (PY142) β-catenin and dephospho S/T β-catenin (a classical Wnt transducer) in glioma biopsies, GBM cell lines and biopsy-derived glioma cell cultures. We found that PY142 β-catenin mainly localizes in the nucleus and signals through transcriptional activation in GBM cells. Tissue microarray analysis confirmed strong nuclear PY142 β-catenin immunostaining in astrocytoma and GBM biopsies. By contrast, active β-catenin showed nuclear localization only in GBM samples. Western blot analysis of tumor biopsies further indicated that PY142 and active β-catenin accumulate independently, correlating with the expression of Snail/Slug (an epithelial-mesenchymal transition marker) and Cyclin-D1 (a regulator of cell cycle progression), respectively, in high grade astrocytomas and GBMs. Moreover, GBM cells stimulated with HGF showed increasing levels of PY142 β-catenin and Snail/Slug. Importantly, the expression of mutant Y142F β-catenin decreased cell detachment and invasion induced by HGF in GBM cell lines and biopsy-derived cell cultures. Our results identify PY142 β-catenin as a nuclear β-catenin signaling form that downregulates adhesion and promotes GBM cell invasion. PMID:26654598

  17. Hierarchical phosphorylation at N-terminal transformation-sensitive sites in c-Myc protein is regulated by mitogens and in mitosis.

    PubMed Central

    Lutterbach, B; Hann, S R

    1994-01-01

    The N-terminal domain of the c-Myc protein has been reported to be critical for both the transactivation and biological functions of the c-Myc proteins. Through detailed phosphopeptide mapping analyses, we demonstrate that there is a cluster of four regulated and complex phosphorylation events on the N-terminal domain of Myc proteins, including Thr-58, Ser-62, and Ser-71. An apparent enhancement of Ser-62 phosphorylation occurs on v-Myc proteins having a mutation at Thr-58 which has previously been correlated with increased transforming ability. In contrast, phosphorylation of Thr-58 in cells is dependent on a prior phosphorylation of Ser-62. Hierarchical phosphorylation of c-Myc is also observed in vitro with a specific glycogen synthase kinase 3 alpha, unlike the promiscuous phosphorylation observed with other glycogen synthase kinase 3 alpha and 3 beta preparations. Although both p42 mitogen-activated protein kinase and cdc2 kinase specifically phosphorylate Ser-62 in vitro and cellular phosphorylation of Thr-58/Ser-62 is stimulated by mitogens, other in vivo experiments do not support a role for these kinases in the phosphorylation of Myc proteins. Unexpectedly, both the Thr-58 and Ser-62 phosphorylation events, but not other N-terminal phosphorylation events, can occur in the cytoplasm, suggesting that translocation of the c-Myc proteins to the nucleus is not required for phosphorylation at these sites. In addition, there appears to be an unusual block to the phosphorylation of Ser-62 during mitosis. Finally, although the enhanced transforming properties of Myc proteins correlates with the loss of phosphorylation at Thr-58 and an enhancement of Ser-62 phosphorylation, these phosphorylation events do not alter the ability of c-Myc to transactivate through the CACGTG Myc/Max binding site. Images PMID:8035827

  18. Regulation of erythrocyte Na-K-2Cl cotransport by threonine phosphorylation.

    PubMed

    Matskevich, Ioulia; Hegney, Karen L; Flatman, Peter W

    2005-08-01

    A method is described to measure threonine phosphorylation of the Na-K-2Cl cotransporter in ferret erythrocytes using readily available antibodies. We show that most, if not all, cotransporter in these cells is NKCC1, and this was immunoprecipitated with T4. Cotransport rate, measured as 86Rb influx, correlates well with threonine phosphorylation of T4-immunoprecipitated protein. The cotransporter effects large fluxes and is significantly phosphorylated in cells under control conditions. Transport and phosphorylation increase 2.5- to 3-fold when cells are treated with calyculin A or Na+ arsenite. Both fall to 60% control when cell [Mg2+] is reduced below micromolar or when cells are treated with the kinase inhibitors, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine or staurosporine. Importantly, these latter interventions do not abolish either phosphorylation or transport suggesting that a phosphorylated form of the cotransporter is responsible for residual fluxes. Our experiments suggest protein phosphatase 1 (PrP-1) is extremely active in these cells and dephosphorylates key regulatory threonine residues on the cotransporter. Examination of the effects of kinase inhibition after cells have been treated with high concentrations of calyculin indicates that residual PrP-1 activity is capable of rapidly dephosphorylating the cotransporter. Experiments on cotransporter precipitation with microcystin sepharose suggest that PrP-1 binds to a phosphorylated form of the cotransporter.

  19. Phosphorylation of Minichromosome Maintenance 3 (MCM3) by Checkpoint Kinase 1 (Chk1) Negatively Regulates DNA Replication and Checkpoint Activation.

    PubMed

    Han, Xiangzi; Mayca Pozo, Franklin; Wisotsky, Jacob N; Wang, Benlian; Jacobberger, James W; Zhang, Youwei

    2015-05-08

    Mechanisms controlling DNA replication and replication checkpoint are critical for the maintenance of genome stability and the prevention or treatment of human cancers. Checkpoint kinase 1 (Chk1) is a key effector protein kinase that regulates the DNA damage response and replication checkpoint. The heterohexameric minichromosome maintenance (MCM) complex is the core component of mammalian DNA helicase and has been implicated in replication checkpoint activation. Here we report that Chk1 phosphorylates the MCM3 subunit of the MCM complex at Ser-205 under normal growth conditions. Mutating the Ser-205 of MCM3 to Ala increased the length of DNA replication track and shortened the S phase duration, indicating that Ser-205 phosphorylation negatively controls normal DNA replication. Upon replicative stress treatment, the inhibitory phosphorylation of MCM3 at Ser-205 was reduced, and this reduction was accompanied with the generation of single strand DNA, the key platform for ataxia telangiectasia mutated and Rad3-related (ATR) activation. As a result, the replication checkpoint is activated. Together, these data provide significant insights into the regulation of both normal DNA replication and replication checkpoint activation through the novel phosphorylation of MCM3 by Chk1.

  20. AMPK Phosphorylates Desnutrin/ATGL and Hormone-Sensitive Lipase To Regulate Lipolysis and Fatty Acid Oxidation within Adipose Tissue

    PubMed Central

    Kim, Sun-Joong; Tang, Tianyi; Abbott, Marcia; Viscarra, Jose A.; Wang, Yuhui

    2016-01-01

    The role of AMP-activated protein kinase (AMPK) in promoting fatty acid (FA) oxidation in various tissues, such as liver and muscle, has been well understood. However, the role of AMPK in lipolysis and FA metabolism in adipose tissue has been controversial. To investigate the role of AMPK in the regulation of adipose lipolysis in vivo, we generated mice with adipose-tissue-specific knockout of both the α1 and α2 catalytic subunits of AMPK (AMPK-ASKO mice) by using aP2-Cre and adiponectin-Cre. Both models of AMPK-ASKO ablation show no changes in desnutrin/ATGL levels but have defective phosphorylation of desnutrin/ATGL at S406 to decrease its triacylglycerol (TAG) hydrolase activity, lowering basal lipolysis in adipose tissue. These mice also show defective phosphorylation of hormone-sensitive lipase (HSL) at S565, with higher phosphorylation at protein kinase A sites S563 and S660, increasing its hydrolase activity and isoproterenol-stimulated lipolysis. With higher overall adipose lipolysis, both models of AMPK-ASKO mice are lean, having smaller adipocytes with lower TAG and higher intracellular free-FA levels. Moreover, FAs from higher lipolysis activate peroxisome proliferator-activated receptor delta to induce FA oxidative genes and increase FA oxidation and energy expenditure. Overall, for the first time, we provide in vivo evidence of the role of AMPK in the phosphorylation and regulation of desnutrin/ATGL and HSL and thus adipose lipolysis. PMID:27185873

  1. Identification of a novel phosphorylation site, Ser-170, as a regulator of bad pro-apoptotic activity.

    PubMed

    Dramsi, Shaynoor; Scheid, Michael P; Maiti, Arpita; Hojabrpour, Payman; Chen, Xianming; Schubert, Kathryn; Goodlett, David R; Aebersold, Ruedi; Duronio, Vincent

    2002-02-22

    Bad is a pro-apoptotic member of the Bcl-2 family of proteins that is thought to exert a death-promoting effect by heterodimerization with Bcl-X(L), nullifying its anti-apoptotic activity. Growth factors may promote cell survival at least partially through phosphorylation of Bad at one or more of Ser-112, -136, or -155. Our previous work showed that Bad is also phosphorylated in response to cytokines at another site, which we now identify as Ser-170. The functional role of this novel phosphorylation site was assessed by site-directed mutagenesis and analysis of the pro-apoptotic function of Bad in transiently transfected HEK293 and COS-7 cells or by stable expression in the cytokine-dependent cell line, MC/9. In general, mutation of Ser-170 to Ala results in a protein with increased ability to induce apoptosis, similar to the S112A mutant. Mutation of Ser-170 to Asp, mimicking a constitutively phosphorylated site, results in a protein that is virtually unable to induce apoptosis. Similarly, the S112A/S170D double mutant does not cause apoptosis in HEK293 and MC/9 cell lines. These data strongly suggest that phosphorylation of Bad at Ser-170 is a critical event in blocking the pro-apoptotic activity of Bad.

  2. Rad9 BRCT domain interaction with phosphorylated H2AX regulates the G1 checkpoint in budding yeast

    PubMed Central

    Hammet, Andrew; Magill, Christine; Heierhorst, Jörg; Jackson, Stephen P

    2007-01-01

    Phosphorylation of histone H2A or H2AX is an early and sensitive marker of DNA damage in eukaryotic cells, although mutation of the conserved damage-dependent phosphorylation site is well tolerated. Here, we show that H2A phosphorylation is required for cell-cycle arrest in response to DNA damage at the G1/S transition in budding yeast. Furthermore, we show that the tandem BRCT domain of Rad9 interacts directly with phosphorylated H2A in vitro and that a rad9 point mutation that abolishes this interaction results in in vivo phenotypes that are similar to those caused by an H2A phosphorylation site mutation. Remarkably, similar checkpoint defects are also caused by a Rad9 Tudor domain mutation that impairs Rad9 chromatin association already in undamaged cells. These findings indicate that constitutive Tudor domain-mediated and damage-specific BRCT domain–phospho-H2A-dependent interactions of Rad9 with chromatin cooperate to establish G1 checkpoint arrest. PMID:17721446

  3. Novel screening cascade identifies MKK4 as key kinase regulating Tau phosphorylation at Ser422.

    PubMed

    Grueninger, Fiona; Bohrmann, Bernd; Christensen, Klaus; Graf, Martin; Roth, Doris; Czech, Christian

    2011-11-01

    Phosphorylation of Tau at serine 422 promotes Tau aggregation. The kinase that is responsible for this key phosphorylation event has so far not been identified but could be a potential drug target for Alzheimer's disease. We describe here an assay strategy to identify this kinase. Using a combination of screening a library of 65'000 kinase inhibitors and in vitro inhibitor target profiling of the screening hits using the Ambit kinase platform, MKK4 was identified as playing a key role in Tau-S422 phosphorylation in human neuroblastoma cells.

  4. Policies for regulation of direct broadcast satellites

    NASA Astrophysics Data System (ADS)

    Setzer, F. O.; Franca, B. A.; Cornell, N. W.

    1980-09-01

    The potential market for satellite to home television broadcasting was examined and recommendations were made to the FCC concerning appropriate regulatory policies for direct broadcast satellites (DBS) for the period following the 1983 Regional Administrative Radio Conference (RARC). It is concluded that many substitutes for DBS will be available in the market for subscription video programming. Either conventional broadcast regulation, which assumes scarcity of channels, nor common carrier regulation, which assumes monopoly power will be appropriate. The report recommends several functions the Commission should perform because of its spectrum allocation responsibilities, but recommends that the Commission make no rules concerning compatibility, signal quality, ownership of receiving equipment, program content, prices, service offerings, or control of channels.

  5. Phosphorylation-Regulated Transitions in an Oligomeric State Control the Activity of the Sae2 DNA Repair Enzyme

    PubMed Central

    Fu, Qiong; Chow, Julia; Bernstein, Kara A.; Makharashvili, Nodar; Arora, Sucheta; Lee, Chia-Fang; Person, Maria D.; Rothstein, Rodney

    2014-01-01

    In the DNA damage response, many repair and signaling molecules mobilize rapidly at the sites of DNA double-strand breaks. This network of immediate responses is regulated at the level of posttranslational modifications that control the activation of DNA processing enzymes, protein kinases, and scaffold proteins to coordinate DNA repair and checkpoint signaling. Here we investigated the DNA damage-induced oligomeric transitions of the Sae2 protein, an important enzyme in the initiation of DNA double-strand break repair. Sae2 is a target of multiple phosphorylation events, which we identified and characterized in vivo in the budding yeast Saccharomyces cerevisiae. Both cell cycle-dependent and DNA damage-dependent phosphorylation sites in Sae2 are important for the survival of DNA damage, and the cell cycle-regulated modifications are required to prime the damage-dependent events. We found that Sae2 exists in the form of inactive oligomers that are transiently released into smaller active units by this series of phosphorylations. DNA damage also triggers removal of Sae2 through autophagy and proteasomal degradation, ensuring that active Sae2 is present only transiently in cells. Overall, this analysis provides evidence for a novel type of protein regulation where the activity of an enzyme is controlled dynamically by posttranslational modifications that regulate its solubility and oligomeric state. PMID:24344201

  6. A pan-specific antibody for direct detection of protein histidine phosphorylation.

    PubMed

    Kee, Jung-Min; Oslund, Rob C; Perlman, David H; Muir, Tom W

    2013-07-01

    Despite its importance in central metabolism and bacterial cell signaling, protein histidine phosphorylation has remained elusive with respect to its extent and functional roles in biological systems because of the lack of adequate research tools. We report the development of the first pan-phosphohistidine (pHis) antibody using a stable pHis mimetic as the hapten. This antibody was successfully used in ELISA, western blotting, dot blot assays and immunoprecipitation and in detection and identification of histidine-phosphorylated proteins from native cell lysates when coupled with MS analysis. We also observed that the amount of protein pHis in Escherichia coli lysates depends on carbon source and nitrogen availability in the growth medium. In particular, we found that the amount of pHis on phosphoenolpyruvate synthase (PpsA) is sensitive to nitrogen availability in vivo and that α-ketoglutarate inhibits phosphotransfer from phosphorylated PpsA to pyruvate. We expect this antibody to open opportunities for investigating other pHis proteins and their functions.

  7. A Pan-specific Antibody for Direct Detection of Protein Histidine Phosphorylation

    PubMed Central

    Kee, Jung-Min; Oslund, Rob C.; Perlman, David H.; Muir, Tom W.

    2013-01-01

    Despite its importance in central metabolism and bacterial cell signaling, protein histidine phosphorylation has remained elusive with respect to its extent and functional roles in biological systems due to the lack of adequate research tools. We report the development of the first pan-pHis antibody using a stable phosphohistidine (pHis) mimetic as the hapten. This antibody was successfully used in ELISA, Western blot, dot blot, immunoprecipitation, and in detection and identification of histidine-phosphorylated proteins from native cell lysates when coupled with mass spectrometric analysis. We also observed that protein pHis levels in E. coli lysates depend on carbon source and nitrogen availability in the growth media. In particular, we found that pHis levels on PpsA are sensitive to nitrogen availability in vivo and that α-ketoglutarate (α-KG) inhibits phosphotransfer from phosphorylated phosphoenolpyruvate synthase (PpsA) to pyruvate. We expect this antibody to open opportunities for investigating other pHis-proteins and their functions. PMID:23708076

  8. Gonads directly regulate growth in teleosts.

    PubMed

    Bhatta, Sandip; Iwai, Toshiharu; Miura, Chiemi; Higuchi, Masato; Shimizu-Yamaguchi, Sonoko; Fukada, Haruhisa; Miura, Takeshi

    2012-07-10

    In general, there is a relationship between growth and reproduction, and gonads are known to be important organs for growth, but direct evidence for their role is lacking. Here, using a fish model, we report direct evidence that gonads are endocrine organs equal to the pituitary in controlling body growth. Gonadal loss of function, gain of function, and rescue of growth were investigated in tilapia. Gonadectomy experiments were carried out in juvenile males and females. Gonadectomy significantly retarded growth compared with controls; however, this retardation was rescued by the implantation of extirpated gonads. Because gonads express growth hormone, it is possible that gonads control body growth through the secretion of growth hormone and/or other endocrine factors. We propose that gonads are integral players in the dynamic regulation of growth in teleosts.

  9. Relationship between Rap1 protein phosphorylation and regulation of Ca2+ transport in platelets: a new approach.

    PubMed Central

    Magnier, C; Corvazier, E; Aumont, M C; Le Jemtel, T H; Enouf, J

    1995-01-01

    Although the interrelationship between the two messengers Ca2+ and cyclic AMP in platelet function is well documented, its mechanism of action still remains to be established. We investigated here the question of the regulation of platelet Ca(2+)-ATPases by cyclic AMP through the phosphorylation of the Rap1 protein using a pathological model. We first found experimental conditions where Ca(2+)-transport by platelet membrane vesicles appeared to be dependent on the phosphorylation of the Rap1 protein. Then, we studied platelets of patients with congestive heart failure for their expression of the potential 97 kDa Ca(2+)-ATPase target of regulation through the Rap1 protein as well as the phosphorylation of the Rap1 protein using the catalytic subunit of the cyclic AMP-dependent protein kinase (C. Sub.). In the first patients studied, we found no significant modification in the expression of the 97 kDa Ca(2+)-ATPase by Western blotting using the PL/IM 430 monoclonal antibody which specifically recognized this isoform. In contrast, the Rap1 protein was differentially phosphorylated when using 15 micrograms/ml of the C. Sub. These results allowed us to use these pathological platelets to study the relationship between the expression of Rap1 protein and the regulation of Ca2+ transport by selecting a patient with severe heart failure. We could show a decrease in the expression as well as in the phosphorylation of Rap1 protein and demonstrate a lower effect of C. Sub. on Ca2+ transport. Finally, by studying a further series of patients, we could confirm that the decrease in Rap1 protein expression in heart failure, whatever its extent, was variable, and could strictly correlate the expression of Rap1 protein with the stimulatory effect of C. Sub. on Ca2+ transport. Besides the evidence for regulation of the expression of the Rap1 protein in platelets from patients with heart failure, these findings constitute a new approach in favour of the regulation of platelet Ca2

  10. Phosphorylation Regulates Interaction of 210-kDa Myosin Light Chain Kinase N-terminal Domain with Actin Cytoskeleton.

    PubMed

    Vilitkevich, E L; Khapchaev, A Y; Kudryashov, D S; Nikashin, A V; Schavocky, J P; Lukas, T J; Watterson, D M; Shirinsky, V P

    2015-10-01

    High molecular weight myosin light chain kinase (MLCK210) is a multifunctional protein involved in myosin II activation and integration of cytoskeletal components in cells. MLCK210 possesses actin-binding regions both in the central part of the molecule and in its N-terminal tail domain. In HeLa cells, mitotic protein kinase Aurora B was suggested to phosphorylate MLCK210 N-terminal tail at serine residues (Dulyaninova, N. G., and Bresnick, A. R. (2004) Exp. Cell Res., 299, 303-314), but the functional significance of the phosphorylation was not established. We report here that in vitro, the N-terminal actin-binding domain of MLCK210 is located within residues 27-157 (N27-157, avian MLCK210 sequence) and is phosphorylated by cAMP-dependent protein kinase (PKA) and Aurora B at serine residues 140/149 leading to a decrease in N27-157 binding to actin. The same residues are phosphorylated in a PKA-dependent manner in transfected HeLa cells. Further, in transfected cells, phosphomimetic mutants of N27-157 showed reduced association with the detergent-stable cytoskeleton, whereas in vitro, the single S149D mutation reduced N27-157 association with F-actin to a similar extent as that achieved by N27-157 phosphorylation. Altogether, our results indicate that phosphorylation of MLCK210 at distinct serine residues, mainly at S149, attenuates the interaction of MLCK210 N-terminus with the actin cytoskeleton and might serve to regulate MLCK210 microfilament cross-linking activity in cells.

  11. Analysis of a cAMP regulated coactivator family reveals an alternative phosphorylation motif for AMPK family members

    PubMed Central

    Moresco, James J.; Vaughan, Joan M.; Matsumura, Shigenobu; Yates, John R.; Montminy, Marc

    2017-01-01

    The second messenger cAMP stimulates cellular gene expression via the PKA-mediated phosphorylation of the transcription factor CREB and through dephosphorylation of the cAMP-responsive transcriptional coactivators (CRTCs). Under basal conditions, CRTCs are phosphorylated by members of the AMPK family of Ser/Thr kinases and sequestered in the cytoplasm via a phosphorylation-dependent association with 14-3-3 proteins. Increases in cAMP promote the dephosphorylation and nuclear translocation of CRTCs, where they bind to CREB and stimulate relevant target genes. Although they share considerable sequence homology, members of the CRTC family exert non-overlapping effects on cellular gene expression through as yet unidentified mechanisms. Here we show that the three CRTCs exhibit distinct patterns of 14-3-3 binding at three conserved sites corresponding to S70, S171, and S275 (in CRTC2). S171 functions as the gatekeeper site for 14-3-3 binding; it acts cooperatively with S275 in stabilizing this interaction following its phosphorylation by the cAMP-responsive SIK and the cAMP-nonresponsive MARK kinases. Although S171 contains a consensus recognition site for phosphorylation by AMPK family members, S70 and S275 carry variant motifs (MNTGGS275LPDL), lacking basic residues that are otherwise critical for SIK/MARK recognition as well as 14-3-3 binding. Correspondingly, the activity of these motifs differs between CRTC family members. As the variant (SLPDL) motif is present and apparently phosphorylated in other mammalian proteins, our studies suggest that the regulation of cellular targets by AMPK family members is more extensive than previously appreciated. PMID:28235073

  12. Calcineurin B homologous protein 3 negatively regulates cardiomyocyte hypertrophy via inhibition of glycogen synthase kinase 3 phosphorylation.

    PubMed

    Kobayashi, Soushi; Nakamura, Tomoe Y; Wakabayashi, Shigeo

    2015-07-01

    Cardiac hypertrophy is a leading cause of serious heart diseases. Although many signaling molecules are involved in hypertrophy, the functions of some proteins in this process are still unknown. Calcineurin B homologous protein 3 (CHP3)/tescalcin is an EF-hand Ca(2+)-binding protein that is abundantly expressed in the heart; however, the function of CHP3 is unclear. Here, we aimed to identify the cardiac functions of CHP3. CHP3 was expressed in hearts at a wide range of developmental stages and was specifically detected in neonatal rat ventricular myocytes (NRVMs) but not in cardiac fibroblasts in culture. Moreover, knockdown of CHP3 expression using adenoviral-based RNA interference in NRVMs resulted in enlargement of cardiomyocyte size, concomitant with increased expression of a pathological hypertrophy marker ANP. This same treatment elevated glycogen synthase kinase (GSK3α/β) phosphorylation, which is known to inhibit GSK3 function. In contrast, CHP3 overexpression blocked the insulin-induced phosphorylation of GSK3α/β without affecting the phosphorylation of Akt, which is an upstream kinase of GSK3α/β, in HEK293 cells, and it inhibited both IGF-1-induced phosphorylation of GSK3β and cardiomyocyte hypertrophy in NRVMs. Co-immunoprecipitation experiments revealed that GSK3β interacted with CHP3. However, a Ca(2+)-binding-defective mutation of CHP3 (CHP3-D123A) also interacted with GSK3β and had the same inhibitory effect on GSK3α/β phosphorylation, suggesting that the action of CHP3 was independent of Ca(2+). These findings suggest that CHP3 functions as a novel negative regulator of cardiomyocyte hypertrophy via inhibition of GSK3α/β phosphorylation and subsequent enzymatic activation of GSK3α/β.

  13. Tyrosine phosphorylated c-Cbl regulates platelet functional responses mediated by outside-in signaling.

    PubMed

    Buitrago, Lorena; Langdon, Wallace Y; Sanjay, Archana; Kunapuli, Satya P

    2011-11-17

    c-Cbl protein functions as an E3 ligase and scaffolding protein, where 3 residues, Y700, Y731, and Y774, upon phosphorylation, have been shown to initiate several signaling cascades. In this study, we investigated the role of these phospho-tyrosine residues in the platelet functional responses after integrin engagement. We observed that c-Cbl Y700, Y731 and Y774 undergo phosphorylation upon platelet adhesion to immobilized fibrinogen, which was inhibited in the presence of PP2, a pan-src family kinase (SFK) inhibitor, suggesting that c-Cbl is phosphorylated downstream of SFKs. However, OXSI-2, a Syk inhibitor, significantly reduced c-Cbl phosphorylation at residues Y774 and Y700, without affecting Y731 phosphorylation. Interestingly, PP2 inhibited both platelet-spreading on fibrinogen as well as clot retraction, whereas OXSI-2 blocked only platelet-spreading, suggesting a differential role of these tyrosine residues. The physiologic role of c-Cbl and Y731 was studied using platelets from c-Cbl KO and c-Cbl(YF/YF) knock-in mice. c-Cbl KO and c-Cbl(YF/YF) platelets had a significantly reduced spreading over immobilized fibrinogen. Furthermore, clot retraction with c-Cbl KO and c-Cbl(YF/YF) platelets was drastically delayed. These results indicate that c-Cbl and particularly its phosphorylated residue Y731 plays an important role in platelet outside-in signaling contributing to platelet-spreading and clot retraction.

  14. Akt Phosphorylation and Regulation of Transketolase Is a Nodal Point for Amino Acid Control of Purine Synthesis

    PubMed Central

    Saha, Arindam; Connelly, Stephen; Jiang, Jingjing; Zhuang, Shunhui; Amador, Deron T.; Phan, Tony; Pilz, Renate B.; Boss, Gerry R.

    2014-01-01

    SUMMARY The phosphatidylinositol 3-kinase (PI3K)/Akt pathway integrates environmental clues to regulate cell growth and survival. We showed previously that depriving cells of a single essential amino acid rapidly and reversibly arrests purine synthesis. Here we demonstrate that amino acids via mTORC2 and IκB kinase regulate Akt activity, and Akt association and phosphorylation of transketolase (TKT), a key enzyme of the non-oxidative pentose phosphate pathway (PPP). Akt phosphorylates TKT on Thr382, markedly enhancing enzyme activity and increasing carbon flow through the non-oxidative PPP, thereby increasing purine synthesis. Mice fed a lysine-deficient diet for two days show decreased Akt activity, TKT activity, and purine synthesis in multiple organs. These results provide a new mechanism whereby Akt coordinates amino acid availability with glucose utilization, purine synthesis, and RNA and DNA synthesis. PMID:24981175

  15. RIP1 maintains DNA integrity and cell proliferation by regulating PGC-1α-mediated mitochondrial oxidative phosphorylation and glycolysis.

    PubMed

    Chen, W; Wang, Q; Bai, L; Chen, W; Wang, X; Tellez, C S; Leng, S; Padilla, M T; Nyunoya, T; Belinsky, S A; Lin, Y

    2014-07-01

    Aerobic glycolysis or the Warburg effect contributes to cancer cell proliferation; however, how this glucose metabolism pathway is precisely regulated remains elusive. Here we show that receptor-interacting protein 1 (RIP1), a cell death and survival signaling factor, regulates mitochondrial oxidative phosphorylation and aerobic glycolysis. Loss of RIP1 in lung cancer cells suppressed peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) expression, impairing mitochondrial oxidative phosphorylation and accelerating glycolysis, resulting in spontaneous DNA damage and p53-mediated cell proliferation inhibition. Thus, although aerobic glycolysis within a certain range favors cancer cell proliferation, excessive glycolysis causes cytostasis. Our data suggest that maintenance of glycolysis by RIP1 is pivotal to cancer cell energy homeostasis and DNA integrity and may be exploited for use in anticancer therapy.

  16. RIP1 maintains DNA integrity and cell proliferation by regulating PGC-1α-mediated mitochondrial oxidative phosphorylation and glycolysis

    PubMed Central

    Chen, W; Wang, Q; Bai, L; Chen, W; Wang, X; Tellez, C S; Leng, S; Padilla, M T; Nyunoya, T; Belinsky, S A; Lin, Y

    2014-01-01

    Aerobic glycolysis or the Warburg effect contributes to cancer cell proliferation; however, how this glucose metabolism pathway is precisely regulated remains elusive. Here we show that receptor-interacting protein 1 (RIP1), a cell death and survival signaling factor, regulates mitochondrial oxidative phosphorylation and aerobic glycolysis. Loss of RIP1 in lung cancer cells suppressed peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) expression, impairing mitochondrial oxidative phosphorylation and accelerating glycolysis, resulting in spontaneous DNA damage and p53-mediated cell proliferation inhibition. Thus, although aerobic glycolysis within a certain range favors cancer cell proliferation, excessive glycolysis causes cytostasis. Our data suggest that maintenance of glycolysis by RIP1 is pivotal to cancer cell energy homeostasis and DNA integrity and may be exploited for use in anticancer therapy. PMID:24583643

  17. Calcium regulates motility and protein phosphorylation by changing cAMP and ATP concentrations in boar sperm in vitro.

    PubMed

    Li, Xinhong; Wang, Lirui; Li, Yuhua; Zhao, Na; Zhen, Linqing; Fu, Jieli; Yang, Qiangzhen

    2016-09-01

    Considering the importance of calcium (Ca(2+)) in regulating sperm capacitation, hyperactivation and acrosome reaction, little is known about the molecular mechanism of action of this ion in this process. In the present study, assessment of the molecular mechanism from the perspective of energy metabolism occurred. Sperm motility variables were determined using computer-assisted sperm analysis (CASA) and the phosphorylation of PKA substrates, tyrosine residues and AMP-activated protein kinase (AMPK) were analyzed by Western blot. Moreover, intracellular sperm-specific glyceraldehyde 3-phosphatedehydrogenase (GAPDH) activity, 3'-5'-cyclic adenosine monophosphate (cAMP) and adenosine 5'-triphosphate (ATP) concentrations were assessed in boar sperm treated with Ca(2+). Results of the present study indicated that, under greater extracellular Ca(2+)concentrations (≥3.0mM), sperm motility and protein phosphorylation were inhibited. Interestingly, these changes were correlated with that of GAPDH activity, AMPK phosphorylation, cAMP and ATP concentrations. The negative effects of Ca(2+) on these intracellular processes were attenuated by addition of the calmodulin (CaM) inhibitor W7 and the inhibitor of calmodulin-dependent protein kinase (CaMK), KN-93. In the presence of greater extracellular Ca(2+), however, the phosphorylation pathway was suppressed by H-89. Taken together, these results suggested that Ca(2+) had a dual role in regulating boar sperm motility and protein phosphorylation due to the changes of cAMP and ATP concentrations, in response to cAMP-mediated signal transduction and the Ca(2+) signaling cascade. The present study provided some novel insights into the molecular mechanism underlying the effects of Ca(2+) on boar sperm as well as the involvement of energy metabolism in this mechanism.

  18. Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: IMPLICATIONS FOR AMP KINASE.

    PubMed

    Mahapatra, Gargi; Varughese, Ashwathy; Ji, Qinqin; Lee, Icksoo; Liu, Jenney; Vaishnav, Asmita; Sinkler, Christopher; Kapralov, Alexandr A; Moraes, Carlos T; Sanderson, Thomas H; Stemmler, Timothy L; Grossman, Lawrence I; Kagan, Valerian E; Brunzelle, Joseph S; Salomon, Arthur R; Edwards, Brian F P; Hüttemann, Maik

    2017-01-06

    Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr(28), leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (ΔΨm), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr(28) is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr(28) in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr(28) in vivo We conclude that Cytc phosphorylation is mediated in a tissue-specific manner and leads to regulation of electron transport chain flux via "controlled respiration," preventing ΔΨm hyperpolarization, a known cause of ROS and trigger of apoptosis.

  19. Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: IMPLICATIONS FOR AMP KINASE

    SciTech Connect

    Mahapatra, Gargi; Varughese, Ashwathy; Ji, Qinqin; Lee, Icksoo; Liu, Jenney; Vaishnav, Asmita; Sinkler, Christopher; Kapralov, Alexandr A.; Moraes, Carlos T.; Sanderson, Thomas H.; Stemmler, Timothy L.; Grossman, Lawrence I.; Kagan, Valerian E.; Brunzelle, Joseph S.; Salomon, Arthur R.; Edwards, Brian F. P.; Hüttemann, Maik

    2016-10-07

    Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr28, leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc. Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (ΔΨm), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr28 is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr28 in vivo. We conclude that Cytc phosphorylation is mediated in a tissue-specific manner and leads to regulation of electron transport chain flux via “controlled respiration,” preventing ΔΨm hyperpolarization, a known cause of ROS and trigger of apoptosis.

  20. The viral transactivator HBx protein exhibits a high potential for regulation via phosphorylation through an evolutionarily conserved mechanism

    PubMed Central

    2012-01-01

    Background Hepatitis B virus (HBV) encodes an oncogenic factor, HBx, which is a multifunctional protein that can induce dysfunctional regulation of signaling pathways, transcription, and cell cycle progression, among other processes, through interactions with target host factors. The subcellular localization of HBx is both cytoplasmic and nuclear. This dynamic distribution of HBx could be essential to the multiple roles of the protein at different stages during HBV infection. Transactivational functions of HBx may be exerted both in the nucleus, via interaction with host DNA-binding proteins, and in the cytoplasm, via signaling pathways. Although there have been many studies describing different pathways altered by HBx, and its innumerable binding partners, the molecular mechanism that regulates its different roles has been difficult to elucidate. Methods In the current study, we took a bioinformatics approach to investigate whether the viral protein HBx might be regulated via phosphorylation by an evolutionarily conserved mechanism. Results We found that the phylogenetically conserved residues Ser25 and Ser41 (both within the negative regulatory domain), and Thr81 (in the transactivation domain) are predicted to be phosphorylated. By molecular 3D modeling of HBx, we further show these residues are all predicted to be exposed on the surface of the protein, making them easily accesible to these types of modifications. Furthermore, we have also identified Yin Yang sites that might have the potential to be phosphorylated and O-β-GlcNAc interplay at the same residues. Conclusions Thus, we propose that the different roles of HBx displayed in different subcellular locations might be regulated by an evolutionarily conserved mechanism of posttranslational modification, via phosphorylation. PMID:23079056

  1. Glucose Regulated Protein 78 Phosphorylation in Sperm Undergoes Dynamic Changes during Maturation

    PubMed Central

    Lobo, Vivian; Rao, Parimala; Gajbhiye, Rahul; Kulkarni, Vijay; Parte, Priyanka

    2015-01-01

    GRP78, a resident endoplasmic reticulum (ER) chaperone involved in protein transport, folding and assembly, has been reported in sperm. It is shown to be localized in the neck region of human sperm. We have previously reported GRP78 to be less phosphorylated in asthenozoosperm.The present study aimed to determine whether sperm GRP78 undergoes phosphorylation changes during epididymal maturation and whether there are any differences in GRP78 phosphoforms in asthenozoosperm vis-à-vis normozoosperm. Testicular- and cauda epididymal- sperm from adult male Holtzman rats, and semen ejaculates collected from normal and asthenozoospermic individuals were investigated. DIGE carried out to determine phosphorylation of GRP78 in asthenozoosperm and normal sperm reveals a shift in the location of GRP78 of asthenozoosperm towards the alkaline pH, indicative of reduced GRP78 phosphorylation. Immunoprecipitation studies using antibodies specific to GRP78, serine-, threonine-, and tyrosine phosphorylation and Pan phospho antibody demonstrates GRP78 to be phosphorylated at all three residues in rat spermatozoa. Phosphatase assays using Calf intestinal alkaline phosphatase and Lambda protein phosphatase followed by nanofluidic proteomic immunoassay (NIA) show that in rat, GP4.96, GP4.94 and GP4.85 are the three phosphoforms in mature (caudal) sperm as against two phosphoforms GP4.96and GP4.94in immature (testicular) sperm. In mature human sperm GP5.04, GP4.96, and GP4.94were the 3 phosphoforms observed. GP4.94[P = 0.014]andGP5.04 [P = 0.02] are significantly reduced in asthenozoosperm. Ours is the first report indicating GRP78 in sperm to be phosphorylated at serine, threonine and tyrosine residues contrary to published literature reporting GRP78 not to be tyrosine phosphorylated. We report the presence of GRP78 phosphoforms in rat- and human- sperm and our data suggest that GRP78 phosphorylation in sperm undergoes spatial reorganization during epididymal maturation. Significant

  2. Attenuation of Phosphorylation-dependent Activation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Disease-causing Mutations at the Transmission Interface*

    PubMed Central

    Chin, Stephanie; Yang, Donghe; Miles, Andrew J.; Eckford, Paul D. W.; Molinski, Steven; Wallace, B. A.; Bear, Christine E.

    2017-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a multidomain membrane protein that functions as a phosphorylation-regulated anion channel. The interface between its two cytosolic nucleotide binding domains and coupling helices conferred by intracellular loops extending from the channel pore domains has been referred to as a transmission interface and is thought to be critical for the regulated channel activity of CFTR. Phosphorylation of the regulatory domain of CFTR by protein kinase A (PKA) is required for its channel activity. However, it was unclear if phosphorylation modifies the transmission interface. Here, we studied purified full-length CFTR protein using spectroscopic techniques to determine the consequences of PKA-mediated phosphorylation. Synchrotron radiation circular dichroism spectroscopy confirmed that purified full-length wild-type CFTR is folded and structurally responsive to phosphorylation. Intrinsic tryptophan fluorescence studies of CFTR showed that phosphorylation reduced iodide-mediated quenching, consistent with an effect of phosphorylation in burying tryptophans at the transmission interface. Importantly, the rate of phosphorylation-dependent channel activation was compromised by the introduction of disease-causing mutations in either of the two coupling helices predicted to interact with nucleotide binding domain 1 at the interface. Together, these results suggest that phosphorylation modifies the interface between the catalytic and pore domains of CFTR and that this modification facilitates CFTR channel activation. PMID:28003367

  3. Attenuation of Phosphorylation-dependent Activation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Disease-causing Mutations at the Transmission Interface.

    PubMed

    Chin, Stephanie; Yang, Donghe; Miles, Andrew J; Eckford, Paul D W; Molinski, Steven; Wallace, B A; Bear, Christine E

    2017-02-03

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a multidomain membrane protein that functions as a phosphorylation-regulated anion channel. The interface between its two cytosolic nucleotide binding domains and coupling helices conferred by intracellular loops extending from the channel pore domains has been referred to as a transmission interface and is thought to be critical for the regulated channel activity of CFTR. Phosphorylation of the regulatory domain of CFTR by protein kinase A (PKA) is required for its channel activity. However, it was unclear if phosphorylation modifies the transmission interface. Here, we studied purified full-length CFTR protein using spectroscopic techniques to determine the consequences of PKA-mediated phosphorylation. Synchrotron radiation circular dichroism spectroscopy confirmed that purified full-length wild-type CFTR is folded and structurally responsive to phosphorylation. Intrinsic tryptophan fluorescence studies of CFTR showed that phosphorylation reduced iodide-mediated quenching, consistent with an effect of phosphorylation in burying tryptophans at the transmission interface. Importantly, the rate of phosphorylation-dependent channel activation was compromised by the introduction of disease-causing mutations in either of the two coupling helices predicted to interact with nucleotide binding domain 1 at the interface. Together, these results suggest that phosphorylation modifies the interface between the catalytic and pore domains of CFTR and that this modification facilitates CFTR channel activation.

  4. Ras/cAMP-dependent Protein Kinase (PKA) Regulates Multiple Aspects of Cellular Events by Phosphorylating the Whi3 Cell Cycle Regulator in Budding Yeast*

    PubMed Central

    Mizunuma, Masaki; Tsubakiyama, Ryohei; Ogawa, Takafumi; Shitamukai, Atsunori; Kobayashi, Yoshifumi; Inai, Tomomi; Kume, Kazunori; Hirata, Dai

    2013-01-01

    The Start/G1 phase in the cell cycle is an important period during which cells determine their developmental fate, onset of mitotic progression, or the switch to developmental stages in response to both external and internal signals. In the budding yeast Saccharomyces cerevisiae, Whi3, a negative regulator of the G1 cyclins, has been identified as a positive regulator of cell size control and is involved in the regulation of Start. However, the regulatory pathway of Whi3 governing the response to multiple signals remains largely unknown. Here, we show that Whi3 is phosphorylated by the Ras/cAMP-dependent protein kinase (PKA) and that phosphorylation of Ser-568 in Whi3 by PKA plays an inhibitory role in Whi3 function. Phosphorylation of Whi3 by PKA led to its decreased interaction with CLN3 G1 cyclin mRNA and was required for the promotion of G1/S progression. Furthermore, we demonstrate that the phosphomimetic S568D mutation of Whi3 prevented the developmental fate switch to sporulation or invasive growth. Thus, PKA modulated the function of Whi3 by phosphorylation, thus implicating PKA-mediated modulation of Whi3 in multiple cellular events. PMID:23471970

  5. CTR1 phosphorylates the central regulator EIN2 to control ethylene hormone signaling from the ER membrane to the nucleus in Arabidopsis.

    PubMed

    Ju, Chuanli; Yoon, Gyeong Mee; Shemansky, Jennifer Marie; Lin, David Y; Ying, Z Irene; Chang, Jianhong; Garrett, Wesley M; Kessenbrock, Mareike; Groth, Georg; Tucker, Mark L; Cooper, Bret; Kieber, Joseph J; Chang, Caren

    2012-11-20

    The gaseous phytohormone ethylene C(2)H(4) mediates numerous aspects of growth and development. Genetic analysis has identified a number of critical elements in ethylene signaling, but how these elements interact biochemically to transduce the signal from the ethylene receptor complex at the endoplasmic reticulum (ER) membrane to transcription factors in the nucleus is unknown. To close this gap in our understanding of the ethylene signaling pathway, the challenge has been to identify the target of the CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) Raf-like protein kinase, as well as the molecular events surrounding ETHYLENE-INSENSITIVE2 (EIN2), an ER membrane-localized Nramp homolog that positively regulates ethylene responses. Here we demonstrate that CTR1 interacts with and directly phosphorylates the cytosolic C-terminal domain of EIN2. Mutations that block the EIN2 phosphorylation sites result in constitutive nuclear localization of the EIN2 C terminus, concomitant with constitutive activation of ethylene responses in Arabidopsis. Our results suggest that phosphorylation of EIN2 by CTR1 prevents EIN2 from signaling in the absence of ethylene, whereas inhibition of CTR1 upon ethylene perception is a signal for cleavage and nuclear localization of the EIN2 C terminus, allowing the ethylene signal to reach the downstream transcription factors. These findings significantly advance our understanding of the mechanisms underlying ethylene signal transduction.

  6. Phosphorylation regulates proteolytic efficiency of TEV protease detected by a 5(6)-carboxyfluorescein-pyrene based fluorescent sensor.

    PubMed

    He, Yao-Hui; Li, Yan-Mei; Chen, Yong-Xiang

    2016-04-01

    TEV protease is of great importance for in vitro and in vivo site-specific cleavage of proteins. The proteolytic efficiency of TEV protease is often regulated by mutation of the substrate, which is irreversible and hard to be modulated. Herein, a facile and reversible method, based on phosphorylation in the substrate, is developed to regulate the cleavage capability of TEV protease. Phosphorylation at P3 tyrosine hinders the recognition of TEV protease to the substrate by using a robust fluorescent protease sensor. Moreover, the phosphate group can be easily removed by alkaline phosphatases for recovering the proteolytic efficiency of TEV protease. Additionally, 5(6)-carboxyfluorescein and pyrene have been used as high-efficiency mutual fluorophore-quencher pair in the peptide-based protease sensor for the first time, which provides a chance to simultaneously monitor the cleavage process in two respective fluorescence channels. Further studies indicated both dynamic and static components contributing to the mutual quenching system. The phosphorylation-regulated TEV protease proteolysis system can be used in conditional cleavage of protein or peptide tag.

  7. Kid-1 participates in regulating ERK phosphorylation as a part of the circadian clock output in rat kidney.

    PubMed

    Yamato, Masaya; Ishida, Norio; Iwatani, Hirotsugu; Yamato, Masafumi; Rakugi, Hiromi; Ito, Takahito

    2009-01-01

    Circadian clock genes play a role for the regulation of cell cycle, but the factors connecting clock to cell cycle are not fully understood. We found that mRNA of Kid-1--a zinc-finger-type transcriptional repressor was localized to cortical and juxtamedullary segments of tubules but not to glomeruli in the rat kidney. Kid-1 mRNA showed robust circadian oscillation with a peak at ZT16. Under temporal restricted feeding, the phase of the oscillation shifted along with mRNAs of the clock genes--Per1 and Per2. The rhythm of S-phase in cell cycle disappeared in the kidney under the restricted feeding. The level of phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 was rhythmic with a peak at ZT16 in the kidney. We found that knockdown and overexpression of Kid-1 in NRK52E (normal rat kidney epithelial) cells induced and reduced the phosphorylation of ERK1/2, respectively. The data suggest that clock-controlled Kid-1 regulates the cell cycle of proliferating renal tubular epithelial cells through ERK phosphorylation.

  8. Carboxyl-terminal multi-site phosphorylation regulates internalization and desensitization of the human sst2 somatostatin receptor.

    PubMed

    Lehmann, Andreas; Kliewer, Andrea; Schütz, Dagmar; Nagel, Falko; Stumm, Ralf; Schulz, Stefan

    2014-04-25

    The somatostatin receptor 2 (sst2) is the pharmacological target of somatostatin analogs that are widely used in the diagnosis and treatment of human neuroendocrine tumors. We have recently shown that the stable somatostatin analogs octreotide and pasireotide (SOM230) stimulate distinct patterns of sst2 receptor phosphorylation and internalization. Like somatostatin, octreotide promotes the phosphorylation of at least six carboxyl-terminal serine and threonine residues namely S341, S343, T353, T354, T356 and T359, which in turn leads to a robust receptor endocytosis. Unlike somatostatin, pasireotide stimulates a selective phosphorylation of S341 and S343 of the human sst2 receptor followed by a partial receptor internalization. Here, we show that exchange of S341 and S343 by alanine is sufficient to block pasireotide-driven internalization, whereas mutation of T353, T354, T356 and T359 to alanine is required to strongly inhibited both octreotide- and somatostatin-induced internalization. Yet, combined mutation of T353, T354, T356 and T359 is not sufficient to prevent somatostatin-driven β-arrestin mobilization and receptor desensitization. Replacement of all fourteen carboxyl-terminal serine and threonine residues by alanine completely abrogates sst2 receptor internalization and β-arrestin mobilization in HEK293 cells. Together, our findings demonstrate for the first time that agonist-selective sst2 receptor internalization is regulated by multi-site phosphorylation of its carboxyl-terminal tail.

  9. Hydrophobic motif site-phosphorylated protein kinase CβII between mTORC2 and Akt regulates high glucose-induced mesangial cell hypertrophy.

    PubMed

    Das, Falguni; Ghosh-Choudhury, Nandini; Mariappan, Meenalakshmi M; Kasinath, Balakuntalam S; Choudhury, Goutam Ghosh

    2016-04-01

    PKCβII controls the pathologic features of diabetic nephropathy, including glomerular mesangial cell hypertrophy. PKCβII contains the COOH-terminal hydrophobic motif site Ser-660. Whether this hydrophobic motif phosphorylation contributes to high glucose-induced mesangial cell hypertrophy has not been determined. Here we show that, in mesangial cells, high glucose increased phosphorylation of PKCβII at Ser-660 in a phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner. Using siRNAs to downregulate PKCβII, dominant negative PKCβII, and PKCβII hydrophobic motif phosphorylation-deficient mutant, we found that PKCβII regulates activation of mechanistic target of rapamycin complex 1 (mTORC1) and mesangial cell hypertrophy by high glucose. PKCβII via its phosphorylation at Ser-660 regulated phosphorylation of Akt at both catalytic loop and hydrophobic motif sites, resulting in phosphorylation and inactivation of its substrate PRAS40. Specific inhibition of mTORC2 increased mTORC1 activity and induced mesangial cell hypertrophy. In contrast, inhibition of mTORC2 decreased the phosphorylation of PKCβII and Akt, leading to inhibition of PRAS40 phosphorylation and mTORC1 activity and prevented mesangial cell hypertrophy in response to high glucose; expression of constitutively active Akt or mTORC1 restored mesangial cell hypertrophy. Moreover, constitutively active PKCβII reversed the inhibition of high glucose-stimulated Akt phosphorylation and mesangial cell hypertrophy induced by suppression of mTORC2. Finally, using renal cortexes from type 1 diabetic mice, we found that increased phosphorylation of PKCβII at Ser-660 was associated with enhanced Akt phosphorylation and mTORC1 activation. Collectively, our findings identify a signaling route connecting PI3-kinase to mTORC2 to phosphorylate PKCβII at the hydrophobic motif site necessary for Akt phosphorylation and mTORC1 activation, leading to mesangial cell hypertrophy.

  10. Sedimentation studies reveal a direct role of phosphorylation in Smad3:Smad4 homo- and hetero-trimerization.

    PubMed

    Correia, J J; Chacko, B M; Lam, S S; Lin, K

    2001-02-06

    SMAD proteins are known to oligomerize and hetero-associate during their activation and translocation to the nucleus for transcriptional control. Analytical ultracentrifuge studies on Smad3 and Smad4 protein constructs are presented to clarify the model of homo- and hetero-oligomerization and the role of phosphorylation in the activation process. These constructs all exhibit a tendency to form disulfide cross-linked aggregates, primarily dimers, and a strong reducing agent, TCEP, was found to be required to determine the best estimates for reversible association models and equilibrium constants. A Smad4 construct, S4AF, consisting of the middle linker (L) domain and the C-terminal (C) domain, is shown to be a monomer, while a Smad3 construct, S3LC, consisting of the LC domains, is shown to form a trimer with an affinity K(3) = (1.2-3.1) x 10(9) M(-2). A Smad3 construct that mimics phosphorylation at the C-terminal target sequence, S3LC(3E), has 17--35-fold enhanced ability to form trimer over that of the wild-type construct, S3LC. S4AF associates with either S3LC or S3LC(3E) to form a hetero-trimer. In each case, the hetero-trimer is favored over the formation of the homo-trimer. Despite high sequence homology between Smad3 and Smad4, a chimeric Smad4 construct with an engineered Smad3 C-terminal pseudo-phosphorylation sequence, S4AF(3E), shows no tendency to form trimer. This suggests a Smad4-specific sequence insert inhibits homo-trimer formation, or other domains or sequences in S3LC are required in addition to the target sequence to mediate the formation of trimer. These results represent a direct molecular measure of the importance of hetero-trimerization and phosphorylation in the TGF-beta-activated Smad protein signal transduction process.

  11. The Cell Cycle Regulator Phosphorylated Retinoblastoma Protein Is Associated with Tau Pathology in Several Tauopathies

    PubMed Central

    Stone, Jeremy G.; Siedlak, Sandra L.; Tabaton, Massimo; Hirano, Asao; Castellani, Rudy J.; Santocanale, Corrado; Perry, George; Smith, Mark A.; Zhu, Xiongwei; Lee, Hyoung-gon

    2011-01-01

    Retinoblastoma protein (pRb) is a ubiquitous 928 amino acid cell cycle regulatory molecule with diverse biological activities. One critical function of pRb is control of the G1-to-S phase checkpoint of the cell cycle. In the hypophosphorylated state, pRb suppresses the activity of E2F transcription factors thereby inhibiting transcription of cell cycle promoting genes. Upon phosphorylation, primarily by cyclin dependent kinases, phosphorylated pRb dissociates from E2F and permits cell cycle progression. We previously found phosphorylated pRb to be intimately associated with hyperphosphorylated tau-containing neurofibrillary tangles of Alzheimer disease (AD), the pathogenesis of which is believed to involve dysregulation of the cell cycle and marked neuronal death. Here, we used immunohistochemistry to investigate the presence of phosphorylated pRb in other distinct neurodegenerative diseases that share the common characteristic of hyperphosphorylated tau pathology and neuronal loss with AD. We found colocalized labeling of tau pathology and phosphorylated pRb in Pick disease and progressive supranuclear palsy (3 cases each), neurodegeneration with brain iron accumulation type 1 (2 cases) and Parkinson-amyotrophic lateral sclerosis of Guam, subacute sclerosing panencephalitis, frontotemporal dementia and Parkinsonism linked to chromosome 17 and dementia pugilistica (1 case each). These observations further implicate aberrant neuronal cell cycle progression in neurodegenerative diseases, particularly tauopathies, and suggest a novel target for therapeutic intervention. PMID:21666500

  12. Structural Mechanism for Regulation of Bcl-2 protein Noxa by phosphorylation

    NASA Astrophysics Data System (ADS)

    Karim, Christine B.; Michel Espinoza-Fonseca, L.; James, Zachary M.; Hanse, Eric A.; Gaynes, Jeffrey S.; Thomas, David D.; Kelekar, Ameeta

    2015-09-01

    We showed previously that phosphorylation of Noxa, a 54-residue Bcl-2 protein, at serine 13 (Ser13) inhibited its ability to promote apoptosis through interactions with canonical binding partner, Mcl-1. Using EPR spectroscopy, molecular dynamics (MD) simulations and binding assays, we offer evidence that a structural alteration caused by phosphorylation partially masks Noxa’s BH3 domain, inhibiting the Noxa-Mcl-1 interaction. EPR of unphosphorylated Noxa, with spin-labeled amino acid TOAC incorporated within the BH3 domain, revealed equilibrium between ordered and dynamically disordered states. Mcl-1 further restricted the ordered component for non-phosphorylated Noxa, but left the pSer13 Noxa profile unchanged. Microsecond MD simulations indicated that the BH3 domain of unphosphorylated Noxa is housed within a flexible loop connecting two antiparallel β-sheets, flanked by disordered N- and C-termini and Ser13 phosphorylation creates a network of salt-bridges that facilitate the interaction between the N-terminus and the BH3 domain. EPR showed that a spin label inserted near the N-terminus was weakly immobilized in unphosphorylated Noxa, consistent with a solvent-exposed helix/loop, but strongly constrained in pSer13 Noxa, indicating a more ordered peptide backbone, as predicted by MD simulations. Together these studies reveal a novel mechanism by which phosphorylation of a distal serine inhibits a pro-apoptotic BH3 domain and promotes cell survival.

  13. Phosphorylation of plasma membrane aquaporin regulates temperature-dependent opening of tulip petals.

    PubMed

    Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

    2004-05-01

    The opening and closing of tulip petals was reproduced in the dark by changing the temperature from 5 degrees C to 20 degrees C for opening and 20 degrees C to 5 degrees C for closing. The opening process was accompanied by (3)H(2)O transport through the stem from the incubation medium to the petals. A Ca(2+)-channel blocker and a Ca(2+)-chelator inhibited petal opening and (3)H(2)O transport. Several proteins in the isolated plasma membrane fraction were phosphorylated in the presence of 25 micro M Ca(2+) at 20 degrees C. The 31-kDa protein that was phosphorylated, was suggested immunologically as the putative plasma membrane aquaporin (PM-AQP). This phosphorylated PM-AQP clearly reacted with the anti-phospho-Ser. In-gel assay revealed the presence of a 45-kDa Ca(2+)-dependent protein kinase in the isolated plasma membrane. Phosphorylation of the putative PM-AQP was thought to activate the water channel composed of PM-AQP. Dephosphorylation of the phosphorylated PM-AQP was also observed during petal closing at 5 degrees C, suggesting the inactivation of the water channel.

  14. Regulation of phosphorylation of nicotinic acetylcholine receptors in mouse BC3H1 myocytes

    SciTech Connect

    Smith, M.M.; Merlie, J.P.; Lawrence, J.C. Jr.

    1987-09-01

    By using /sup 32/P-labeling methods and performing immunoprecipitations with specific antibodies, the authors have found that three subunits of the nicotinic acetylcholine receptor and phosphorylated in mouse skeletal muscle cells. In nonstimulated cells, the molar ratios of phosphate estimated in ..cap alpha.., ..beta.., and delta subunits were 0.02, 0.05, and 0.5, respectively. All three subunits contained predominantly phosphoserine with some phosphothreonine; the ..beta.., subunit also contained phosphotyrosine. Incubating cells with agents that stimulate cAMP-dependent pathways (isoproterenol, forskolin, 8-Br-cAMP) increased the phosphorylation of the delta subunit by 50%, but phosphate labeling of the ..beta.. subunit was depressed by a third. In contrast, when cells were incubated with the divalent cation ionophores A-23187 or ionomycin, phosphorylation of both the delta and ..beta.. subunits increased. The results indicate that acetylcholine receptors are phosphorylated to significant levels in skeletal muscle cells and that cAMP-dependent and Ca/sup 2 +/-dependent pathways exist for controlling the phosphorylation state of the receptor subunits.

  15. KSR1 is a functional protein kinase capable of serine autophosphorylation and direct phosphorylation of MEK1

    SciTech Connect

    Goettel, Jeremy A.; Liang, Dongchun; Hilliard, Valda C.; Edelblum, Karen L.; Broadus, Matthew R.; Gould, Kathleen L.; Hanks, Steven K.; Polk, D. Brent

    2011-02-15

    The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway is a highly conserved signaling pathway that regulates diverse cellular processes including differentiation, proliferation, and survival. Kinase suppressor of Ras-1 (KSR1) binds each of the three ERK cascade components to facilitate pathway activation. Even though KSR1 contains a C-terminal kinase domain, evidence supporting the catalytic function of KSR1 remains controversial. In this study, we produced recombinant wild-type or kinase-inactive (D683A/D700A) KSR1 proteins in Escherichia coli to test the hypothesis that KSR1 is a functional protein kinase. Recombinant wild-type KSR1, but not recombinant kinase-inactive KSR1, underwent autophosphorylation on serine residue(s), phosphorylated myelin basic protein (MBP) as a generic substrate, and phosphorylated recombinant kinase-inactive MAPK/ERK kinase-1 (MEK1). Furthermore, FLAG immunoprecipitates from KSR1{sup -/-} colon epithelial cells stably expressing FLAG-tagged wild-type KSR1 (+KSR1), but not vector (+vector) or FLAG-tagged kinase-inactive KSR1 (+D683A/D700A), were able to phosphorylate kinase-inactive MEK1. Since TNF activates the ERK pathway in colon epithelial cells, we tested the biological effects of KSR1 in the survival response downstream of TNF. We found that +vector and +D683A/D700A cells underwent apoptosis when treated with TNF, whereas +KSR1 cells were resistant. However, +KSR1 cells were sensitized to TNF-induced cell loss in the absence of MEK kinase activity. These data provide clear evidence that KSR1 is a functional protein kinase, MEK1 is an in vitro substrate of KSR1, and the catalytic activities of both proteins are required for eliciting cell survival responses downstream of TNF.

  16. Replicative Functions of Minute Virus of Mice NS1 Protein Are Regulated In Vitro by Phosphorylation through Protein Kinase C

    PubMed Central

    Nüesch, Jürg P. F.; Dettwiler, Sabine; Corbau, Romuald; Rommelaere, Jean

    1998-01-01

    NS1, the major nonstructural protein of the parvovirus minute virus of mice, is a multifunctional phosphoprotein which is involved in cytotoxicity, transcriptional regulation, and initiation of viral DNA replication. For coordination of these various functions during virus propagation, NS1 has been proposed to be regulated by posttranslational modifications, in particular phosphorylation. Recent in vitro studies (J. P. F. Nüesch, R. Corbau, P. Tattersall, and J. Rommelaere, J. Virol. 72:8002–8012, 1998) provided evidence that distinct NS1 activities, notably the intrinsic helicase function, are modulated by the phosphorylation state of the protein. In order to study the dependence of the initiation of viral DNA replication on NS1 phosphorylation and to identify the protein kinases involved, we established an in vitro replication system that is devoid of endogenous protein kinases and is based on plasmid substrates containing the minimal left-end origins of replication. Cellular components necessary to drive NS1-dependent rolling-circle replication (RCR) were freed from endogenous serine/threonine protein kinases by affinity chromatography, and the eukaryotic DNA polymerases were replaced by the bacteriophage T4 DNA polymerase. While native NS1 (NS1P) supported RCR under these conditions, dephosphorylated NS1 (NS1O) was impaired. Using fractionated HeLa cell extracts, we identified two essential protein components which are able to phosphorylate NS1O, are enriched in protein kinase C (PKC), and, when present together, reactivate NS1O for replication. One of these components, containing atypical PKC, was sufficient to restore NS1O helicase activity. The requirement of NS1O reactivation for characteristic PKC cofactors such as Ca2+/phosphatidylserine or phorbol esters strongly suggests the involvement of this protein kinase family in regulation of NS1 replicative functions in vitro. PMID:9811734

  17. Crystal structure of Bacillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity

    DOE PAGES

    Hammerstrom, Troy G.; Horton, Lori B.; Swick, Michelle C.; ...

    2014-12-30

    The Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthesis operon. AtxA activity is elevated during growth in media containing glucose and CO2/bicarbonate, and there is a positive correlation between the CO2/bicarbonate signal, AtxA activity, and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA-binding domains (HTH1 and HTH2) and phosphoenolpyruvate: carbohydrate phosphotransferase system-regulated domains (PRD1 and PRD2) are apparent. We tested AtxA variants containing single and double phosphomimetic (His → Asp) and phosphoablative (His → Ala) aminomore » acid changes for activity in B. anthracis cultures and for protein-protein interactions in cell lysates. Reduced activity of AtxA H199A, lack of multimerization and activity of AtxAH379D variants, and predicted structural changes associated with phosphorylation support a model for control of AtxA function. We propose that (1) in the AtxA dimer, phosphorylation of H199 in PRD1 affects HTH2 positioning, influencing DNA-binding; and (2) phosphorylation of H379 in PRD2 disrupts dimer formation. In conclusion, the AtxA structure is the first reported high-resolution full-length structure of a PRD-containing regulator and can serve as a model for proteins of this family, especially those that link virulence to bacterial metabolism.« less

  18. Crystal structure of Bacillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity

    SciTech Connect

    Hammerstrom, Troy G.; Horton, Lori B.; Swick, Michelle C.; Joachimiak, Andrzej; Osipiuk, Jerzy; Koehler, Theresa M.

    2014-12-30

    The Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthesis operon. AtxA activity is elevated during growth in media containing glucose and CO2/bicarbonate, and there is a positive correlation between the CO2/bicarbonate signal, AtxA activity, and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA-binding domains (HTH1 and HTH2) and phosphoenolpyruvate: carbohydrate phosphotransferase system-regulated domains (PRD1 and PRD2) are apparent. We tested AtxA variants containing single and double phosphomimetic (His → Asp) and phosphoablative (His → Ala) amino acid changes for activity in B. anthracis cultures and for protein-protein interactions in cell lysates. Reduced activity of AtxA H199A, lack of multimerization and activity of AtxAH379D variants, and predicted structural changes associated with phosphorylation support a model for control of AtxA function. We propose that (1) in the AtxA dimer, phosphorylation of H199 in PRD1 affects HTH2 positioning, influencing DNA-binding; and (2) phosphorylation of H379 in PRD2 disrupts dimer formation. In conclusion, the AtxA structure is the first reported high-resolution full-length structure of a PRD-containing regulator and can serve as a model for proteins of this family, especially those that link virulence to bacterial metabolism.

  19. Crystal structure of Bacillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity.

    PubMed

    Hammerstrom, Troy G; Horton, Lori B; Swick, Michelle C; Joachimiak, Andrzej; Osipiuk, Jerzy; Koehler, Theresa M

    2015-02-01

    The Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthetic operon. AtxA activity is elevated during growth in media containing glucose and CO(2)/bicarbonate, and there is a positive correlation between the CO(2)/bicarbonate signal, AtxA activity and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA-binding domains (HTH1 and HTH2) and phosphoenolpyruvate: carbohydrate phosphotransferase system-regulated domains (PRD1 and PRD2) are apparent. We tested AtxA variants containing single and double phosphomimetic (His→Asp) and phosphoablative (His→Ala) amino acid changes for activity in B. anthracis cultures and for protein-protein interactions in cell lysates. Reduced activity of AtxA H199A, lack of multimerization and activity of AtxAH379D variants, and predicted structural changes associated with phosphorylation support a model for control of AtxA function. We propose that (i) in the AtxA dimer, phosphorylation of H199 in PRD1 affects HTH2 positioning, influencing DNA-binding; and (ii) phosphorylation of H379 in PRD2 disrupts dimer formation. The AtxA structure is the first reported high-resolution full-length structure of a PRD-containing regulator, and can serve as a model for proteins of this family, especially those that link virulence to bacterial metabolism.

  20. Phosphorylation of sterol regulatory element binding protein-1a by protein kinase A (PKA) regulates transcriptional activity.

    PubMed

    Dong, Qingming; Giorgianni, Francesco; Deng, Xiong; Beranova-Giorgianni, Sarka; Bridges, Dave; Park, Edwards A; Raghow, Rajendra; Elam, Marshall B

    2014-07-11

    The counter-regulatory hormone glucagon inhibits lipogenesis via downregulation of sterol regulatory element binding protein 1 (SREBP-1). The effect of glucagon is mediated via protein kinase A (PKA). To determine if SREBP-1 is a direct phosphorylation target of PKA, we conducted mass spectrometry analysis of recombinant n-terminal SREBP-1a following PKA treatment in vitro. This analysis identified serines 331/332 as bona-fide phosphorylation targets of PKA. To determine the functional consequences of phosphorylation at these sites, we constructed mammalian expression vector for both nSREBP-1a and 1c isoforms in which the candidate PKA phosphorylation sites were mutated to active phosphomimetic or non-phosphorylatable amino acids. The transcriptional activity of SREBP was reduced by the phosphomimetic mutation of S332 of nSREBP-1a and the corresponding serine (S308) of nSREBP-1c. This site is a strong candidate for mediating the negative regulatory effect of glucagon on SREBP-1 and lipogenesis.

  1. Phosphorylation on Ser-279 and Ser-282 of connexin43 regulates endocytosis and gap junction assembly in pancreatic cancer cells

    PubMed Central

    Johnson, Kristen E.; Mitra, Shalini; Katoch, Parul; Kelsey, Linda S.; Johnson, Keith R.; Mehta, Parmender P.

    2013-01-01

    The molecular mechanisms regulating the assembly of connexins (Cxs) into gap junctions are poorly understood. Using human pancreatic tumor cell lines BxPC3 and Capan-1, which express Cx26 and Cx43, we show that, upon arrival at the cell surface, the assembly of Cx43 is impaired. Connexin43 fails to assemble, because it is internalized by clathrin-mediated endocytosis. Assembly is restored upon expressing a sorting-motif mutant of Cx43, which does not interact with the AP2 complex, and by expressing mutants that cannot be phosphorylated on Ser-279 and Ser-282. The mutants restore assembly by preventing clathrin-mediated endocytosis of Cx43. Our results also document that the sorting-motif mutant is assembled into gap junctions in cells in which the expression of endogenous Cx43 has been knocked down. Remarkably, Cx43 mutants that cannot be phosphorylated on Ser-279 or Ser-282 are assembled into gap junctions only when connexons are composed of Cx43 forms that can be phosphorylated on these serines and forms in which phosphorylation on these serines is abolished. Based on the subcellular fate of Cx43 in single and contacting cells, our results document that the endocytic itinerary of Cx43 is altered upon cell–cell contact, which causes Cx43 to traffic by EEA1-negative endosomes en route to lysosomes. Our results further show that gap-junctional plaques formed of a sorting motif–deficient mutant of Cx43, which is unable to be internalized by the clathrin-mediated pathway, are predominantly endocytosed in the form of annular junctions. Thus the differential phosphorylation of Cx43 on Ser-279 and Ser-282 is fine-tuned to control Cx43’s endocytosis and assembly into gap junctions. PMID:23363606

  2. Phosphorylation of the Drosophila transient receptor potential ion channel is regulated by the phototransduction cascade and involves several protein kinases and phosphatases.

    PubMed

    Voolstra, Olaf; Bartels, Jonas-Peter; Oberegelsbacher, Claudia; Pfannstiel, Jens; Huber, Armin

    2013-01-01

    Protein phosphorylation plays a cardinal role in regulating cellular processes in eukaryotes. Phosphorylation of proteins is controlled by protein kinases and phosphatases. We previously reported the light-dependent phosphorylation of the Drosophila transient receptor potential (TRP) ion channel at multiple sites. TRP generates the receptor potential upon stimulation of the photoreceptor cell by light. An eye-enriched protein kinase C (eye-PKC) has been implicated in the phosphorylation of TRP by in vitro studies. Other kinases and phosphatases of TRP are elusive. Using phosphospecific antibodies and mass spectrometry, we here show that phosphorylation of most TRP sites depends on the phototransduction cascade and the activity of the TRP ion channel. A candidate screen to identify kinases and phosphatases provided in vivo evidence for an involvement of eye-PKC as well as other kinases and phosphatases in TRP phosphorylation.

  3. Phosphorylation of Ser-204 and Tyr-405 in human malonyl-CoA decarboxylase expressed in silkworm Bombyx mori regulates catalytic decarboxylase activity.

    PubMed

    Hwang, In-Wook; Makishima, Yu; Suzuki, Tomohiro; Kato, Tatsuya; Park, Sungjo; Terzic, Andre; Chung, Shin-Kyo; Park, Enoch Y

    2015-11-01

    Decarboxylation of malonyl-CoA to acetyl-CoA by malonyl-CoA decarboxylase (MCD; EC 4.1.1.9) is a vital catalytic reaction of lipid metabolism. While it is established that phosphorylation of MCD modulates the enzymatic activity, the specific phosphorylation sites associated with the catalytic function have not been documented due to lack of sufficient production of MCD with proper post-translational modifications. Here, we used the silkworm-based Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid system to express human MCD (hMCD) and mapped phosphorylation effects on enzymatic function. Purified MCD from silkworm displayed post-translational phosphorylation and demonstrated coherent enzymatic activity with high yield (-200 μg/silkworm). Point mutations in putative phosphorylation sites, Ser-204 or Tyr-405 of hMCD, identified by bioinformatics and proteomics analyses reduced the catalytic activity, underscoring the functional significance of phosphorylation in modulating decarboxylase-based catalysis. Identified phosphorylated residues are distinct from the decarboxylation catalytic site, implicating a phosphorylation-induced global conformational change of MCD as responsible in altering catalytic function. We conclude that phosphorylation of Ser-204 and Tyr-405 regulates the decarboxylase function of hMCD leveraging the silkworm-based BmNPV bacmid expression system that offers a fail-safe eukaryotic production platform implementing proper post-translational modification such as phosphorylation.

  4. Phosphorylation of the Bin, Amphiphysin, and RSV161/167 (BAR) domain of ACAP4 regulates membrane tubulation

    PubMed Central

    Zhao, Xuannv; Wang, Dongmei; Liu, Xing; Liu, Lifang; Song, Zhenwei; Zhu, Tongge; Adams, Gregory; Gao, Xinjiao; Tian, Ruijun; Huang, Yuejia; Chen, Runhua; Wang, Fengsong; Liu, Dong; Yu, Xue; Chen, Yong; Chen, Zhengjun; Teng, Maikun; Ding, Xia; Yao, Xuebiao

    2013-01-01

    ArfGAP With Coiled-Coil, Ankyrin Repeat And PH Domains 4 (ACAP4) is an ADP-ribosylation factor 6 (ARF6) GTPase-activating protein essential for EGF-elicited cell migration. However, how ACAP4 regulates membrane dynamics and curvature in response to EGF stimulation is unknown. Here, we show that phosphorylation of the N-terminal region of ACAP4, named the Bin, Amphiphysin, and RSV161/167 (BAR) domain, at Tyr34 is necessary for EGF-elicited membrane remodeling. Domain structure analysis demonstrates that the BAR domain regulates membrane curvature. EGF stimulation of cells causes phosphorylation of ACAP4 at Tyr34, which subsequently promotes ACAP4 homodimer curvature. The phospho-mimicking mutant of ACAP4 demonstrates lipid-binding activity and tubulation in vitro, and ARF6 enrichment at the membrane is associated with ruffles of EGF-stimulated cells. Expression of the phospho-mimicking ACAP4 mutant promotes ARF6-dependent cell migration. Thus, the results present a previously undefined mechanism by which EGF-elicited phosphorylation of the BAR domain controls ACAP4 molecular plasticity and plasma membrane dynamics during cell migration. PMID:23776207

  5. Ubiquitin-specific peptidase 20 regulates Rad17 stability, checkpoint kinase 1 phosphorylation and DNA repair by homologous recombination.

    PubMed

    Shanmugam, Ilanchezhian; Abbas, Mohammad; Ayoub, Farhan; Mirabal, Susan; Bsaili, Manal; Caulder, Erin K; Weinstock, David M; Tomkinson, Alan E; Hromas, Robert; Shaheen, Monte

    2014-08-15

    Rad17 is a subunit of the Rad9-Hus1-Rad1 clamp loader complex, which is required for Chk1 activation after DNA damage. Rad17 has been shown to be regulated by the ubiquitin-proteasome system. We have identified a deubiquitylase, USP20 that is required for Rad17 protein stability in the steady-state and post DNA damage. We demonstrate that USP20 and Rad17 interact, and that this interaction is enhanced by UV exposure. We show that USP20 regulation of Rad17 is at the protein level in a proteasome-dependent manner. USP20 depletion results in poor activation of Chk1 protein by phosphorylation, consistent with Rad17 role in ATR-mediated phosphorylation of Chk1. Similar to other DNA repair proteins, USP20 is phosphorylated post DNA damage, and its depletion sensitizes cancer cells to damaging agents that form blocks ahead of the replication forks. Similar to Chk1 and Rad17, which enhance recombinational repair of collapsed replication forks, we demonstrate that USP20 depletion impairs DNA double strand break repair by homologous recombination. Together, our data establish a new function of USP20 in genome maintenance and DNA repair.

  6. H2AX phosphorylation regulated by p38 is involved in Bim expression and apoptosis in chronic myelogenous leukemia cells induced by imatinib.

    PubMed

    Dong, Yaqiong; Xiong, Min; Duan, Lianning; Liu, Ze; Niu, Tianhui; Luo, Yuan; Wu, Xinpin; Xu, Chengshan; Lu, Chengrong

    2014-08-01

    Increasing evidence suggests that histone H2AX plays a critical role in regulation of tumor cell apoptosis and acts as a novel human tumor suppressor protein. However, the action of H2AX in chronic myelogenous leukemia (CML) cells is unknown. The detailed mechanism and epigenetic regulation by H2AX remain elusive in cancer cells. Here, we report that H2AX was involved in apoptosis of CML cells. Overexpression of H2AX increased apoptotic sensitivity of CML cells (K562) induced by imatinib. However, overexpression of Ser139-mutated H2AX (blocking phosphorylation) decreased sensitivity of K562 cells to apoptosis. Similarly, knockdown of H2AX made K562 cells resistant to apoptotic induction. These results revealed that the function of H2AX involved in apoptosis is strictly related to its phosphorylation (Ser139). Our data further indicated that imatinib may stimulate mitogen-activated protein kinase (MAPK) family member p38, and H2AX phosphorylation followed a similar time course, suggesting a parallel response. H2AX phosphorylation can be blocked by p38 siRNA or its inhibitor. These data demonstrated that H2AX phosphorylation was regulated by p38 MAPK pathway in K562 cells. However, the p38 MAPK downstream, mitogen- and stress-activated protein kinase-1 and -2, which phosphorylated histone H3, were not required for H2AX phosphorylation during apoptosis. Finally, we provided epigenetic evidence that H2AX phosphorylation regulated apoptosis-related gene Bim expression. Blocking of H2AX phosphorylation inhibited Bim gene expression. Taken together, these data demonstrated that H2AX phosphorylation regulated by p38 is involved in Bim expression and apoptosis in CML cells induced by imatinib.

  7. The MYC-Associated Protein CDCA7 Is Phosphorylated by AKT To Regulate MYC-Dependent Apoptosis and Transformation

    PubMed Central

    Gill, R. Montgomery; Gabor, Timothy V.; Couzens, Amber L.

    2013-01-01

    Cell division control protein A7 (CDCA7) is a recently identified target of MYC-dependent transcriptional regulation. We have discovered that CDCA7 associates with MYC and that this association is modulated in a phosphorylation-dependent manner. The prosurvival kinase AKT phosphorylates CDCA7 at threonine 163, promoting binding to 14-3-3, dissociation from MYC, and sequestration to the cytoplasm. Upon serum withdrawal, induction of CDCA7 expression in the presence of MYC sensitized cells to apoptosis, whereas CDCA7 knockdown reduced MYC-dependent apoptosis. The transformation of fibroblasts by MYC was reduced by coexpression of CDCA7, while the non-MYC-interacting protein Δ(156–187)-CDCA7 largely inhibited MYC-induced transformation. These studies provide insight into a new mechanism by which AKT signaling to CDCA7 could alter MYC-dependent growth and transformation, contributing to tumorigenesis. PMID:23166294

  8. SpoIIE governs the phosphorylation state of a protein regulating transcription factor sigma F during sporulation in Bacillus subtilis.

    PubMed Central

    Arigoni, F; Duncan, L; Alper, S; Losick, R; Stragier, P

    1996-01-01

    Cell-specific activation of the transcription factor sigma F during sporulation in Bacillus subtilis is controlled by a regulatory pathway involving the proteins SpoIIE, SpoIIAA, and SpoIIAB. SpoIIAB is an antagonist of sigma F, and SpoIIAA, which is capable of overcoming SpoIIAB-mediated inhibition of sigma F, is an antagonist of SpoIIAB. SpoIIAA is, in turn, negatively regulated by SpoIIAB, which phosphorylates SpoIIAA on serine 58. SpoIIAA is also positively regulated by SpoIIE, which dephosphorylates SpoIIAA-P, the phosphorylated form of SpoIIAA. Here, isoelectric focusing and Western blot analysis were used to examine the phosphorylation state of SpoIIAA in vivo. SpoIIAA was found to be largely in the phosphorylated state during sporulation in wild-type cells but a significant portion of the protein that was unphosphorylated could also be detected. Consistent with the idea that SpoIIE governs dephosphorylation of SpoIIAA-P, SpoIIAA was entirely in the phosphorylated state in spoIIE mutant cells. Conversely, overexpression of spoIIE led to an increase in the ratio of unphosphorylated SpoIIAA to SpoIIAA-P and caused inappropriate activation of sigma F in the predivisional sporangium. We also show that a mutant form of SpoIIAA (SpoIIAA-S58T) in which serine 58 was replaced with threonine was present exclusively as SpoIIAA-P, a finding that confirms previous biochemical evidence that the mutant protein is an effective substrate for the SpoIIAB kinase but that SpoIIAA-S58T-P cannot be dephosphorylated by SpoIIE. We conclude that SpoIIE plays a crucial role in controlling the phosphorylation state of SpoIIAA during sporulation and thus in governing the cell-specific activation of sigma F. Images Fig. 1 Fig. 2 Fig. 4 PMID:8622920

  9. Vimentin expression influences flow dependent VASP phosphorylation and regulates cell migration and proliferation

    SciTech Connect

    Lund, Natalie; Henrion, Daniel; Tiede, Petra; Ziche, Marina; Schunkert, Heribert; Ito, Wulf D.

    2010-05-07

    The cytoskeleton plays a central role for the integration of biochemical and biomechanical signals across the cell required for complex cellular functions. Recent studies indicate that the intermediate filament vimentin is necessary for endothelial cell morphogenesis e.g. in the context of leukocyte transmigration. Here, we present evidence, that the scaffold provided by vimentin is essential for VASP localization and PKG mediated VASP phosphorylation and thus controls endothelial cell migration and proliferation. Vimentin suppression using siRNA technique significantly decreased migration velocity by 50% (videomicroscopy), diminished transmigration activity by 42.5% (Boyden chamber) and reduced proliferation by 43% (BrdU-incorporation). In confocal microscopy Vimentin colocalized with VASP and PKG in endothelial cells. Vimentin suppression was accompanied with a translocation of VASP from focal contacts to the perinuclear region. VASP/Vimentin and PKG/Vimentin colocalization appeared to be essential for proper PKG mediated VASP phosphorylation because we detected a diminished expression of PKG and p{sup Ser239}-VASP in vimentin-suppressed cells, Furthermore, the induction of VASP phosphorylation in perfused arteries was markedly decreased in vimentin knockout mice compared to wildtypes. A link is proposed between vimentin, VASP phosphorylation and actin dynamics that delivers an explanation for the important role of vimentin in controlling endothelial cell morphogenesis.

  10. Subcellular Localization and Ser-137 Phosphorylation Regulate Tumor-suppressive Activity of Profilin-1*

    PubMed Central

    Diamond, Marc I.; Cai, Shirong; Boudreau, Aaron; Carey, Clifton J.; Lyle, Nicholas; Pappu, Rohit V.; Swamidass, S. Joshua; Bissell, Mina; Piwnica-Worms, Helen; Shao, Jieya

    2015-01-01

    The actin-binding protein profilin-1 (Pfn1) inhibits tumor growth and yet is also required for cell proliferation and survival, an apparent paradox. We previously identified Ser-137 of Pfn1 as a phosphorylation site within the poly-l-proline (PLP) binding pocket. Here we confirm that Ser-137 phosphorylation disrupts Pfn1 binding to its PLP-containing ligands with little effect on actin binding. We find in mouse xenografts of breast cancer cells that mimicking Ser-137 phosphorylation abolishes cell cycle arrest and apoptotic sensitization by Pfn1 and confers a growth advantage to tumors. This indicates a previously unrecognized role of PLP binding in Pfn1 antitumor effects. Spatial restriction of Pfn1 to the nucleus or cytoplasm indicates that inhibition of tumor cell growth by Pfn1 requires its nuclear localization, and this activity is abolished by a phosphomimetic mutation on Ser-137. In contrast, cytoplasmic Pfn1 lacks inhibitory effects on tumor cell growth but rescues morphological and proliferative defects of PFN1 null mouse chondrocytes. These results help reconcile seemingly opposed cellular effects of Pfn1, provide new insights into the antitumor mechanism of Pfn1, and implicate Ser-137 phosphorylation as a potential therapeutic target for breast cancer. PMID:25681442

  11. MOF phosphorylation by ATM regulates 53BP1-mediated double-strand break repair pathway choice.

    PubMed

    Gupta, Arun; Hunt, Clayton R; Hegde, Muralidhar L; Chakraborty, Sharmistha; Chakraborty, Sharmistha; Udayakumar, Durga; Horikoshi, Nobuo; Singh, Mayank; Ramnarain, Deepti B; Hittelman, Walter N; Namjoshi, Sarita; Asaithamby, Aroumougame; Hazra, Tapas K; Ludwig, Thomas; Pandita, Raj K; Tyler, Jessica K; Pandita, Tej K

    2014-07-10

    Cell-cycle phase is a critical determinant of the choice between DNA damage repair by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Here, we report that double-strand breaks (DSBs) induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF colocalizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S and G2 phase but not G1 phase cells. Expression of MOF-T392A also blocks the reduction in DSB-associated 53BP1 seen in wild-type S/G2 phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair, and decreased cell survival following irradiation. These data support a model whereby ATM-mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2 phase.

  12. Phosphorylation Regulates the Endocytic Function of the Yeast Dynamin-Related Protein Vps1.

    PubMed

    Smaczynska-de Rooij, Iwona I; Marklew, Christopher J; Allwood, Ellen G; Palmer, Sarah E; Booth, Wesley I; Mishra, Ritu; Goldberg, Martin W; Ayscough, Kathryn R

    2015-12-28

    The family of dynamin proteins is known to function in many eukaryotic membrane fusion and fission events. The yeast dynamin-related protein Vps1 functions at several stages of membrane trafficking, including Golgi apparatus to endosome and vacuole, peroxisomal fission, and endocytic scission. We have previously shown that in its endocytic role, Vps1 functions with the amphiphysin heterodimer Rvs161/Rvs167 to facilitate scission and release of vesicles. Phosphoproteome studies of Saccharomyces cerevisiae have identified a phosphorylation site in Vps1 at serine 599. In this study, we confirmed this phosphorylation event, and we reveal that, like Rvs167, Vps1 can be phosphorylated by the yeast cyclin-associated kinase Pho85 in vivo and in vitro. The importance of this posttranslational modification was revealed when mutagenesis of S599 to a phosphomimetic or nonphosphorylatable form caused defects in endocytosis but not in other functions associated with Vps1. Mutation to nonphosphorylatable valine inhibited the Rvs167 interaction, while both S599V and S599D caused defects in vesicle scission, as shown by both live-cell imaging and electron microscopy of endocytic invaginations. Our data support a model in which phosphorylation and dephosphorylation of Vps1 promote distinct interactions and highlight the importance of such regulatory events in facilitating sequential progression of the endocytic process.

  13. Novel Regulation of Parkin Function Through c-Abl-Mediated Tyrosine Phosphorylation: Implications for Parkinson's Disease

    PubMed Central

    Imam, Syed Z.; Zhou, Qing; Yamamoto, Ayako; Valente, Anthony J.; Ali, Syed F.; Bains, Mona; Roberts, James L.; Kahle, Philipp J.; Clark, Robert A.; Li, Senlin

    2011-01-01

    Mutations in parkin, an E3 ubiquitin ligase, are most common cause of autosomal-recessive Parkinson's disease (PD). Here, we show that the stress-signaling non-receptor tyrosine-kinase c-Abl links parkin to sporadic forms of PD via tyrosine phosphorylation. Under oxidative and dopaminergic stress, c-Abl was activated in cultured neuronal cells and in striatum of adult C57 mice. Activated c-Abl was found in the striatum of PD patients. Concomitantly, parkin was tyrosine-phosphorylated, causing loss ofit's ubiquitin ligase and cytoprotective activities, and the accumulation of parkin substrates, AIMP2 (p38/JTV-1) and FBP-1. STI-571, a selective c-Abl inhibitor, prevented tyrosine phosphorylation of parkin and restored its E3 ligase activity and cytoprotective function both in vitro and in vivo. Our results suggest that tyrosine phosphorylation of parkin by c-Abl is a major post-translational modification that leads to loss of parkin function and disease progression in sporadic PD. Moreover, inhibition of c-Abl offers new therapeutic opportunities for blocking PD progression. PMID:21209200

  14. Regulation of CD3-induced phospholipase C-gamma 1 (PLC gamma 1) tyrosine phosphorylation by CD4 and CD45 receptors.

    PubMed Central

    Kanner, S B; Deans, J P; Ledbetter, J A

    1992-01-01

    Stimulation of the signal transduction cascade in T cells through the T-cell receptor (CD3) coincides with activation of the phosphatidylinositol-phospholipase C (PI-PLC) pathway. activation of phospholipase C-gamma 1 (PLC gamma 1) occurs through tyrosine phosphorylation in T cells following surface ligation of CD3 receptors with CD3-specific monoclonal antibodies (mAb). Here we show that cross-linking of CD4 molecules with CD3 augments the tyrosine phosphorylation of PLC gamma 1, while co-ligation of CD3 with CD45 (a receptor tyrosine phosphatase) results in reduced PLC gamma 1 tyrosine phosphorylation. Mobilization of intracellular calcium correlated with the extent of PLC gamma 1 tyrosine phosphorylation, indicating that PLC gamma 1 enzymatic activity in T cells may be regulated by its phosphorylation state. The time-course of PLC gamma 1 tyrosine phosphorylation in cells stimulated by soluble anti-CD3 was transient and closely paralleled that of calcium mobilization, while the kinetics in cells stimulated by immobilized anti-CD3 were prolonged. The PI-PLC pathway in T cells was not stimulated by tyrosine phosphorylation of PLC gamma 2, a homologue of PLC gamma 1, demonstrating the strict regulation of PLC gamma isoform usage in CD3-stimulated T cells. A 35,000/36,000 MW tyrosine phosphorylated protein in T cells formed stable complexes with PLC gamma 1, and its tyrosine phosphorylation was co-regulated with that of PLC gamma 1 by CD4 and CD45 receptors. Enzymatic activation and tyrosine phosphorylation of PLC gamma 1 occurs during growth factor stimulation of fibroblasts, where PLC gamma 1 exists in multi-component complexes. The observation that PLC gamma 1 exists in complexes with unique tyrosine phosphorylated proteins in T cells suggests that haematopoietic lineage-specific proteins associated with PLC gamma 1 may play roles in cellular signalling. Images Figure 1 Figure 4 PMID:1533389

  15. Regulation of neuronal mRNA translation by CaM-kinase I phosphorylation of eIF4GII.

    PubMed

    Srivastava, Taasin; Fortin, Dale A; Nygaard, Sean; Kaech, Stefanie; Sonenberg, Nahum; Edelman, Arthur M; Soderling, Thomas R

    2012-04-18

    Ca²⁺/calmodulin-dependent kinases (CaMKs) are essential for neuronal development and plasticity, processes requiring de novo protein synthesis. Roles for CaMKs in modulating gene transcription are well established, but their involvement in mRNA translation is evolving. Here we report that activity-dependent translational initiation in cultured rat hippocampal neurons is enhanced by CaMKI-mediated phosphorylation of Ser1156 in eukaryotic initiation factor eIF4GII (4GII). Treatment with bicuculline or gabazine to enhance neuronal activity promotes recruitment of wild-type 4GII, but not the 4GII S1156A mutant or 4GI, to the heterotrimeric eIF4F (4F) complex that assembles at the 5' cap structure (m⁷GTP) of mRNA to initiate ribosomal scanning. Recruitment of 4GII to 4F is suppressed by pharmacological inhibition (STO-609) of CaM kinase kinase, the upstream activator of CaMKI. Post hoc in vitro CaMKI phosphorylation assays confirm that activity promotes phosphorylation of S1156 in transfected 4GII in neurons. Changes in cap-dependent and cap-independent translation were assessed using a bicistronic luciferase reporter transfected into neurons. Activity upregulates cap-dependent translation, and RNAi knockdown of CaMKIβ and γ isoforms, but not α or δ, led to its attenuation as did blockade of NMDA receptors. Furthermore, RNAi knockdown of 4GII attenuates cap-dependent translation and reduces density of dendritic filopodia and spine formation without effect on dendritic arborization. Together, our results provide a mechanistic link between Ca²⁺ influx due to neuronal activity and regulation of cap-dependent RNA translation via CaMKI activation and selective recruitment of phosphorylated 4GII to the 4F complex, which may function to regulate activity-dependent changes in spine density.

  16. Rho-ROCK-dependent ezrin-radixin-moesin phosphorylation regulates Fas-mediated apoptosis in Jurkat cells.

    PubMed

    Hébert, Marylise; Potin, Sophie; Sebbagh, Michaël; Bertoglio, Jacques; Bréard, Jacqueline; Hamelin, Jocelyne

    2008-11-01

    Upon engagement by its ligand, the Fas receptor (CD95/APO-1) is oligomerized in a manner dependent on F-actin. It has been shown that ezrin, a member of the ERM (ezrin-radixin-moesin) protein family can link Fas to the actin cytoskeleton. We show herein that in Jurkat cells, not only ezrin but also moesin can associate with Fas. The same observation was made in activated human peripheral blood T cells. Fas/ezrin or moesin (E/M) association increases in Jurkat cells following Fas triggering and occurs concomitantly with the formation of SDS- and 2-ME-stable high molecular mass Fas aggregates. Ezrin and moesin have to be present together for the formation of Fas aggregates since down-regulation of either ezrin or moesin expression with small interfering RNAs completely inhibits Fas aggregate formation. Although FADD (Fas-associated death domain protein) and caspase-8 associate with Fas in the absence of E/M, subsequent events such as caspase-8 activation and sensitivity to apoptosis are decreased. During the course of Fas stimulation, ezrin and moesin become phosphorylated, respectively, on T567 and on T558. This phosphorylation is mediated by the kinase ROCK (Rho-associated coiled coil-containing protein kinase) I subsequently to Rho activation. Indeed, inhibition of either Rho or ROCK prevents ezrin and moesin phosphorylation, abrogates the formation of Fas aggregates, and interferes with caspase-8 activation. Thus, phosphorylation of E/M by ROCK is involved in the early steps of apoptotic signaling following Fas triggering and regulates apoptosis induction.

  17. Calcium/calmodulin dependent protein kinase II regulates the phosphorylation of cyclic AMP-responsive element-binding protein of spinal cord in rats following noxious stimulation.

    PubMed

    Fang, Li; Wu, Jing; Zhang, Xuan; Lin, Qing; Willis, William D

    2005-02-01

    We have previously reported that intradermal capsaicin injection causes the phosphorylation of cyclic adenosine monophosphate-responsive element-binding protein (CREB) in the spinal cord of rats. The present study was designed to investigate the role of calcium/camodulin protein dependent protein kinase II (CaM kinase II) in the regulation of phosphorylation of CREB after capsaicin injection. We found that capsaicin injection produces a significant upregulation of phosphorylated CREB in the spinal cord of rat. Intrathecal treatment with a CaM kinase II inhibitor, KN-93, significantly blocked the increased phosphorylation of CREB, but did not affect the CREB protein itself. These results suggest that increased phosphorylation of CREB protein may contribute to central sensitization following acute peripheral noxious stimuli, and the effect may be regulated through the activation of CaM kinase cascades.

  18. Molecular Insights of p47phox Phosphorylation Dynamics in the Regulation of NADPH Oxidase Activation and Superoxide Production*

    PubMed Central

    Meijles, Daniel N.; Fan, Lampson M.; Howlin, Brendan J.; Li, Jian-Mei

    2014-01-01

    Phagocyte superoxide production by a multicomponent NADPH oxidase is important in host defense against microbial invasion. However inappropriate NADPH oxidase activation causes inflammation. Endothelial cells express NADPH oxidase and endothelial oxidative stress due to prolonged NADPH oxidase activation predisposes many diseases. Discovering the mechanism of NADPH oxidase activation is essential for developing novel treatment of these diseases. The p47phox is a key regulatory subunit of NADPH oxidase; however, due to the lack of full protein structural information, the mechanistic insight of p47phox phosphorylation in NADPH oxidase activation remains incomplete. Based on crystal structures of three functional domains, we generated a computational structural model of the full p47phox protein. Using a combination of in silico phosphorylation, molecular dynamics simulation and protein/protein docking, we discovered that the C-terminal tail of p47phox is critical for stabilizing its autoinhibited structure. Ser-379 phosphorylation disrupts H-bonds that link the C-terminal tail to the autoinhibitory region (AIR) and the tandem Src homology 3 (SH3) domains, allowing the AIR to undergo phosphorylation to expose the SH3 pocket for p22phox binding. These findings were confirmed by site-directed mutagenesis and gene transfection of p47phox−/− coronary microvascular cells. Compared with wild-type p47phox cDNA transfected cells, the single mutation of S379A completely blocked p47phox membrane translocation, binding to p22phox and endothelial O2⨪ production in response to acute stimulation of PKC. p47phox C-terminal tail plays a key role in stabilizing intramolecular interactions at rest. Ser-379 phosphorylation is a molecular switch which initiates p47phox conformational changes and NADPH oxidase-dependent superoxide production by cells. PMID:24970888

  19. Molecular insights of p47phox phosphorylation dynamics in the regulation of NADPH oxidase activation and superoxide production.

    PubMed

    Meijles, Daniel N; Fan, Lampson M; Howlin, Brendan J; Li, Jian-Mei

    2014-08-15

    Phagocyte superoxide production by a multicomponent NADPH oxidase is important in host defense against microbial invasion. However inappropriate NADPH oxidase activation causes inflammation. Endothelial cells express NADPH oxidase and endothelial oxidative stress due to prolonged NADPH oxidase activation predisposes many diseases. Discovering the mechanism of NADPH oxidase activation is essential for developing novel treatment of these diseases. The p47(phox) is a key regulatory subunit of NADPH oxidase; however, due to the lack of full protein structural information, the mechanistic insight of p47(phox) phosphorylation in NADPH oxidase activation remains incomplete. Based on crystal structures of three functional domains, we generated a computational structural model of the full p47(phox) protein. Using a combination of in silico phosphorylation, molecular dynamics simulation and protein/protein docking, we discovered that the C-terminal tail of p47(phox) is critical for stabilizing its autoinhibited structure. Ser-379 phosphorylation disrupts H-bonds that link the C-terminal tail to the autoinhibitory region (AIR) and the tandem Src homology 3 (SH3) domains, allowing the AIR to undergo phosphorylation to expose the SH3 pocket for p22(phox) binding. These findings were confirmed by site-directed mutagenesis and gene transfection of p47(phox-/-) coronary microvascular cells. Compared with wild-type p47(phox) cDNA transfected cells, the single mutation of S379A completely blocked p47(phox) membrane translocation, binding to p22(phox) and endothelial O2(·-) production in response to acute stimulation of PKC. p47(phox) C-terminal tail plays a key role in stabilizing intramolecular interactions at rest. Ser-379 phosphorylation is a molecular switch which initiates p47(phox) conformational changes and NADPH oxidase-dependent superoxide production by cells.

  20. Regulation of Nociceptive Plasticity Threshold and DARPP-32 Phosphorylation in Spinal Dorsal Horn Neurons by Convergent Dopamine and Glutamate Inputs

    PubMed Central

    Buesa, Itsaso; Aira, Zigor

    2016-01-01

    Dopamine can influence NMDA receptor function and regulate glutamate-triggered long-term changes in synaptic strength in several regions of the CNS. In spinal cord, regulation of the threshold of synaptic plasticity may determine the proneness to undergo sensitization and hyperresponsiveness to noxious input. In the current study, we increased endogenous dopamine levels in the dorsal horn by using re-uptake inhibitor GBR 12935. During the so-induced hyperdopaminergic transmission, conditioning low-frequency (1 Hz) stimulation (LFS) to the sciatic nerve induced long-term potentiation (LTP) of C-fiber-evoked potentials in dorsal horn neurons. The magnitude of LTP was attenuated by blockade of either dopamine D1-like receptors (D1LRs) by with SCH 23390 or NMDA receptor subunit NR2B with antagonist Ro25-6981. Conditioning LFS during GBR 12935 administration increased phosphorylation of dopamine- and cAMP-regulated phosphoprotein of Mr 32kDa (DARPP-32) at threonine 34 residue in synaptosomal (P3) fraction of dorsal horn homogenates, as assessed by Western blot analysis, which was partially prevented by NR2B blockade prior to conditioning stimulation. Conditioning LFS also was followed by higher co-localization of phosphorylated form of NR2B at tyrosine 1472 and pDARPP-32Thr34- with postsynaptic marker PSD-95 in transverse L5 dorsal horn sections. Such increase could be significantly attenuated by D1LR blockade with SCH 23390. The current results support that coincidental endogenous recruitment of D1LRs and NR2B in dorsal horn synapses plays a role in regulating afferent-induced nociceptive plasticity. Parallel increases in DARPP-32 phosphorylation upon LTP induction suggests a role for this phosphoprotein as intracellular detector of convergent D1L- and NMDA receptor activation. PMID:27610622

  1. Regulation of p27Kip1 phosphorylation and G1 cell cycle progression by protein phosphatase PPM1G

    PubMed Central

    Sun, Chuang; Wang, Gaohang; Wrighton, Katharine H; Lin, Han; Songyang, Zhou; Feng, Xin-Hua; Lin, Xia

    2016-01-01

    The cell cycle, an essential process leading to the cell division, is stringently controlled by the key cell cycle regulators, cyclin-CDK complexes, whose activity is further regulated by a variety of mechanisms. p27Kip1 is a cyclin-CDK inhibitor that arrests the cell cycle at the G1 phase by blocking the activation of cyclin E-CDK2 complex, preventing the improper entry to the cell cycle. Dysfunction of p27 has been frequently observed in many types of human cancers, resulting from p27 protein degradation and cytoplasmic mislocalization, which are highly regulated by the phosphorylation status of p27. Although the kinases that phosphorylate p27 have been extensively studied, phosphatases that dephosphorylate p27 remain to be elucidated. By using genomic phosphatase screening, we identified a PPM family phosphatase, PPM1G, which could reduce p27 phosphorylation at T198. We further confirmed that PPM1G is a novel p27 phosphatase by demonstrating that PPM1G can interact with and dephosphorylate p27 in cells and in vitro. Functionally, ectopic expression of PPM1G enhanced p27 protein stability and delayed cell cycle progression from G1 to S phase. In accordance, knockdown of PPM1G accelerated p27 degradation during G1 phase and rendered cells resistant to the cell cycle arrest induced by serum deprivation. Mechanistically, PPM1G inhibited the interaction of p27 to 14-3-3θ, a chaperone protein that facilitates p27 nuclear export. Knockdown of PPM1G promoted the cytoplasmic localization of p27. Taken together, our studies identified PPM1G as a novel regulator of p27 that dephosphorylates p27 at T198 site and, together with p27 kinases, PPM1G controls cell cycle progression by maintaining the proper level of p27 protein. PMID:27822412

  2. Structure, function, and evolution of plant NIMA-related kinases: implication for phosphorylation-dependent microtubule regulation.

    PubMed

    Takatani, Shogo; Otani, Kento; Kanazawa, Mai; Takahashi, Taku; Motose, Hiroyasu

    2015-11-01

    Microtubules are highly dynamic structures that control the spatiotemporal pattern of cell growth and division. Microtubule dynamics are regulated by reversible protein phosphorylation involving both protein kinases and phosphatases. Never in mitosis A (NIMA)-related kinases (NEKs) are a family of serine/threonine kinases that regulate microtubule-related mitotic events in fungi and animal cells (e.g. centrosome separation and spindle formation). Although plants contain multiple members of the NEK family, their functions remain elusive. Recent studies revealed that NEK6 of Arabidopsis thaliana regulates cell expansion and morphogenesis through β-tubulin phosphorylation and microtubule destabilization. In addition, plant NEK members participate in organ development and stress responses. The present phylogenetic analysis indicates that plant NEK genes are diverged from a single NEK6-like gene, which may share a common ancestor with other kinases involved in the control of microtubule organization. On the contrary, another mitotic kinase, polo-like kinase, might have been lost during the evolution of land plants. We propose that plant NEK members have acquired novel functions to regulate cell growth, microtubule organization, and stress responses.

  3. Strong regulation of slow anion channels and abscisic acid signaling in guard cells by phosphorylation and dephosphorylation events.

    PubMed Central

    Schmidt, C; Schelle, I; Liao, Y J; Schroeder, J I

    1995-01-01

    Recent evidence suggests that slow anion channels in guard cells need to be activated to trigger stomatal closing and efficiently inactivated during stomatal opening. The patch-clamp technique was employed here to determine mechanisms that produce strong regulation of slow anion channels in guard cells. MgATP in guard cells, serving as a donor for phosphorylation, leads to strong activation of slow anion channels. Slow anion-channel activity was almost completely abolished by removal of cytosolic ATP or by the kinase inhibitors K-252a and H7. Nonhydrolyzable ATP, GTP, and guanosine 5'-[gamma-thio]triphosphate did not replace the ATP requirement for anion-channel activation. In addition, down-regulation of slow anion channels by ATP removal was inhibited by the phosphatase inhibitor okadaic acid. Stomatal closures in leaves induced by the plant hormone abscisic acid (ABA) and malate were abolished by kinase inhibitors and/or enhanced by okadaic acid. These data suggest that ABA signal transduction may proceed by activation of protein kinases and inhibition of an okadaic acid-sensitive phosphatase. This modulation of ABA-induced stomatal closing correlated to the large dynamic range for up- and down-regulation of slow anion channels by opposing phosphorylation and dephosphorylation events in guard cells. The presented opposing regulation by kinase and phosphatase modulators could provide important mechanisms for signal transduction by ABA and other stimuli during stomatal movements. PMID:11607582

  4. Algal dual-specificity tyrosine phosphorylation-regulated kinase, triacylglycerol accumulation regulator1, regulates accumulation of triacylglycerol in nitrogen or sulfur deficiency.

    PubMed

    Kajikawa, Masataka; Sawaragi, Yuri; Shinkawa, Haruka; Yamano, Takashi; Ando, Akira; Kato, Misako; Hirono, Masafumi; Sato, Naoki; Fukuzawa, Hideya

    2015-06-01

    Although microalgae accumulate triacylglycerol (TAG) and starch in response to nutrient-deficient conditions, the regulatory mechanisms are poorly understood. We report here the identification and characterization of a kinase, triacylglycerol accumulation regulator1 (TAR1), that is a member of the yeast (Saccharomyces cerevisiae) Yet another kinase1 (Yak1) subfamily in the dual-specificity tyrosine phosphorylation-regulated kinase family in a green alga (Chlamydomonas reinhardtii). The kinase domain of TAR1 showed auto- and transphosphorylation activities. A TAR1-defective mutant, tar1-1, accumulated TAG to levels 0.5- and 0.1-fold of those of a wild-type strain in sulfur (S)- and nitrogen (N)-deficient conditions, respectively. In N-deficient conditions, tar1-1 showed more pronounced arrest of cell division than the wild type, had increased cell size and cell dry weight, and maintained chlorophyll and photosynthetic activity, which were not observed in S-deficient conditions. In N-deficient conditions, global changes in expression levels of N deficiency-responsive genes in N assimilation and tetrapyrrole metabolism were noted between tar1-1 and wild-type cells. These results indicated that TAR1 is a regulator of TAG accumulation in S- and N-deficient conditions, and it functions in cell growth and repression of photosynthesis in conditions of N deficiency.

  5. Phosphorylation of intracellular proteins related to the multihormonal regulation of prolactin: comparison of normal anterior pituitary cells in culture with the tumor-derived GH cell lines

    SciTech Connect

    Beretta, L.; Boutterin, M.C.; Sobel, A.

    1988-01-01

    We have previously identified a group of cytoplasmic phosphoproteins (proteins 1-11) whose phosphorylation could be related, on a pharmacological basis, to the multihormonal regulation of PRL synthesis and release in the anterior pituitary tumor-derived GH cell lines. Phosphoproteins with identical migration properties on two-dimensional electrophoresis gels were also detectable in normal rat anterior pituitary cells in culture. We designed appropriate culture and (/sup 32/P) phosphate-labeling conditions allowing to analyze the regulation of the phosphorylation of these proteins in normal pituitary cells. TRH, 12-O-tetradecanoylphorbol-13-acetate, and vasoactive intestinal peptide induced the same qualitative changes in phosphorylation of proteins 1-11 in normal as in GH cells. Quantitative differences observed are most likely due to the heterogeneity of primary pituitary cultures. Phosphorylation changes affecting proteins 14-16, not previously detected in GH cells, were also observed with normal anterior pituitary cells. GH cell lines have lost the sensitivity of pituitary lactotrophs for dopamine, an important physiological inhibitor of PRL synthesis and release. In normal anterior pituitary cells in culture, dopamine inhibited also the TRH-stimulated phosphorylation of proteins 1-10, thus strengthening the correlation between phosphorylation of these proteins and multihormonal regulation of pituitary cell functions. Our results indicate: 1) that the same phosphoproteins as in GH cells are related to the multihormonal regulation of nontumoral, normal anterior pituitary cells in culture; 2) that dopamine acts by interfering with the phosphorylation of these proteins.

  6. Dopamine D2 receptors are involved in the regulation of Fyn and metabotropic glutamate receptor 5 phosphorylation in the rat striatum in vivo.

    PubMed

    Mao, Li-Min; Wang, John Q

    2016-04-01

    Fyn, a major Src family kinase (SFK) member that is densely expressed in striatal neurons, is actively involved in the regulation of cellular and synaptic activities in local neurons. This SFK member is likely regulated by dopamine signaling through a receptor mechanism involving dopamine D2 receptors (D2Rs). This study characterizes the D2R-dependent regulation of Fyn in the rat striatum in vivo. Moreover, we explore whether D2Rs regulate metabotropic glutamate receptor 5 (mGluR5) in its tyrosine phosphorylation and whether the D2R-SFK pathway modulates trafficking of mGluR5. We found that blockade of D2Rs by systemic administration of a D2R antagonist, eticlopride, substantially increased SFK phosphorylation in the striatum. This increase was a transient and reversible event. The eticlopride-induced SFK phosphorylation occurred predominantly in immunopurified Fyn but not in another SFK member, Src. Eticlopride also elevated tyrosine phosphorylation of mGluR5. In parallel, eticlopride enhanced synaptic delivery of active Fyn and mGluR5. Pretreatment with an SFK inhibitor blocked the eticlopride-induced tyrosine phosphorylation and synaptic trafficking of mGluR5. These results indicate that D2Rs inhibit SFK (mainly Fyn) phosphorylation in the striatum. D2Rs also inhibit tyrosine phosphorylation and synaptic recruitment of mGluR5 through a signaling mechanism likely involving Fyn.

  7. CK2-dependent C-terminal phosphorylation at T{sup 30} directs the nuclear transport of TSPY protein

    SciTech Connect

    Krick, Roswitha; Aschrafi, Amaz; Hasguen, Dilek; Arnemann, Joachim |. E-mail: Joachim_Arnemann@web.de

    2006-03-10

    TSPY (testis-specific protein, Y-encoded) is a member of the greater SET/NAP family of molecules with various functions, e.g., in chromatin remodeling, regulation of gene expression, and has been implicated to play a role in the malignant development of gonadoblastoma, testicular and prostate cancer. Here we demonstrate that the C-terminus has a functional role for the nucleo-cytoplasmatic shuttling of the TSPY protein. Using various combinations of in vitro mutagenesis and enhanced green fluorescent protein reporter gene-expression experiments we were able to show that while the deletion of C-terminus leads to a decreased stability and enhanced degradation of the protein, the selective mutation of a C-terminal CK2 phosphorylation site (T{sup 30}) prevents the TSPY protein from entering the nucleus. We conclude tha