scholarly journals Regulation of Raf-1 and Raf-1 mutants by Ras-dependent and Ras-independent mechanisms in vitro.

1995 ◽  
Vol 15 (8) ◽  
pp. 4125-4135 ◽  
Author(s):  
P Dent ◽  
D B Reardon ◽  
D K Morrison ◽  
T W Sturgill
Keyword(s):  
Protein Kinase ◽  
Plasma Membranes ◽  
De Novo ◽  
Serine Phosphorylation ◽  
Mitogen Activated Protein Kinase ◽  
Significant Activity ◽  
Dependent Manner ◽  
Wild Type ◽  
Independent Manner

The serine/threonine kinase Raf-1 functions downstream from Ras to activate mitogen-activated protein kinase kinase, but the mechanisms of Raf-1 activation are incompletely understood. To dissect these mechanisms, wild-type and mutant Raf-1 proteins were studied in an in vitro system with purified plasma membranes from v-Ras- and v-Src-transformed cells (transformed membranes). Wild-type (His)6- and FLAG-Raf-1 were activated in a Ras- and ATP-dependent manner by transformed membranes; however, Raf-1 proteins that are kinase defective (K375M), that lack an in vivo site(s) of regulatory tyrosine (YY340/341FF) or constitutive serine (S621A) phosphorylation, that do not bind Ras (R89L), or that lack an intact zinc finger (CC165/168SS) were not. Raf-1 proteins lacking putative regulatory sites for an unidentified kinase (S259A) or protein kinase C (S499A) were activated but with apparently reduced efficiency. The kinase(s) responsible for activation by Ras or Src may reside in the plasma membrane, since GTP loading of plasma membranes from quiescent NIH 3T3 cells (parental membranes) induced de novo capacity to activate Raf-1. Wild-type Raf-1, possessing only basal activity, was not activated by parental membranes in the absence of GTP loading. In contrast, Raf-1 Y340D, possessing significant activity, was, surprisingly, stimulated by parental membranes in a Ras-independent manner. The results suggest that activation of Raf-1 by phosphorylation may be permissive for further modulation by another membrane factor, such as a lipid. A factor(s) extracted with methanol-chloroform from transformed membranes or membranes from Sf9 cells coexpressing Ras and SrcY527F significantly enhanced the activity of Raf-1 Y340D or active Raf-1 but not that of inactive Raf-1. Our findings suggest a model for activation of Raf-1, wherein (i) Raf-1 associates with Ras-GTP, (ii) Raf-1 is activated by tyrosine and/or serine phosphorylation, and (iii) Raf-1 activity is further increased by a membrane cofactor.

scholarly journals Pentachloronitrobenzene alters progesterone production and primordial follicle recruitment in cultured granulosa cells and rat ovary†

2019 ◽  
Vol 102 (2) ◽  
pp. 511-520
Author(s):  
Yanrong Kuai ◽  
Xiaobo Gao ◽  
Huixia Yang ◽  
Haiyan Luo ◽  
Yang Xu ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Granulosa Cells ◽  
Crop Production ◽  
Follicular Development ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Serum Progesterone ◽  
Primordial Follicles ◽  
Progesterone Production

Abstract Pentachloronitrobenzene (PCNB) is an organochlorine fungicide widely used for crop production and has become an environmental concern. Little is known about the effect of PCNB on ovarian steroidogenesis and follicular development. We found that PCNB stimulated Star expression and progesterone production in cultured rat granulosa cells in a dose-dependent manner. PCNB activated mitogen-activated protein kinase (MAPK3/1) extracellulat regulated kinase (ERK1/2), thus inhibition of either protein kinase A (PKA) or MAPK3/1 signaling pathway significantly attenuated progesterone biosynthesis caused by PCNB, suggesting that PCNB induced progesterone production by activating the cyclic adenosine monophosphate (cAMP/PKA) and MAPK3/1 signaling pathways. Further investigation demonstrated that PCNB induced Star expression and altered MAPK3/1 signaling in ovary tissues of immature SD rats treated with PCNB at the dose of 100, 200, or 300 mg/kg by daily gavage for 7 days, while serum progesterone level was dose-dependently decreased. We demonstrated that PCNB exposure accelerated the recruitment of primordial follicles into the growing follicle pool in ovary tissues, accompanied by increased levels of anti-Mullerian hormone (AMH) in both ovary tissues and serum. Taken together, our data demonstrate for the first time that PCNB stimulated Star expression, altered MAPK3/1 signaling and progesterone production in vivo and in vitro, and accelerated follicular development with a concomitant increase in AMH in ovary tissues and serum. Our findings provide novel insight into the toxicity of PCNB to animal ovary function.

scholarly journals Modulation of Virulence by Two Acidified Nitrite-Responsive Loci of Salmonella enterica Serovar Typhimurium

2003 ◽  
Vol 71 (6) ◽  
pp. 3196-3205 ◽  
Author(s):  
Charles C. Kim ◽  
Denise Monack ◽  
Stanley Falkow
Keyword(s):  
Salmonella Enterica ◽  
Fluorescent Protein ◽  
Dependent Manner ◽  
Wild Type ◽  
Independent Manner ◽  
Inducible Promoters ◽  
Serovar Typhimurium ◽  
Bacterial Genes ◽  
Green Fluorescent

ABSTRACT Two acidified nitrite-inducible genes of Salmonella enterica serovar Typhimurium were identified with a green fluorescent protein-based promoter-trap screen. The nitrite-inducible promoters were located upstream of loci that we designated nipAB and nipC, which correspond to hcp-hcr (hybrid cluster protein) of Escherichia coli and norA of Alcaligenes eutrophus, respectively. Maximal induction of the promoters by nitrite was dependent on pH. The nipAB promoter was regulated by oxygen in an Fnr-dependent manner. The nipC promoter was also regulated by oxygen but in an Fnr-independent manner. The promoters were upregulated in activated RAW264.7 macrophage-like cells, which produce NO via the inducible nitric oxide synthase (iNOS), and the induction was inhibited by aminoguanidine, an inhibitor of iNOS. Although the nipAB and nipC mutants displayed no defects under a variety of in vitro conditions or in tissue culture infections, they exhibited lower oral 50% lethal doses (LD50s) than did the wild type in C57BL/6J mouse infections. The lower LD50s reflected an unexpected increased ability of small inoculating doses of the mutant bacteria to cause lethal infection 2 to 3 weeks after challenge, compared to a similar challenge dose of wild-type bacteria. We conclude that these genes are regulated by physiological nitrogen oxides and that the absence of these bacterial genes in some way diminishes the ability of mice to clear a low dose infection.

Taurine Activates BMP-2/Wnt3a-Mediated Osteoblast Differentiation and Mineralization via Akt and MAPK Signaling

2020 ◽  
Author(s):  
Minsu PARK ◽  
Hyeon Kyeong CHOI ◽  
Jeung Hee AN
Keyword(s):  
Protein Kinase ◽  
Osteoblast Differentiation ◽  
Integration Site ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Bone Morphogenetic Protein 2 ◽  
Dependent Manner ◽  
Ovx Rats

Background: We aimed to elucidate the preventive effects of taurine against osteopenia in ovariectomized (OVX) rats and the mechanisms by which taurine regulates osteoblastogenesis in vitro and in vivo. Methods: The effects of the taurine on human osteoblast MG-63 cell differentiation and osteoblastogenesis effect in OVX rat were examined Konkuk University in 2018 by evaluating osteoblast differentiation, and expression of osteoblast-specific factors by western blotting analysis. Results: Taurine supplementation significantly improved alkaline phosphatase (ALP) activity and mineralization in a concentration-dependent manner. Further, taurine induced the expression of osteogenic growth factors such as bone morphogenetic protein-2 (BMP-2), runt-related transcription factor 2 (RUNX2), small mothers against decapentaplegic 1/5/8 (SMAD1/5/8), wingless-type MMTV integration site family member 3A (Wnt3a), and collagen type 1 (COL-1) via mitogen-activated protein kinase (MAPK) and serine/threonine protein kinase (Akt). Moreover, the RUNX2 activity of the taurine-treated group was enhanced by proteinprotein interactions such as Wnt3a-induced p-AKT/RUNX2 and BMP-mediated SMADs/MAPK/RUNX2 interactions. Conclusion: Our in vitro and in vivo results suggested that taurine can be considered as a potential therapeutic candidate agent for preventing bone loss in postmenopausal osteoporosis.

scholarly journals Phosphoprotein Enriched in Astrocytes-15 kDa Expression Inhibits Astrocyte Migration by a Protein Kinase Cδ-dependent Mechanism

2006 ◽  
Vol 17 (12) ◽  
pp. 5141-5152 ◽  
Author(s):  
François Renault-Mihara ◽  
Frédéric Beuvon ◽  
Xavier Iturrioz ◽  
Brigitte Canton ◽  
Sophie De Bouard ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Fluorescent Protein ◽  
Constitutive Expression ◽  
Mitogen Activated Protein Kinase ◽  
Wild Type ◽  
Bryostatin 1 ◽  
Calcium Calmodulin Dependent ◽  
Mitogen Activated Protein ◽  
Green Fluorescent

Phosphoprotein enriched in astrocytes-15 kDa (PEA-15), a phosphoprotein enriched in astrocytes, inhibits both apoptosis and proliferation in normal and cancerous cells. Here, analysis of PEA-15 expression in glioblastoma organotypic cultures revealed low levels of PEA-15 in tumor cells migrating away from the explants, regardless of the expression levels in the originating explants. Because glioblastomas are highly invasive primary brain tumors that can originate from astrocytes, we explored the involvement of PEA-15 in the control of astrocyte migration. PEA-15−/− astrocytes presented an enhanced motility in vitro compared with their wild-type counterparts. Accordingly, NIH-3T3 cells transfected by green fluorescent protein-PEA-15 displayed a reduced migration. Reexpression of PEA-15 restored PEA-15−/− astrocyte motility to wild-type levels. Pharmacological manipulations excluded a participation of extracellular signal-regulated kinase/mitogen-activated protein kinase, phosphatidylinositol 3-kinase/Akt, and calcium/calmodulin-dependent protein kinase II in this effect of PEA-15. In contrast, treatment by bisindolylmaleimide, Gö6976, and rottlerin, and chronic application of phorbol 12-myristate 13-acetate and/or bryostatin-1 indicated that PKCδ mediated PEA-15 inhibition of astrocyte migration. PEA-15−/− astrocytes constitutively expressed a 40-kDa form of PKCδ that was down-regulated upon PEA-15 reexpression. Together, these data reveal a new function for PEA-15 in the inhibitory control of astrocyte motility through a PKCδ-dependent pathway involving the constitutive expression of a catalytic fragment of PKCδ.

scholarly journals Progesterone secretion by luteinizing human granulosa cells: a possible cAMP-dependent but PKA-independent mechanism involved in its regulation

2004 ◽  
Vol 183 (1) ◽  
pp. 51-60 ◽  
Author(s):  
E C Chin ◽  
D R E Abayasekara
Keyword(s):  
Protein Kinase ◽  
Adenylate Cyclase ◽  
Granulosa Cells ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Nucleotide Exchange ◽  
Independent Manner ◽  
Human Granulosa Cells ◽  
Progesterone Secretion ◽  
Dose Dependent

The corpus luteum formed after luteinization of follicular cells secretes progesterone under the control of luteinizing hormone (LH). Binding of LH to its G-protein-coupled receptor leads to the activation of the adenylate cyclase/ cyclic AMP (cAMP)/cAMP-dependent protein kinase (PKA) signalling pathway. The identification of a new class of cAMP-binding proteins termed ‘guanine nucleotide exchange factors’ (cAMP-GEFs) provides a means by which changes in cAMP could yield actions that are independent of PKA. Hence, in this study, we have explored the hypothesis that steroidogenesis in luteinizing cells is mediated in both a cAMP/PKA-dependent and cAMP-dependent, but PKA-independent, manner. Human granulosa cells were isolated from follicular aspirates of women undergoing assisted conception. Luteinizing human granulosa cells were cultured for up to 3 days in the presence of human (h)LH and the adenylate cyclase activator forskolin in the added presence or absence of increasing doses of the PKA inhibitors H89 (N-[2-(4-bromocinnamylamino)ethyl] 5-isoquinoline) and PKI (myristoylated protein kinase A inhibitor amide 14–22) or the cAMP antagonist, Rp-cAMP. Agonist-stimulated progesterone secretion was inhibited in a dose-dependent manner by the PKA inhibitors and the cAMP antagonist, with decreasing sensitivity as luteinization progressed. Pretreatment of granulosa cells for 4 h with human (h)LH reduced the effectiveness of H89 in inhibiting progester-one secretion. Under basal conditions, cAMP-GEFI expression increased progressively throughout culture, and this could be further enhanced when cells were incubated with increasing doses of LH and forskolin. Furthermore, incubation of cells in the presence of increasing concentrations of the novel cAMP-GEF-specific cAMP analogue, 8 CPT-2 ME-cAMP (8-(4-chloro-phenylthio)-2′-0-methyladenosine-3′,5′-cyclic monophosphate), increased progesterone secretion in a dose-dependent manner. The results show that increases in cAMP generated by LH and forskolin, in addition to activating PKA, also induce increases in cAMP-GEFI protein expression in luteinizing human granulosa cells. In addition, activation of cAMP-GEFI results in increased progesterone secretion. Hence, increases in cAMP lead to the activation of PKA-dependent, as well as PKA-independent but cAMP-dependent (via cAMP-GEFI), signalling mechanisms. Since cAMP-GEFs have the capacity to activate the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-K)/protein kinase B (PKB) signalling pathways, these may provide the potential mechanisms by which cAMP-dependent but PKA-independent progesterone synthesis is regulated.

scholarly journals Characterization of a serum response factor-like protein in Saccharomyces cerevisiae, Rlm1, which has transcriptional activity regulated by the Mpk1 (Slt2) mitogen-activated protein kinase pathway.

1997 ◽  
Vol 17 (5) ◽  
pp. 2615-2623 ◽  
Author(s):  
Y Watanabe ◽  
G Takaesu ◽  
M Hagiwara ◽  
K Irie ◽  
K Matsumoto
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Map Kinase ◽  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Mads Box ◽  
Mitogen Activated Protein

The Mpk1 (Slt2) mitogen-activated protein (MAP) kinase has been implicated in several biological processes in Saccharomyces cerevisiae. The Rlm1 protein, a member of the MADS box family of transcription factors, functions downstream of Mpk1 in the pathway. To characterize the role of Rlm1 in mediating the transcriptional activation by the Mpk1 pathway, we constructed a LexA-Rlm1 deltaN chimera in which sequences, including the MADS box domain of the Rlm1 protein, were replaced by the LexA DNA binding domain and tested the ability of this chimera to activate a LexA operator-controlled reporter gene. In this assay, the Rlm1 protein was found to activate transcription in a manner regulated by the Mpk1 pathway. The Mpk1 protein kinase phosphorylated Rlm1 deltaN in vitro and the LexA-Rlm1 deltaN chimera protein was phosphorylated in vivo in a Mpk1-dependent manner. These results suggest that Mpk1 regulates the transcriptional activity of Rlm1 by directly phosphorylating it. We identified a Mpk1-like protein kinase, Mlp1, as an Rlm1-associated protein by using the yeast two-hybrid system. Overexpression of MLP1 suppresses the caffeine-sensitive phenotype of the bck1 delta mutation. The additivity of the mlp1 delta defect with the Mpk1 delta defect with regard to the caffeine sensitivity, combined with the results of genetic epistasis experiments, suggested that the activity of Rlm1 is regulated independently by Mpk1 MAP kinase and the Mlp1 MAP kinase-like kinase.

scholarly journals Kv4.2 is a locus for PKC and ERK/MAPK cross-talk

2009 ◽  
Vol 417 (3) ◽  
pp. 705-715 ◽  
Author(s):  
Laura A. Schrader ◽  
Yajun Ren ◽  
Feng Cheng ◽  
Dui Bui ◽  
J. David Sweatt ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cross Talk ◽  
Glutathione Transferase ◽  
Mitogen Activated Protein Kinase ◽  
K Channel ◽  
Wild Type ◽  
Membrane Excitability ◽  
Significant Difference ◽  
Pkc Activation

Transient outward K+ currents are particularly important for the regulation of membrane excitability of neurons and repolarization of action potentials in cardiac myocytes. These currents are modulated by PKC (protein kinase C) activation, and the K+- channel subunit Kv4.2 is a major contributor to these currents. Furthermore, the current recorded from Kv4.2 channels expressed in oocytes is reduced by PKC activation. The mechanism underlying PKC regulation of Kv4.2 currents is unknown. In the present study, we determined that PKC directly phosphorylates the Kv4.2 channel protein. In vitro phosphorylation of the intracellular N- and C-termini of Kv4.2 GST (glutathione transferase) tagged fusion protein revealed that the C-terminal of Kv4.2 was phosphorylated by PKC, whereas the N-terminal was not. Amino acid mapping and site-directed mutagenesis revealed that the phosphorylated residues on the Kv4.2 C-terminal were Ser447 and Ser537. A phospho-site-specific antibody showed that phosphorylation at the Ser537 site was increased in the hippocampus in response to PKC activation. Surface biotinylation experiments revealed that mutation to alanine of both Ser447 and Ser537 in order to block phosphorylation at both of the PKC sites increased surface expression compared with wild-type Kv4.2. Electrophysiological recordings of the wild-type and both the alanine and aspartate mutant Kv4.2 channels expressed with KChIP3 (Kv4 channel-interacting protein 3) revealed no significant difference in the half-activation or half-inactivation voltage of the channel. Interestingly, Ser537 lies within a possible ERK (extracellular-signal-regulated kinase)/MAPK (mitogen-activated protein kinase) recognition (docking) domain in the Kv4.2 C-terminal sequence. We found that phosphorylation of Kv4.2 by PKC enhanced ERK phosphorylation of the channel in vitro. These findings suggest the possibility that Kv4.2 is a locus for PKC and ERK cross-talk.

scholarly journals S6K1−/−/S6K2−/− Mice Exhibit Perinatal Lethality and Rapamycin-Sensitive 5′-Terminal Oligopyrimidine mRNA Translation and Reveal a Mitogen-Activated Protein Kinase-Dependent S6 Kinase Pathway

2004 ◽  
Vol 24 (8) ◽  
pp. 3112-3124 ◽  
Author(s):  
Mario Pende ◽  
Sung Hee Um ◽  
Virginie Mieulet ◽  
Melanie Sticker ◽  
Valerie L. Goss ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cell Cycle Progression ◽  
Mammalian Target Of Rapamycin ◽  
Mrna Translation ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Wild Type ◽  
Mitogen Activated Protein ◽  
Terminal Oligopyrimidine ◽  
Perinatal Lethality

ABSTRACT Activation of 40S ribosomal protein S6 kinases (S6Ks) is mediated by anabolic signals triggered by hormones, growth factors, and nutrients. Stimulation by any of these agents is inhibited by the bacterial macrolide rapamycin, which binds to and inactivates the mammalian target of rapamycin, an S6K kinase. In mammals, two genes encoding homologous S6Ks, S6K1 and S6K2, have been identified. Here we show that mice deficient for S6K1 or S6K2 are born at the expected Mendelian ratio. Compared to wild-type mice, S6K1−/− mice are significantly smaller, whereas S6K2 −/− mice tend to be slightly larger. However, mice lacking both genes showed a sharp reduction in viability due to perinatal lethality. Analysis of S6 phosphorylation in the cytoplasm and nucleoli of cells derived from the distinct S6K genotypes suggests that both kinases are required for full S6 phosphorylation but that S6K2 may be more prevalent in contributing to this response. Despite the impairment of S6 phosphorylation in cells from S6K1 −/−/S6K2 −/− mice, cell cycle progression and the translation of 5′-terminal oligopyrimidine mRNAs were still modulated by mitogens in a rapamycin-dependent manner. Thus, the absence of S6K1 and S6K2 profoundly impairs animal viability but does not seem to affect the proliferative responses of these cell types. Unexpectedly, in S6K1 −/−/S6K2 −/− cells, S6 phosphorylation persisted at serines 235 and 236, the first two sites phosphorylated in response to mitogens. In these cells, as well as in rapamycin-treated wild-type, S6K1 −/−, and S6K2 −/− cells, this step was catalyzed by a mitogen-activated protein kinase (MAPK)-dependent kinase, most likely p90rsk. These data reveal a redundancy between the S6K and the MAPK pathways in mediating early S6 phosphorylation in response to mitogens.

STAT1 mediates differentiation of chronic lymphocytic leukemia cells in response to Bryostatin 1

Blood ◽  
2003 ◽  
Vol 102 (8) ◽  
pp. 3016-3024 ◽  
Author(s):  
Traci E. Battle ◽  
David A. Frank
Keyword(s):  
Transcription Factors ◽  
Protein Kinase ◽  
Chronic Lymphocytic Leukemia ◽  
Immunoglobulin M ◽  
Lymphocytic Leukemia ◽  
Serine Phosphorylation ◽  
Mitogen Activated Protein Kinase ◽  
Bryostatin 1 ◽  
Induced Differentiation

Abstract Bryostatin 1 is known to exhibit in vitro and in vivo activity against chronic lymphocytic leukemia (CLL) cells by inducing their further maturation into plasmalike cells. Signal transducer and activator of transcription (STAT) proteins play a central role in B-lymphocyte growth and function and are aberrantly phosphorylated on serine residues in CLL cells. To determine whether STAT transcription factors are important in Bryostatin 1–induced differentiation of CLL cells, primary CLL cells were examined for signaling events following exposure to Bryostatin 1 in vitro. Western analysis and electrophoretic mobility shift assays revealed that Bryostatin 1 induced tyrosine phosphorylation and DNA binding of STAT1, yet there was no effect on constitutive serine phosphorylation of STAT1. Bryostatin 1–induced STAT1 activation occurred in a manner that was dependent on protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and Janus tyrosine kinase (JAK) activation. Evidence indicates that Bryostatin 1 induces STAT1 activation through an interferon γ (IFNγ) autocrine loop. However, STAT1 activation by IFNγ stimulation alone was not sufficient to induce differentiation. This insufficiency is due to the broader effect on gene expression caused by Bryostatin 1 compared with IFNγ, as demonstrated by microarray analysis. Both up-regulation of CD22 expression and immunoglobulin M (IgM) production, markers of CLL differentiation, were inhibited by a decoy oligonucleotide for STAT1, indicating that STAT1 is necessary for Bryostatin 1–induced differentiation of CLL cells. This study implicates STAT transcription factors as important mediators of Bryostatin 1–induced differentiation of CLL cells and could possibly lead to improved therapeutic approaches for the treatment of CLL.

Functional Regulation of the GPIb-IX-V Complex by 14-3-3ζ: Role of Protein Kinase A Phosphorylation of Multiple GPIbα Cytoplasmic Residues.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3654-3654
Author(s):  
Adam D. Munday ◽  
Christopher M. Diangco ◽  
Jose A. Lopez
Keyword(s):  
Amino Acids ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Platelet Function ◽  
De Novo ◽  
Cytoplasmic Domain ◽  
Wild Type ◽  
Functional Regulation ◽  
Kinase A

Abstract Platelet adhesion to the site of vascular injury is essential to prevent blood loss. The initial step of adhesion is mediated by the glycoprotein (GP) Ib-IX-V complex on the platelet surface, binding von Willebrand factor (VWF) on the exposed subendothelium. This interaction is transitory, resulting in platelet rolling, and elicits “inside-out” activation of the integrin αIIβ3, thus instigating stable arrest of platelets on fibrinogen and/or VWF and their subsequent spreading and aggregation. The GPIb-IX-V complex consists of 4 polypeptides: GPIbα disulfide linked to GPIbβ, GPIX, and GPV. While recent effort has focused on elucidation of GPIb-IX-V-generated signals, much remains to be learned. Each component of the complex is a type I transmembrane protein, possessing a C-terminal cytoplasmic tail. Of these, GPIbα’s is the longest at 96 amino acids, and associates with both signaling molecules (PI 3-kinase and Src kinase) and structural proteins (filamin A and 14-3-3ζ). Yet, the GPIbα cytoplasmic sequence lacks domains used by other receptors to recruit signaling molecules. Its only tyrosine residue, at amino acid 605, is not within a known consensus sequence for phosphorylation. However, 10 serine and 8 threonine residues are contained within the cytoplasmic domain. Of these, S587, S590, and S609 are known to be stably phosphorylated in resting platelets and to facilitate 14-3-3ζ binding. S609 does not reside in a consensus motif for phosphorylation, whereas S590 and S587 are within consensus motifs for Casein kinase I and the cAMP-dependent protein kinase A (PKA), respectively. Two other residues, T547 and S566, also reside within consensus sites for PKA. PKA has previously been demonstrated to phosphorylate S166 of GPIbβ and thereby inhibit platelet function. We hypothesized that phosphorylation of GPIbα by PKA also regulates platelet function and 14-3-3ζ binding. To test this, we produced a recombinant protein comprising the cytoplasmic domain of GPIbα (amino acids 515–610) fused to glutathione S-transferase at its N-terminus and evaluated the ability of PKA to phosphorylate the protein in vitro. Once we established that PKA could indeed phosphorylate the protein, we produced the recombinant in bacteria also expressing the PKA catalytic domain in an effort to phosphorylate the recombinant GPIbα cytoplasmic domain de novo to avoid cumbersome in vitro phosphorylations and increase the yield. Analysis using a phosphoS609 antibody demonstrated that GPIbα was phosphorylated on S609. We also examined 14-3-3 binding to wild type and mutant GPIbα expressed as part of the GPIb-IX complex in CHO cells (CHOαβIX) by evaluating which proteins were pulled down with GST-14-3-3ζ from lysates. 14-3-3ζ was able to pull down wild-type GPIbα, but only 5–10% as much of GPIbα S609A. Combined mutation of T547 and S609, each to A, completely abrogated 14-3-3ζ binding, as did combined mutation to A of T547, S566, S587, S590 and S609. Interestingly, approximately 20% residual binding was observed for GPIbα S587A/S590A and GPIbα T547A/S587A/S590A/S609A. These results indicate that PKA phosphorylates T547, S566, S587, S590 and S609 in vitro and at least S609 de novo in bacteria. They also demonstrate that 14-3-3ζ can associate with the cytoplasmic tail of GPIbα via residues T547, S566, S587, S590 and S609. This suggests a complex pattern of functional regulation of the GPIb-IX-V complex by PKA mediated through differential binding of 14-3-3ζ, involving both GPIbα and GPIbβ.

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