Molecular analysis of pleckstrin: The major protein kinase c substrate of platelets

Tyers, Michael; Haslam, Richard J.; Rachubinski, Richard A.; Harley, Calvin B.

doi:10.1002/jcb.240400202

Cited by 64 publications

(40 citation statements)

References 47 publications

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“…5,43 When expressed in cell lines, pleckstrin-1 causes rearrangements of the cytoskeleton and morphologic changes such as membrane ruffles and cell spreading. These effects are dependent on phosphorylation of pleckstrin and the N-terminal PH (N-PH) domain.…”

Section: Discussionmentioning

confidence: 99%

PI3K regulates pleckstrin-2 in T-cell cytoskeletal reorganization

Bach

Kerr

Wang

et al. 2006

Blood

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Pleckstrin-2 is composed of 2 pleckstrin homology (PH) domains and a disheveled-Egl-10-pleckstrin (DEP) domain. A lipid-binding assay revealed that pleckstrin-2 binds with greatest affinity to D3 and D5 phosphoinositides. Pleckstrin-2 expressed in Jurkat T cells bound to the cellular membrane and enhanced actindependent spreading only after stimulation of the T-cell antigen receptor or the integrin ␣4␤1. A pleckstrin-2 variant containing point mutations in both PH domains failed to associate with the Jurkat membrane and had no effect on spreading under the same conditions. Although still membrane bound, a pleckstrin-2 variant containing point mutations in the DEP domain demonstrated a decreased ability to induce membrane ruffles and spread. Pleckstrin-2 also colocalized with actin at the immune synapse and integrin clusters via its PH domains. Although pleckstrin-2 can bind to purified D3 and D5 phosphoinositides, the intracellular membrane association of pleckstrin-2 and cell spreading are dependent on D3 phosphoinositides, because these effects were disrupted by pharmacologic inhibition of phosphatidylinositol 3-kinase (PI3K). Our results indicate that pleckstrin-2 uses its modular domains to bind to membraneassociated phosphatidylinositols generated by PI3K, whereby it coordinates with the actin cytoskeleton in lymphocyte spreading and immune synapse formation. IntroductionPleckstrin-1, the platelet and leukocyte C kinase substrate, is the major substrate of protein kinase C (PKC) in platelets, monocytes, macrophages, lymphocytes, and granulocytes. 1 Its 350 amino acid sequence can be divided into 3 motifs: PH domains at the aminoand carboxy-termini of the molecule and an intervening DEP domain. 2-4 A short stretch of amino acids between the aminoterminal PH domain and the DEP domain contains 3 sites of phosphorylation (Ser113, Thr114, and Ser117) by PKC, which are essential for the function of pleckstrin-1. [5][6][7] PH domains have been identified in approximately 252 other human proteins (Simple Modular Architecture Research Tool [SMART] database) and are found in many molecules involved in cellular signaling, cytoskeletal organization, membrane trafficking, and phospholipid modification. 8 Previous results have suggested that PH domains mediate binding of their host proteins to certain phosphoinositides. [9][10][11] Consistent with this hypothesis, nearly all PH domain-containing proteins require membrane association for their function in signal transduction, including pathways that contribute to cytoskeletal assembly, membrane budding, and fusion. 4,7,[12][13][14][15] DEP homology domains are present in numerous signaling proteins, 3,16 including 67 found in the human genome (SMART database). The DEP domain is a protein module of approximately 100 amino acids, first found in the signaling proteins disheveled, Egl-10, and pleckstrin. 3 The domain is also present in certain kinases, regulators of G-protein signaling proteins (RGSs), and Epac, the cyclic adenosine monophosphateregulated guanine nucleotide ...

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Section: Discussionmentioning

confidence: 99%

PI3K regulates pleckstrin-2 in T-cell cytoskeletal reorganization

Bach

Kerr

Wang

et al. 2006

Blood

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“…1) as well as weakly phosphorylated P47 protein (or pleckstrin) (Fig. 2) [17,18]. P47 is a major and specific substrate for kinase C in platelets [18] which is easily detectable among total phosphoproteins (Figs.…”

Section: Nhei Is Phosphorylated In Response To Platelet Activating Agmentioning

confidence: 99%

The Na⁺/H⁺ exchanger is phosphorylated in human platelets in response to activating agents

Livné¹,

Sardet²,

Pouysségur³

1991

FEBS Letters

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z-Thrombin, phorbol esters (PMA) and I,t-diacylglycerol (DAG), three activators of the amiloride-sensitive Na+/H+ exchange in human platelets, rapidly increase the intracellular pH and the level of phosphorylation of the Na'/H+ cxchangcr protein (NHEI). This stimulatory effect is sup pressed by staurosporine, a potent kinase inhibitor, and increased by okadaic acid. a potent inhibitor of phosphatase 1 and 2A. The modulations of NHEI phosphorylation by these factors correlate well with their cfTects on platelet pH. Thus. we conclude that in platelets (i) Na*/H+ exchange is mediated by NHE 1, and (ii) platelet activating agents stimulate NHEI via the modulation of the kinase/phosphatase equilibrium.

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“…Overall, there was a shift of many proteins present towards a more acidic region after CRP-stimulation, which is consistent with protein phosphorylation. This is exemplified by pleckstrin, which has six potential phosphorylation sites for protein kinase C [11]. Pleckstrin is present as an array of spots under basal conditions which move to the left upon stimulation by CRP (see Supplementary Table 2 and Fig.…”

Section: Differential Proteome Analysis Of Basal Versus Crp-activatedmentioning

confidence: 99%

A global proteomics approach identifies novel phosphorylated signaling proteins in GPVI‐activated platelets: Involvement of G6f, a novel platelet Grb2‐binding membrane adapter

et al. 2006

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Collagen-related peptide (CRP) stimulates powerful activation of platelets through the GPVI-FcR γ-chain complex. We have combined proteomics and traditional biochemistry approaches to study the proteome of CRP-activated platelets, focusing in detail on tyrosine phosphorylation. In two separate approaches, phosphotyrosine immunoprecipitations followed by 1D-PAGE, and 2-DE, were used for protein separation. Proteins were identified by MS. By following these approaches, 96 proteins were found to undergo post-translational modification in response to CRP in human platelets, including 11 novel platelet proteins such as Dok-1, SPIN90, osteoclast stimulating factor 1, and β-Pix. Interestingly, the type I transmembrane protein G6f was found to be specifically phosphorylated on Tyr-281 in response to platelet activation by CRP, providing a docking site for the adapter Grb2. G6f tyrosine phoshporylation was also found to take place in response to collagen, although not in response to the G protein-coupled receptor agonists, thrombin and ADP. Further, we also demonstrate for the first time that Grb2 and its homologue Gads are tyrosinephosphorylated in CRP-stimulated platelets. This study provides new insights into the mechanism of platelet activation through the GPVI collagen receptor, helping to build the basis for the development of new drug targets for thrombotic disease.

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Molecular analysis of pleckstrin: The major protein kinase c substrate of platelets

Cited by 64 publications

References 47 publications

PI3K regulates pleckstrin-2 in T-cell cytoskeletal reorganization

PI3K regulates pleckstrin-2 in T-cell cytoskeletal reorganization

The Na⁺/H⁺ exchanger is phosphorylated in human platelets in response to activating agents

A global proteomics approach identifies novel phosphorylated signaling proteins in GPVI‐activated platelets: Involvement of G6f, a novel platelet Grb2‐binding membrane adapter

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Molecular analysis of pleckstrin: The major protein kinase c substrate of platelets

Cited by 64 publications

References 47 publications

PI3K regulates pleckstrin-2 in T-cell cytoskeletal reorganization

PI3K regulates pleckstrin-2 in T-cell cytoskeletal reorganization

The Na+/H+ exchanger is phosphorylated in human platelets in response to activating agents

A global proteomics approach identifies novel phosphorylated signaling proteins in GPVI‐activated platelets: Involvement of G6f, a novel platelet Grb2‐binding membrane adapter

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The Na⁺/H⁺ exchanger is phosphorylated in human platelets in response to activating agents