Effects of collagen, ionophore A23187 and prostaglandin E1 on the phosphorylation of specific proteins in blood platelets

Haslam, Richard J.; Lynham, James A.; Fox, Joan E.B.

doi:10.1042/bj1780397

Cited by 154 publications

(72 citation statements)

References 34 publications

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“…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][3][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.…”

Section: Introductionmentioning

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: Introductionmentioning

confidence: 99%

PI3K regulates pleckstrin-2 in T-cell cytoskeletal reorganization

Bach

Kerr

Wang

et al. 2006

Blood

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“…Platelet activation, by many external stimuli, is associated with phosphorylation and dephosphorylation reactions (Lyons et al, 1975;Haslam & Lynham, 1977;Haslam et al, 1979;Daniel et al, 1981;Imaoka et al, 1983;Schafer et al, 1986;Touqui et al, 1986). This has led us to study the possibility that the dephosphorylating enzyme alkaline phosphatase, by removing phosphate groups from the external platelet membrane, might modulate the platelet function.…”

Section: Introductionmentioning

confidence: 99%

Alkaline phosphatase prevents platelet stimulation by thromboxane‐mimetics

Hatmi

Haye

Gavaret

et al. 1991

British J Pharmacology

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1 The effects of alkaline phosphatase on platelet aggregation, secretion and thromboxane B2 (TxB2) generation induced by the full dose-range of common platelet agonists were studied in human platelet-rich plasma and washed platelets. 2 Platelet aggregation and adenosine 5'-triphosphate (ATP) secretion induced by threshold and supramaximal concentrations of arachidonate and stable TxA2 and prostaglandin endoperoxide-mimetics (compounds U46619 and EP171) were abolished in the presence of alkaline phosphatase (0.5-1 u ml 1), even though the synthesis of TxB2 persisted. In contrast, platelet aggregation by PAF-acether and by supramaximal concentrations of thrombin as well as the primary wave of aggregation by adenosine diphosphate (ADP) and adrenaline were unaffected by alkaline phosphatase under conditions where the secondary wave of aggregation by ADP was blocked. 3 Alkaline phosphatase, unlike prostacyclin, failed to raise the adenosine 3': 5'-cyclic monophosphate (cyclic AMP) content of the platelets. Also, the pretreatment of platelets by inorganic phosphate or by ATP plus creatine phosphate/creatine phosphokinase reversed the inhibitory effect of alkaline phosphatase.4 Experiments performed in the guinea-pig in vivo showed that alkaline phosphatase was effective on thrombocytopenia induced by arachidonate. 5 Our results provide the first direct evidence for a specific inhibitory effect of alkaline phosphatase at a site sensitive to TxA2 and prostaglandin endoperoxides and suggest that its phosphorylation/ dephosphorylation state may play an important role in modulating platelet activation. These results also suggest the presence of ecto-protein kinases on membrane platelets.

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“…We previously reported that thrombin treatment of intact 32PO43-_loaded human platelets resulted in phosphorylation of two specific peptides of M, 41,000 and 20,000 (termed P7P and P9P, respectively) as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)' (8). The time-course of phosphorylation was similar to that of serotonin secretion (release I [9]); this suggests that phosphorylation of these two peptides may be important in induction of the release reaction.…”

Section: Introductionmentioning

confidence: 99%

Interaction of Ca2+ and protein phosphorylation in the rabbit platelet release reaction.

Lyons¹,

Shaw²

1980

J. Clin. Invest.

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A B S T RA C T Ca2+ flux and protein phosphorylation have been implicated as playing an important role in the induction ofthe platelet release reaction. However, the interactions between Ca2+, protein phosphorylation, and the release reaction have been difficult to study because secretion in human platelets is independent of extracellular Ca2+. Thus, we studied rabbit platelets, which, unlike human platelets, require extracellular Ca2+ for serotonin release to occur. Thrombin, basophil platelet-activating factor (PAF), or ionophore A23187 treatment of intact 32PO43-_loaded rabbit platelets resulted in a 200-400% increase in phosphorylation of two peptides of Mr 41,000 and 20,000, designated as P7P and P9P, respectively. These peptides were similar in all respects to the peptides phosphorylated in thrombin-treated human platelets. When Ca2+ was replaced in the medium by EGTA, (a) thrombin-and PAF-induced rabbit platelet [3H]serotonin release was inhibited by 60-75%, whereas ionophore-induced release was blocked completely; (b) thrombin-, PAF-, or ionophore-induced P9P phosphorylation was inhibited by 60%; and (c) ionophore-induced P7P phosphorylation was decreased by 60%, whereas that caused by thrombin or PAF was decreased by only 20%. At 0.25-0.5 U/ml of thrombin, phosphorylation preceded [3H]serotonin release with the time for half-maximal release being 26.0±1.3 s SE (n = 3) and the time for half-maximal phosphorylation being 12.3±1.3 s SE (n = 3) for P7P and 3.7±0.17 s SE (n = 3) for P9P. P9P phosphorylation was significantly inhibited (P < 0.015) by removal of Ca2+ from the medium at a time point before any thrombin-or ionophore-induced serotonin release was detectable. Thus, our data suggest that Ca2+ flux precedes the onset of serotonin secretion and that the

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Effects of collagen, ionophore A23187 and prostaglandin E1 on the phosphorylation of specific proteins in blood platelets

Cited by 154 publications

References 34 publications

PI3K regulates pleckstrin-2 in T-cell cytoskeletal reorganization

PI3K regulates pleckstrin-2 in T-cell cytoskeletal reorganization

Alkaline phosphatase prevents platelet stimulation by thromboxane‐mimetics

Interaction of Ca2+ and protein phosphorylation in the rabbit platelet release reaction.

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