Interplay between the tyrosine kinases Chk, Csk and phosphatase PTPRJ is critical for regulating platelets in mice (original) (raw)
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Blood, 2020
The Src family kinases (SFKs) Src, Lyn, and Fyn are essential for platelet activation and also involved in megakaryocyte (MK) development and platelet production. Platelet SFKs are inhibited by C-terminal Src kinase (Csk), which phosphorylates a conserved tyrosine in their C-terminal tail, and are activated by the receptor-type tyrosine phosphatase PTPRJ (CD148, DEP-1), which dephosphorylates the same residue. Deletion of Csk and PTPRJ in the MK lineage in mice results in increased SFK activity, but paradoxically hypoactive platelets resulting from negative feedback mechanisms, including upregulation of Csk homologous kinase (Chk) expression. Here, we investigate the role of Chk in platelets, functional redundancy with Csk, and the physiological consequences of ablating Chk, Csk, and PTPRJ in mice. Platelet count was normal in Chk knockout (KO) mice, reduced by 92% in Chk;Csk double KO (DKO) mice, and partially rescued in Chk;Csk;Ptprj triple KO (TKO) mice. Megakaryocyte numbers wer...
Maintenance of murine platelet homeostasis by the kinase Csk and the phosphatase CD148
Blood, 2018
Src family kinases (SFKs) coordinate the initiating and propagating activation signals in platelets, however it remains unclear how they are regulated. Here we show that ablation of C-terminal Src kinase (Csk) and receptor-like protein tyrosine-phosphatase CD148 in mice results in a dramatic increase in platelet SFK activity, demonstrating that these proteins are essential regulators of platelet reactivity. Paradoxically, Csk/CD148-deficient mice exhibit reduced in vivo and ex vivo thrombus formation and increased bleeding following injury, rather than a prothrombotic phenotype. This is a consequence of multiple negative feedback mechanisms, including downregulation of the immunoreceptor tyrosine-based activation motif (ITAM)- and hemi-ITAM-containing receptors GPVI-FcR γ-chain and CLEC-2, respectively and upregulation of the immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B and its interaction with the tyrosine phosphatases Shp1 and Shp2. Results from ...
2009
Platelets play a fundamental role in hemostasis and thrombosis. They are also involved in pathologic conditions resulting from blocked blood vessels, including myocardial infarction and ischemic stroke. Platelet adhesion, activation, and aggregation at sites of vascular injury are regulated by a diverse repertoire of tyrosine kinase-linked and G proteincoupled receptors. Src family kinases (SFKs) play a central role in initiating and propagating signaling from several platelet surface receptors; however, the underlying mechanism of how SFK activity is regulated in platelets remains unclear. CD148 is the only receptor-like protein tyrosine phosphatase identified in platelets to date. In the present study, we show that mutant mice lacking CD148 exhibited a bleeding tendency and defective arterial thrombosis. Basal SFK activity was found to be markedly reduced in CD148-deficient platelets, resulting in a global hyporesponsiveness to agonists that signal through SFKs, including collagen and fibrinogen. G protein-coupled receptor responses to thrombin and other agonists were also marginally reduced. These results highlight CD148 as a global regulator of platelet activation and a novel antithrombotic drug target. (Blood. 2009;113: 4942-4954)
Blood, 2009
The tyrosine phosphatase CD148 is an essential positive regulator of http://bloodjournal.hematologylibrary.org/cgi/content/full/113/20/4942 Updated information and services can be found at: (49 articles) Thrombosis and Hemostasis (36 articles) Platelets and Thrombopoiesis collections: Blood Articles on similar topics may be found in the following http://bloodjournal.hematologylibrary.org/misc/rights.dtl#repub\_requests
Proceedings of the National Academy of Sciences, 1991
Activation of platelets with thrombin and other agonists causes a rapid increase in the phosphorylation of multiple proteins on tyrosine. To identify candidate proteintyrosine kinases (PIKs; EC 2.7.1.112) that may be responsible for these phosphorylation events, we analyzed the expression of seven Src-family PTKs and examined the association of these kinases with known platelet membrane glycoproteins. Five Src-related PTKs were detected in platelets: pp60SRCC, pp6OFYN,
European Journal of Biochemistry, 1994
We have previously reported that a non-receptor-type protein-tyrosine kinase p7PYk, exists in both membrane and cytosolic fractions in porcine platelets and is activated after thrombin stimulation. To facilitate the understanding of the function of p7PYk, we have investigated the topological features, kinase activities and the interaction with another signal-transducing molecule, namely phosphatidylinositol 3-kinase, during platelet activation. Membrane and cytosolic fractions were separated from thrombin-treated porcine platelets, and the amount of p7Pyk was quantified by the immunoblot technique or the kinase activity of each fraction was determined by an immunoprecipitation kinase assay. After stimulation by thrombin, cytosolic p7Pyk rapidly translocated to the membrane fraction within 10 s and there was also a significant increase in the amount of p7Pyk in the cytoskeletal fraction. The autophosphorylation activity of membrane-associated p7Pyk significantly increased approximately tenfold and reached a maximum at 10 s ; the activity subsequently decreased to almost the basal level within 120 s. For similar time courses, association of p7PYk with phosphatidylinositol 3-kinase and tyrosine phosphorylation of ~7 2 '~~ were observed. These results suggest that translocation, activation, and association of p72'yk with transducing molecules such as phosphatidylinositol 3-kinase, events which occur during platelet activation, may participate in early signal-transduction events. Blood platelets, which are terminally differentiated nonproliferative cells devoid of growth or the capability cell division, contain exceptionally high levels of protein-tyrosine kinase activity in both particulate and cytosolic fractions [l-31. Several studies have recently established that various agonists such as thrombin, collagen, platelet-activating factor, and vasopressin, stimulate protein-tyrosine phosphorylation in platelets [4-61. These observations suggested that proteintyrosine kinase may regulate certain cellular processes in platelets that are distinct from cell growth. We previously reported a porcine gene encoding a nonreceptor-type 72-kDa protein-tyrosine kinase (~7 2 "~) which has unique structural characteristics, possessing the second src homology region 2 (SH2) domain instead of SH3 in its amino-acid sequence [7]. The expression of p7PYk has been demonstrated in porcine splenocytes, platelets, peripheral blood lymphocytes, polymorphonuclear leukocytes and tonsilocytes [8, 91. Recently, we showed that ~7 2 " ' was observed in both particulate and cytosolic fractions of platelets and was rapidly activated following thrombin stimulation [ 8,
Mechanism of Activation and Functional Role of Protein Kinase Cη in Human Platelets
Journal of Biological Chemistry, 2009
The novel class of protein kinase C (nPKC) isoform is expressed in platelets, but not much is known about its activation and function. In this study, we investigated the mechanism of activation and functional implications of nPKC using pharmacological and gene knockout approaches. nPKC was phosphorylated (at Thr-512) in a time-and concentration-dependent manner by 2MeSADP. Pretreatment of platelets with MRS-2179, a P2Y 1 receptor antagonist, or YM-254890, a G q blocker, abolished 2MeSADP-induced phosphorylation of nPKC. Similarly, ADP failed to activate nPKC in platelets isolated from P2Y 1 and G q knockout mice. However, pretreatment of platelets with P2Y 12 receptor antagonist, AR-C69331MX did not interfere with ADP-induced nPKC phosphorylation. In addition, when platelets were activated with 2MeSADP under stirring conditions, although nPKC was phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by activated integrin ␣ IIb  3 mediated outside-in signaling. Moreover, in the presence of SC-57101, a ␣ IIb  3 receptor antagonist, nPKC dephosphorylation was inhibited. Furthermore, in murine platelets lacking PP1c␥, a catalytic subunit of serine/threonine phosphatase, ␣ IIb  3 failed to dephosphorylate nPKC. Thus, we conclude that ADP activates nPKC via P2Y 1 receptor and is subsequently dephosphorylated by PP1␥ phosphatase activated by ␣ IIb  3 integrin. In addition, pretreatment of platelets with-RACK antagonistic peptides, a specific inhibitor of nPKC, inhibited ADP-induced thromboxane generation. However, these peptides had no affect on ADP-induced aggregation when thromboxane generation was blocked. In summary, nPKC positively regulates agonist-induced thromboxane generation with no effects on platelet aggregation.