Regular articleSuppression of Protein Kinase C Is Associated with Inhibition of PYK2 Tyrosine Phosphorylation and Enhancement of PYK2 Interaction with Src in Thrombin-Activated Platelets
Section snippets
Materials
Ro31-8220, a specific PKC inhibitor 20, 21, was a gift from Rosche Products (Welwyn Garden City, Herts, UK). The following materials were obtained from indicated suppliers: thrombin (Green Cross, Osaka, Japan); anti-PYK2 polyclonal antibody, anti-Src monoclonal antibody (MoAb), and anti-phosphotyrosine MoAb (4G10) (Upstate Biotechnology, Lake Placid, NY); anti-PYK2 MoAb, anti-p130Cas MoAb, and anti-phosphotyrosine MoAb (PY20) (Transduction Laboratories, Lexington, KY); anti-Syk MoAb and
Results and Discussion
We first investigated PYK2 tyrosine phosphorylation in thrombin-stimulated platelets by immunoprecipitation with anti-PYK2 polyclonal antibody and Western blotting with antiphosphotyrosine antibody. As reported previously [19], PYK2 underwent rapid tyrosine phosphorylation, with a maximum level obtained as early as 2 minutes after stimulation with thrombin (Figure 1). Thrombin is a potent physiologic agonist and stimulates most of the signaling pathways in platelets; for example, Ca2+
Acknowledgements
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan.
References (32)
- et al.
Activation of Fcγ RII induces tyrosine phosphorylation of multiple proteins including FcγRII
J Biol Chem
(1992) - et al.
Glycoprotein Ib-von Willebrand factor interactions activate tyrosine kinases in human platelets
Blood
(1997) - et al.
Identification and characterization of a novel related adhesion focal tyrosine kinase (RAFTK) from megakaryocytes and brain
J Biol Chem
(1995) - et al.
Cloning and characterization of cell adhesion kinase β, a novel protein-tyrosine kinase of the focal adhesion kinase subfamily
J Biol Chem
(1995) - et al.
Involvement of p130Cas and p105HEF1, a novel Cas-like docking protein, in a cytoskeleton-dependent signaling pathway initiated by ligation of integrin or antigen receptor on human B cells
J Biol Chem
(1997) - et al.
The related adhesion focal tyrosine kinase is tyrosine-phosphorylated after β1-integrin stimulation in B cells and binds to p130cas
J Biol Chem
(1997) - et al.
Characterization of RAFTK, a novel focal adhesion kinase, and its integrin-dependent phosphorylation and activation in megakaryocytes
Blood
(1996) - et al.
Tyrosine phosphorylation of the related adhesion focal tyrosine kinase in megakaryocytes upon stem cell factor and phorbol myristate acetate stimulation and its association with paxillin
J Biol Chem
(1997) - et al.
Ras-dependent mitogen-activated protein kinase activation by G protein-coupled receptors
J Biol Chem
(1997) - et al.
Activation of P2Y2 receptors by UTP and ATP stimulates mitogen-activated kinase activity through a pathway that involves related adhesion focal tyrosine kinase and protein kinase C
J Biol Chem
(1998)
Tyrosine phosphorylation of the novel protein-tyrosine kinase RAFTK during an early phase of platelet activation by an integrin glycoprotein IIb-IIIa-independent mechanism
J Biol Chem
Potent selective inhibitors of protein kinase C
FEBS Lett
Anti-CD9 monoclonal antibody activates p72syk in human platelets
J Biol Chem
Synthesis of phosphatidylinositol 3,4-bisphosphate is regulated by protein-tyrosine phosphorylation but the p85α subunit of phosphatidylinositol 3-kinase may not be a target for tyrosine kinases in thrombin-stimulated human platelets
Biochim Biophys Acta
Cytosolic phospholipase A2 is phosphorylated in collagen- and thrombin-stimulated human platelets independent of protein kinase C and mitogen-activated protein kinase
J Biol Chem
Use and specificity of staurosporine, UCN-01, and calphostin C as protein kinase inhibitors
Methods Enzymol
Cited by (15)
Distinct role of Pyk2 in mediating thromboxane generation downstream of both G<inf>12/13</inf> and integrin αIIbβ3 in platelets
2013, Journal of Biological ChemistryCitation Excerpt :They have also shown that Pyk2 tyrosine phosphorylation is regulated by calcium and is mediated through PKC pathways. However, it has been also reported that PKC activation, but not calcium mobilization, is involved in Pyk2 phosphorylation in thrombin-activated platelets (16). Stimulation of human platelets with von Willebrand factor also induces the rapid phosphorylation of Pyk2, which is not affected by either calcium chelation or PKC inhibition (17).
Impaired thrombin-induced platelet activation and thrombus formation in mice lacking the Ca<sup>2+</sup>-dependent tyrosine kinase Pyk2
2013, BloodCitation Excerpt :In thrombin- and VWF-activated platelets, Pyk2 interacts with the actin-based cytoskeleton.9,11 Other studies have documented its association with PI3K, Cas, Shc, and Hic-5.15-18 Thrombin-induced Pyk2 activation was found to be regulated by Ca2+ and protein kinase C, as well as by cytoskeleton reorganization.9,10,18
Reciprocal cross-talk between P2Y<inf>1</inf> and P2Y<inf>12</inf> receptors at the level of calcium signaling in human platelets
2004, BloodCitation Excerpt :There are a number of ways in which P2Y1may activate Src, although these are not investigated here. It has been shown that calcium-dependent tyrosine kinase (CADTK or Pyk2) can activate Src downstream of an increase in cytosolic calcium,53,54and Src has been shown to be linked to GPCR activation through binding to arrestins.35 In conclusion,Figure 7Csummarizes the findings that P2Y12may regulate the P2Y1-activated calcium response to ADP through activation of PI3K and inhibition of adenylate cyclase.
CrkL is an adapter for Wiskott-Aldrich syndrome protein and Syk
2001, BloodCitation Excerpt :However, because both CrkL and WASP are incorporated into the actin-rich platelet cytoskeleton after platelet aggregation, it is possible that the CrkL-WASP complex plays a role in cell adhesion structures.15,17 Although platelets lack paxillin, they do express cas and paxillin-like Hic-5, which may provide the crucial scaffold for CrkL in the cytoskeleton of platelets.33,39 The possibility that Hic-5, WASP, CrkL, and syk may colocalize in platelets was supported by the observations that all these proteins redistribute to the platelet cytoskeleton after platelet aggregation (Figure 4).
Rhodocytin induces platelet aggregation by interacting with glycoprotein Ia/IIa (GPIa/IIa, integrin α <inf>2</inf>β <inf>1</inf>). Involvement of GPIa/IIa-associated Src and protein tyrosine phosphorylation
2001, Journal of Biological ChemistryCitation Excerpt :Recently, several groups reported that Cas (Crk-associated protein), an adapter protein of 130 kDa, and FAK are involved in cell activation induced by cross-linking of β1 integrin or cell adhesion to extracellular matrix including collagen in other cells (30, 31, 39). Recently, we identified Cas in platelets (33) and found that it undergoes changes upon platelet activation (34). Therefore, we investigated tyrosine phosphorylation of Cas and FAK upon rhodocytin stimulation.