Functional Interaction of Protein Kinase Cα with the Tyrosine Kinases Syk and Src in Human Platelets

Abstract

There is a high degree of cross-talk between tyrosine phosphorylation and the serine/threonine phosphorylation signaling pathways. Here we show a physical and functional interaction between the classical protein kinase C isoform (cPKC), PKC␣, and two major nonreceptor tyrosine kinases in platelets, Syk and Src. In the presence of the cPKC-selective inhibitor Gö 6976, platelet 5-hydroxytryptamine release was abolished in response to co-activation of glycoproteins VI and Ib-IX-V by the snake venom alboaggregin A, whereas platelet aggregation was substantially inhibited. Of the two platelet cPKCs, PKC␣ but not PKC␤ was activated, occurring in an Syk-and phospholipase C-dependent manner. Syk and PKC␣ associate in a stimulation-dependent manner, requiring Syk but not PKC activity. PKC␣ and Syk also co-translocate from the cytosol to the plasma membrane upon platelet activation, in a manner dependent upon the activities of both kinases. Although PKC␣ is phosphorylated on tyrosine downstream of Syk, we provide evidence against phosphorylation of Syk by PKC␣, consistent with a lack of effect of PKC␣ inhibition on Syk activity. PKC␣ also associates with Src; although in contrast to interaction with Syk, PKC␣ activity is required for the association of these kinases but not the stimulation-induced translocation of Src to the cell membrane. Finally, the activity of Src is negatively regulated by PKC, as shown by potentiation of Src activity in the presence of the PKC inhibitors GF109203X or Gö 6976. Therefore, there is a complex interplay between PKC␣, Syk, and Src involving physical interaction, phosphorylation, translocation within the cell, and functional activity regulation.

Tyrosine Phosphorylation on Spleen Tyrosine Kinase (Syk) Is Differentially Regulated in Human and Murine Platelets by Protein Kinase C Isoforms

“…tested, it is reasonable to think that the nonspecific effects of the inhibitor might be affecting other tyrosine kinases. With respect to GFX, Pula et al (14) used a much higher concentration than ours (20 M versus 5 M), which might explain the observed difference.…”

“…The notion that PKC regulates Syk activity has been described in endothelial cells as in these cells, a PKC␦-dependent serine phosphorylation on Syk regulates thrombin-induced ICAM expression (13). In contrast, Pula et al (14) postulated that active Syk is required for PKC␣ activation in platelets, locating Syk upstream of PKC␣. A growing body of evidence suggests a close correlation between PKC-dependent serine/threonine phosphorylation and tyrosine phosphorylation events (15)(16)(17).…”

Androgen Induction of Prostate Cancer Cell Invasion Is Mediated by Ezrin

“…Previous studies have shown that T567A-ezrin mutant is poorly phosphorylated in Tyr-145 tyrosine (16,55), a residue located in the N-terminal domain of the protein. Here, we demonstrate that the T567A-ezrin mutant is poorly phosphorylated in Tyr-353 in response to androgen, suggesting that tyrosine kinase activity toward this residue also requires the conformational changes induced by Thr-567 phosphorylation.…”

Fibroblast Growth Factor Receptor 2 Promotes Osteogenic Differentiation in Mesenchymal Cells via ERK1/2 and Protein Kinase C Signaling

“…However, we found that molecular inhibition of PKC␣ but not ERK1/2 reduced osteoblast gene expression in MT FGFR2 cells, suggesting that ERK1/2 does not act downstream of PKC␣ in these cells. Functional interactions may also occur between PKC␣ and the tyrosine kinase Src (65,66). In osteoblasts, PKC␣ is known to activate Src signaling (67)(68)(69).…”