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

• Published in: The Journal of biological chemistry 2013-10, Vol.288 (40), p.29160-29169
• Main Authors: Buitrago, Lorena, Bhavanasi, Dheeraj, Dangelmaier, Carol, Manne, Bhanu Kanth, Badolia, Rachit, Borgognone, Alessandra, Tsygankov, Alexander Y., McKenzie, Steven E., Kunapuli, Satya P.
• Format: Article
• Language: English
• Subjects: Adenosine Diphosphate - pharmacology; Animals; Blood Platelets - drug effects; Blood Platelets - enzymology; Enzyme Activation - drug effects; Feedback, Physiological - drug effects; GPVI; Humans; Intracellular Signaling Peptides and Proteins - metabolism; Isoenzymes - metabolism; Lectins, C-Type - metabolism; Membrane Glycoproteins - metabolism; Mice; Mice, Inbred C57BL; Phospholipase C gamma - metabolism; Phosphorylation - drug effects; Phosphotyrosine - metabolism; Phosphotyrosine Signaling; Platelet Aggregation - drug effects; Platelets; Protein Kinase C (PKC); Protein Kinase C beta - antagonists & inhibitors; Protein Kinase C beta - metabolism; Protein Kinase Inhibitors - pharmacology; Protein Phosphorylation; Protein-tyrosine Kinase (Tyrosine Kinase); Protein-Tyrosine Kinases - metabolism; Receptors, Fc - metabolism; Signal Transduction; Syk; Syk Kinase
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.464107

PKC regulating Syk activity has been demonstrated in other cells but is unknown in platelets. Results: PKCs regulate tyrosine phosphorylation and activity of Syk. Conclusion: PKCβ-dependent differential regulation of Syk activity is seen in human but not in murine platelets. Significance: Understanding this human pathway of platelet regulation might aid in development of anti-platelet therapy. Protein kinase C (PKC) isoforms differentially regulate platelet functional responses downstream of glycoprotein VI (GPVI) signaling, but the role of PKCs regulating upstream effectors such as Syk is not known. We investigated the role of PKC on Syk tyrosine phosphorylation using the pan-PKC inhibitor GF109203X (GFX). GPVI-mediated phosphorylation on Syk Tyr-323, Tyr-352, and Tyr-525/526 was rapidly dephosphorylated, but GFX treatment inhibited this dephosphorylation on Tyr-525/526 in human platelets but not in wild type murine platelets. GFX treatment did not affect tyrosine phosphorylation on FcRγ chain or Src family kinases. Phosphorylation of Lat Tyr-191 and PLCγ2 Tyr-759 was also increased upon treatment with GFX. We evaluated whether secreted ADP is required for such dephosphorylation. Exogenous addition of ADP to GFX-treated platelets did not affect tyrosine phosphorylation on Syk. FcγRIIA- or CLEC-2-mediated Syk tyrosine phosphorylation was also potentiated with GFX in human platelets. Because potentiation of Syk phosphorylation is not observed in murine platelets, PKC-deficient mice cannot be used to identify the PKC isoform regulating Syk phosphorylation. We therefore used selective inhibitors of PKC isoforms. Only PKCβ inhibition resulted in Syk hyperphosphorylation similar to that in platelets treated with GFX. This result indicates that PKCβ is the isoform responsible for Syk negative regulation in human platelets. In conclusion, we have elucidated a novel pathway of Syk regulation by PKCβ in human platelets.