Differential dephosphorylation of the Protein Kinase C-zeta (PKCζ) in an integrin αIIbβ3-dependent manner in platelets

Protein kinase C-zeta (PKC zeta), an atypical isoform of the PKC family of protein serine/threonine kinases, is expressed in human platelets. However, the mechanisms of its activation and the regulation of its activity in platelets are not known. We have found that under basal resting conditions, PKC zeta has a high phosphorylation status at the activation loop threonine 410 (T410) and the turn motif (autophosphorylation site) threonine 560 (T560), both of which have been shown to be important for its catalytic activity. After stimulation with agonist under stirring conditions, the T410 residue was dephosphorylated in a time- and concentration-dependent manner, while the T560 phosphorylation remained unaffected. The 1410 dephosphorylation could be significantly prevented by blocking the binding of fibrinogen to integrin alpha IIb beta 3 with an antagonist, SC-57101; or by okadaic acid used at concentrations that inhibits protein serine/threonine phosphatases PP1 and PP2A in vitro. The dephosphorylation of T410 residue on PKC zeta was also observed in PP1c gamma null murine platelets after agonist stimulation, suggesting that other isoforms of PP1c or another phosphatase could be responsible for this dephosphorylation event. We conclude that human platelets express PKC zeta, and it may be constitutively phosphorylated at the activation loop threonine 410 and the turn motif threonine 560 under basal resting conditions, which are differentially dephosphorylated by outside-in signaling. This differential dephosphorylation of PKC zeta might be an important regulatory mechanism for platelet functional responses. (C) 2011 Elsevier Inc. All rights reserved.