An essential role for autophosphorylation in the dissociation of activated protein kinase C from the plasma membrane

The cellular localization of protein kinase C (PKC) is intimately associated with the regulation of its biological activity. Previously we have demonstrated that the redistribution of PKC to the plasma membrane in response to physiological stimuli is followed by a rapid returning of PKC back to the cytoplasm (Feng, X., Zhang, J., Barak, L. S., Meyer, T., Caron, M. G., and Hannun, Y. A. (1998) J. Biol. Chem. 273, 10755-10762). Although the process of PKC membrane targeting has been extensively studied, the molecular mechanism underlying the dissociation of membrane-bound PKC remains unclear. In the present study, by examining the dynamic distribution of wild- type PKC βII and its kinase-deficient mutant (K371R), we demonstrate that kinase activity is required for PKC membrane dissociation. Moreover, the inability of PKC βII(K371R) to dissociate from the plasma membrane in cells overexpressing wild-type PKC βII suggests that autophosphorylation activity of the kinase might be essential for its membrane dissociation. This was further supported by mutational analysis of two in vivo autophosphorylation sites on PKC βII. The replacement of Ser⁶⁶⁰ or Thr⁶⁴¹ by alanine (S660A or T641A) was found to synergistically reduce the reversal of PKC βII membrane translocation, whereas the replacement of the same amino acids by glutamic acid (S660E or T641E), an amino acid commonly used to mimic phosphate, results in mutants behaving similar to wild-type PKC βII. These findings point to an essential role for autophosphorylation in the dissociation of activated PKC from the plasma membrane and suggest that, like PKC membrane translocation, the returning of PKC to the cytoplasm after its activation is also delicately regulated.