Unique phosphorylation of protein kinase C-alpha in PC12 cells induces resistance to translocation and down-regulation.

Cell exposure to phorbol ester stimulates translocation and activation of protein kinase C (PKC), ultimately followed by its down-regulation. Upon activation, PKC-alpha, the best studied isotype of the PKC family, undergoes changes in its phosphorylation state. With a two-dimensional immunoblot procedure we have previously shown the existence in PC12 cells of several multiply phosphorylated forms of PKC-alpha, whose number increases in response to phorbol esters (Gatti, A., Wang, X., and Robinson, P. J. (1996) Biochim. Biophys. Acta 1313, 111-118). Using the same experimental system, here we report that besides the predominant pool of 80-kDa PKC-alpha forms that respond to phorbol ester by translocating to the cell membranes and down-regulating, there is a small pool of cytosolic 82-kDa PKC-alpha forms that are characterized by a more acidic pI and by an unique resistance to phorbol ester-mediated translocation and down-regulation. The appearance of similarly slower migrating and more acidic PKC-alpha forms is reproduced upon in vitro autophosphorylation in the presence of phosphatidylserine and phorbol ester, but not in the presence of calcium. These results suggest that site-specific transphosphorylation or autophosphorylation of this kinase may regulate its subcellular localization and susceptibility to down-regulation.