Multisite dephosphorylation and desensitization of conventional protein kinase C isotypes

The generation of antisera specific for the priming phosphorylation sites o n protein kinase C alpha (PKC alpha) has permitted analysis of the dephosph orylation of these sites in relation to the downregulation of the protein. It was demonstrated that these priming sites are subject to agonist-induced dephosphorylation, consistent with inactivation of the protein. Further, t he process is shown to be blocked by a PKC inhibitor, indicating a requirem ent for PKC catalytic activity. This was corroborated by showing that a con stitutively active fragment of PKC alpha is able to stimulate the dephospho rylation of wild-type PKC alpha in transfected cells. Consistent with a mem brane-traffic event, the process controlled by PKC that leads to dephosphor ylation is shown to be temperature-sensitive and to correlate with transien t accumulation of PKC alpha on cytoplasmic vesicular structures. It was est ablished that the dephosphorylation of priming sites in PKC alpha is not un ique and occurs with other conventional PKC isotypes, demonstrating that th is is a general desensitization process for this subclass of kinases. The p hysiological importance of this desensitization is evidenced by the behavio ur of PKC beta 1 in U937 cells, where dephosphorylation of the activation l oop site is shown to be a function of cell density.