Multisite dephosphorylation and desensitization of conventional protein kinase C isotypes

The generation of antisera specific for the priming phosphorylation sites on protein kinase C alpha (PKC alpha) has permitted analysis of the dephosphorylation 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, the process is shown to be blocked by a PKC inhibitor, indicating a requirement for PKC catalytic activity. This was corroborated by showing that a constitutively active fragment of PKC alpha is able to stimulate the dephosphorylation of wild-type PKC alpha in transfected cells. Consistent with a membrane-traffic event, the process controlled by PKC that leads to dephosphorylation is shown to be temperature-sensitive and to correlate with transient accumulation of PKC alpha on cytoplasmic vesicular structures. It was established that the dephosphorylation of priming sites in PKC alpha is not unique and occurs with other conventional PKC isotypes, demonstrating that this is a general desensitization process for this subclass of kinases. The physiological importance of this desensitization is evidenced by the behaviour of PKC beta 1 in U937 cells, where dephosphorylation of the activation loop site is shown to be a function of cell density.