Phosphorylation of threonine 638 critically controls the dephosphorylation and inactivation of protein kinase C alpha

Background: It has been widely reported that multisite phosphorylation plays an essential role in the regulation of protein kinases. However, our understanding of how these events modify protein function in vitro and in vivo is poorly understood. Protein kinase C (PKC) affords an interesting example of how phosphorylation control is coupled to effector control. PKC is acutely regulated by the second messenger diacylglycerol; however, it is also known to undergo multisite phosphorylation. Previously, we and others have shown that one site in the 'activation loop' of PKC alpha (a threonine residue at position 497; T497) and PKC beta (T500) is essential for the catalytic competence of these proteins. More recently, a carboxy-terminal site (T638 in PKC alpha) has been implicated. In this report, we investigate the role of this site and its interaction with the catalytic core site. Results: We have analyzed mutant PKC alpha proteins, in which amino-acid substitutions were made at the T638 site, and shown that phosphorylation at this site affects the conformation of the protein, as judged by thermal stability, and sensitivity to oxidation, trypsin and phosphatase treatment. This supersensitivity to dephosphorylation in vitro was also seen in an agonist-dependent context in vivo. We have also shown that phosphorylation of this site is not essential for catalytic activity of the purified protein. The molecular basis of the control operating through the T638 site was provided by the evidence of a functional interaction with the previously described catalytic core site, T497. This interrelationship was further established by the demonstration that the E497 mutant protein had a thermal instability and phosphatase supersensitivity similar to that of the A638 and E638 mutants. Conclusions: The T638 phosphorylation site is not required for the catalytic function of PKC alpha per se, but serves to control the duration of activation by regulating the rate of dephosphorylation and inactivation of the protein. This is achieved through the cooperative interaction between the T638 and T497 sites; if either of these residues is not phosphorylated, the protein is supersensitive to phosphatase action. This model of PKC alpha function is likely to be of general significance to the protein kinase superfamily, where similarly juxtaposed sites exist. We conclude that dephosphorylation of PKC alpha, and, by inference, other protein kinases, is regulated by multisite phosphorylation.