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 vit ro and in vivo is poorly understood. Protein kinase C (PKC) affords an interesting example of how phosphorylation control is coupled to effe ctor control. PKC is acutely regulated by the second messenger diacylg lycerol; however, it is also known to undergo multisite phosphorylatio n. Previously, we and others have shown that one site in the 'activati on loop' of PKC alpha (a threonine residue at position 497; T497) and PKC beta (T500) is essential for the catalytic competence of these pro teins. 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 ana lyzed mutant PKC alpha proteins, in which amino-acid substitutions wer e made at the T638 site, and shown that phosphorylation at this site a ffects the conformation of the protein, as judged by thermal stability , and sensitivity to oxidation, trypsin and phosphatase treatment. Thi s supersensitivity to dephosphorylation in vitro was also seen in an a gonist-dependent context in vivo. We have also shown that phosphorylat ion of this site is not essential for catalytic activity of the purifi ed protein. The molecular basis of the control operating through the T 638 site was provided by the evidence of a functional interaction with the previously described catalytic core site, T497. This interrelatio nship was further established by the demonstration that the E497 mutan t protein had a thermal instability and phosphatase supersensitivity s imilar to that of the A638 and E638 mutants. Conclusions: The T638 pho sphorylation site is not required for the catalytic function of PKC al pha per se, but serves to control the duration of activation by regula ting the rate of dephosphorylation and inactivation of the protein. Th is is achieved through the cooperative interaction between the T638 an d T497 sites; if either of these residues is not phosphorylated, the p rotein is supersensitive to phosphatase action. This model of PKC alph a function is likely to be of general significance to the protein kina se superfamily, where similarly juxtaposed sites exist. We conclude th at dephosphorylation of PKC alpha, and, by inference, other protein ki nases, is regulated by multisite phosphorylation.