PKD autoinhibition in <i>trans</i> regulates activation loop autophosphorylation in <i>cis</i>.

Abstract

Phosphorylation is a ubiquitous mechanism by which signals are transduced in cells. Protein kinases, enzymes that catalyze the phosphotransfer reaction are, themselves, often regulated by phosphorylation. Paradoxically, however, a substantial fraction of more than 500 human protein kinases are capable of catalyzing their own activation loop phosphorylation. Commonly, these kinases perform this autophosphorylation reaction in <i>trans</i>, whereby transient dimerization leads to the mutual phosphorylation of the activation loop of the opposing protomer. In this study, we demonstrate that protein kinase D (PKD) is regulated by the inverse mechanism of dimerization-mediated <i>trans</i>-autoinhibition, followed by activation loop autophosphorylation in <i>cis</i>. We show that PKD forms a stable face-to-face homodimer that is incapable of either autophosphorylation or substrate phosphorylation. Dissociation of this <i>trans</i>-autoinhibited dimer results in activation loop autophosphorylation, which occurs exclusively in <i>cis</i>. Phosphorylation serves to increase PKD activity and prevent <i>trans</i>-autoinhibition, thereby switching PKD on. Our findings not only reveal the mechanism of PKD regulation but also have profound implications for the regulation of many other eukaryotic kinases.