Many eukaryotic protein kinases are activated by phosphorylation of their activation loop. Some protein kinases can autoactivate by phosphorylating their own activation loop. This reaction is widely believed to occur exclusively in trans (by a copy of the same protein) and has been observed in a wide array of kinases that undergo stimulus-induced dimerization. Here, we describe the inverse mechanism for activation of protein kinase D (PKD), an essential kinase involved in membrane trafficking. We show that inactive PKD is dimeric and that its activation depends on dissociation of its kinase domains followed by activation loop autophosphorylation in cis (by itself). Nature has, therefore, found two solutions to the same problem that are simply the inverse of one another.

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 trans, 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 trans-autoinhibition, followed by activation loop autophosphorylation in cis. We show that PKD forms a stable face-to-face homodimer that is incapable of either autophosphorylation or substrate phosphorylation. Dissociation of this trans-autoinhibited dimer results in activation loop autophosphorylation, which occurs exclusively in cis. Phosphorylation serves to increase PKD activity and prevent trans-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