Abstract
Protein kinase D (PKD) is acutely activated by two tightly coupled events: binding to the second messenger diacylglycerol (DAG) followed by novel protein kinase C (nPKC) phosphorylation at the activation loop and autophosphorylation at the C terminus. Thus, phosphorylation serves as a widely accepted measure of PKD activity. Here we show that treatment of cells with PKD inhibitors paradoxically promotes agonist-dependent activation loop phosphorylation, thus uncoupling phosphorylation from activation. This inhibitor-induced enhancement of phosphorylation differs mechanistically from that previously reported for PKC and Akt, for which active-site inhibitors stabilize a phosphatase-resistant conformation. Rather, a conformational reporter reveals that inhibitor binding induces a conformational change, resulting in relocalization of PKD to basal DAG pools, where it is more readily phosphorylated by nPKCs. These findings illustrate the diverse conformational effects that small molecules exert on their target proteins, underscoring the importance of using caution when interpreting kinase activity from phosphorylation state.