Phosphorylation is a ubiquitous mechanism by which signals are transduced in cells. Protein kinases, enzymes that catalyze the phospho-transfer reaction are, themselves, often regulated by phosphorylation. Paradoxically, however, a substantial fraction of the 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 auto- 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 have profound implications for the regulation of many other eukaryotic kinases.

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.

Wrapped writing mode is a simple, inexpensive approach to multiphoton stereolithography. Standard ∼10 µm thin cling foil shields the objective from direct contact with the photoresist, without compromising writing resolution. A diffraction limited lateral voxel width below 150 nm was demonstrated through ray tracing simulations and electron microscopy using standard polymer photoresist. Wrapped mode, like dip-in printing, is not limited by the objective working distance height. Its utility to prototype new print resists was validated through custom aqueous protein, silver nitrate, and black epoxy based formulations.