Structure, Function, and Regulation of Protein Kinase D

Protein kinase D (PKD) is a membrane-activated kinase that controls a multitude of cell signaling pathways in response to the membrane-embedded lipid signaling molecule diacyglycerol (DAG). By phosphorylating downstream effector molecules, PKD controls essential processes such as secretion of proteins to the extracellular environment, intracellular transport of molecules, and changes in cell shape. While the specific regions of PKD that respond to DAG have been identified, the mechanisms by which they activate the kinase are not known. This proposal seeks to address these questions using complementary structural, biophysical, biochemical, and cell biological approaches. We hypothesize that PKD is activated by a process of auto-phosphorylation in which the activating phosphate group is transferred from ATP onto a neighboring molecule of PKD. We present evidence to suggest that this process is mediated by a previously unidentified domain of unknown structure and function in the regulatory portion of PKD. Interestingly, this same domain appears to play an important role in stabilizing the monomeric, inactive state of PKD. This proposal will build on these findings with the goal of elucidating how PKD is both activated in the presence of a stimulus and, conversely, maintained in an inactive state in its absence. The proposal will exploit a wide range of high- and low-resolution structural and biophysical techniques to obtain information on the biochemical and physical properties of PKD. To investigate PKD function in cells, we will use state-of-the-art fluorescence imaging techniques in live cells that will provide spatial, temporal, and local proximity information on inactive and actively signaling PKD. Biochemically, we will characterize PKD in vitro using purified components. To translate our findings into a cellular context, we will collaborate with the lab of Dr. Romeo Ricci (IGBMC, Strasbourg, France) to probe the role of PKD in stimulated insulin and cytokine secretion. Whilst PKD dimerization and transactivation have been suggested before, the molecular evidence underpinning such a mechanism has been conspicuously absent. Identification of a novel dimerization domain in PKD distinguishes PKD from its hereto more famous cousin, PKC. The successful execution of this proposal is therefore expected to mark a milestone in our understanding of this under-appreciated, but critical signaling enzyme.

Organisms are composed of millions of specialized cells that carry out critical functions to maintain organismal viability and respond to environmental cues. Within each cell, growth, proliferation, differentiation, and metabolism, as well as shape and motility must be regulated to ensure that physiological processes occur with high fidelity and at appropriate locations in both space and time. At the heart of the regulation of many of these processes are protein kinases, enzymes which catalyze the addition of a small chemical group, phosphate, to their targets, thereby influencing their function. We have systematically investigated how a number of these protein kinases are controlled at cellular membranes, which represent important surfaces within the cell upon which these reactions are initiated, scaffolded, and controlled in both space and time. The primary focus of this work was protein kinase D (PKD). We have discovered that this essential protein kinase, which controls the transport and trafficking of membrane-bound cargo within the cell, is regulated by a unique mechanism. We have identified and characterized a novel dimerization domain in PKD that mediates dimerization (two copies that self-associate) and inhibition of PKD. When PKD is activated by membranes containing the lipid diacylglycerol (DAG), the kinase domains within PKD dimers are dissociated from one another, leading to an autocatalytic event that installs a single phosphate group on a serine residue in its activation loop. This regulatory switch accomplishes two things: (1) it prevents the inhibitory self-association of the kinase domains and (2) it increases the catalytic activity of PKD. Our findings are conceptually exciting, because the mechanism is the inverse of the classical mechanism of kinase activation by ligand-induced, dimerization-mediated trans-autophosphorylation, most often associated with receptor tyrosine kinases. We will now, in follow-up work, extend this concept to other kinases to determine whether this mechanism is unique to PKD or more prevalent within the protein kinase superfamily than previously thought. This grant has partially funded other work dedicated to understanding the principles of membrane-regulated signal transduction. We have discovered and characterized mechanisms of autoinhibition and/or trans-autoactivation in the kinases Akt, PDK1, and Sgk3, as well as extended a regulatory model for the DMPK kinases that we previously published for Rho-associated coiled-coil kinase (ROCK). These works have revealed common principles of membrane-regulated kinase activation and membrane-scaffolding of protein kinases, as well as cis and trans-autoactivation. In summary, our work provides important new insights into the regulation of a number of essential cellular switches and provided a compelling new conceptual framework for the further investigation of other cellular switches.