Lipid-activated kinases in cell shape and motility

In order to grow, divide, and replicate, cells must be capable of changing their shape. In addition, many cells carry out very specialized functions: cytotoxic T-lymphocytes must be capable of engaging their target and delivering cytotoxic enzymes in a controlled and specific manner; axonal projections must be able to navigate towards their target; and pancreatic cells must be capable of secreting hormones into the bloodstream. The actomyosin cytoskeleton allows cells to change their shape in response to environmental signals: migration, adhesion, cytokinesis, chemotaxis, and vesicular transport all rely on the coordinated remodeling of structural components within the cell. In order to regulate these processes, the cell must be able to respond to extracellular cues, transduce the signal across the plasma membrane, and drive the appropriate intracellular responses. Lipid-activated kinases, in particular those also activated by small GTPases, control essential aspects of actin and myosin function in eukaryotic cells. Pathophysiological processes, including cancer cell metastasisand neurodegeneration, are associated with cytoskeletal dysfunction. Though many of the substrate effector molecules have been identified and, in many cases the specific amino acid that is phosphorylated, the mechanisms by which membrane-based signals are transduced by these kinases are not understood. The dearth of structural information on the full-length kinases involved has, to date, hindered a more complete understanding of how these processes are regulated. This proposal is concerned with addressing this deficit in our understanding by taking an integrated biophysical, biochemical, and cell biological approach to studying the full-length kinases of the ROCK and MRCK families of AGC kinases. We will seek answers to the following questions: what is the oligomeric state of the active and inactive kinases in cells? What are the lipid second messengers for these kinases, and how is membrane-engagement coupled to kinase activation? Finally, we will address how these kinases integrate multiple membrane-based signals, including proteins and lipids, to elicit a specific downstream response. We hope to shed light on the details of the molecular communication between membrane and cytoskeleton, a process that is critical for the proper function of all eukaryotic cells.

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 scaffolded and coordinated. We have discovered that ROCK, a key protein kinase that controls cellular shape and motility, is regulated by the spatial organization of its kinase domain with respect to the membrane. ROCK contains a specialized domain called a coiled-coil domain, in between its membrane-binding domains and its kinase domains. This coiled-coil, which is evolutionarily conserved in length, acts as a molecular ruler to position the kinase domains at a fixed position. Shortening of this coiled-coil has no effect on the intrinsic catalytic activity of the enzyme, but completely abrogates its function in the cell. This represents a new paradigm in kinase regulation, wherein the activity of the kinase depends only on the local proximity and presentation of its target. Akt is a lipid-activated protein kinase that controls cell growth, proliferation, and metabolism. Akt is activated by a combination of binding to a membrane-embedded small molecule called PIP3 and phosphorylation by two other protein kinases. We have discovered that Akt is directly activated by physically engaging PIP3 and that phosphorylation cannot override this requirement. Our work implies that Akt activity is restricted to membranes within the cell that contain either PIP3 or a related signaling molecule called PI(3,4)P2. This has important implications for the control of cellular processes by Akt in both space and time. Furthermore, our work allows us to understand how rare mutations in human overgrowth disorders and cancer cause the hyperactivation of Akt, leading to over-proliferation of cells and tissues. Protein kinase D (PKD) is a signaling enzyme that controls the secretion of cellular products from an intracellular compartment called the trans-Golgi network (TGN). We have identified and determined the structure of a novel domain in PKD, called a ubiquitin-like domain (ULD). The ULD in PKD mediates its dimerization, a process in which two molecules of PKD interact to form a pair, and this is essential for its activation. In summary, our work provides important new insights into the regulation of a number of essential cellular signaling molecules.