LIM Domain Kinases: Regulation and Substrate Recognition ABSTRACT Eukaryotic cells interpret extracellular and intrinsic cues to effect remodeling of the actin cytoskeleton, a process critical for controlling cell morphology, movement, and invasiveness. Tight control of signaling pathways impinging on the cytoskeleton is therefore essential to normal development and homeostasis. The RHO family GTPases RHO, RAC and CDC42 each directly activate kinases (RHO kinases, PAKs, and MRCKs) in a spatially restricted manner that in turn directly phosphorylate and activate the LIM domain kinases (LIMK1 and LIMK2). These kinase signaling cascades ultimately converge on phosphorylation of the cofilin/ADF (actin- depolymerizing factor) group of proteins, key molecules that mediate remodeling of actin filaments. Over the previous two periods we have leveraged the highly complementary expertise of our two laboratories to provide significant advances in two main areas: understanding the specificity and regulation of p21-activated kinases (PAKs) and revealing the basis for selective targeting of cofilin by LIMKs. We will now target our efforts toward answering outstanding questions that remain regarding regulation and function of LIMKs. Our preliminary data suggest that an intramolecular interaction between a LIM-PDZ module and the kinase domain, potentially involving evolutionarily conserved binding surfaces, is responsible for suppressing LIMK catalytic activity. Combining biophysical, biochemical, and cell-based approaches, we will address the hypothesis that disruption of this interaction results in activation of the LIM kinases, and we will reveal the structural basis for LIMK autoregulation. We will further investigate recent evidence that LIMKs can phosphorylate both Ser and Tyr residues by X-ray crystallography of LIMK-substrate complexes and molecular dynamics simulations. In this way LIMKs will serve as a general model for understanding substrate recognition by the various “dual specificity” kinase families. Finally, we will investigate the myotonic dystrophy related CDC42-binding protein kinases (MRCKs), a major group of LIMK activating kinases downstream of the GTPase CDC42, about which little is currently known. We will use structural, biophysical and biochemical approaches to define the basic architecture of MRCKβ studies and to probe how its activation is coupled to interactions with LIMKs through substrate adaptor proteins. Overall, our studies will provide a substantial advance in our molecular level understanding of signaling pathways downstream of the RHO family GTPases that impinge on regulation of the actin cytoskeleton.