LIM domain kinases: regulation and substrate recognition

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.

Grant Number: 5R01GM102262-12
NIH Institute/Center: NIH
Principal Investigator: Titus Boggon