Title: Specificity and regulation in a protein kinase cascade affecting the actin cytoskeleton 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. In this proposal we will investigate mechanisms underlying specificity and regulation in protein kinase signaling cascades converging on phosphorylation of the cofilin/ADF (actin-depolymerizing factor) group of proteins, key molecules that mediate remodeling of actin filaments. The RHO family GTPases RHO, RAC and CDC42 each directly activate kinases (ROCK, PAK1 and PAK4, respectively) in a spatially restricted manner that in turn directly phosphorylate and activate LIM kinases. The LIM kinases are exquisitely specific in their ability to phosphorylate cofilin/ADF proteins at Ser3, which inactivates cofilin/ADF by causing their dissociation from actin. This signaling cascade is tightly controlled through multiple mechanisms, including substrate specificity of both the upstream kinases and of LIM kinases themselves, and through autoregulation of the LIM kinases. The major goal of this proposal is to discover the molecular basis for specificity and regulation in this biologically important pathway. In our preliminary studies we have determined the X-ray crystal structure of LIM kinase 1 in complex with its substrate, cofilin. Therefore in Aim 1 we build on this result to test the hypothesis that the exquisite substrate specificity of LIM kinases for cofilin is defined by a novel kinase- substrate interaction by probing biochemical, catalytic and biophysical effects, and functional impact, of disrupting the crystallographically defined LIMK1:cofilin interface. In our preliminary studies we have also begun to map the molecular level details of the autoregulatory head-tail interaction of LIMK1, and so in Aim 2 we will utilize a range of structural, biochemical and biophysical techniques obtain a significantly improved understanding of this head-tail interaction and will then examine the effect of targeted mutations on interdomain interactions, on kinase activity in vitro, and on function in cells. Lastly, in the previous grant period we discovered a novel mechanism for autoinhibiton of type II PAKs through an N-terminal pseudosubstrate sequence.
In Aim 3 we will test the hypothesis that type II PAK activation is mediated by direct engagement of this pseudosubstrate sequence by specific SH3 domains, and perform structural, biochemical, and cellular studies of SH3-PAK4 interactions.
This aim will thereby provide molecular level details of type II PAK regulation that have remained obscure. In this Multi-PI proposal, the Boggon and Turk laboratories will conduct a highly collaborative structure-directed functional study to provide a significantly improved understanding of the molecular mechanisms that govern LIM kinase-mediated control of the actin cytoskeleton and of the general rules that govern cellular outcomes in protein kinase signaling.

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Tight control of signaling pathways impinging on the actin cytoskeleton are essential to normal development and homeostasis. The goal of the application is to discover the molecular basis for specificity and regulation in this biologically important pathway focused on the LIM kinase proteins and understanding how the p21- activated kinases are regulated.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular and Integrative Signal Transduction Study Section (MIST)
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Falcon-Morales, Edgardo
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Yale University
Schools of Medicine
New Haven
United States
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Stiegler, Amy L; Boggon, Titus J (2018) The N-Terminal GTPase Domain of p190RhoGAP Proteins Is a PseudoGTPase. Structure 26:1451-1461.e4
Miller, Chad J; Turk, Benjamin E (2018) Homing in: Mechanisms of Substrate Targeting by Protein Kinases. Trends Biochem Sci 43:380-394
Ha, Byung Hak; Boggon, Titus J (2018) CDC42 binds PAK4 via an extended GTPase-effector interface. Proc Natl Acad Sci U S A 115:531-536
Zhang, Eric Y; Ha, Byung Hak; Boggon, Titus J (2018) PAK4 crystal structures suggest unusual kinase conformational movements. Biochim Biophys Acta Proteins Proteom 1866:356-365
Ha, Byung Hak; Boggon, Titus J (2018) The crystal structure of pseudokinase PEAK1 (Sugen kinase 269) reveals an unusual catalytic cleft and a novel mode of kinase fold dimerization. J Biol Chem 293:1642-1650
Li, Xiaofeng; Tao, Yeqing; Murphy, James W et al. (2017) The repeat region of cortactin is intrinsically disordered in solution. Sci Rep 7:16696
Chen, Catherine; Nimlamool, Wutigri; Miller, Chad J et al. (2017) Rational Redesign of a Functional Protein Kinase-Substrate Interaction. ACS Chem Biol 12:1194-1198
Stiegler, Amy L; Boggon, Titus J (2017) p190RhoGAP proteins contain pseudoGTPase domains. Nat Commun 8:506
Morse, Elizabeth M; Sun, Xiaowen; Olberding, Jordan R et al. (2016) PAK6 targets to cell-cell adhesions through its N-terminus in a Cdc42-dependent manner to drive epithelial colony escape. J Cell Sci 129:380-93
Miller, Chad J; Turk, Benjamin E (2016) Rapid Identification of Protein Kinase Phosphorylation Site Motifs Using Combinatorial Peptide Libraries. Methods Mol Biol 1360:203-16

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