Integrin-Linked Kinase Is a Functional Mn 2 +-Dependent Protein Kinase that Regulates Glycogen Synthase Kinase-3 b ( GSK-3 b ) Phosphorylation

Background: Integrin-linked kinase (ILK) is a highly evolutionarily conserved, multi-domain signaling protein that localizes to focal adhesions, myofilaments and centrosomes where it forms distinct multi-protein complexes to regulate cell adhesion, cell contraction, actin cytoskeletal organization and mitotic spindle assembly. Numerous studies have demonstrated that ILK can regulate the phosphorylation of various protein and peptide substrates in vitro, as well as the phosphorylation of potential substrates and various signaling pathways in cultured cell systems. Nevertheless, the ability of ILK to function as a protein kinase has been questioned because of its atypical kinase domain. Methodology/Principal Findings: Here, we have expressed full-length recombinant ILK, purified it to . 94% homogeneity, and characterized its kinase activity. Recombinant ILK readily phosphorylates glycogen synthase kinase-3 (GSK-3) peptide and the 20-kDa regulatory light chains of myosin (LC20). Phosphorylation kinetics are similar to those of other active kinases, and mutation of the ATP-binding lysine (K220 within subdomain 2) causes marked reduction in enzymatic activity. We show that ILK is a Mn-dependent kinase (the Km for MnATP is ,150-fold less than that for MgATP). Conclusions/Significance: Taken together, our data demonstrate that ILK is a bona fide protein kinase with enzyme kinetic properties similar to other active protein kinases. Citation: Maydan M, McDonald PC, Sanghera J, Yan J, Rallis C, et al. (2010) Integrin-Linked Kinase Is a Functional Mn-Dependent Protein Kinase that Regulates Glycogen Synthase Kinase-3b (GSK-3b) Phosphorylation. PLoS ONE 5(8): e12356. doi:10.1371/journal.pone. 0012356 Editor: Stefan Wölfl, Universität Heidelberg, Germany Received May 13, 2010; Accepted July 29, 2010; Published August 23, 2010 Copyright: 2010 Maydan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Canadian Cancer Society Research Institute (SD), Cancer Research UK (DIH) and the Canadian Institutes of Health Research (MPW). SignalChem Inc (JS and JY) produced and provided the recombinant proteins used in the study. As such, they were involved in data collection and preparation of the manuscript as it relates to the production of these recombinant proteins. The remaining funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: JS and JY of SignalChem Inc. produced and provided the recombinant proteins used in the study. Their involvement is part of a scientific collaboration and these individuals did not derive financial benefit from their role in the study. SD confirms that their involvement in the study does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: sdedhar@bccrc.ca