Phosphorylation of RACK1 on tyrosine 52 by c-Abl is required for IGF-I-mediated regulation of focal adhesion kinase (FAK)

Kiely, P. A., Baillie, G. S. , Barrett, R., Buckley, D. A., Adams, D. R., Houslay, M. D. and O'Connor, R. (2009) Phosphorylation of RACK1 on tyrosine 52 by c-Abl is required for IGF-I-mediated regulation of focal adhesion kinase (FAK). Journal of Biological Chemistry, 284, pp. 20263-20274. (doi:10.1074/jbc.M109.017640) (PMID:19423701) (PMCID:PMC2740452)

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Abstract

Focal Adhesion Kinase (FAK) activity is controlled by growth factors and adhesion signals in tumor cells. The scaffolding protein RACK1 (Receptor for Activated C Kinases) integrates Insulin-like Growth Factor I (IGF-I) and integrin signalling, but whether RACK1 is required for FAK function is unknown. Here we show that association of FAK with RACK1 is required for both FAK phosphorylation and dephosphorylation in response to IGF-I. Suppression of RACK1 by siRNA ablates FAK phosphorylation, and reduces cell adhesion, cell spreading and clonogenic growth. Peptide array and mutagenesis studies localise the FAK-binding interface to blades I-III of the RACK1 a-propeller and specifically identify a set of basic and hydrophobic amino acids (Arg47, Tyr52, Arg57, Arg60, Phe65, Lys127 and Lys130) as key determinants for association with FAK. Mutation of tyrosine 52 alone is sufficient to disrupt interaction of RACK1 with FAK in cells where endogenous RACK1 is suppressed by siRNA. Cells expressing a Y52F mutant RACK1 are impaired in adhesion, growth and foci formation. Comparative analyses of homology models and crystal structures for RACK1 orthologues suggest a role for Tyr52 as a site for phosphorylation that induces conformational change in RACK1, switching the protein into a FAK-binding state. Tyrosine 52 is further shown to be phosphorylated by c-Abl kinase, and the c-Abl inhibitor STI571 disrupts FAK interaction with RACK1. We conclude that FAK association with RACK1 is regulated by phosphorylation of Tyr52. Our data reveal a novel mechanism whereby IGF-I and c-Abl control RACK1 association with FAK to facilitate adhesion signalling

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Houslay, Professor Miles and Baillie, Professor George
Authors: Kiely, P. A., Baillie, G. S., Barrett, R., Buckley, D. A., Adams, D. R., Houslay, M. D., and O'Connor, R.
Subjects:Q Science > QH Natural history > QH345 Biochemistry
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
College of Medical Veterinary and Life Sciences > Institute of Neuroscience and Psychology
Journal Name:Journal of Biological Chemistry
Journal Abbr.:J Biol Chem.
Publisher:American Society for Biochemistry and Molecular Biology, Inc.
ISSN:0021-9258
ISSN (Online):1083-351X
Published Online:07 May 2009

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
438301Phosphodiesterase-4 isoforms - intracellular targeting, regulation and potential therapeutic targetsMiles HouslayMedical Research Council (MRC)G0600765Institute of Neuroscience and Psychology
374281Protein interactions and compartmentalisation in cell signallingWalter KolchMedical Research Council (MRC)G0400053/69186Biochemistry & Cell Biology
432501Transatlantic networks of excellence in cardiovascular diseaseMiles HouslayFoundation Leducq (LEDUCQ-VIL)06 CVD 02Institute of Neuroscience and Psychology
421571thera-cAMP: identification of therapeutic molecules to target compartmentalised cAMP signalling networks in human diseaseMiles HouslayEuropean Commission (EC)UNSPECIFIEDInstitute of Neuroscience and Psychology
374281Protein interactions and compartmentalisation in cell signallingWalter KolchMedical Research Council (MRC)G0400053/69186MCSB - BIOCHEMISTRY & CELL BIOLOGY