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• W2022274575 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 signaling, but whether RACK1 is required for FAK function is unknown. Here we show that association of FAK with RACK1 is required for both FAK phos pho ryl a tion and dephos pho ryl a tion in response to IGF-I. Suppression of RACK1 by small interfering RNA ablates FAK phos pho ryl a tion and reduces cell adhesion, cell spreading, and clonogenic growth. Peptide array and mutagenesis studies localize the FAK binding interface to blades I-III of the RACK1 β-propeller and specifically identify a set of basic and hydrophobic amino acids (Arg-47, Tyr-52, Arg-57, Arg-60, Phe-65, Lys-127, and Lys-130) 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 small interfering RNA. 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 Tyr-52 as a site for phos pho ryl a tion that induces conformational change in RACK1, switching the protein into a FAK binding state. Tyrosine 52 is further shown to be phos pho ryl a ted 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 phos pho ryl a tion of Tyr-52. Our data reveal a novel mechanism whereby IGF-I and c-Abl control RACK1 association with FAK to facilitate adhesion signaling. 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 signaling, but whether RACK1 is required for FAK function is unknown. Here we show that association of FAK with RACK1 is required for both FAK phos pho ryl a tion and dephos pho ryl a tion in response to IGF-I. Suppression of RACK1 by small interfering RNA ablates FAK phos pho ryl a tion and reduces cell adhesion, cell spreading, and clonogenic growth. Peptide array and mutagenesis studies localize the FAK binding interface to blades I-III of the RACK1 β-propeller and specifically identify a set of basic and hydrophobic amino acids (Arg-47, Tyr-52, Arg-57, Arg-60, Phe-65, Lys-127, and Lys-130) 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 small interfering RNA. 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 Tyr-52 as a site for phos pho ryl a tion that induces conformational change in RACK1, switching the protein into a FAK binding state. Tyrosine 52 is further shown to be phos pho ryl a ted 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 phos pho ryl a tion of Tyr-52. Our data reveal a novel mechanism whereby IGF-I and c-Abl control RACK1 association with FAK to facilitate adhesion signaling. RACK1 2The abbreviations used are: RACK1receptor for activated C kinasesiRNAsmall interfering RNAERKextracellular signal-regulated kinaseGSTglutathione S-transferaseFAKfocal adhesion kinaseIGF-IRinsulin-like growth factor I receptorEGFepidermal growth factorPBSphosphate-buffered salineHAhemagglutinin. is a tryptophan-aspartate (WD) repeat containing protein that acts as a scaffolding protein in a wide array of signaling events (1McCahill A. Warwicker J. Bolger G.B. Houslay M.D. Yarwood S.J. Mol. Pharmacol. 2002; 62: 1261-1273Crossref PubMed Scopus (333) Google Scholar, 2Sklan E.H. Podoly E. Soreq H. Prog. Neurobiol. 2006; 78: 117-134Crossref PubMed Scopus (110) Google Scholar). It has been reported to both regulate and promote cell migration in different cell types (3Buensuceso C.S. Woodside D. Huff J.L. Plopper G.E. O'Toole T.E. J. Cell Sci. 2001; 114: 1691-1698Crossref PubMed Google Scholar, 4Cox E.A. Bennin D. Doan A.T. O'Toole T. Huttenlocher A. Mol. Biol. Cell. 2003; 14: 658-669Crossref PubMed Scopus (123) Google Scholar, 5Kiely P.A. O'Gorman D. Luong K. Ron D. O'Connor R. Mol. Cell. Biol. 2006; 26: 4041-4051Crossref PubMed Scopus (85) Google Scholar). RACK1 scaffolds proteins at focal adhesions and is capable of mediating both focal adhesion assembly and disassembly (4Cox E.A. Bennin D. Doan A.T. O'Toole T. Huttenlocher A. Mol. Biol. Cell. 2003; 14: 658-669Crossref PubMed Scopus (123) Google Scholar, 6Doan A.T. Huttenlocher A. Exp. Cell Res. 2007; 313: 2667-2679Crossref PubMed Scopus (51) Google Scholar, 7Onishi I. Lin P.J. Diering G.H. Williams W.P. Numata M. Cell. Signal. 2007; 19: 194-203Crossref PubMed Scopus (31) Google Scholar). RACK1 also scaffolds core kinases of the ERK pathway in response to adhesion signals and modulates the phosphorylation of focal adhesion proteins including focal adhesion kinase (FAK) and paxillin (8Mamidipudi V. Chang B.Y. Harte R.A. Lee K.C. Cartwright C.A. FEBS Lett. 2004; 567: 321-326Crossref PubMed Scopus (18) Google Scholar, 9Vomastek T. Iwanicki M.P. Schaeffer H.J. Tarcsafalvi A. Parsons J.T. Weber M.J. Mol. Cell. Biol. 2007; 27: 8296-8305Crossref PubMed Scopus (66) Google Scholar). In transformed cells RACK1 integrates signaling from the IGF-I receptor (IGF-IR) and β1 integrin by forming a scaffolding complex that includes these receptors as well as signaling molecules that promote cell migration (5Kiely P.A. O'Gorman D. Luong K. Ron D. O'Connor R. Mol. Cell. Biol. 2006; 26: 4041-4051Crossref PubMed Scopus (85) Google Scholar, 10Hermanto U. Zong C.S. Li W. Wang L.H. Mol. Cell. Biol. 2002; 22: 2345-2365Crossref PubMed Scopus (174) Google Scholar, 11Lynch L. Vodyanik P.I. Boettiger D. Guvakova M.A. Mol. Biol. Cell. 2005; 16: 51-63Crossref PubMed Google Scholar). Cooperation between IGF-IR and β1 integrin signaling is essential for growth of certain tumors (12Goel H.L. Breen M. Zhang J. Das I. Aznavoorian-Cheshire S. Greenberg N.M. Elgavish A. Languino L.R. Cancer Res. 2005; 65: 6692-6700Crossref PubMed Scopus (64) Google Scholar), and we propose that RACK1 has an important role in this. receptor for activated C kinase small interfering RNA extracellular signal-regulated kinase glutathione S-transferase focal adhesion kinase insulin-like growth factor I receptor epidermal growth factor phosphate-buffered saline hemagglutinin. The interaction of RACK1 with the IGF-IR requires integrins to be ligated and also requires a domain in the C terminus of the IGF-IR that is essential for IGF-IR function in anchorage-independent growth, cell survival, and cell migration (13Leahy M. Lyons A. Krause D. O'Connor R. J. Biol. Chem. 2004; 279: 18306-18313Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 14O'Connor R. Kauffmann-Zeh A. Liu Y. Lehar S. Evan G.I. Baserga R. Blättler W.A. Mol. Cell. Biol. 1997; 17: 427-435Crossref PubMed Scopus (242) Google Scholar). Ligand-mediated activation of the IGF-IR leads to recruitment of certain proteins to RACK1 such as IRS-1, β1 integrin, and dissociation of other proteins from RACK1 such as PP2A and Src. Competitive binding to RACK1 occurs for some of these proteins. For example, IGF-I-mediated dissociation of PP2A from RACK1 is required for recruitment of β1 integrin, and both PP2A and β1 integrin compete for binding to tyrosine 302 in RACK1 (5Kiely P.A. O'Gorman D. Luong K. Ron D. O'Connor R. Mol. Cell. Biol. 2006; 26: 4041-4051Crossref PubMed Scopus (85) Google Scholar, 15Kiely P.A. Baillie G.S. Lynch M.J. Houslay M.D. O'Connor R. J. Biol. Chem. 2008; 283: 22952-22961Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). RACK1 is located in areas of cell protrusion that are rich in paxillin (4Cox E.A. Bennin D. Doan A.T. O'Toole T. Huttenlocher A. Mol. Biol. Cell. 2003; 14: 658-669Crossref PubMed Scopus (123) Google Scholar, 7Onishi I. Lin P.J. Diering G.H. Williams W.P. Numata M. Cell. Signal. 2007; 19: 194-203Crossref PubMed Scopus (31) Google Scholar) and can increase the phosphorylation of FAK (7Onishi I. Lin P.J. Diering G.H. Williams W.P. Numata M. Cell. Signal. 2007; 19: 194-203Crossref PubMed Scopus (31) Google Scholar). FAK is a well characterized kinase in mediating integrin signaling and is associated with the enhanced migratory potential of several cancer cell types (16Mitra S.K. Schlaepfer D.D. Curr. Opin. Cell Biol. 2006; 18: 516-523Crossref PubMed Scopus (1194) Google Scholar, 17Mon N.N. Ito S. Senga T. Hamaguchi M. Ann. N. Y. Acad. Sci. 2006; 1086: 199-212Crossref PubMed Scopus (62) Google Scholar, 18Schlaepfer D.D. Hou S. Lim S.T. Tomar A. Yu H. Lim Y. Hanson D.A. Uryu S.A. Molina J. Mitra S.K. J. Biol. Chem. 2007; 282: 17450-17459Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). FAK is phosphorylated on tyrosine 397 in response to the clustering of integrins (for review, see Ref. 19Schlaepfer D.D. Mitra S.K. Curr. Opin. Genet. Dev. 2004; 14: 92-101Crossref PubMed Scopus (357) Google Scholar) or by activation of the EGF and platelet-derived growth factor receptors (20Hsia D.A. Mitra S.K. Hauck C.R. Streblow D.N. Nelson J.A. Ilic D. Huang S. Li E. Nemerow G.R. Leng J. Spencer K.S. Cheresh D.A. Schlaepfer D.D. J. Cell Biol. 2003; 160: 753-767Crossref PubMed Scopus (459) Google Scholar, 21Li W. Duzgun A. Sumpio B.E. Basson M.D. Am. J. Physiol. Gastrointest. Liver Physiol. 2001; 280: G75-G87Crossref PubMed Google Scholar, 22Wang J.G. Miyazu M. Xiang P. Li S.N. Sokabe M. Naruse K. Life Sci. 2005; 76: 2817-2825Crossref PubMed Scopus (65) Google Scholar, 23Webb D.J. Donais K. Whitmore L.A. Thomas S.M. Turner C.E. Parsons J.T. Horwitz A.F. Nat. Cell Biol. 2004; 6: 154-161Crossref PubMed Scopus (1076) Google Scholar). This results in recruitment of Src and subsequent phosphorylation of target proteins that are associated with focal adhesion formation and activation of mitogen-activated protein kinase pathways. FAK becomes rapidly dephosphorylated when cells are detached, and this is thought to be essential for focal adhesion dissolution and cell migration. FAK dephosphorylation can be stimulated by IGF-I (5Kiely P.A. O'Gorman D. Luong K. Ron D. O'Connor R. Mol. Cell. Biol. 2006; 26: 4041-4051Crossref PubMed Scopus (85) Google Scholar, 24Guvakova M.A. Surmacz E. Exp. Cell Res. 1999; 251: 244-255Crossref PubMed Scopus (77) Google Scholar, 25Kiely P.A. Leahy M. O'Gorman D. O'Connor R. J. Biol. Chem. 2005; 280: 7624-7633Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 26Mañes S. Mira E. Gómez-Mouton C. Zhao Z.J. Lacalle R.A. Martínez-A C. Mol. Cell. Biol. 1999; 19: 3125-3135Crossref PubMed Scopus (220) Google Scholar, 27Mauro L. Sisci D. Bartucci M. Salerno M. Kim J. Tam T. Guvakova M.A. Ando S. Surmacz E. Exp. Cell Res. 1999; 252: 439-448Crossref PubMed Scopus (60) Google Scholar). Interestingly, we have observed that IGF-I-mediated dephosphorylation of FAK is enhanced in cells overexpressing RACK1, which also have enhanced migratory potential and increased activation of mitogen-activated protein kinase pathways (28Kiely P.A. Sant A. O'Connor R. J. Biol. Chem. 2002; 277: 22581-22589Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). However, it is not known how the phosphorylation and subsequent dephosphorylation of FAK are coordinated. In particular, the role of RACK1 in regulation of FAK phosphorylation remains undefined. Here we investigated this in the context of IGF-I and adhesion signaling by determining the role of RACK1 in FAK function. Recombinant IGF-I and EGF were purchased from Pepro Tech. Inc. (Rocky Hill, NJ). The anti-RACK1 and anti-Abl (554148) were from BD Transduction Laboratories. The anti-IGF-IR polyclonal antibody and anti c-Abl antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-phospho-Akt, anti-Akt, and anti-phospho FAK (Tyr-397) polyclonal antibodies were from Cell Signaling Technology (Beverly, MA). The anti-Shc polyclonal antibodies, the anti-phosphotyrosine monoclonal antibody 4G10, anti-ERK-2, anti-FAK antibodies, and recombinant glutathione S-transferase (GST)-Crk was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). The anti-actin monoclonal antibody was from Sigma. Recombinant GST-FAK and anti-c-Abl (OP14) were purchased from Calbiochem. STI571 was a kind gift from Novartis Pharma AG, (Basel, Switzerland). The MCF-7 breast carcinoma cell line and R− cell line (the mouse embryonic fibroblast cell line derived from the IGF-IR knock out mouse (29Sell C. Dumenil G. Deveaud C. Miura M. Coppola D. DeAngelis T. Rubin R. Efstratiadis A. Baserga R. Mol. Cell. Biol. 1994; 14: 3604-3612Crossref PubMed Scopus (504) Google Scholar) was maintained in Dulbecco's modified Eagle's medium (BioWhittaker, Verviers, Belgium) were supplemented with 10% (v/v) fetal bovine serum, 10 mml-Glu, and 5 mg/ml penicillin/streptomycin. R− cells were transiently transfected with pcDNA3/IGF-IR WT or IGF-IR Y1250F/Y1251F or empty pcDNA3 vectors (8 μg of DNA) using Lipofectamine transfection reagent (Invitrogen). After 24 h in culture the transfected cells were seeded into 10-cm plates and cultured for an additional 18 h. For analysis of signaling responses in adherent cells, the cells were then washed with PBS and starved from serum for 4 h before stimulation with IGF-I or EGF for the indicated times. For analysis of signaling responses in non-adherent cells (Fig. 1A) confluent R− cells were detached with trypsin/EDTA and then washed with PBS. Cells were resuspended in serum-free medium and maintained in suspension for 4 h before stimulation with IGF-I for the indicated times. siRNAs targeted to human RACK1 were purchased from Ambion. The sequences are: siRNA ID 135615 (siRNA 1) 5′-ccaucaagcuauggaauactt-3′ (sense), 5′-guauuccauagcuugauggtt-3′ (antisense); siRNA ID 135616 (siRNA 2) 5′-gcuauggaauacccugggutt-3′ (sense), 5′-acccaggguauuccauagctt-3′ (antisense); siRNA ID135617 (siRNA 3) 5′-ccuuuacacgcuagauggutt-3′ (sense), 5′-accaucuagcguguaaaggtg-3′ (antisense). MCF-7 cells were seeded at 50% confluency and transfected with 100 μm siRNA using the OligofectAMINE transfection reagent (Invitrogen) as described previously (5Kiely P.A. O'Gorman D. Luong K. Ron D. O'Connor R. Mol. Cell. Biol. 2006; 26: 4041-4051Crossref PubMed Scopus (85) Google Scholar). After 24 h the cells were starved and stimulated as described above or lifted using trypsin/EDTA and replated for cell assays. Cellular protein extracts were prepared by washing cells with PBS and then scraping into lysis buffer consisting of 10 mm Tris HCl, pH 7.4, 150 mm NaCl, 1% Nonidet P-40 plus the tyrosine phosphatase inhibitor Na3VO4 (1 mm), and the protease inhibitors phenylmethylsulfonyl fluoride (1 mm), pepstatin (1 μm), and aprotinin (1.5 μg/ml). After incubation at 4 °C for 20 min nuclear and cellular debris were removed by microcentrifugation at 14,000 rpm for 15 min at 4 °C. For immunoprecipitation of endogenous proteins from stimulated or unstimulated cells, cells were initially precleared using bovine serum albumin-coated protein G-agarose beads (15 μl of beads per 400 μg of total protein in 700 μl of lysis buffer) by incubation at 4 °C for 1 h with gentle rocking. The lysates were recovered from the beads by centrifugation at 3000 rpm for 3 min and transferred to fresh tubes for incubation with primary antibody (3 μg of each antibody) overnight at 4 °C with gentle rocking. Immune complexes were obtained by adding 20 μl of protein G-agarose beads for 3 h at 4 °C, washing (×3) with ice-cold lysis buffer, and removal from the beads by boiling for 5 min in 20 μl of 2× SDS-PAGE sample buffer before electrophoresis and Western blot analysis. For RACK1 immunoprecipitations, 700 μg of protein was incubated with 1 μg of anti-RACK1 IgM antibodies, 5 μg of goat anti-mouse IgM Fab fragment, 30 μl of protein G-agarose beads, 500 μl of immunoprecipitation buffer (1 mm EGTA, 1 mm EDTA, 10 mm Tris-HCl, pH 7.4, 150 mm NaCl, and 1% deoxycholate together with the tyrosine phosphatase inhibitor Na3VO4 (1 mm) and protease inhibitors as described previously (Kiely et al. (25Kiely P.A. Leahy M. O'Gorman D. O'Connor R. J. Biol. Chem. 2005; 280: 7624-7633Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar)). Protein samples were resolved by SDS-PAGE on 4–20% gradient gels and then transferred to nitrocellulose membranes, which were blocked for 1 h at room temperature in Tris-buffered saline containing 0.05% Tween 20 and 5% milk (w/v). Primary antibody incubations were overnight at 4 °C. Secondary antibody incubations were at room temperature for 1 h. Where indicated, membranes were stripped in 62.5 mm Tris-Cl, 1% SDS, and 0.7% 2-mercaptoethanol for 30 min at 50 °C followed by extensive washing in 0.2 and 0.05% Tris-buffered saline containing 0.05% Tween 20. Secondary antibodies conjugated with horseradish peroxidase were used for detection of Shc with enhanced chemiluminescence (Super Signal from Pierce). In all other cases we used Alexa Fluor 680- and 800-coupled anti-rabbit and anti-mouse secondary antibodies (LI-COR Biosciences) for detection with the Odyssey® infrared imaging system (LI-COR Biosciences). Plates (96 wells) were coated with collagen I (10 μg/ml) overnight at 4 °C and washed twice with 2× PBS and then blocked with 2.5% bovine serum albumin for 2 h at 37 °C. MCF-7 cells transfected with siRNA targeting RACK1 or a control siRNA for 24 h were-serum starved for 4 h before harvesting with trypsin/EDTA, washing with serum-free media (SFM), and resuspending in SFM to give a final density of 2.0 × 105 cells/ml. This cell suspension (100 μl/2.0 × 104 cells) was plated onto collagen-coated plates and allowed to attach for the indicated times at 37 °C. Unbound cells were removed by inverting and gentle washing in PBS before the cells were fixed in methanol at −20 °C for 5 min. Cells were stained with 0.1% crystal violet and measured by reading the absorbance at 595 nm. To assess cell spreading, MCF-7 cells (transfected with siRNA targeting RACK1 or a control siRNA) were harvested with trypsin/EDTA and replated onto multiple wells of a collagen-coated 24-well plate. After 8 h the cells were inspected and photographed. MCF-7 cells transfected with siRNA targeting RACK1 or a control siRNA were harvested after 24 h with trypsin/EDTA, washed in serum-free medium, and resuspended in Dulbecco's modified Eagle's medium/10% fetal bovine serum at a final density of 3.0 × 104 cells/well in multiple wells of a 24-well plate. At regular intervals (48, 72, and 96 h post-transfection) the cells were removed from triplicate wells and counted using a hemocytometer and trypan blue exclusion. To assess plating efficiency cells were seeded in triplicate wells of a 6-well plate at 500 cells/well in 3 ml of Dulbecco's modified Eagle's medium, 10% fetal bovine serum and cultured for 14 days. Cells were fixed in methanol at −20 °C for 5 min and stained with Giemsa. Colonies were examined, counted, and photographed. Peptides arrays of RACK1 in nitrocellulose were generated as previously described (30Frank R. J. Immunol. Methods. 2002; 267: 13-26Crossref PubMed Scopus (616) Google Scholar, 31Frank R. Overwin H. Methods Mol. Biol. 1996; 66: 149-169PubMed Google Scholar, 32Kramer A. Schneider-Mergener J. Methods Mol. Biol. 1998; 87: 25-39PubMed Google Scholar). Essentially, scanning libraries of overlapping 25-mer peptides covering the entire sequence of RACK1 were produced by automatic SPOT synthesis and synthesized on continuous cellulose membrane supports on Whatman 50 cellulose using Fmoc (9-fluorenylmethyloxycarbonyl) chemistry with the AutoSpot-Rosbot ASS 222 (Intavis Bioanalytical Instruments). The interaction of GST, GST-FAK, and c-Abl with the RACK1 array was investigated by overlaying the cellulose membranes with 10 μg/ml concentrations of each recombinant protein. Bound protein was detected with specific rabbit antisera for each protein and a secondary anti-rabbit antibody coupled with horseradish peroxidase. Once the binding site of FAK on the full-length RACK1 array was determined, specific alanine scanning substitution arrays were generated for selected peptides using the same synthesis procedure. c-Abl kinase was first immunoprecipitated from 500 μg of cell lysate by incubating 40 μl of protein G-Sepharose beads and 3 μg of anti-c-Abl (K-12) overnight. Beads were washed twice in lysis buffer and once with the kinase buffer (20 mm HEPES, pH 7.1, 150 mm NaCl, 1% Triton X-100, 10% glycerol, 1 mm MgCl2, 2 mm MnCl2, 1 mm phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, and 100 μm sodium vanadate). The in vitro kinase reaction was performed using 10 μg of GST-Crk as an exogenous substrate in the presence of 10 μm ATP and 10 μCi of [γ-32P]ATP at 30 °C for 15 min. The reaction mixture was then subjected to SDS-PAGE, Western blotting, and autoradiography phosphorimaging analysis. To assess the phosphorylation of peptide arrays, the membrane was placed in membrane phosphorylation buffer (20 mm HEPES, pH 7.4, 100 mm NaCl, 5 mm MgCl2, 1 mm dithiothreitol, 0.2 mg/ml bovine serum albumin for 1 h at room temperature. The membrane was blocked overnight at 4 °C in membrane phosphorylation buffer containing 1 mg/ml bovine serum albumin, 100 μm ATP. Each membrane was phosphorylated in 20 ml of membrane phosphorylation buffer containing 50 μm ATP and 10 μCi of [γ-32P]ATP with and without kinase at 30 °C for 30 min. After extensive washing with 1 m NaCl (10 × 10 min), H2O (3 × 5 min), 5% H3PO4 (3 × 15 min), and H2O (3 × 5 min), the membrane was exposed by autoradiography phosphorimaging analysis. We have shown previously that FAK phosphorylation on Tyr-397 is increased in RACK1-overexpressing cells and that IGF-I induced FAK dephosphorylation is accelerated (25Kiely P.A. Leahy M. O'Gorman D. O'Connor R. J. Biol. Chem. 2005; 280: 7624-7633Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). We, therefore, investigated whether RACK1 is required for FAK phosphorylation and dephosphorylation by cell adhesion and IGF-I stimulation, respectively. To do this we suppressed RACK1 expression with siRNA in MCF-7 cells. In cells transfected with control siRNA, FAK phosphorylation on Tyr-397 was evident in adherent serum-starved cell cultures and was decreased after IGF-I stimulation. However, in cells transfected with three different siRNAs targeting RACK1, FAK phosphorylation on Tyr-397 was not detectable in either the presence or absence of IGF-I (Fig. 1A). This indicates that FAK phosphorylation on Tyr-397 requires RACK1. We next investigated whether FAK phosphorylation was dependent on an interaction between RACK1 and the IGF-IR. To test this, R− cells were transfected with either wild type IGF-IR (IGF-IR WT) or an IGF-IR mutant (Y1250F/Y1251F) that does not interact with RACK1 and does not promote cell migration (25Kiely P.A. Leahy M. O'Gorman D. O'Connor R. J. Biol. Chem. 2005; 280: 7624-7633Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). As can be seen in Fig. 1B, FAK phosphorylation was decreased in response to IGF-I in cells expressing wild type IGF-IR but was unresponsive to IGF-I in cells expressing the Y1250F/Y1251F mutant. Moreover, when R− cells expressing WT IGF-IR were maintained in suspension culture, FAK phosphorylation was not detected (Fig. 1B). These data confirm that FAK phosphorylation requires cell adhesion and demonstrate that IGF-I-mediated dephosphorylation of FAK requires association of RACK1 with the IGF-IR. These results led us to hypothesize that FAK association with RACK1 may be regulated by IGF-I. To test this we investigated the co-immunoprecipitation of FAK with RACK1 in MCF-7 cells, R+ cells, and R− cells. FAK was found to be constitutively associated with RACK1 in immunoprecipitates from all three cell lines (Fig. 1C). This suggests that the association is not dependent on the presence of the IGF-IR. However, although RACK1-associated FAK was phosphorylated in serum-starved cultures with all cell lines, IGF-I-mediated dephosphorylation of FAK was observed in both MCF-7 and R+ cells but not in R− cells. This indicates that phosphorylation of FAK in response to integrin ligation and dephosphorylation of FAK in response to IGF-I, both, occur when RACK1 is associated with the IGF-IR. We next investigated whether suppression of RACK1 would affect the ability of MCF-7 cells to attach and spread on collagen and proliferate under different conditions. In cells with RACK1 suppressed, attachment to collagen was decreased by almost 50% in comparison to control siRNA- transfected MCF-7 cells (Fig. 2A). Furthermore, as can be seen in Fig. 2B, cell spreading after 8 h of attachment was greatly impaired compared with untransfected or control cells. Although cells in which RACK1 was suppressed were attached and viable, they clearly occupied a smaller surface area than control cells (Fig. 2B). Proliferation was assessed in normal monolayer cultures and in plating efficiency assays, which measure the ability to form foci at low density, a feature of transformed cells. In monolayer cultures the proliferation rate of MCF-7 cells with RACK1 suppressed was reduced by 60% compared with controls (Fig. 2C). In plating efficiency assays these cells also yielded fewer and smaller foci than control cells (Fig. 2D). Taken together, these data indicate that RACK1 is required for FAK phosphorylation, cell attachment, initiation of cell spreading, proliferation, and foci formation. The data suggest that RACK1 may be required for the initial stages of cell attachment necessary for both proliferation and migration. Our co-immunoprecipitation results suggest that FAK is constitutively associated with RACK1 (Fig. 1C). To determine whether this is a direct interaction and to define the site of interaction, we employed peptide arrays, which we have recently used to identify the binding site for PP2A and β1 integrin on RACK1 (15Kiely P.A. Baillie G.S. Lynch M.J. Houslay M.D. O'Connor R. J. Biol. Chem. 2008; 283: 22952-22961Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) and sites for RACK1 and β arrestin2 on PDE4D5 (33Bolger G.B. Baillie G.S. Li X. Lynch M.J. Herzyk P. Mohamed A. Mitchell L.H. McCahill A. Hundsrucker C. Klussmann E. Adams D.R. Houslay M.D. Biochem. J. 2006; 398: 23-36Crossref PubMed Scopus (133) Google Scholar, 34Baillie G.S. Adams D.R. Bhari N. Houslay T.M. Vadrevu S. Meng D. Li X. Dunlop A. Milligan G. Bolger G.B. Klussmann E. Houslay M.D. Biochem. J. 2007; 404: 71-80Crossref PubMed Scopus (75) Google Scholar). A library of overlapping peptides (25-mers) each shifted by 5 amino acids encompassing the entire sequence of RACK1 (60 in all) was spot-synthesized on nitrocellulose membranes to generate RACK1 peptide arrays. These arrays were then probed with recombinant GST or GST-FAK. GST did not bind to any peptide spot within the RACK1 array. GST-FAK bound to a number of peptides with the positive reactions indicated by dark spots (8, 9, 12, 22, and 23) (Fig. 3A). All of these peptides are derived from the RACK1 sequence spanning WD repeats 1–3. To identify the specific amino acids within these peptides required for FAK binding we generated an array of peptides derived from the 25-mer parent peptides corresponding to spot 10 (Thr-46 to Val-70) and spot 22 (Lys-106 to Lys-130). For each parent peptide, 25 progeny were generated where each new peptide in the array had a single alanine substitution in successive amino acids in the sequence. These two alanine-scanning peptide arrays were then probed with recombinant GST-FAK. Results showed that FAK binding to these peptides was either severely attenuated or ablated by alanine substitution of Arg-47, Tyr-52, Arg-57, Arg-60, and Phe-65 in peptide 10 and by alanine substitution of Lys-127 and Lys-130 in peptide 22 (Fig. 3B). Dual substitution of the positively charged Arg-57 and Arg-60 in peptide 10 and the positively charged Lys-127 and Lys-130 in peptide 22 completely ablated binding of FAK. Dual substitutions of either Val-69 and Val-70 or His-62 and His-64 in peptide 10 and Lys-106 and Asp-107 or Ser-114 and Ser-115 in peptide 22 had no effect on the binding of FAK to RACK1 and, thus, provide internal controls. The interaction of FAK with the peptides was quantified by densitometry and is presented in Fig. 3B as a percentage of binding of FAK to the control parent peptide. Together these analyses confirm that the binding of FAK to RACK1 is direct and demonstrates that key amino acids required for binding are located in the RACK1 sequence immediately after WD1 and running into WD2 as well as into WD3. During the course of this work no crystal structure was available for RACK1, although we had previously developed a seven-bladed β-propeller RACK1 model based on homologous proteins (35Steele M.R. McCahill A. Thompson D.S. MacKenzie C. Isaacs N.W. Houslay M.D. Bolger G.B. Cell. Signal. 2001; 13: 507-513Crossref PubMed Scopus (63) Google Scholar). To direct subsequent mutagenesis studies, this model was used to assess the likely surface exposure of array peptide residues implicated in FAK binding. The clustered residues from peptide 10 (Arg-47, Tyr-52, Arg-57, and Arg-60) were predicted to occupy surface-exposed positions on a single β-hairpin motif (Fig. 4Ai), and peptides encompassing this region might reasonably preserve the native structure of the intact protein. In contrast, Phe-65 fell within a loop region in WD2, and its structural organization in the array peptides and mimicry of the intact protein was considered to be l" @default.
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• W2022274575 title "Phosphorylation of RACK1 on Tyrosine 52 by c-Abl Is Required for Insulin-like Growth Factor I-mediated Regulation of Focal Adhesion Kinase" @default.
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