Role of Focal Adhesion Kinase Ser-732 Phosphorylation in Centrosome Function during Mitosis
2009; Elsevier BV; Volume: 284; Issue: 14 Linguagem: Inglês
10.1074/jbc.m809040200
ISSN1083-351X
AutoresAnn Y.J. Park, Tang‐Long Shen, Shu Chien, Jun‐Lin Guan,
Tópico(s)Cell Adhesion Molecules Research
ResumoFocal adhesion kinase (FAK) is the major cytoplasmic tyrosine kinase in focal adhesions and a critical mediator of integrin signaling in a variety of cells, including endothelial cells (ECs). Here we describe a new function for FAK in the regulation of centrosome functions in a Ser-732 phosphorylation-dependent manner during mitosis. Deletion of FAK in primary ECs causes increases in centrosome numbers, multipolar and disorganized spindles, and unaligned chromosomes during mitosis. Re-expression of wild-type FAK, but not S732A mutant, rescued these mitotic defects, suggesting a role for Ser-732 phosphorylation in the regulation of centrosomal functions. Consistent with this possibility, Ser-732-phosphorylated FAK was found to co-localize in centrosomes in mitotic cells. FAK also associated with cytoplasmic dynein in a Ser-732 phosphorylation-dependent manner. Further analysis in FAK-null primary ECs showed that S732A mutant could rescue EC migration but not proliferation or tubulogenesis in vitro. Last, we showed that deletion of FAK in ECs reduced tumor angiogenesis in vivo, which could be restored by re-expression of wild-type FAK but not S732A mutant. Together, these studies demonstrated a novel role for Ser-732 phosphorylation of FAK in the regulation of centrosome function during mitosis, which may contribute to EC proliferation and angiogenesis. Focal adhesion kinase (FAK) is the major cytoplasmic tyrosine kinase in focal adhesions and a critical mediator of integrin signaling in a variety of cells, including endothelial cells (ECs). Here we describe a new function for FAK in the regulation of centrosome functions in a Ser-732 phosphorylation-dependent manner during mitosis. Deletion of FAK in primary ECs causes increases in centrosome numbers, multipolar and disorganized spindles, and unaligned chromosomes during mitosis. Re-expression of wild-type FAK, but not S732A mutant, rescued these mitotic defects, suggesting a role for Ser-732 phosphorylation in the regulation of centrosomal functions. Consistent with this possibility, Ser-732-phosphorylated FAK was found to co-localize in centrosomes in mitotic cells. FAK also associated with cytoplasmic dynein in a Ser-732 phosphorylation-dependent manner. Further analysis in FAK-null primary ECs showed that S732A mutant could rescue EC migration but not proliferation or tubulogenesis in vitro. Last, we showed that deletion of FAK in ECs reduced tumor angiogenesis in vivo, which could be restored by re-expression of wild-type FAK but not S732A mutant. Together, these studies demonstrated a novel role for Ser-732 phosphorylation of FAK in the regulation of centrosome function during mitosis, which may contribute to EC proliferation and angiogenesis. Focal adhesion kinase (FAK) 2The abbreviations used are: FAK, focal adhesion kinase; EC, endothelial cell; ROCK, Rho-dependent kinase; GFP, green fluorescent protein; shRNA, short hairpin RNA. is a cytoplasmic tyrosine kinase that is a major mediator of signal transduction by integrins and also participates in signaling by other cell surface receptors in a variety of cells, including endothelial cells (ECs) (1Parsons J.T. J. Cell Sci. 2003; 116: 1409-1416Crossref PubMed Scopus (1136) Google Scholar, 2Schlaepfer D.D. Mitra S.K. Curr. Opin. Genet. Dev. 2004; 14: 92-101Crossref PubMed Scopus (355) Google Scholar, 3Siesser P.M. Hanks S.K. Clin. Cancer Res. 2006; 12: 3233-3237Crossref PubMed Scopus (146) Google Scholar, 4Schaller M.D. Biochim. Biophys. Acta. 2001; 1540: 1-21Crossref PubMed Scopus (504) Google Scholar). In most adherent cells FAK is activated upon integrin-mediated cell adhesion to extracellular matrix proteins through disruption of an intramolecular inhibitory interaction between its amino-terminal FERM domain and the kinase domain (5Cooper L.A. Shen T.L. Guan J.L. Mol. Cell. Biol. 2003; 23: 8030-8041Crossref PubMed Scopus (145) Google Scholar, 6Lietha D. Cai X. Ceccarelli D.F. Li Y. Schaller M.D. Eck M.J. Cell. 2007; 129: 1177-1187Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). Once it is activated, FAK undergoes autophosphorylation at Tyr-397, which creates a binding site for several SH2 (Src homology 2) domain-containing proteins including Src family kinases (1Parsons J.T. J. Cell Sci. 2003; 116: 1409-1416Crossref PubMed Scopus (1136) Google Scholar, 2Schlaepfer D.D. Mitra S.K. Curr. Opin. Genet. Dev. 2004; 14: 92-101Crossref PubMed Scopus (355) Google Scholar). The associated Src could then phosphorylate additional sites on FAK, including residues Tyr-576 and Tyr-577 in its activation loop, to further activate FAK and Tyr-925 to create binding sites for the adaptor molecule Grb2 (7Owen J.D. Ruest P.J. Fry D.W. Hanks S.K. Mol. Cell. Biol. 1999; 19: 4806-4818Crossref PubMed Scopus (340) Google Scholar, 8Calalb M.B. Polte T.R. Hanks S.K. Mol. Cell. Biol. 1995; 15: 954-963Crossref PubMed Google Scholar, 9Schlaepfer D.D. Hanks S.K. Hunter T. van der Geer P. Nature. 1994; 372: 786-791Crossref PubMed Scopus (1442) Google Scholar). FAK also functions as a scaffold to mediate Src family kinase phosphorylation of several proteins, including paxillin (10Schaller M.D. Parsons J.T. Mol. Cell. Biol. 1995; 15: 2635-2645Crossref PubMed Scopus (500) Google Scholar, 11Burridge K. Turner C.E. Romer L.H. J. Cell Biol. 1992; 119: 893-903Crossref PubMed Scopus (1180) Google Scholar), p130cas (12Vuori K. Hirai H. Aizawa S. Ruoslahti E. Mol. Cell. Biol. 1996; 16: 2606-2613Crossref PubMed Google Scholar, 13Ruest P.J. Shin N.Y. Polte T.R. Zhang X. Hanks S.K. Mol. Cell. Biol. 2001; 21: 7641-7652Crossref PubMed Scopus (131) Google Scholar), and endophilin A2 (14Wu X. Gan B. Yoo Y. Guan J.L. Dev. Cell. 2005; 9: 185-196Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar), which bound to the carboxyl-terminal region of FAK. This cascade of phosphorylation events and protein-protein interactions has been shown to trigger several signaling pathways in the regulation of a variety of cellular functions in different cells. Besides these well characterized tyrosine phosphorylations, recent studies have identified FAK phosphorylation on several serine residues (15Ma A. Richardson A. Schaefer E.M. Parsons J.T. Mol. Biol. Cell. 2001; 12: 1-12Crossref PubMed Scopus (77) Google Scholar, 16Grigera P.R. Jeffery E.D. Martin K.H. Shabanowitz J. Hunt D.F. Parsons J.T. J. Cell Sci. 2005; 118: 4931-4935Crossref PubMed Scopus (44) Google Scholar). In the post-mitotic neurons, Ser-732 has been shown to be phosphorylated by Cdk5, which plays an important role in microtubule organization and proper nuclear movement during neuronal migration (17Xie Z. Sanada K. Samuels B.A. Shih H. Tsai L.H. Cell. 2003; 114: 469-482Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 18Xie Z. Tsai L.H. Cell Cycle. 2004; 3: 108-110Crossref PubMed Google Scholar). Indeed, Ser-732-phosphorylated FAK is enriched in centrosome-associated microtubule fork that abuts the nucleus and a perinuclear region around the centrosome, consistent with its regulation of these functions in neurons. Ser-732 of FAK has also been shown to be phosphorylated by Rho-dependent kinase (ROCK) in ECs, which has been suggested to play a role in vascular endothelial growth factor-stimulated EC migration (19Le Boeuf F. Houle F. Sussman M. Huot J. Mol. Biol. Cell. 2006; 17: 3508-3520Crossref PubMed Scopus (50) Google Scholar). In addition to Ser-732, Ser-722, Ser-843, and Ser-910 in the carboxyl-terminal domain of FAK have also been found to be phosphorylated and regulate cell spreading and migration in recent studies (16Grigera P.R. Jeffery E.D. Martin K.H. Shabanowitz J. Hunt D.F. Parsons J.T. J. Cell Sci. 2005; 118: 4931-4935Crossref PubMed Scopus (44) Google Scholar, 20Jacamo R. Jiang X. Lunn J.A. Rozengurt E. J. Cell. Physiol. 2007; 210: 436-444Crossref PubMed Scopus (38) Google Scholar, 21Jiang X. Sinnett-Smith J. Rozengurt E. Cell. Signal. 2007; 19: 1000-1010Crossref PubMed Scopus (39) Google Scholar, 22Villa-Moruzzi E. Biochem. J. 2007; 408: 7-18Crossref PubMed Scopus (17) Google Scholar, 23Bianchi M. De Lucchini S. Marin O. Turner D.L. Hanks S.K. Villa-Moruzzi E. Biochem. J. 2005; 391: 359-370Crossref PubMed Scopus (79) Google Scholar, 24Hunger-Glaser I. Fan R.S. Perez-Salazar E. Rozengurt E. J. Cell. Physiol. 2004; 200: 213-222Crossref PubMed Scopus (64) Google Scholar). Despite these findings, our understanding of serine phosphorylation of FAK is very limited in contrast to the wealth of information on the regulation and function of tyrosine phosphorylation of FAK. In particular, it is not clear whether and how serine phosphorylation is involved in the regulation of cell cycle progression and proliferation by FAK. Focal adhesion localization of FAK in adherent cells is essential for its functions in the regulation of cell migration as well as proliferation (1Parsons J.T. J. Cell Sci. 2003; 116: 1409-1416Crossref PubMed Scopus (1136) Google Scholar, 2Schlaepfer D.D. Mitra S.K. Curr. Opin. Genet. Dev. 2004; 14: 92-101Crossref PubMed Scopus (355) Google Scholar). During mitosis, however, focal adhesion complexes dissociate as cells round up and detach from extracellular matrix. Interestingly, serine phosphorylation of FAK is increased during mitosis, and this has been suggested to cause FAK dissociation from p130Cas and Src to inactivate signaling at focal adhesions (25Yamakita Y. Totsukawa G. Yamashiro S. Fry D. Zhang X. Hanks S.K. Matsumura F. J. Cell Biol. 1999; 144: 315-324Crossref PubMed Scopus (106) Google Scholar), although the relevant sites of phosphorylation were not mapped in this study. It is not known whether FAK is localized to any specific subcellular structures and/or plays a role in mitosis and whether these are regulated by serine phosphorylation of FAK in mitotic cells. Consistent with its critical importance in the regulation of various cellular functions, deletion of FAK gene leads to early embryonic lethality at embryonic day 8.5 (E8.5) due to defects in the axial mesodermal tissues including the cardiovascular system with incomplete development of both the blood vessels and the heart (26Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1584) Google Scholar). Using a conditional mouse KO approach, we and others have recently shown a role of FAK in vascular angiogenesis through its regulation of multiple functions of ECs including their survival, proliferation, migration, and tubulogenesis (27Shen T.L. Park A.Y. Alcaraz A. Peng X. Jang I. Koni P. Flavell R.A. Gu H. Guan J.L. J. Cell Biol. 2005; 169: 941-952Crossref PubMed Scopus (243) Google Scholar, 28Braren R. Hu H. Kim Y.H. Beggs H.E. Reichardt L.F. Wang R. J. Cell Biol. 2006; 172: 151-162Crossref PubMed Scopus (190) Google Scholar, 29Weis S.M. Lim S.T. Lutu-Fuga K.M. Barnes L.A. Chen X.L. Gothert J.R. Shen T.L. Guan J.L. Schlaepfer D.D. Cheresh D.A. J. Cell Biol. 2008; 181: 43-50Crossref PubMed Scopus (127) Google Scholar). The availability of the floxed FAK mice and ECs isolated from these mice also allowed us to investigate FAK signaling pathways involved in the regulation of EC functions and angiogenesis in vivo by a reconstitution strategy, where endogenous FAK is deleted via recombinant adenoviruses encoding Cre followed by re-expression of FAK or its various mutants in ECs both in vitro and in vivo. In this study we present data showing a novel function of FAK in the regulation of centrosomal functions in a Ser-732 phosphorylation-dependent manner in ECs during mitosis, which plays a role in the regulation of EC proliferation and tubulogenesis in vitro and tumor angiogenesis in vivo. Recombinant Adenoviruses-Recombinant adenoviruses encoding Cre recombinase or lacZ control were purchased from Gene Transfer Vector Core (University of Iowa, Iowa City, IA). The recombinant adenoviruses encoding FAK (Ad-FAK), its kinase-defective (Ad-KD), Y397F (Ad-Y397F), P712A/P715A (Ad-P712A/P715A), and S732A (Ad-S732A) mutants, or GFP control (Ad-GFP) were generated using the Adeasy-1 system (Stratagene) according to the manufacturer's instruction. Isolation and Infection of ECs-ECs were isolated from 4-6-week-old homozygous FAK floxed mice using the magnetic bead (Dyanbead M-450; Dynal Corp.) purification protocol with rat anti-mouse PECAM-1 (BD Biosciences), as described previously (27Shen T.L. Park A.Y. Alcaraz A. Peng X. Jang I. Koni P. Flavell R.A. Gu H. Guan J.L. J. Cell Biol. 2005; 169: 941-952Crossref PubMed Scopus (243) Google Scholar, 30Cattelino A. Liebner S. Gallini R. Zanetti A. Balconi G. Corsi A. Bianco P. Wolburg H. Moore R. Oreda B. Kemler R. Dejana E. J. Cell Biol. 2003; 162: 1111-1122Crossref PubMed Scopus (262) Google Scholar, 31Peng X. Ueda H. Zhou H. Stokol T. Shen T.L. Alcaraz A. Nagy T. Vassalli J.D. Guan J.L. Cardiovasc. Res. 2004; 64: 421-430Crossref PubMed Scopus (88) Google Scholar). EC population was ∼90% pure as determined by anti-PECAM-1 staining. Isolated ECs were infected at a multiplicity of infection of 100 with Ad-lacZ or Ad-Cre. To increase efficiency, a second infection was performed after 9-12 h and incubated for 48 h. For the rescue experiments, cells infected with Ad-Cre were re-infected with recombinant adenoviruses encoding FAK, its mutants, or Ad-GFP 2 days after infection of Ad-Cre at a multiplicity of infection of 100. No detectable cell toxicity was observed. Cell Culture and Transfections-Isolated ECs were cultured on a 0.1% gelatin (Sigma-Aldrich)-coated dish in high glucose Dulbecco's modified Eagle's medium supplemented with 20% fetal calf serum (Hyclone), endothelial mitogen (Biomedical Technologies), and heparin (100 μg/ml; Sigma-Aldrich) (27Shen T.L. Park A.Y. Alcaraz A. Peng X. Jang I. Koni P. Flavell R.A. Gu H. Guan J.L. J. Cell Biol. 2005; 169: 941-952Crossref PubMed Scopus (243) Google Scholar, 31Peng X. Ueda H. Zhou H. Stokol T. Shen T.L. Alcaraz A. Nagy T. Vassalli J.D. Guan J.L. Cardiovasc. Res. 2004; 64: 421-430Crossref PubMed Scopus (88) Google Scholar). 293T, HeLa, murine embryonic fibroblast (MEF), and Cos-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. 293T cells were transfected with Cdk5, ROCK1, or control shRNA (University of Michigan Comprehensive Cancer Center shRNA Core Facility) for 3 days by use of Lipofectamine following the manufacturer's instructions. Flow Cytometry Analysis-ECs were fixed with 70% ice-cold ethanol at 4 °C for more than 2 h. After fixation cells were stained with 50 μg/ml propidium iodide (Sigma-Aldrich) with 100 μg/ml RNase A in phosphate-buffered saline containing 0.1% Triton X-100. Flow cytometry analysis was performed by a BD Biosciences BD-LSR II flow cytometer. Immunofluorescence Staining-Cells were processed for immunofluorescence staining as described previously (32Cary L.A. Chang J.F. Guan J.L. J. Cell Sci. 1996; 109: 1787-1794Crossref PubMed Google Scholar). The primary antibodies used were anti-Ser(P)-732 FAK (BioSource; 1:100), anti-α-tubulin (Zymed Laboratories Inc.; 1:50), anti-γ-tubulin (Sigma-Aldrich; 1:100), anti-dynein intermediate chain (Sigma-Aldrich; 1:100) and anti-BrdUrd (Sigma-Aldrich; 1:50). Fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratory; 1:200) and Texas Red-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratory; 1:200) were used as the secondary antibodies. Cells were examined by a microscope (model BX41; Olympus) with an UplanF1 × 40/0.75 objective lens at room temperature. The images were captured using a camera (model DP70; Olympus) with DP Controller, Version 1.2.1.108. Immunoprecipitation and Western Blotting-Immunoprecipitation and Western blotting analysis was performed as described previously (32Cary L.A. Chang J.F. Guan J.L. J. Cell Sci. 1996; 109: 1787-1794Crossref PubMed Google Scholar). Antibodies used are anti-FAK (C20; Santa Cruz Biotechnology, Inc.), anti-vinculin (Sigma-Aldrich), anti-actin (Santa Cruz Biotechnology, Inc.), anti-Myc (9E10; Santa Cruz Biotechnology, Inc.), anti-dynein intermediate chain (clone 70.1; Sigma-Aldrich), anti-cdk5 (C8; Santa Cruz Biotechnology, Inc.), and anti-ROCK1 (H-85; Santa Cruz Biotechnology, Inc.). BrdUrd Incorporation Assay-ECs were serum-starved for 18 h to arrest the cells in G0. A BrdUrd incorporation assay was performed as described previously (27Shen T.L. Park A.Y. Alcaraz A. Peng X. Jang I. Koni P. Flavell R.A. Gu H. Guan J.L. J. Cell Biol. 2005; 169: 941-952Crossref PubMed Scopus (243) Google Scholar). Three independent experiments were performed, and the percentage of cells positive for BrdUrd was quantified using a microscope (model BX41; Olympus) with UplanF1 × 10/0.3 objective lens at room temperature. Approximately 100 cells were examined for each condition in each independent experiment. Wound Closure Cell Migration Assay-ECs were plated on gelatin-coated dishes (60 mm) and stimulated with 50 ng/ml vascular endothelial growth factor and then subjected to assays. Wound closure assays were performed as described previously (27Shen T.L. Park A.Y. Alcaraz A. Peng X. Jang I. Koni P. Flavell R.A. Gu H. Guan J.L. J. Cell Biol. 2005; 169: 941-952Crossref PubMed Scopus (243) Google Scholar, 33Liang C.C. Park A.Y. Guan J.L. Nat. Protoc. 2007; 2: 329-333Crossref PubMed Scopus (3057) Google Scholar). Tube Formation Assay-ECs were plated on a layer of Matrigel (growth factor-reduced, BD Biosciences) and allowed to form a tubular structure as described previously (27Shen T.L. Park A.Y. Alcaraz A. Peng X. Jang I. Koni P. Flavell R.A. Gu H. Guan J.L. J. Cell Biol. 2005; 169: 941-952Crossref PubMed Scopus (243) Google Scholar). Tubulogenesis in each condition was examined on a microscope (model IX70; Olympus) with a UplanF1 × 10/0.3 objective lens and photographed with a progressive 3CCD camera (Sony) at room temperature. The length and branch points were determined using Image-Pro Plus, Version 3.0.00.00 as described previously (34Haskell H. Natarajan M. Hecker T.P. Ding Q. Stewart Jr., J. Grammer J.R. Gladson C.L. Clin. Cancer Res. 2003; 9: 2157-2165PubMed Google Scholar). Matrigel Plug Assays-Matrigel (growth factor-reduced, BD Biosciences) was supplemented with 5 × 108 plaque-forming units/ml recombinant adenoviruses and 5 × 105 B16F10 melanoma cells in a final volume of 0.5 ml. Matrigel mixture was then injected subcutaneously into the flank region of 8-week-old floxed FAK mice. Mice were sacrificed 10 days after injection, and Matrigel weights were determined. Vascularization in Matrigel plugs was visualized by immunohistological examination using anti-PECAM-1 antibody (M-20, 1:200 dilution; Santa Cruz Biotechnology, Inc.) as described previously (27Shen T.L. Park A.Y. Alcaraz A. Peng X. Jang I. Koni P. Flavell R.A. Gu H. Guan J.L. J. Cell Biol. 2005; 169: 941-952Crossref PubMed Scopus (243) Google Scholar). They were then examined under a microscope (model BX41; Olympus) with an UplanF1 × 10/0.3 objective lens at room temperature, and the images were captured using a camera (model DP70; Olympus) with DP Controller, Version 1.2.1.108. Five representative images were obtained from each Matrigel plug, and vessel density was quantified using Image-Pro Plus, Version 3.0.00.00. Deletion of FAK Causes Spindle and Centrosomal Abnormalities in Mitotic ECs-Previous studies of EC-specific FAK knock-out mice and isolated ECs showed a role of FAK in angiogenesis and vascular development due to its essential role in the regulation of EC functions, including proliferation, migration, cell survival, and tubulogenesis (27Shen T.L. Park A.Y. Alcaraz A. Peng X. Jang I. Koni P. Flavell R.A. Gu H. Guan J.L. J. Cell Biol. 2005; 169: 941-952Crossref PubMed Scopus (243) Google Scholar). To further investigate mechanisms of FAK in the regulation of EC proliferation, we performed flow cytometric analyses to get more detailed information on the cell cycle profile in FAK-/- ECs. Primary ECs were isolated from floxed FAK mice and were infected by a recombinant adenovirus encoding Cre recombinase (Ad-Cre) to delete endogenous FAK or by a control recombinant adenovirus encoding lacZ to produce FAK-/- ECs or control FAK+/+ ECs, respectively, as described previously (27Shen T.L. Park A.Y. Alcaraz A. Peng X. Jang I. Koni P. Flavell R.A. Gu H. Guan J.L. J. Cell Biol. 2005; 169: 941-952Crossref PubMed Scopus (243) Google Scholar). Analysis of these cells by flow cytometry showed an altered cell cycle profile for FAK-/- ECs compared with the control FAK+/+ ECs (Fig. 1A). In consistent with our previous results showing an increased apoptosis in FAK-/- ECs, a significant increase in SubG1 population was found for these cells compared with FAK+/+ ECs (from about 10 to 22%). We also found a significantly increased G2/M population in FAK-/- ECs compared with FAK+/+ ECs (from about 20 to 32%), suggesting a possibly increased mitotic arrest upon FAK deletion in ECs. To investigate the mechanisms of mitotic abnormalities in FAK-/- ECs, we examined the effect of FAK deletion on mitotic spindle organization and chromosome alignment and segregation in these cells. As shown in Fig. 1B, normal mitotic spindles and chromosome alignment were detected in FAK+/+ ECs during mitosis by staining for α-tubulin and DNA, respectively (a, f, and k). In contrast, the analysis of FAK-/- ECs revealed various defects including multiple and randomly positioned spindles (b-d), loosely congregated chromosomes (g-i), and unaligned chromosomes (i and n, arrowheads). In some cells anaphase proceeded with an unattached chromosome (j and o, arrows). Because centrosomes are the primary microtubule organization center and play an essential role in mitotic spindle organization and chromosome segregation, we next evaluated possible defects in centrosome organizations in FAK-/- ECs by immunostaining for γ-tubulin, a centrosome marker. Fig. 1C shows that whereas FAK+/+ ECs have a typical staining of two centrosomes on the opposite sides of the condensed chromosomes during mitosis (a, f, and k), deletion of FAK in ECs (FAK-/- ECs) caused various centrosomal defects in the number, size, and position of centrosomes (b-e), which are associated with abnormal chromosome condensation in metaphase (g-i) and segregation in anaphase (j). Quantitation of ∼200 mitotic cells for each group showed centrosomal defects in about 60% of FAK-/- ECs compared with less than 10% of the control FAK+/+ ECs (Fig. 2B). Together, these results suggest that FAK plays a role in the regulation of mitotic spindle assembly, chromosome alignment and centrosome integrity during mitosis and that the deregulation of these functions caused by the deletion of FAK may be responsible for mitotic arrest in FAK-/- ECs. Ser-732 Phosphorylation of FAK Is Required for Its Regulation of Centrosome Function during Mitosis in Primary ECs-To investigate the mechanisms of FAK regulation of centrosome function during mitosis, we generated recombinant adenoviruses encoding several FAK mutants and analyzed their ability to rescue the mitotic defects in ECs upon deletion of endogenous FAK. Primary ECs isolated from floxed FAK mice were infected by Ad-Cre to delete endogenous FAK followed by infection with recombinant adenoviruses encoding FAK (Ad-FAK), kinase-defective (Ad-KD), Tyr-397 to Phe (Ad-Y397F), Pro-712 and Pro-715 to Ala (Ad-P712A/P715A), or Ser-732 to Ala (Ad-S732A) mutant. As expected, infection of Ad-Cre, but not Ad-LacZ, resulted in the deletion of FAK (see Fig. 1A, right panel). Re-infection of FAK-/- ECs with recombinant adenoviruses encoding FAK or its mutants led to expression of exogenous FAK and mutants to comparable levels in these cells (Fig. 2A). As expected, restoration of FAK expression in FAK-/- ECs significantly rescued the centrosomal abnormalities caused by deletion of endogenous FAK (Fig. 2B). Consistent with previous studies on the critical roles of Tyr-397 in FAK downstream signaling pathways initiated by autophosphorylation of this site, re-expression of either FAK Y397F or KD mutant did not rescue the centrosomal defects (Fig. 2B). In contrast to well characterized tyrosine phosphorylation of FAK, the role of serine phosphorylation of FAK is relatively less investigated, although several serine residues of FAK have also been shown to be phosphorylated (15Ma A. Richardson A. Schaefer E.M. Parsons J.T. Mol. Biol. Cell. 2001; 12: 1-12Crossref PubMed Scopus (77) Google Scholar, 16Grigera P.R. Jeffery E.D. Martin K.H. Shabanowitz J. Hunt D.F. Parsons J.T. J. Cell Sci. 2005; 118: 4931-4935Crossref PubMed Scopus (44) Google Scholar). In particular, phosphorylation of Ser-732 in FAK by Cdk5 has been shown to play a role in nuclear translocation during neuronal migration through regulation of microtubule networks (17Xie Z. Sanada K. Samuels B.A. Shih H. Tsai L.H. Cell. 2003; 114: 469-482Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). As the centrosome-associated microtubule structure, the mitotic spindle, plays a crucial role during mitosis in non-neuronal cells, we examined the potential role of Ser-732 phosphorylation in FAK regulation of centrosome function by analysis of FAK S732A mutant in the rescue experiments. We found that re-expression of S732A mutant did not rescue centrosomal abnormalities in FAK-/- ECs (Fig. 2B). Analysis of another FAK mutant, P712A/P715A, which is deficient in binding to p130Cas, rescued centrosomal defects in FAK-/- ECs to a comparable level as the wild-type FAK (Fig. 2B), suggesting that FAK signaling through p130Cas is not involved in the regulation of centrosome function. Together, these mutational analyses suggest a novel role of Ser-732 phosphorylation in mediating FAK regulation of centrosome functions during mitosis. Localization of Ser-732-phosphorylated FAK in Centrosomes during Mitosis-FAK is localized in focal adhesions in adherent cells, which are disassembled during mitosis. Previous studies have shown an increase of serine phosphorylation of FAK concomitant with cell rounding-up and disassembly of focal adhesions in mitotic cells (25Yamakita Y. Totsukawa G. Yamashiro S. Fry D. Zhang X. Hanks S.K. Matsumura F. J. Cell Biol. 1999; 144: 315-324Crossref PubMed Scopus (106) Google Scholar). It is not clear, however, whether serine-phosphorylated FAK are evenly distributed in the cytoplasm or are localized to particular subcellular structures in mitotic cells. In light of the above observation suggesting a potential role of Ser-732 phosphorylation of FAK in the integrity of centrosomes, we examined the possibility of a centrosomal localization of Ser-732-phosphorylated FAK in mitotic ECs. Primary FAK+/+ ECs at various phases of mitosis were subjected to double-label immunofluorescent staining with antibodies against phospho-Ser-732 of FAK (Ser(P)-732) and the centrosomal marker γ-tubulin. As shown in Fig. 3A, Ser-732-phosphorylated FAK was detected in the centrosomes throughout mitosis. The lack of staining in FAK-/- ECs by anti-Ser(P)-732 confirmed the specificity of the antibody against FAK Ser(P)-732 (Fig. 3B). Furthermore, localization of Ser-732-phosphorylated FAK in the centrosomes was also detected in several other cell types, including murine embryonic fibroblasts, COS7 cells (Fig. 3C), and HeLa cells (Fig. 3D). These results suggest that FAK may regulate centrosome functions through acting on some components of centrosomes directly in a Ser-732 phosphorylation-dependent manner during mitosis. Ser-732 Phosphorylation-dependent Association of FAK with Cytoplasmic Dynein-To explore potential FAK targets, we examined various proteins localized in centrosomes for their potential association with FAK in a Ser-732 phosphorylation-dependent manner. FAK-/- ECs were infected with Ad-FAK, Ad-S732A, or Ad-GFP as a control, and lysates from these cells were immunoprecipitated by anti-Myc for the Myc-tagged FAK and S732A mutant and their associated proteins. Analysis of the immunoprecipitates by anti-dynein showed that it was associated with wild-type FAK but not S732A mutant (Fig. 4A). Previous studies suggested that Ser-732 of FAK can be phosphorylated by Cdk5 and ROCK1 in different cells (17Xie Z. Sanada K. Samuels B.A. Shih H. Tsai L.H. Cell. 2003; 114: 469-482Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 19Le Boeuf F. Houle F. Sussman M. Huot J. Mol. Biol. Cell. 2006; 17: 3508-3520Crossref PubMed Scopus (50) Google Scholar). We, therefore, examined the effect of down-regulation of Cdk5 and ROCK1 on the association of FAK with cytoplasmic dynein. Lysates were prepared from 293T cells that had been transfected with vectors encoding Cdk5 shRNA, ROCK1 shRNA, or the vector alone control. Fig. 4B shows that interaction of FAK with cytoplasmic dynein was reduced by knockdown of expression of Cdk5, but not ROCK1, when compared with cells treated with control shRNA. Last, colocalization of Ser-732-phosphorylated FAK with dynein at centrosomes was also confirmed by double-label immunofluorescent staining of mitotic cells (Fig. 4C). Together, these results suggest that Cdk5-dependent Ser-732 phosphorylation of FAK and its binding to cytoplasmic dynein may play a role in the regulation of centrosome function during mitosis. Ser-732 Phosphorylation of FAK Is Required for Cell Proliferation and Tubulogenesis in Primary ECs-Our previous studies showed that inactivation of FAK in primary ECs caused increased apoptosis, reduced proliferation and migration, and reduced capillary formation on Matrigel, suggesting essential function of FAK in the regulation of multiple EC activities (27Shen T.L. Park A.Y. Alcaraz A. Peng X. Jang I. Koni P. Flavell R.A. Gu H. Guan J.L. J. Cell Biol. 2005; 169: 941-952Crossref PubMed Scopus (243) Google Scholar). The inability of S732A mutant to rescue the centrosomal defects in FAK-/- ECs raised the possibi
Referência(s)