Comprehensive Analysis of Phosphorylation Sites in Tensin1 Reveals Regulation by p38MAPK
2010; Elsevier BV; Volume: 9; Issue: 12 Linguagem: Inglês
10.1074/mcp.m110.003665
ISSN1535-9484
AutoresEmily H. Hall, Jeremy L. Balsbaugh, Kristie L. Rose, Jeffrey Shabanowitz, Donald F. Hunt, David L. Brautigan,
Tópico(s)Cellular Mechanics and Interactions
ResumoTensin1 is the archetype of a family of focal adhesion proteins. Tensin1 has a phosphotyrosine binding domain that binds the cytoplasmic tail of β-integrin, a Src homology 2 domain that binds focal adhesion kinase, p130Cas, and the RhoGAP called deleted in liver cancer-1, a phosphatase and tensin homology domain that binds protein phosphatase-1α and other regions that bind F-actin. The association between tensin1 and these partners affects cell polarization, migration, and invasion. In this study we analyzed the phosphorylation of human S-tag-tensin1 expressed in HEK293 cells by mass spectrometry. Peptides covering >90% of the sequence initially revealed 50 phosphorylated serine/phosphorylated threonine (pSer/pThr) but no phosphorylated tyrosine (pTyr) sites. Addition of peroxyvanadate to cells to inhibit protein tyrosine phosphatases exposed 10 pTyr sites and addition of calyculin A to cells to inhibit protein phosphatases type 1 and 2A gave a total of 62 pSer/pThr sites. We also characterized two sites modified by O-linked N-acetylglucosamine. Tensin1 F302A, which does not bind protein phosphatase-1, showed > twofold enhanced phosphorylation of seven sites. The majority of pSer/pThr have adjacent proline (Pro) residues and we show endogenous p38 mitogen activated protein kinase (MAPK) associated with and phosphorylated tensin1 in an in vitro kinase assay. Recombinant p38α MAPK also phosphorylated S-tag-tensin1, resulting in decreased binding with deleted in liver cancer-1. Activation of p38 MAPK in cells by sorbitol-induced hyperosmotic stress increased phosphorylation of S-tag-tensin1, which reduced binding to deleted in liver cancer-1 and increased binding to endogenous pTyr proteins, including p130Cas and focal adhesion kinase. These data demonstrate that tensin1 is extensively phosphorylated on Ser/Thr residues in cells and phosphorylation by p38 MAPK regulates the specificity of the tensin1 Src homology 2 domain for binding to different proteins. Tensin1 provides a hub for connecting signaling pathways involving p38 MAP kinase, tyrosine kinases and RhoGTPases. Tensin1 is the archetype of a family of focal adhesion proteins. Tensin1 has a phosphotyrosine binding domain that binds the cytoplasmic tail of β-integrin, a Src homology 2 domain that binds focal adhesion kinase, p130Cas, and the RhoGAP called deleted in liver cancer-1, a phosphatase and tensin homology domain that binds protein phosphatase-1α and other regions that bind F-actin. The association between tensin1 and these partners affects cell polarization, migration, and invasion. In this study we analyzed the phosphorylation of human S-tag-tensin1 expressed in HEK293 cells by mass spectrometry. Peptides covering >90% of the sequence initially revealed 50 phosphorylated serine/phosphorylated threonine (pSer/pThr) but no phosphorylated tyrosine (pTyr) sites. Addition of peroxyvanadate to cells to inhibit protein tyrosine phosphatases exposed 10 pTyr sites and addition of calyculin A to cells to inhibit protein phosphatases type 1 and 2A gave a total of 62 pSer/pThr sites. We also characterized two sites modified by O-linked N-acetylglucosamine. Tensin1 F302A, which does not bind protein phosphatase-1, showed > twofold enhanced phosphorylation of seven sites. The majority of pSer/pThr have adjacent proline (Pro) residues and we show endogenous p38 mitogen activated protein kinase (MAPK) associated with and phosphorylated tensin1 in an in vitro kinase assay. Recombinant p38α MAPK also phosphorylated S-tag-tensin1, resulting in decreased binding with deleted in liver cancer-1. Activation of p38 MAPK in cells by sorbitol-induced hyperosmotic stress increased phosphorylation of S-tag-tensin1, which reduced binding to deleted in liver cancer-1 and increased binding to endogenous pTyr proteins, including p130Cas and focal adhesion kinase. These data demonstrate that tensin1 is extensively phosphorylated on Ser/Thr residues in cells and phosphorylation by p38 MAPK regulates the specificity of the tensin1 Src homology 2 domain for binding to different proteins. Tensin1 provides a hub for connecting signaling pathways involving p38 MAP kinase, tyrosine kinases and RhoGTPases. Tensin1 is a protein localized at focal adhesions that acts as a scaffold for signaling (1.Chen H. Ishii A. Wong W.K. Chen L.B. Lo S.H. Molecular characterization of human tensin.Biochem J. 2000; 351: 403-411Crossref PubMed Scopus (61) Google Scholar). The tensin1 phosphotyrosine binding (PTB) 1The abbreviations used are:PTBPhosphotyrosine bindingMAPKmitogen activated protein kinaseSH2Src Homology 2FAKfocal adhesion kinaseDLC-1deleted in liver cancer-1PTENphosphatase and tensin homologuePP1protein phosphatase 1Serserine residueThrthreonine residueTyrtyrosine residueProproline residuepphosphorylated residuewtwild-typeIMACimmobilized metal affinity chromatographyLCliquid chromatographyMSmass spectrometryICRion cyclotron resonanceCADcollisionally activated dissociationETDelectron transfer dissociationLTQLinear Trap QuadrupoleFTFourier transform. domain binds the cytoplasmic tail of β-integrin (2.Calderwood D.A. Fujioka Y. de Pereda J.M. Garcia-Alvarez B. Nakamoto T. Margolis B. McGlade C.J. Liddington R.C. Ginsberg M.H. Integrin beta cytoplasmic domain interactions with phosphotyrosine-binding domains: a structural prototype for diversity in integrin signaling.Proc Natl Acad Sci U S A. 2003; 100: 2272-2277Crossref PubMed Scopus (333) Google Scholar), presumed to be the basis for focal adhesion localization. Human tensin1 interacts with actin by capping the barbed ends and cross-linking actin filaments through two different actin binding regions (3.Lo S.H. Tensin.Int J Biochem Cell Biol. 2004; 36: 31-34Crossref PubMed Scopus (159) Google Scholar). Actin binding regions were identified in chicken tensin1 at residues 1–263, 263–463, and 889–1143 (4.Lo S.H. Janmey P.A. Hartwig J.H. Chen L.B. Interactions of tensin with actin and identification of its three distinct actin-binding domains.J Cell Biol. 1994; 125: 1067-1075Crossref PubMed Scopus (131) Google Scholar). The C terminus region of tensin1, as well as family members tensin2, tensin3, and c-ten, has adjacent Src homology 2 (SH2) and PTB domains that interact with the tyrosine phosphorylated proteins Dok2 and PDK1 (5.Wavreille A.S. Pei D. A chemical approach to the identification of tensin-binding proteins.ACS Chem Biol. 2007; 2: 109-118Crossref PubMed Scopus (24) Google Scholar) as well as PI3 kinase, p130Cas, and focal adhesion kinase (FAK) (6.Davis S. Lu M.L. Lo S.H. Lin S. Butler J.A. Druker B.J. Roberts T.M. An Q. Chen L.B. Presence of an SH2 domain in the actin-binding protein tensin.Science. 1991; 252: 712-715Crossref PubMed Scopus (159) Google Scholar), thereby posing a role for tensin1 in multiple signal transduction pathways. The N-terminal region of tensin1 contains a domain that is related in sequence to the tumor suppressor protein and PIP3 phosphatase called phosphatase and tensin homologue (PTEN) (3.Lo S.H. Tensin.Int J Biochem Cell Biol. 2004; 36: 31-34Crossref PubMed Scopus (159) Google Scholar). This domain of tensin1 binds the alpha isoform of protein phosphatase 1 (PP1) (7.Eto M. Kirkbride J. Elliott E. Lo S.H. Brautigan D.L. Association of the tensin N-terminal protein-tyrosine phosphatase domain with the alpha isoform of protein phosphatase-1 in focal adhesions.J Biol Chem. 2007; 282: 17806-17815Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar), the major protein Ser/Thr phosphatase in cells that regulates a variety of signaling pathways. The SH2 domain of tensin1 also associates with a RhoGAP protein called deleted in liver cancer-1 (DLC-1) but does not require Tyr phosphorylation of DLC-1 (8.Liao Y.C. Si L. deVere White R.W. Lo S.H. The phosphotyrosine-independent interaction of DLC-1 and the SH2 domain of cten regulates focal adhesion localization and growth suppression activity of DLC-1.J Cell Biol. 2007; 176: 43-49Crossref PubMed Scopus (126) Google Scholar). DLC-1 has a role in cell migration and is a negative regulator of tumor formation (8.Liao Y.C. Si L. deVere White R.W. Lo S.H. The phosphotyrosine-independent interaction of DLC-1 and the SH2 domain of cten regulates focal adhesion localization and growth suppression activity of DLC-1.J Cell Biol. 2007; 176: 43-49Crossref PubMed Scopus (126) Google Scholar, 9.Liao Y.C. Lo S.H. Deleted in liver cancer-1 (DLC-1): a tumor suppressor not just for liver.Int J Biochem Cell Biol. 2008; 40: 837-843Crossref PubMed Scopus (97) Google Scholar, 10.Qian X. Li G. Asmussen H.K. Asnaghi L. Vass W.C. Braverman R. Yamada K.M. Popescu N.C. Papageorge A.G. Lowy D.R. Oncogenic inhibition by a deleted in liver cancer gene requires cooperation between tensin binding and Rho-specific GTPase-activating protein activities.Proc Natl Acad Sci U S A. 2007; 104: 9012-9017Crossref PubMed Scopus (128) Google Scholar). Human breast carcinoma, prostate carcinoma, head and neck squamous cell carcinoma, and melanoma all exhibit reduced expression of tensin1, suggesting a tumor suppressor action (11.Rhodes D.R. Yu J. Shanker K. Deshpande N. Varambally R. Ghosh D. Barrette T. Pandey A. Chinnaiyan A.M. ONCOMINE: a cancer microarray database and integrated data-mining platform.Neoplasia. 2004; 6: 1-6Crossref PubMed Google Scholar). In addition, various cancer cell lines do not express detectable levels of tensin1 protein relative to normal fibroblasts that have abundant expression (1.Chen H. Ishii A. Wong W.K. Chen L.B. Lo S.H. Molecular characterization of human tensin.Biochem J. 2000; 351: 403-411Crossref PubMed Scopus (61) Google Scholar, 7.Eto M. Kirkbride J. Elliott E. Lo S.H. Brautigan D.L. Association of the tensin N-terminal protein-tyrosine phosphatase domain with the alpha isoform of protein phosphatase-1 in focal adhesions.J Biol Chem. 2007; 282: 17806-17815Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Re-expression of tensin1 in cancer cells promoted formation of focal adhesions (4.Lo S.H. Janmey P.A. Hartwig J.H. Chen L.B. Interactions of tensin with actin and identification of its three distinct actin-binding domains.J Cell Biol. 1994; 125: 1067-1075Crossref PubMed Scopus (131) Google Scholar) and decreased migration and invasion of MDA MB 231 human breast cancer cells (12.Hall E.H. Daugherty A.E. Choi C.K. Horwitz A.F. Brautigan D.L. Tensin1 requires protein phosphatase-1alpha in addition to RhoGAP DLC-1 to control cell polarization, migration, and invasion.J Biol Chem. 2009; 284: 34713-34722Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Taken together, these studies support a model for tensin1 as a tumor suppressor that acts as a scaffold protein for various signaling enzymes.Tensin1 was first shown to be tyrosine phosphorylated following concentration by immunoprecipitation and immunoblotting with a pTyr antibody (6.Davis S. Lu M.L. Lo S.H. Lin S. Butler J.A. Druker B.J. Roberts T.M. An Q. Chen L.B. Presence of an SH2 domain in the actin-binding protein tensin.Science. 1991; 252: 712-715Crossref PubMed Scopus (159) Google Scholar). Tyrosine phosphorylation of tensin1 was only detected if fibroblasts were plated on fibronectin, laminin, or vitronectin (13.Bockholt S.M. Burridge K. Cell spreading on extracellular matrix proteins induces tyrosine phosphorylation of tensin.J Biol Chem. 1993; 268: 14565-14567Abstract Full Text PDF PubMed Google Scholar), suggesting that tensin1 tyrosine phosphorylation depends on integrin-mediated signaling. Jiang et al. (14.Jiang B. Yamamura S. Nelson P.R. Mureebe L. Kent K.C. Differential effects of platelet-derived growth factor isotypes on human smooth muscle cell proliferation and migration are mediated by distinct signaling pathways.Surgery. 1996; 120 (discussion 432): 427-431Abstract Full Text PDF PubMed Scopus (65) Google Scholar) showed increased tyrosine phosphorylation of tensin1 when cells were treated with platelet-derived growth factor. In addition, epidermal growth factor treatment of human gastric epithelial cells stimulated tyrosine phosphorylation of tensin1 and this stimulation was inhibited with the nonsteroidal anti-inflammatory drug indomethacin (15.Szabo I.L. Pai R. Jones M.K. Ehring G.R. Kawanaka H. Tarnawski A.S. Indomethacin delays gastric restitution: association with the inhibition of focal adhesion kinase and tensin phosphorylation and reduced actin stress fibers.Exp Biol Med (Maywood). 2002; 227: 412-424Crossref PubMed Scopus (22) Google Scholar). Cells transformed by the oncogene p210BCR/ABL contained tyrosine phosphorylated tensin1 (16.Salgia R. Brunkhorst B. Pisick E. Li J.L. Lo S.H. Chen L.B. Griffin J.D. Increased tyrosine phosphorylation of focal adhesion proteins in myeloid cell lines expressing p210BCR/ABL.Oncogene. 1995; 11: 1149-1155PubMed Google Scholar). Treatment of rat aortic smooth muscle cells with angiotensin or thrombin also showed an increase in tensin1 tyrosine phosphorylation (17.Ishida T. Ishida M. Suero J. Takahashi M. Berk B.C. Agonist-stimulated cytoskeletal reorganization and signal transduction at focal adhesions in vascular smooth muscle cells require c-Src.J Clin Invest. 1999; 103: 789-797Crossref PubMed Scopus (143) Google Scholar). Rapid turnover of pTyr by phosphatases presumably keeps tensin1 pTyr levels low in cells following stimulation. Different publications report tensin1 is phosphorylated on Ser and Thr residues, but data supporting these claims was not shown (1.Chen H. Ishii A. Wong W.K. Chen L.B. Lo S.H. Molecular characterization of human tensin.Biochem J. 2000; 351: 403-411Crossref PubMed Scopus (61) Google Scholar, 3.Lo S.H. Tensin.Int J Biochem Cell Biol. 2004; 36: 31-34Crossref PubMed Scopus (159) Google Scholar, 18.Auger K.R. Songyang Z. Lo S.H. Roberts T.M. Chen L.B. Platelet-derived growth factor-induced formation of tensin and phosphoinositide 3-kinase complexes.J Biol Chem. 1996; 271: 23452-23457Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 19.Lee S.B. Cho K.S. Kim E. Chung J. blistery encodes Drosophila tensin protein and interacts with integrin and the JNK signaling pathway during wing development.Development. 2003; 130: 4001-4010Crossref PubMed Scopus (39) Google Scholar). Phosphoproteomics implementing shotgun mass spectrometry techniques have turned up as many as 20 pTyr, 30 pSer, and 8 pThr peptides from human tensin (www.phosphosite.org). However, to date no comprehensive analysis of tensin1 phosphorylation has been reported.We previously identified residue F302 in the KVEF motif in tensin1 as necessary for PP1α binding (12.Hall E.H. Daugherty A.E. Choi C.K. Horwitz A.F. Brautigan D.L. Tensin1 requires protein phosphatase-1alpha in addition to RhoGAP DLC-1 to control cell polarization, migration, and invasion.J Biol Chem. 2009; 284: 34713-34722Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Tensin1 F302A showed a reduced electrophoretic mobility in SDS-PAGE compared with tensin1 wild type, suggesting an increase in tensin1 phosphorylation because of absence of bound PP1. We also observed less DLC-1 binding to tensin1 F302A, but it is not known whether this was because of an increase in tensin1 phosphorylation (12.Hall E.H. Daugherty A.E. Choi C.K. Horwitz A.F. Brautigan D.L. Tensin1 requires protein phosphatase-1alpha in addition to RhoGAP DLC-1 to control cell polarization, migration, and invasion.J Biol Chem. 2009; 284: 34713-34722Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The tensin1 F302A did not suppress cancer cell invasion like tensin1 wild type (12.Hall E.H. Daugherty A.E. Choi C.K. Horwitz A.F. Brautigan D.L. Tensin1 requires protein phosphatase-1alpha in addition to RhoGAP DLC-1 to control cell polarization, migration, and invasion.J Biol Chem. 2009; 284: 34713-34722Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), and this could be because of loss of PP1 binding, or less DLC-1 binding, or changes in phosphorylation.In the present study we comprehensively analyze the phosphorylation of human S-tag-tensin1. Addition of phosphatase inhibitors to cells is shown to enhance phosphorylation to yield a total of 62 Ser/Thr phosphorylation sites and expose 10 Tyr sites not otherwise seen. The majority of Ser/Thr sites have adjacent proline residues and we identify p38α MAPK activity associated with tensin1. The p38MAPK phosphorylation of tensin1 alters binding of DLC-1, p130Cas and FAK. Our results demonstrate that tensin1 is extensively phosphorylated on Ser/Thr residues in addition to Tyr residues and this phosphorylation alters association with its SH2 domain binding partners.EXPERIMENTAL PROCEDURESCell Culture and ReagentsHEK-293 and HEK293T cells were grown in modified Eagle's medium and Dulbecco's modified Eagle's medium, respectively, supplemented with 10% fetal bovine serum (Invitrogen). All cell cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2. S-protein agarose and S-TagTM monoclonal antibody were purchased from Novagen (Madison, WI). The anti-DLC-1 mouse monoclonal antibody was purchased from BD Transduction Laboratories, the anti-phospho-tyrosine (pTyr) mouse monoclonal antibody 4G10 was purchased from Millipore (Billerica, MA), and the p38MAPK, phospho-p38MAPK, and phospho-threonine-proline antibodies were purchased from Cell Signaling (Danvers, MA). The p130Cas and FAK antibodies were generous gifts from Dr. Amy Bouton (University of Virginia). The secondary antibody Alexa Fluor® 680 goat anti-mouse IgG was purchased from Invitrogen and the goat anti-mouse HRP was purchased from Thermo Scientific.Full length pTriEX4-(S-tag)-tensin1 was mutated to F302A by polymerase chain reaction as previously described (12.Hall E.H. Daugherty A.E. Choi C.K. Horwitz A.F. Brautigan D.L. Tensin1 requires protein phosphatase-1alpha in addition to RhoGAP DLC-1 to control cell polarization, migration, and invasion.J Biol Chem. 2009; 284: 34713-34722Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar).Affinity purification of S-tagged Tensin1 and Tensin1 F302AFor each experiment HEK293T cells were plated onto five 15-cm plates, grown to 80% confluency and transiently transfected with pTriEx4-tensin1 wild type (wt) or F302A according to manufacturer's instructions with Arrest-InTM Transfection Reagent (Open Biosystems). Cells were treated with DMSO as control or 25 nm calyculin A for 30 min. Calyculin caused the cells to detach and they were collected by centrifugation at 1500 × g for 5 min. For the identification of Tyr phosphorylation, cells were treated with 1 mm peroxyvanadate for 30 min prior to scraping in a total of 3 ml ice-cold MS lysis buffer (50 mm Tris-HCl, pH 7.9, 1% IGEPAL CA-630, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 20 mm β-glycerophosphate, 1× protease inhibitor mixture V (Calbiochem), and 1 mm dithiothreitol). The cell suspension was centrifuged at 16,000 × g for 15 min in a microcentrifuge and the supernatant (extract) incubated with 60 μl of S-protein agarose for 3 h. The agarose beads were washed twice by centrifugation with MS lysis buffer containing 500 mm NaCl and no protease inhibitors, twice with 50 mm Tris, pH 7.9, 500 mm NaCl, and once with Tris buffer, 150 mm NaCl.LC-MS/MS Analysis of S-tagged Tensin1 and Tensin1 F302AEach sample of purified tensin1 was reduced and carbamidomethylated at room temperature using dithiothreitol (Sigma Aldrich, St. Louis, MO) and iodoacetamide (Sigma Aldrich), respectively, then subjected to proteolytic digestion (1:20 enzyme to substrate) by trypsin (Promega, Madison, WI) as previously reported (20.Schroeder M.J. Shabanowitz J. Schwartz J.C. Hunt D.F. Coon J.J. A neutral loss activation method for improved phosphopeptide sequence analysis by quadrupole ion trap mass spectrometry.Anal Chem. 2004; 76: 3590-3598Crossref PubMed Scopus (335) Google Scholar). An additional chymotrypsin (Roche, Penzberg, Germany) digest using similar conditions was used to characterize modified sites in tryptic peptide comprised of residues Asp1199 to Arg1248. For mass spectrometric analysis, a fraction of the digest was pressure loaded onto a precolumn (360 μm o.d. × 150 μm i.d. fused silica capillary) packed with 5 cm of C18 reverse-phase resin (5–20 μm diameter, 120 Å pore size, YMC Co., Ltd., Kyoto, Japan). Following a desalting rinse using 0.1 m acetic acid, the precolumn was connected to an analytical column (360 μm o.d. × 50 μm i.d.) packed with 8 cm of C18 reverse-phase resin (5 μm diameter, 120 Å pore size, YMC Co., Ltd.) and equipped with an electrospray emitter tip (21.Udeshi N.D. Compton P.D. Shabanowitz J. Hunt D.F. Rose K.L. Methods for analyzing peptides and proteins on a chromatographic timescale by electron-transfer dissociation mass spectrometry.Nat Protoc. 2008; 3: 1709-1717Crossref PubMed Scopus (75) Google Scholar). For liquid chromatography-tandem MS (LC-MS/MS) analysis, the tryptic peptides were gradient eluted at a flow rate of 60 nL/min using an LC gradient previously described (22.Syka J.E. Coon J.J. Schroeder M.J. Shabanowitz J. Hunt D.F. Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry.Proc Natl Acad Sci U S A. 2004; 101: 9528-9533Crossref PubMed Scopus (1985) Google Scholar). MS mass analyses were acquired in high-resolution Fourier transform mass analyzers (FT or Orbitrap) whereas MS2 spectra were measured in linear ion traps of LTQ-FT-ion cyclotron resonance and LTQ-Orbitrap hybrid instruments (Thermo Fisher Scientific, Bremen, Germany) using collisionally activated dissociation (CAD) and front end electron transfer dissociation. Mass analyses were completed using a method consisting of one high resolution MS1 scan (resolving power of 60,000 at m/z 400) followed by 8 to 10 data dependent low resolution MS2 scans acquired in the LTQ ion trap. Data dependence parameters were set as follows: repeat count of 2, repeat duration of 30s, exclusion list duration of 20s. Electron transfer dissociation (ETD) MS2 parameters were set as follows: 30 ms reaction time, 3 m/z precursor isolation window, charge state rejection "on" for +1 and +2 charge state precursor ions, 2 × 105 FTMS full automated gain control target, 1 × 104 ITMSn automated gain control target, 2 × 105 reagent target with azulene as the electron transfer reagent.Differential Phosphorylation AnalysisPrior to phosphopeptide enrichment using immobilized metal affinity chromatography (IMAC), tensin1 wt and tensin1 F302A tryptic peptides were differentially esterified on acidic residues using d0-methanolic HCl and d3-methanolic HCl, respectively to reduce nonspecific binding to the IMAC column (23.Ficarro S. Chertihin O. Westbrook V.A. White F. Jayes F. Kalab P. Marto J.A. Shabanowitz J. Herr J.C. Hunt D.F. Visconti P.E. Phosphoproteome analysis of capacitated human sperm. Evidence of tyrosine phosphorylation of a kinase-anchoring protein 3 and valosin-containing protein/p97 during capacitation.J Biol Chem. 2003; 278: 11579-11589Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar). Esterified peptides from both tensin1 protein samples were then combined and simultaneously enriched for phosphorylated peptides using IMAC methods previously described (24.Zarling A.L. Polefrone J.M. Evans A.M. Mikesh L.M. Shabanowitz J. Lewis S.T. Engelhard V.H. Hunt D.F. Identification of class I MHC-associated phosphopeptides as targets for cancer immunotherapy.Proc Natl Acad Sci U S A. 2006; 103: 14889-14894Crossref PubMed Scopus (131) Google Scholar). The LC-MS/MS methods were identical to those described above. Phosphorylation relative abundance comparisons between samples, namely the d3/d0 ratios, were calculated on the basis of the abundance of the C12 isotope peak within the charge envelope of each interrogated phosphorylated peptide.Mass Spectrometry Data AnalysisPrior to using the Open Mass Spectrometry Search Algorithm (version 2.1.1) to search against a human tensin1 and tensin1 F302A protein sequence database (gi 187954597, Accession number: BC140942.1, NCBI), MS/MS peak lists were generated using Bioworks Browser (version 3.3.1 SP1). For both CAD (b- and y-type ions) and ETD (c- and z-type ions) data set searches, Open Mass Spectrometry Search Algorithm mass tolerances were ± 0.01 Da and ± 0.35 Da for precursor and product ion masses, respectively. The Open Mass Spectrometry Search Algorithm provided the removal of reduced charge species from ETD peak lists prior to searching. All database searches were completed selecting trypsin for enzyme specificity and included the following variable modifications: carbamidomethylation of Cys, oxidation of Met, phosphorylated Ser, Thr, and Tyr as well as O-GlcNAcylation of Ser and Thr. Missed cleavage count was set to three. The Open Mass Spectrometry Search Algorithm search results for complementary CAD and ETD data sets were confirmed by manual interpretation of the MS/MS spectra.Analysis of S-tag-Tensin1 by ImmunoblottingHEK293 cells transfected with pTriEx4-tensin1 or tensin1 F302A plasmid were untreated, or treated with either dimethyl sulfoxide, or 25 nm calyculin A (Calbiochem), collected, and lysed on ice for 15 min in Golden lysis buffer (20 mm Tris-HCl, pH 7.9, 0.137 m NaCl, 1 mm EGTA, 5 mm EDTA, 10% glycerol, 1% Triton X-100, 10 mm NaF, 1 mm sodium pyrophosphate, 1 mm β-glycerophosphate, 1 mm Na3VO4, and 1× protease inhibitor mixture set V). Following centrifugation at 16,000 × g for 20 min whole cell extracts were resolved on a 4%–15% gradient Criterion Tris-HCl polyacrylamide gel (BioRad Laboratories), proteins transferred to a nitrocellulose membrane and immunoblotted anti-phospho-ThrPro, anti-phospho-Tyr, or anti-S-tag. Immunoblots were scanned by the Odyssey Infrared Imaging System (LiCor®) and analyzed with the Odyssey Application Software version 2.0.41 (LiCor®).In Vitro Kinase AssaysHEK293 cells were transiently transfected with pTriEx4-tensin1 or mock transfected, the medium removed by aspiration, and cells lysed on ice for 15 min in 0.4 ml Golden lysis buffer or in RIPA buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1% IGEPAL CA-630, 0.25% deoxycholate, 0.1% SDS, 20 mm β-glycerophosphate, 1 mm EDTA, and 0.4 mm Pefabloc). Cells were scraped from the plates and following centrifugation the extracts were incubated with S-protein agarose for 3 h and the beads recovered and washed with Golden buffer or RIPA buffer by centrifugation. S-protein agarose beads were incubated in kinase reaction buffer alone (50 mm Tris-HCl, pH 7.5, 0.1% 2-mercaptoethanol, 0.2 mm Na3VO4, and 1 mm dithiothreitol), with 10 μm kinase inhibitors (GSKβ3 inhibitor II, SB220025, or staurosporine), or with 100 ng of p38α MAPK purified kinase. The 32P-ATP was added in a concentrated Mg/Mn buffer to give final concentrations of 10 mm MgCl2, 1 mm MnCl2, 0.1 mm ATP, and 5 mm β-glycerophosphate. Samples were incubated at 30 °C for the specified time points, boiled for 5 min following adding SDS sample buffer, and proteins resolved by SDS-PAGE on 12% gels that were dried and imaged with the PhosphorImager 445S1 (Molecular Probes, Carlsbad, CA).DLC-1 Binding AssaysFor DLC-1 binding experiments S-tag-tensin1 was prepared as described above and incubated ± p38 MAPK with nonradioactive ATP. Separate plates of HEK293 cells transfected with pcDNA3-DLC-1 (a generous gift from Dr. D. Lowy, National Institutes of Health) were extracted with Golden lysis buffer and the extract incubated with the S-tag-tensin1 on beads for 2 h. Following incubation the S-protein agarose beads were washed, boiled in SDS sample buffer, and the proteins resolved a on a 4%–15% gradient gel, transferred to nitrocellulose and immunoblotted for DLC-1 and anti-S-tag. S-tag immunoblots were scanned by the Odyssey Infrared Imaging System (LiCor) and analyzed with the Odyssey Application Software version 2.0.41 (LiCor). DLC-1 immunoblots were detected with enhanced chemiluminescence, exposure of x-ray film and analyzed with Image J software.Hyperosmotic Sorbitol Treatment of CellsHEK293 cells were transfected with pTriEx4-tensin1 or mock transfected with reagent alone. Following 24 h cells were treated with 0.4 m sorbitol for 20 min to activate p38 MAPK (25.Prickett T.D. Brautigan D.L. Cytokine activation of p38 mitogen-activated protein kinase and apoptosis is opposed by alpha-4 targeting of protein phosphatase 2A for site-specific dephosphorylation of MEK3.Mol Cell Biol. 2007; 27: 4217-4227Crossref PubMed Scopus (47) Google Scholar), lysed on ice for 15 min in 400 μl Golden lysis buffer, and the lysates centrifuged at 16,000 × g for 15 min. Supernatants were incubated with S-protein agarose for 3 h at 4 °C, and the beads washed three times with Golden lysis buffer by cenrtrifugation. DLC-1 binding was assayed as described above. Samples were washed 3× in Golden lysis buffer and proteins eluted and resolved on 4%–15% Criterion precast gradient gels (BioRad) and transferred to a nitrocellulose membrane. Membranes were immunoblotted with anti-DLC-1 and anti-S-tag. Proteins bound to S-protein agarose were immunoblotted with anti-phospho-threonine-proline, anti-phospho-tyrosine, anti-p130Cas, or anti-FAK antibodies. Extracts from cells with and without sorbitol treatment were immunoblotted with p38 and phospho-p38 antibodies. All immunoblots except DLC-1 were scanned by the Odyssey Infrared Imaging System (LiCor) and analyzed with the Odyssey Application Software version 2.0.41 (LiCor).RESULTSComprehensive Analysis of Human Tensin1 PhosphorylationInitial purifications using green fluorescent protein-tagged tensin1 recovered from cell extracts by immunoprecipitation revealed elution of multiple contaminating proteins, in addition to the immunoglobulin itself, which frustrated attempts to achieve complete sequence coverage of tensin1 by mass spectrometry. As an alternative approach HEK293 cells were transfected with pTri-Ex4-(S-tag) tensin1 to express full le
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