Adhesion or Plasmin Regulates Tyrosine Phosphorylation of a Novel Membrane Glycoprotein p80/gp140/CUB Domain-containing Protein 1 in Epithelia
2004; Elsevier BV; Volume: 279; Issue: 15 Linguagem: Inglês
10.1074/jbc.m309678200
ISSN1083-351X
AutoresTod A. Brown, Tai Mei Yang, Tatiana Zaitsevskaia, Yuping Xia, Clarence A. Dunn, Randy O. Sigle, Beatrice S. Knudsen, William G. Carter,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoSuspension of cultured human foreskin keratinocytes (HKs) with trypsin phosphorylates tyrosine residues on an 80-kDa membrane glycoprotein, p80 (Xia, Y., Gil, S. G., and Carter, W. G. (1996) J. Cell Biol. 132, 727–740). Readhesion dephosphorylates p80. Sequencing of a p80 cDNA established identity to CUB domain-containing protein 1 (CDCP1), a gene elevated in carcinomas. CDCP1/p80 cDNA encodes three extracellular CUB domains, a transmembrane domain, and two putative cytoplasmic Tyr phosphorylation sites. Treatment of adherent HKs with suramin, a heparin analogue, or inhibitors of phosphotyrosine phosphatases (PTPs; vanadate or calpeptin) increases phosphorylation of p80 and a novel 140-kDa membrane glycoprotein, gp140. Phosphorylated gp140 was identified as a trypsin-sensitive precursor to p80. Identity was confirmed by digestion and phosphorylation studies with recombinant gp140-GFP. Plasmin, a serum protease, also converts gp140 to p80, providing biological significance to the cleavage in wounds. Phosphorylation of gp140 and p80 are mediated by Src family kinases at multiple Tyr residues including Tyr734. Dephosphorylation is mediated by PTP(s). Conversion of gp140 to p80 prolongs phosphorylation of p80 in response to suramin and changes in adhesion. This distinguishes gp140 and p80 and explains the relative abundance of phosphorylated p80 in trypsinized HKs. We conclude that phosphorylation of gp140 is dynamic and balanced by Src family kinase and PTPs yielding low equilibrium phosphorylation. We suggest that the balance is altered by conversion of gp140 to p80 and by adhesion, providing a novel transmembrane phosphorylation signal in epithelial wounds. Suspension of cultured human foreskin keratinocytes (HKs) with trypsin phosphorylates tyrosine residues on an 80-kDa membrane glycoprotein, p80 (Xia, Y., Gil, S. G., and Carter, W. G. (1996) J. Cell Biol. 132, 727–740). Readhesion dephosphorylates p80. Sequencing of a p80 cDNA established identity to CUB domain-containing protein 1 (CDCP1), a gene elevated in carcinomas. CDCP1/p80 cDNA encodes three extracellular CUB domains, a transmembrane domain, and two putative cytoplasmic Tyr phosphorylation sites. Treatment of adherent HKs with suramin, a heparin analogue, or inhibitors of phosphotyrosine phosphatases (PTPs; vanadate or calpeptin) increases phosphorylation of p80 and a novel 140-kDa membrane glycoprotein, gp140. Phosphorylated gp140 was identified as a trypsin-sensitive precursor to p80. Identity was confirmed by digestion and phosphorylation studies with recombinant gp140-GFP. Plasmin, a serum protease, also converts gp140 to p80, providing biological significance to the cleavage in wounds. Phosphorylation of gp140 and p80 are mediated by Src family kinases at multiple Tyr residues including Tyr734. Dephosphorylation is mediated by PTP(s). Conversion of gp140 to p80 prolongs phosphorylation of p80 in response to suramin and changes in adhesion. This distinguishes gp140 and p80 and explains the relative abundance of phosphorylated p80 in trypsinized HKs. We conclude that phosphorylation of gp140 is dynamic and balanced by Src family kinase and PTPs yielding low equilibrium phosphorylation. We suggest that the balance is altered by conversion of gp140 to p80 and by adhesion, providing a novel transmembrane phosphorylation signal in epithelial wounds. Wounding of quiescent epidermis activates changes in adhesion and cell signaling resulting in keratinocyte migration, basement membrane (BM) 1The abbreviations used are: BM, basement membrane; Ab, polyclonal antibody; CDCP1, CUB domain-containing protein 1; FAK, focal adhesion kinase; P-FAK, phospho-FAK; P-p80 and P-gp140, phospho-p80 and phospho-gp140, respectively; HK, human keratinocyte; LC MS/MS, liquid chromatography-coupled mass spectrometry/mass spectrometry; NEM, N-ethylmaleimide; mAb, monoclonal antibody; PTP, phosphotyrosine phosphatase; SFK, Src family kinase; WGA, wheat germ agglutinin; HD, hemidesmosome; HPLC, high pressure liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS, mass spectrometry; GFP, green fluorescent protein; uPA, urokinase plasminogen activator; uPAR, uPA receptor. repair, and wound closure (1Martin P. Science. 1997; 276: 75-81Crossref PubMed Scopus (3762) Google Scholar, 2Woodley D.T. O'Toole E. Nadelman C.M. Li W. Progress Dermatol. 1999; 33: 1-12Google Scholar, 3Nguyen B.P. Gil S.G. Ryan M.C. Carter W.G. Curr. Opin. Cell Biol. 2000; 12: 554-562Crossref PubMed Scopus (224) Google Scholar). Based on an in vitro model for wound activation, we reported that trypsin detachment of cultured human foreskin keratinocytes (HKs) promotes phosphorylation of tyrosine residues on an 80-kDa membrane glycoprotein (p80) (4Xia Y. Gil S.G. Carter W.G. J. Cell Biol. 1996; 132: 727-740Crossref PubMed Scopus (116) Google Scholar). Phosphorylated p80 (P-p80) in suspended HKs is dephosphorylated upon readhesion to laminin 5 via integrins α6β4 and α3β1. In work here, we purified and characterized p80. We wished to understand whether phosphorylated p80 identified in an in vitro deadhesion/readhesion screen (4Xia Y. Gil S.G. Carter W.G. J. Cell Biol. 1996; 132: 727-740Crossref PubMed Scopus (116) Google Scholar) might be involved in physiologically significant wound activation. Quiescent epidermis adheres to laminin 5 in the BM via integrin α6β4 in hemidesmosome (HD) cell junctions. Wounding epidermis generates leading and following subpopulations of keratinocytes at the wound margin (5Lampe P.D. Nguyen B.P. Gil S. Usui M. Olerud J. Takada Y. Carter W.G. J. Cell Biol. 1998; 143: 1735-1747Crossref PubMed Scopus (143) Google Scholar). Leading keratinocytes migrate over exposed dermal collagen via integrin α2β1 and fibronectin via integrin α5β1. However, leading cells also deposit laminin 5 as a provisional BM and interact with these deposits via integrin α3β1 (5Lampe P.D. Nguyen B.P. Gil S. Usui M. Olerud J. Takada Y. Carter W.G. J. Cell Biol. 1998; 143: 1735-1747Crossref PubMed Scopus (143) Google Scholar, 6Nguyen B.P. Gil S.G. Carter W.G. J. Biol. Chem. 2000; 275: 31896-31907Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 7Frank D. Carter W.G. J. Cell Sci. 2004; 117 (in press)Crossref PubMed Scopus (144) Google Scholar). The interaction of leading cells with deposited laminin 5 generates distinct transmembrane signals when compared with interaction with dermal ligands. For example, adhesion of HKs to laminin 5 via α3β1 promotes gap junction intercellular communication when compared with adhesion to collagen or fibronectin (5Lampe P.D. Nguyen B.P. Gil S. Usui M. Olerud J. Takada Y. Carter W.G. J. Cell Biol. 1998; 143: 1735-1747Crossref PubMed Scopus (143) Google Scholar). Interactions of leading cells with collagen are inhibited by toxin B, an inhibitor of Rho GTPases, which does not inhibit the phosphatidylinositol 3-kinase-dependent adhesion or spreading on laminin 5. Deposition of laminin 5 over exposed dermal collagen switches adhesion and spreading from toxin B-sensitive to toxin B-resistant (6Nguyen B.P. Gil S.G. Carter W.G. J. Biol. Chem. 2000; 275: 31896-31907Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Cell interaction with laminin 5 (6Nguyen B.P. Gil S.G. Carter W.G. J. Biol. Chem. 2000; 275: 31896-31907Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) or laminin 10 (8Gu J. Sumida Y. Sanzen N. Sekiguchi K. J. Biol. Chem. 2001; 276: 27090-27097Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar) via α3β1 assembles minimal focal adhesions with low phosphorylation of focal adhesion kinase (FAK) when compared with adhesion on dermal ligands. Consistently, we also found that adhesion to dermal ligands was less effective than laminin 5 in regulating phosphorylation of p80 (4Xia Y. Gil S.G. Carter W.G. J. Cell Biol. 1996; 132: 727-740Crossref PubMed Scopus (116) Google Scholar). Dephosphorylation of P-p80 upon adhesion to laminin 5, but not collagen, is resistant to cytochalasin D, an inhibitor of the actin cytoskeleton. Vanadate, an inhibitor of PTPs, prevents dephosphorylation of P-p80 upon adhesion, indicating that a PTP is required for the dephosphorylation. Phosphorylated p80 is detected in primary cultures of many epithelial cells (epidermal, esophageal, cervical, and gastric) but not primary cultures of fibroblasts or some immortalized epithelial cell populations (4Xia Y. Gil S.G. Carter W.G. J. Cell Biol. 1996; 132: 727-740Crossref PubMed Scopus (116) Google Scholar). We concluded that adhesion-dependent phosphorylation and dephosphorylation of p80 on laminin 5 may report unique cell signals and functions in many epithelia. Here, we describe the purification of P-p80 from trypsin-suspended HKs and its characterization. The results integrate five novel observations. (i) A novel 140-kDa transmembrane glycoprotein, gp140, is identified as the trypsin-sensitive precursor to p80 and contains extracellular CUB protein-protein interaction domains. (ii) gp140/p80 is identified as the product of the CUB domain-containing protein 1 (CDCP1) gene that is overexpressed in human colorectal and lung cancers (9Scherl-Mostageer M. Sommergruber W. Abseher R. Hauptmann R. Ambros P. Schweifer N. Oncogene. 2001; 20: 4402-4408Crossref PubMed Scopus (110) Google Scholar), is a marker for hematopoietic stem cells, (10Conze T. Lammers R. Kuci S. Scherl-Mostageer M. Schweifer N. Kanz L. Buhring H.J. Ann. N. Y. Acad. Sci. 2003; 996: 222-226Crossref PubMed Scopus (46) Google Scholar) and is identical to SIMA135, a membrane glycoprotein elevated in metastatic human tumor cells (11Hooper J.D. Zijlstra A. Aimes R.T. Liang H. Claassen G.F. Tarin D. Testa J.E. Quigley J.P. Oncogene. 2003; 22: 1783-1794Crossref PubMed Scopus (109) Google Scholar). (iii) Tyrosine phosphorylation of p80 is mediated by Src family kinase(s) on several tyrosines including tyrosine 734 upon detachment of HKs with trypsin or treatment with suramin, a membrane-impermeable polysulfonated napthylurea (12Dhar S. Gullbo J. Csoka K. Eriksson E. Nilsson K. Nickel P. Larsson R. Nygren P. Eur. J. Cancer. 2000; 36: 803-809Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). (iv) Significantly, trypsin conversion of gp140 to p80 is duplicated by serum plasmin. (v) This conversion decreases the rate of dephosphorylation of p80 relative to gp140 in response to detachment or suramin. The studies here suggest roles for plasmin and adhesion in regulating phosphorylation of gp140/p80 in epidermal wounds. Antibodies and Other Reagents—Anti-phosphotyrosine monoclonal antibodies (mAbs) 4G10 (Upstate Biotechnology, Inc., Lake Placid, NY) and PY20 (Oncogene Research Products, San Diego, CA), anti-green fluorescent protein (GFP) clone JL-8 (BD Biosciences), and rabbit antibodies against FAK Tyr(P)397 (catalog no. 44-624) and FAK Tyr(P)861 (catalog no. 44-626; BIOSOURCE, Hopkinton, MA) were purchased from the designated suppliers and used according to the manufacturers' suggestions. Phosphopeptides from gp140/p80 were synthesized by BIOSOURCE according to our specifications. Suramin, calpeptin, dephostatin, SU6656, PP2, ALLN (proteosome inhibitor), XAMR 0721 (suramin analogue), and PPADS (purinergic receptor antagonist) were purchased from either Calbiochem or Alexis Biochemicals (San Diego, CA). Plasmin, thrombin, wheat germ agglutinin (WGA), protein G-agarose, trypan blue, NF023 (purinergic receptor antagonist), and p-nitrophenyl phosphate were from Sigma. Cell Culture, Drug Treatments, and Cell Extraction—HKs were isolated and grown in monolayer culture as previously described (13Boyce S.T. Ham R.G. J. Tissue Culture Methods. 1985; 9: 83-93Crossref Scopus (258) Google Scholar), using defined medium (KGM, Cambrex, San Diego, CA). For phosphorylation studies, HKs were (i) treated with or without inhibitors for 30 min at the designated concentrations prior to (ii) treatment with or without suramin (35 μm) for 20 min and (iii) extraction with 2% Triton X-100/phosphate-buffered saline/1–2 mm each phenylmethylsulfonyl fluoride, N-ethylmaleimide (NEM), sodium fluoride (NaF), and sodium orthovanadate (Na3VO4; collectively buffer A) for 30 min at 4 °C. The Triton-soluble extract was collected by centrifugation, and the Triton-resistant pellet was extracted with 2 m urea, 1 m sodium chloride plus 1–2 mm each phenylmethylsulfonyl fluoride, NEM, NaF, and Na3VO4 to collect the Triton-insoluble cellular material. Phosphatase Assay—Total phosphatase activity in lysates of HKs was assayed as described (14Gil-Henn H. Elson A. J. Biol. Chem. 2003; 278: 15579-15586Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) using p-nitrophenyl phosphate as substrate. Tyrosine phosphatase activity (vanadate-inhibitable) was calculated as the difference between total phosphatase activities of a given sample measured with and without pervanadate. Purification of Tyrosine-phosphorylated p80 and gp140 —p80 and gp140 were purified from HKs using similar protocols as outlined below. However, p80 was purified from HKs suspended with trypsin-EDTA to induce tyrosine phosphorylation of p80 as previously described (4Xia Y. Gil S.G. Carter W.G. J. Cell Biol. 1996; 132: 727-740Crossref PubMed Scopus (116) Google Scholar). In contrast, gp140 was purified from adherent HKs that were treated with suramin to induce tyrosine phosphorylation of gp140 and p80. Adherent HKs were removed from culture dishes with a cell scraper. Trypsin-EDTA or scrape suspended HKs were homogenized with a Dounce homogenizer in phosphate-buffered saline plus 1–2 mm each phenylmethylsulfonyl fluoride, NEM, NaF and Na3VO4. Unless specifically stated, all subsequent steps used buffers that included these protease and phosphatase inhibitors. Total cellular membrane and cytosolic fractions were collected from the homogenate after centrifugation for 1 h at 100,000 × g. The membrane fraction was extracted with buffer A for 30 min at 4 °C and centrifuged (4 min, 2000 × g). The soluble material was incubated with WGA-agarose for 1 h at 4 °C; unbound or loosely attached protein was washed away with 1% Empigen BB, 50 mm NaCl, 50 mm Tris, pH 7.4; and the bound material was eluted with 400 mm N-acetylglucosamine, 0.1% Triton X-100, 50 mm Tris, pH 7.4. Phosphotyrosine-containing proteins were isolated from the WGA-eluted material by immunoprecipitation with anti-phosphotyrosine mAb (4G10-agarose; Upstate Biotechnology, Inc., Lake Placid, NY) and eluted with 8 m urea with 0.1% Triton X-100 detergent, 20 mm Tris, pH 7.4. The eluted phosphoproteins were further purified by preparative SDS-PAGE and analyzed as follows. Partial Amino Acid Sequencing of Purified p80 —Partial amino acid sequencing of purified p80 was performed by the Harvard microsequencing facility under the direction of William Lane. The purified p80 protein in gel was transferred to polyvinylidene difluoride membranes (24 nmol of purified p80 based on AA analysis). Trypsin-digestion released peptides were fractionated by reverse phase HPLC on a Zorbax C18 column, and peaks of candidate peptides were analyzed by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) to obtain mass number and homogeneity followed by sequencing by phenylthiohydantoin derivatization. This provided amino acid sequence from five different p80 peptides (peptides 65, 98, 38, 118, and 31; see "Results" and Fig. 2). Identification of GP140 by Mass Spectrometry—Identification of gp140 was performed in the FHCRC mass spectrometry facility by Philip Gafkin and Angela Norbeck. The SDS-PAGE-purified gp140 was subject to in-gel trypsin digestion, and the soluble samples were desalted using Millipore μC18 ZipTips and dried. The sample was then resuspended in 5 μl of water and analyzed by LC MS/MS with a ThermoFinnigan LCQ DECA XP mass spectrometer (15Gatlin C.L. Kleemann G.R. Hays L.G. Link A.J. Yates III, J.R. Anal. Biochem. 1998; 263: 93-101Crossref PubMed Scopus (354) Google Scholar). Data were collected in the data-dependent mode in which an MS scan was followed by MS/MS scans of the three most abundant ions from the preceding MS scan. The MS/MS data were searched against the NCBI nonredundant protein data base and a human subset of this data base using SEQUEST™ software. The resulting peptide matches were scored by SEQUEST™, and protein identifications were considered valid if the identified protein contained at least two peptides with Xcorr scores above 2.0 and the identification did not appear in a control sample from a blank portion of the gel. Preparation of Nondegenerated Oligonucleotide Probe from p80 Peptide Sequence—An RT-PCR protocol was used to obtain a nondegenerate oligonucleotide probe from amino acid sequence of p80 peptide 65. Degenerate oligonucleotide primers were synthesized based on the amino acid sequence of p80 peptide 65 and were kinase-labeled with [γ-32P]ATP. The labeled primers were used in RT-PCR reactions with total RNA from HKs, isolated with Trizol (Invitrogen). For synthesis of the first strand cDNA, labeled degenerate antisense primer B (RAANACYTCNGGRTTGGT) was used followed by PCR amplification with a second labeled degenerate sense primer A (GTNGARTAYTAYATHCC). Both labeled primers were run in two PCRs run in parallel. The PCR products were then identified by autoradiography following electrophoresis on a 12% sequencing gel. One labeled band with approximate size of 45 bp was detected in each reaction. The DNA fragment was eluted from the gel with buffer (10 mm Tris-HCl, pH 7.5, 1 mm EDTA, 200 mm NaCl). The purified 45-bp fragment was subcloned into PCRII vector and sequenced. Double-stranded DNA sequencing was performed on purified DNA using dye terminators (Applied Biosystems, Inc.). Screening of cDNA Library and Sequencing-selected cDNA Clones— The p80A/B oligonucleotide probe was labeled by random priming (109 cpm/μg DNA; Stratagene) and used to screen a human keratinocyte λgtll cDNA library. For screening, phage plaques were immobilized on nitrocellulose (Schleicher & Schüll) membranes and prehybridized at 37 °C for 4 h in buffer containing 50% formamide, 5× SSPE, 1× Denhardt's solution, 0.1% SDS, and 100 μg/ml denatured salmon sperm DNA. Blocked filters were incubated with labeled p80A/B probe at 37 °C for 14 h and washed (2× SSC and 0.1% SDS, room temperature), and positive plaques were purified. Phage were grown in Y1090 bacteria, and DNA was isolated (Lambda Preps DNA Purification, Promega, CA) and digested with EcoRI to release insert, which was gel-purified and transferred to BlueScript vector for sequencing. This screening approach identified and sequenced a single 2.0-kb p80 cDNA. Construction and Expression of GFP-gp140 —A full-length gp140/CDCP1 cDNA was constructed by sequential insertion of three partial cDNAs into the multiple cloning site of an pEGFP-N1 plasmid (Clontech). The correct sequence of the full-length cDNA was confirmed. The resulting full-length gp140/CDCP1 cDNA without a stop codon was connected in frame at its 3′-end to the 5′-end of GFP. In this orientation, expression of gp140 would orient the GFP at the cytoplasmic, carboxyl-terminal tail of gp140. Therefore, the GFP would remain after proteolytic conversion of gp140-GFP to p80-GFP. The gp140-pEGFP-N1 plasmid was transfected into 293T cells or mouse keratinocytes with LipofectAMINE Plus reagent (Invitrogen). Mouse keratinocytes were grown in KGM (Cambrex). 293T cells were grown in Dulbecco's modified Eagle's medium without serum after transfection as indicated. Transient transfection of the gp140-pEGFP-N1 plasmid in cells produced GFP-gp140 on the surface of cells. However, stable transfection by selection in G418 was not successful. Apparently, toxicity of the GFP-gp140 construct limited stable transfection. Western Blotting—Western blotting was performed with specific primary antibodies after transfer of proteins to nitrocellulose and the blocking of nonspecific binding sites with either 0.5% heat-denatured bovine serum albumin, 1% polyvinylpyrrolidone (PVP-40; Sigma), and 0.1% Triton X-100 in phosphate-buffered saline (chemiluminescence) or LiCor blocking buffer/phosphate-buffered saline (1:1; LiCor infrared imager). Primary antibodies were then either incubated with species-specific horseradish peroxidase-conjugated secondary antibodies (Dako or Cappel) and visualized by enhanced chemiluminescence (Amersham Biosciences) or incubated with species-specific Alexa Fluor 680- or IRDye800-conjugated secondary Abs (Molecular Probes or Rockland, respectively) and visualized with a LiCor Odyssey infrared imager (LiCor, Lincoln, NE). Immunostaining—Confluent cultures of HKs were incubated with and without PP2 kinase inhibitor (1 μm for 30 min) followed by or not followed by suramin (35 μm for 10 min). Cells were fixed in 2% formaldehyde in 0.1 m sodium cacodylate, 0.1 m sucrose, pH 7.2, containing both vanadate and NEM to inhibit phosphatases and then permeabilized with 0.1% Triton X-100 detergent. Cells were stained with Ab FAK Tyr(P)861 with and without synthetic gp140 phosphopeptide P-Y734 (10 μg/ml), and bound Ab was detected with rhodamine-conjugated secondary Ab. Images were collected with a Zeiss fluorescence microscope with a Photometric SenSys cooled CCD digital camera (Roper Industries, Trenton, NJ) using MetaMorph software (Universal Imaging Corp., Downingtown, PA). Adhesion Regulates Phosphorylation and Dephosphorylation of p80 —As previously shown (4Xia Y. Gil S.G. Carter W.G. J. Cell Biol. 1996; 132: 727-740Crossref PubMed Scopus (116) Google Scholar), deadhesion of HKs with trypsin induces tyrosine phosphorylation of a prominent 80-kDa membrane glycoprotein, p80, and decreases phosphorylation of FAK (Fig. 1, lanes 1 and 2). Subsequent readhesion of the suspended HKs onto laminin 5 via integrins α6β4 and α3β1 for 10 min increases phosphorylation of FAK and dephosphorylates p80 (Fig. 1, lane 3). This indicates that phosphorylation of p80 occurs upon trypsin suspension and dephosphorylation occurs upon readhesion. Vanadate addition prevents the dephosphorylation of p80 upon readhesion, indicating that dephosphorylation by a PTP is the primary reason for the disappearance of P-p80 (4Xia Y. Gil S.G. Carter W.G. J. Cell Biol. 1996; 132: 727-740Crossref PubMed Scopus (116) Google Scholar). Surprisingly, in the subsequent 1–6 h, p80 is transiently rephosphorylated and then dephosphorylated (Fig. 1, lanes 4–6). This indicates that phosphorylation-dephosphorylation of p80 also occurs in adherent HKs possibly as a result of transient changes in spreading or migration. These changes in phosphorylation of p80 raised questions regarding its identity and the outside-in signals that regulate tyrosine phosphorylation of p80. Below, we purify and characterize p80, evaluate tyrosine kinase and PTP that balance phosphorylation of p80, and evaluate proteolysis in regulating phosphorylation. Purification of P-p80 from Trypsin-detached HKs and Partial Amino Acid Sequencing—Candidate studies failed to identify p80, necessitating purification of P-p80 for characterization. HKs were suspended with trypsin/EDTA to promote phosphorylation of p80. The P-p80 was isolated by (i) preparation of a membrane-rich fraction, (ii) extraction of the membranes with Triton X-100 detergent to obtain a soluble membrane extract, (iii) sequential purification of P-p80 from the extract on immobilized WGA to collect membrane glycoproteins followed by elution and affinity purification on immobilized anti-phosphotyrosine antibodies (PY20 or 4G10), and (iv) preparative SDS-PAGE. The purified P-p80 was detected with silver stain and digested with trypsin, peptides were fractionated by HPLC, and peptides were assigned mass by MALDI-TOF MS and amino acid-sequenced. This provided amino acid sequences for five p80 tryptic peptides: (peptide 65, NH2-EERVEYYIPGSTTNPEVFK-COOH, mass = 2261.3 Da; peptide 98, NH2-FAPSFRQEAXXX-COOH, mass = 2771.6 Da; peptide 38, NH2-EEGVFTVTPDTK-COOH, mass = ∼1 kDa; peptide 118, NH2-XYSLQVPSDILHLPVELXDFXXK-COOH, mass = 2723.3 Da; peptide 31, NH2-GPAVGI-COOH, mass = ∼1 kDa). See Fig. 2 (P80 peptides identified) for localization of the peptides within the p80 amino acid sequence. Sequencing of a p80 cDNA Encoding a Transmembrane CUB Domain Protein—A nondegenerate p80 oligonucleotide probe was prepared based on the sequence of p80 peptide 65 and used to screen a HK cDNA library. The peptide 65 oligonucleotide probe identified a single 2-kb cDNA. This cDNA was cloned and sequenced (GenBank™ accession number AY375452). The nucleotide sequence of the 2-kb p80 cDNA was translated in all three reading frames, one of which encoded 716 amino acids, including four of the five p80 tryptic peptides (peptides 65, 98, 118, and 38; Fig. 2) but not peptide 31 (Fig. 2). This established that the p80 cDNA encodes most of the 80-kDa protein that was purified and sequenced. In this reading frame, an in-frame stop codon was present in the p80 cDNA sequence at positions 2155–2157. PCR experiments that followed indicated that the stop codon was an error in the p80 cDNA possibly originating in the cDNA library used for screening and identifying the original p80 cDNA. The 716 amino acids included 26 residues upstream of the initiating methionine identified by a Kozak consensus sequence. Numbering from the initiating methionine, the coding sequence includes 691 amino acids and the following protein motifs (Fig. 2): an amino-terminal signal peptide with a predicted cleavage site that would yield a phenylalanine amino terminus, three extracellular CUB protein-protein interaction domains (16Bork P. Beckmann G. J. Mol. Biol. 1993; 231: 539-545Crossref PubMed Scopus (521) Google Scholar), 11 potential N-glycosylation sites consistent with the known binding of p80 to WGA, and a transmembrane domain with a short carboxyl-terminal cytoplasmic sequence. However, there were no detectable cytoplasmic tyrosine phosphorylation sites. This is inconsistent with the observed tyrosine phosphorylation of p80. Further, the absence of p80 peptide 31 in the identified 716-amino acid sequence suggested that we did not have a full-length cDNA or the complete amino acid sequence of the carboxyl terminus of p80. p80 Is a Fragment of CDCP1 Containing Cytoplasmic Tyrosine Phosphorylation Sites—Nucleotide sequence of the 2.0-kb p80 partial cDNA was compared with the human genome data base (available on the World Wide Web www.ncbi.nlm.nih.gov) and identified nearly identical sequences that localized to chromosome 3p21. Significantly, published gene sequence designated CUB domain containing protein 1 (CDCP1; accession number NM022842) and derived amino acid sequence (accession number AA033397) (9Scherl-Mostageer M. Sommergruber W. Abseher R. Hauptmann R. Ambros P. Schweifer N. Oncogene. 2001; 20: 4402-4408Crossref PubMed Scopus (110) Google Scholar) was 99% identical to the amino acid sequence of p80. Only Arg525 of p80 was distinct from Gln525 in CDCP1. Both MS-based amino acid sequencing of p80 (peptide 98) and LC MS/MS-based sequencing of peptides in gp140 confirmed that amino acid 525 was Arg (see sequence of peptides P6, 7 in Fig. 2 and peptides 6 and 7 under gp140 peptides in Fig. 4B). Consistently, a second published sequence for SIMA135 (11Hooper J.D. Zijlstra A. Aimes R.T. Liang H. Claassen G.F. Tarin D. Testa J.E. Quigley J.P. Oncogene. 2003; 22: 1783-1794Crossref PubMed Scopus (109) Google Scholar) (accession number AAK02058) was 100% identical to derived p80 amino acid sequence. Two additional unpublished but submitted sequences (accession numbers BAB15511 and AA033397) were compared and found to be nearly identical to p80 when using the MULTALIN alignment program (available on the World Wide Web at npsapbil.ibcp.fr/cgi-bin/align_multalin.pl). The CDCP1 gene was originally identified based on its elevated expression in human gastric and lung carcinomas (9Scherl-Mostageer M. Sommergruber W. Abseher R. Hauptmann R. Ambros P. Schweifer N. Oncogene. 2001; 20: 4402-4408Crossref PubMed Scopus (110) Google Scholar). Although no protein was identified, the CDCP1 gene encodes a putative transmembrane protein of 836 amino acids that extends 145 amino acids beyond the carboxyl terminal end of the 691 amino acids encoded by the p80 cDNA (Fig. 2). The additional 145 amino acids include the missing p80 nucleotide sequence for peptide 31 in the p80 cDNA. This established that p80 is CDCP1. The additional 145 residues contain 5 tyrosine residues. Two of these tyrosine residues define canonical phosphorylation sites for tyrosine kinases (residues 734 and 806; Fig. 2). In addition, two type II Src homology 3 ligand-binding sites (residues 716–721 and 772–777) were identified. The 836 amino acids and glycosylation sites of the CDCP1 gene should generate a glycoprotein considerably larger than p80. This raised the possibility that a larger or precursor form of p80 exists that is encoded by the CDCP1 gene. Suramin Promotes Tyrosine Phosphorylation of p80 and gp140 —We wished to determine whether a possible trypsin-sensitive precursor to p80 existed and whether phosphorylation of p80 might be regulated by known signaling pathways. Soluble factors (growth factors or cytokines: transforming growth factor β1, epidermal growth factor, keratinocyte growth factor, hepatocyte growth factor, interferon-γ) or drugs that regulate protein kinases (retinoic acid, TPA, suramin) were evaluated. The compounds were added to both adherent and trypsin/EDTA-suspended HKs. Effects of the compounds on phosphorylation of p80 were detected by Western blotting with anti-phosphotyrosine mAbs (PY20 or 4G10). Most of these compounds had no detectable or inconsistent effects on phosphorylation of p80. In contrast, suramin, a polysulfonated napthylurea with multiple reported activities, stimulated a rapid and selective phosphoryla
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