HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 279, Issue 15, 14772-14783, April 9, 2004

This Article Abstract Full Text (PDF) All Versions of this Article: 279/15/14772    most recent M309678200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Brown, T. A. Articles by Carter, W. G. Search for Related Content PubMed PubMed Citation Articles by Brown, T. A. Articles by Carter, W. G. Social Bookmarking                      What's this?

Adhesion or Plasmin Regulates Tyrosine Phosphorylation of a Novel Membrane Glycoprotein p80/gp140/CUB Domain-containing Protein 1 in Epithelia^*

Tod A. Brown, Tai Mei Yang, Tatiana Zaitsevskaia, Yuping Xia, Clarence A. Dunn¶, Randy O. Sigle, Beatrice Knudsen, and William G. Carter¶||

From the Fred Hutchinson Cancer Research Center, Seattle, Washington 98109 and the ^¶Department of Pathobiology, University of Washington, Seattle, Washington 98195

Received for publication, September 2, 2003 , and in revised form, January 20, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Wounding of quiescent epidermis activates changes in adhesion and cell signaling resulting in keratinocyte migration, basement membrane (BM)¹ repair, and wound closure (1–3).

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) (4). Phosphorylated p80 (P-p80) in suspended HKs is dephosphorylated upon readhesion to laminin 5 via integrins ₆₄ and ₃₁. In work here, we purified and characterized p80. We wished to understand whether phosphorylated p80 identified in an in vitro deadhesion/readhesion screen (4) might be involved in physiologically significant wound activation.

Quiescent epidermis adheres to laminin 5 in the BM via integrin ₆₄ in hemidesmosome (HD) cell junctions. Wounding epidermis generates leading and following subpopulations of keratinocytes at the wound margin (5). Leading keratinocytes migrate over exposed dermal collagen via integrin ₂₁ and fibronectin via integrin ₅₁. However, leading cells also deposit laminin 5 as a provisional BM and interact with these deposits via integrin ₃₁ (5–7). 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 ₃₁ promotes gap junction intercellular communication when compared with adhesion to collagen or fibronectin (5). 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 (6). Cell interaction with laminin 5 (6) or laminin 10 (8) via ₃₁ 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 (4). 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 (4). 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 (9), is a marker for hematopoietic stem cells, (10) and is identical to SIMA135, a membrane glycoprotein elevated in metastatic human tumor cells (11). (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 (12). (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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 (13), 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 (14) 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 (4). 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 x g. The membrane fraction was extracted with buffer A for 30 min at 4 °C and centrifuged (4 min, 2000 x 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).

View larger version (23K): [in this window] [in a new window]   FIG. 2. Model for p80/gp140/CDCP1 identifying protein motifs, subdomains, and sequenced peptides. Peptides sequenced (P65, P118, etc.) in purified p80 are indicated at the right (P80 peptides identified). Peptides sequenced (P1, P2, etc.) from purified gp140 are indicated at the left (gp140 peptides identified). N39, N122, etc. indicates glycosylated asparagine residues. Y734 and Y806 identify possible phosphorylated tyrosine residues. An approximate location for cleavage of gp140 by trypsin generating p80 is indicated. Functional domains (signal peptide, CUB domain, etc.) are as indicated.

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 [-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 gtll 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, 5x SSPE, 1x 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 (2x 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).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Adhesion Regulates Phosphorylation and Dephosphorylation of p80 —As previously shown (4), 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 ₆₄ and ₃₁ 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 (4). 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.

View larger version (65K): [in this window] [in a new window]   FIG. 1. Adhesion regulates phosphorylation and dephosphorylation of p80. Adherent HKs (A, lane 1) were suspended by digestion with trypsin-EDTA (S, lane 2) and then readhered (Re-adherent, lanes 3–6) to laminin 5 surfaces for the indicated time periods (10 min, 30 min, etc.). Cells were extracted with Triton X-100 detergent containing vanadate, NEM, and NaF as phosphatase inhibitors (Buffer A). Soluble cell extracts (75 µg of protein) were fractionated by SDS-PAGE, transferred to nitrocellulose, and Western blotted with mAbs against phosphotyrosine residues (4G10). Migration of P-p80 is indicated (P80). Migration of tyrosine-phosphorylated FAK and epidermal growth factor receptor (EGFR) were established by immunoblotting or immunoprecipitation with specific antibodies. *, an uncharacterized phosphorylated protein migrating above FAK.

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^TM accession number AY375452 [GenBank] ). 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 (16), 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 [PDB] . Significantly, published gene sequence designated CUB domain containing protein 1 (CDCP1; accession number NM022842) and derived amino acid sequence (accession number AA033397 [GenBank] ) (9) 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 (11) (accession number AAK02058 [GenBank] was 100% identical to derived p80 amino acid sequence. Two additional unpublished but submitted sequences (accession numbers BAB15511 [GenBank] and AA033397 [GenBank] ) 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).

View larger version (43K): [in this window] [in a new window]   FIG. 4. A, purification of P-gp140 from suramin-treated adherent HKs. P-gp140 was purified utilizing the basic protocol described for purification of P-p80 (see "Experimental Procedures") except that suramin-treated adherent HKs were used. Lanes 1–8 are immunoblots with anti-phosphotyrosine mAb (4G10) of the cell extracts prepared as follows. HKs were Dounce-homogenized in buffer A (without Triton X-100 detergent), centrifuged (100,000 x g) to collect a membrane-rich pellet. The soluble cyotosolic fraction (C, lane 1) contains 50% of the total cell protein but no gp140. The membrane pellet was resuspended in buffer A with Triton X-100 and then recentrifuged to collect the soluble membrane fraction containing gp140 (M, lane 2). The soluble membrane extract was incubated with immobilized WGA, and an unbound (U, lane 3) and a bound fraction (E, lane 4; eluted with 400 mM N-acetylglucosamine) were collected. The eluted gp140 was incubated with immobilized anti-phosphotyrosine mAb (4G10), and unbound (U, lane 5) and bound fractions (lanes 6–8; eluted with 8 M urea containing 0.1% Triton X-100) were collected. A silver stain of pooled fractions is shown (lane 9). The eluted gp140 was gel-purified on preparative SDS-PAGE gel and analyzed by LC-MS/MS. B, identification of gp140 as CDCP1 by LC MS/MS. In-gel purified gp140 and a negative control gel sample were trypsin-digested and analyzed by LC MS/MS. The MS scan was followed by MS/MS scans of the three most abundant ions from the MS scan that were not present in the negative control samples. The identified peptides unique to the gp140 sample were searched against the NCBI nonredundant protein data base. The peptide matches were considered valid if the identified protein contained at least two peptides with Xcorr scores above 2.0. We obtained results on two separate purifications of gp140 (Samples A and B), both indicating that gp140 is CDCP1.

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 1, epidermal growth factor, keratinocyte growth factor, hepatocyte growth factor, interferon-) 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 phosphorylation of an 80-kDa protein (Fig. 3A, lanes 2 and 4). The phosphorylated 80-kDa protein induced by suramin (35 µM) co-migrated with P-p80 induced by trypsin detachment (Fig. 3A, lane 5). Incubation times as short as 1 min and as long as 30 min had similar activating effects on phosphorylation of p80. The identity of the detachment-dependent p80 and the suramin-dependent 80-kDa protein is established below, and for continuity both shall be referred to here as p80.

View larger version (33K): [in this window] [in a new window]   FIG. 3. A, suramin promotes tyrosine phosphorylation of gp140, a trypsin-sensitive precursor of p80. Adherent (lanes 1 and 2) or trypsin-EDTA rounded HKs (lanes 3 and 4) were treated with (+) or without (–) suramin (35 µM, 15 min). For comparison, trypsin-EDTA-suspended HKs (lane 5) were prepared without suramin and are shown to indicate the migration of P-p80 generated by suspension alone. HKs were extracted with Triton X-100 plus vanadate and Western blotted with anti-phosphotyrosine mAb (4G10). B, time course re-expression of gp140 after conversion with trypsin. Adherent HKs (lane 1) or trypsin/EDTA-suspended HKs (lane 2) were treated with suramin for 15 min prior to extraction with Triton X-100 detergent (buffer A). Trypsin/EDTA suspended HKs (lanes 3–10) were readhered on laminin 5 surfaces for the indicated times (0.5 h, 1 h, etc.) and then were either incubated (+, lanes 3–9) or not (–, lane 10) with suramin for the last 15 min of the readhesion period. All HKs were extracted with Triton X-100 (buffer A) and immunoblotted with an anti-FAK Tyr(P)861 Ab. This Ab cross-reacts with P-gp140 and P-p80 and is characterized in Fig. 6. P-gp140 reappeared 2–4 h after the initial trypsin cleavage.

In a series of controls (results not shown), cycloheximide, an inhibitor of protein translation, blocked re-expression of gp140 after trypsin digestion. Trypsin-suspended HKs re-expressed gp140 even when left in suspension, indicating that re-expression of gp140 did not require readhesion. Treatment of adherent HKs with Ca²⁺ (1 mM) for 24 h prior to trypsin digestion or inclusion of Ca²⁺ (1 mM) or EDTA (5 mM) during the trypsin digestion had no effect on levels of phosphorylated p80 or gp140 induced by suramin. Suramin treatment of cultured mouse keratinocytes also promoted tyrosine phosphorylation of p80 and gp140 and had similar effects on cultures of esophageal and cervical epithelial cells but not fibroblasts. This indicates that P-gp140/P-p80 is widely expressed in epithelium. Suramin homologues were also found to promote phosphorylation of p80 and gp140: PPADS and NF023, antagonists of ATP binding to purinergic receptors, duplicated the effects of suramin and promoted phosphorylation of p80 and gp140. Surprisingly, the membrane-impermeable dye trypan blue, used routinely to assay cell viability, is also an analogue of suramin and also induced phosphorylation of gp140/p80. XAMR 0721, a nonsymmetric suramin analogue containing only one of the two polysulfonated arms (17), failed to duplicate the effects of suramin. This established that suramin or trypan blue promotes phosphorylation of gp140 and p80 by interacting with an extracellular binding site(s) or receptor(s). The results also suggested that trypsin may convert gp140 to p80.

Purification of P-gp140 and Identification as a Trypsin-sensitive Precursor to p80 —gp140 was purified and identified by LC MS/MS to determine whether it is a trypsin-sensitive precursor to p80 and the native form of CDCP1. Adherent HKs were treated with suramin to promote phosphorylation of gp140. P-gp140 was purified utilizing a variation of the protocol used for purification of P-p80. A membrane-rich fraction of suramin-treated HKs containing P-gp140 was solubilized with Triton X-100 detergent and sequentially purified with immobilized WGA and then with immobilized anti-phosphotyrosine mAb (4G10) and finally with preparative SDS-PAGE (Fig. 4A, lanes 2, 4, and 6). In-gel purified gp140 was compared with blank lanes of the same gel by LC MS/MS. CDCP1 was the only protein identified in the gp140 samples above common background proteins (keratins, serum albumin, titin). Identical results were obtained in two separate purifications/identifications of P-gp140 (Fig. 4B, Sample A and Sample B). The most abundant peptide ions in the two samples of P-gp140 (Fig. 4B) were searched against the NCBI nonredundant data base. This search identified gp140 as CDCP1. The localization of the identified peptides within gp140/CDCP1 is summarized in Fig. 2. We conclude that cell surface gp140 is the native protein encoded by the CDCP1 gene and a trypsin-sensitive precursor to p80. Trypsin removes 35 kDa of peptide and 25 kDa of carbohydrate from the amino terminus of gp140 to generate transmembrane p80 (see Fig. 2). p80 tryptic peptide 65 was the most amino-terminal peptide identified in p80 after trypsin digestion of gp140 in HKs. Therefore, tryptic cleavage must occur at an unidentified site of gp140 on the amino-terminal side of peptide 65 in CUB domain 1.

SFKs Phosphorylate and PTPs Dephosphorylate gp140/p80 —Suramin may increase phosphorylation of gp140 by activating a tyrosine kinase(s) and/or inhibiting a PTP(s). Kinases that might mediate tyrosine phosphorylation of p80 and gp140 were examined with selective inhibitors. Inhibitors of Src family kinases (SFKs), PP2 (18) and SU6656 (19), selectively reduced phosphorylation of p80 and gp140 in suramin-treated adherent HKs (Fig. 5A, lanes 3 and 4). Consistently, PP2 and SU6656 also inhibited phosphorylation of p80 in trypsin-suspended HKs (results not shown). In contrast, neither inhibitor blocked phosphorylation of FAK. Additional kinase inhibitors including wortmannin (phosphatidylinositol 3-kinase; Fig. 5A, lane 5) and others (piceatannol, genistein, herbimycin A, calphostin C, quercetin, and tyrphostins 1478, 1295, 1024, and 490; results not shown), had either no effect or minimal effects on phosphorylation of gp140 or p80. A concentration curve for PP2 and SU6656 (Fig. 5B) indicated half-maximal inhibition at 2 nM and 100 nM, respectively, for inhibition of p80 phosphorylation when detected by Western blotting with anti-phosphotyrosine mAb (4G10).

View larger version (18K): [in this window] [in a new window]   FIG. 5. A, PP2 and SU6656 block phosphorylation of gp140/p80. Adherent HKs were incubated without (–) or with (+) the indicated kinase inhibitors for 20 min prior to the addition of suramin (35 µM, an additional 20 min). All HKs were extracted with Triton X-100 plus vanadate (buffer A) and immunoblotted with anti-phosphotyrosine mAb 4G10. Only SU6656 (lane 3) and PP2 (lane 4) significantly inhibited gp140/p80 phosphorylation. B, concentration curves for inhibition of p80 phosphorylation by PP2 and SU6656. Adherent HKs were treated for 30 min with the indicated concentrations of PP2 or SU6656, and then suramin was added (an additional 15 min). Cells were extracted with Triton X-100 (buffer A), and extracts were subjected to SDS-PAGE and Western blotting with anti-phosphotyrosine mAb (4G10). The blots for P-p80 are shown above the quantitation curves for P-p80. The P-p80 band was chosen for its lack of nearby overlapping bands and quantified by densitometry using Scion Image software (Scion Corp., Frederick, MD). The concentration of drug resulting in a 50% inhibition of maximal phosphorylation (IC50) was estimated to be 2 nM (PP2) and 100 nM (SU6656), respectively. C, inhibition of PTPs with vanadate or calpeptin duplicate suramin to phosphorylate gp140. Adherent HKs were either untreated (lane 2) or treated for 20 min with 35 µM suramin (lane 1), 1 mM sodium orthovanadate (VO 4; lanes 3 and 4), 100 µg/ml calpeptin (lane 5), or 20 µM dephostatin (lane 6). Cells were extracted with Triton X-100 detergent (buffer A), and extracts were subjected to SDS-PAGE and immunoblotted with anti-phosphotyrosine mAb (4G10). The cells used for lane 3 were pretreated with 5 µM SFK inhibitor SU6656 for 20 min prior to orthovanadate incubation, thus preventing phosphotyrosine accumulation.

Suramin increased phosphorylation of gp140 and p80 via SFKs but did not increase phosphorylation of other SFK substrates including FAK (Fig. 3A, compare P-FAK in lanes 1 and 2). This indicated that suramin was not generally activating SFKs. We confirmed this finding by blotting FAK with a rabbit polyclonal antibody against an Src phosphorylation site, phosphotyrosine residue 861 in FAK (FAK Tyr(P)⁸⁶¹ Ab; Bio-Source). Although the anti-FAK Tyr(P)⁸⁶¹ Ab reacts with P-FAK, it also cross-reacts with P-p80 and P-gp140 after treatment with suramin (Fig. 6, lanes 1 and 7). In contrast, Abs against phosphotyrosine residue 397 in FAK (Ab FAK Tyr(P)³⁹⁷; BioSource) did not cross-react with P-p80 or P-gp140 (Fig. 6, lane 13).

View larger version (57K): [in this window] [in a new window]   FIG. 6. Anti-FAK Tyr(P)861 antibody cross-reacts with Tyr734 of P-gp140 and P-p80. Adherent (adherent) or trypsin rounded (+trypsin) HKs were treated with suramin (15 min, 35 µM) and extracted with buffer A. The extracts were fractionated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibody against FAK (Tyr(P)861) (pY861) (lanes 1–12) or FAK (Tyr(P)397) (pY397) (lanes 13–16). The anti-FAK (Tyr(P)861) Ab, but not the anti-FAK (Tyr(P)397) Ab, cross-reacted with P-gp140 and P-p80. Where indicated, a 100-fold molar excess of a synthetic phosphopeptide from FAK (Tyr(P)861; pY861) or the identical nonphosphorylated peptide sequence (Tyr861; Y861) was added during the antibody incubation. Synthetic phosphopeptides from gp140 (Tyr(P)734, Tyr(P)743, and Tyr(P)801 (pY734, pY743, and pY801)) were also tested. Only the gp140 Tyr(P)734 phosphopeptide had inhibitory activity toward P-gp140/P-p80 and was specific for Ab FAK Tyr(P)861. Note that the phosphopeptide from gp140 (pY734) had no effect on the interaction of anti-FAK (pY397) with FAK.

View larger version (54K): [in this window] [in a new window]   FIG. 7. Expression of P-gp140-GFP in 293T cells. 293T cells were transiently transfected with nothing (lane 1), GFP cDNA (lane 2), or gp140-GFP cDNA (lane 3) and then cultured in the absence of serum. Triton X-100 extracts (buffer A) were prepared from the cells, fractionated by SDS-PAGE, and then detected by protein stain (A) or immunoblotted with anti-GFP Ab JL-8 (B) and anti-FAK Tyr(P)861 Ab (C) that cross-reacts with gp140 Tyr(P)734. Where indicated, the 293T cells were treated before extraction with suramin (35 µM for 10 min), trypsin for detachment, or mechanical agitation for detachment. gp140-GFP is detected as a single 165-kDa band by both anti-GFP and anti-FAK Tyr(P)861 Abs and only in cells transfected with gp140-GFP. Tryspin converts gp140-GFP to p80-GFP, detaches cells, and dephosphorylates non-cleaved gp140-GFP but does not dephosphorylate p80-GFP. Suramin increases phosphorylation of gp140-GFP above base-line phosphorylation. Mechanical detachment by shaking resulted in the dephosphorylation of gp140-GFP.

P-gp140/P-p80 and gp140-GFP Localize to the Plasma Membrane—gp140-GFP localized to the plasma membrane when expressed in mouse keratinocytes (Fig. 8A) or 293T cells (results not shown). Therefore, we determined whether immunostaining of HKs with Ab FAK Tyr(P)⁸⁶¹ could detect a subcellular localization for P-gp140/P-p80 and whether suramin and PP2 could regulate the subcellular localization. Adherent HKs were treated with or without suramin, fixed, and permeabilized and then stained with Ab FAK Tyr(P)⁸⁶¹ that reacts with gp140 phosphotyrosine 734 (Fig. 8B). Without suramin, there was minimal organized staining with Ab FAK Tyr(P)⁸⁶¹ in the apical-lateral focal planes of the adherent HKs (Fig. 8B, a). In controls (results not shown), Ab FAK Tyr(P)⁸⁶¹ stained the expected focal adhesions in the basal focal plane co-localized with anti-FAK Ab staining. However, in the presence of suramin, Ab FAK Tyr(P)⁸⁶¹ stained plasma membrane on lateral and apical plasma membranes of the adherent HKs above the basal focal plane and distinct from anti-FAK staining in focal adhesions (Fig. 8B, b). Phosphopeptide gp140 Tyr(P)⁷³⁴ blocked the apical and lateral membrane staining (Fig. 8B, c). Suramin-induced staining at lateral membranes was inhibited by pretreatment of cells with PP2, consistent with phosphorylation of Tyr⁷³⁴ being mediated by SFKs (Fig. 8B, d). We conclude that phosphorylation of gp140/CDCP1 at tyrosine 734 in the plasma membrane of confluent HKs is in a state of dynamic equilibrium mediated by SFK and PTP activities generating a low net phosphorylation of gp140 unless suramin is added.

View larger version (79K): [in this window] [in a new window]   FIG. 8. Localization of gp140 in keratinocytes. A, mouse keratinocytes were transfected with a gp140-GFP cDNA, and then gp140-GFP protein was detected in the plasma membrane in live cells. B, HKs grown on glass coverslips were incubated with or without suramin (35 µM, 20 min), fixed (2% formaldehyde in cacodylate buffer containing vanadate and NEM), permeabilized (0.1% Triton X-100 for 1 min), and then stained with Ab FAK Tyr(P)861. All photos were taken in a focal plane vertically midway through the cells. a, in the absence of suramin, staining was localized to focal adhesions (not visible) determined by double staining with anti-FAK antibody. b, in the presence of suramin, staining was elevated in the apical and lateral areas of the plasma membrane and not co-distributed with FAK in the basal focal plane. c, treatment of HKs with phosphopeptide gp140 Tyr(P)734 before (>) reaction with Ab FAK Tyr(P)861 blocked staining of apical/lateral areas. d, treatment of HKs with PP2 to inhibit SFKs before (>) the addition of suramin inhibited the apical/lateral plasma membrane staining.

View larger version (37K): [in this window] [in a new window]   FIG. 9. Phosphorylation of Tyr734 is distinct in gp140 and p80. A, dephosphorylation of gp140 and p80 by PTP after removal of suramin. HKs were grown for 3 days to generate predominantly gp140 or trypsin-rounded to generate p80. The adherent or rounded cells were treated with a pulse of suramin (20 min, 35 µM), washed to remove suramin, and cultured for the indicated times. In controls, identical cultures of the adherent HKs were treated similarly, except that suramin was also added during the final 15 min prior to extraction (+Suramin) to show the presence of gp140 protein. Extracts were prepared with buffer A, fractionated by SDS-PAGE, and Western blotted with Ab FAK Tyr(P)861. P-gp140 was dephosphorylated in less than 1 h, whereas P-p80 was dephosphorylated between 4 and 6 h. P-FAK detected with Ab FAK Tyr(P)861 was unaffected. Thus, P-gp140 and P-p80 are dephosphorylated by a PTP at different rates, explaining the relative ease of detection of P-p80 in cultured HKs. B, concentration of suramin to promote phosphorylation of gp140 is distinct from p80. As indicated, adherent HKs were treated with various concentrations of suramin for 20 min prior to extraction, SDS-PAGE, transfer to nitrocellulose, and analysis of P-gp140 and P-p80 levels by immunoblotting with the FAK Tyr(P)861 Ab. Band intensity was quantified using LiCor Odyssey software. p80 and gp140 exhibited distinct sensitivities to suramin concentration. The half-maximal stimulation of phosphorylation (SC50) occurred at 1.5 µM (gp140) and 40 µM (p80).

Adhesion-dependent Phosphorylation of gp140 Is Distinct from p80 —We determined whether adhesion also regulates phosphorylation of gp140 differently than phosphorylation of p80. Evaluating this difference is complicated by the fact that trypsin used to suspend cells also converts gp140 to p80. To overcome this problem, we used the following approaches. First, we found that suspension of HKs with EDTA failed to induce phosphorylation of gp140, whereas suramin treatment of the EDTA-suspended cells still induced phosphorylation of gp140 (data not shown). In contrast, trypsin-detachment of HKs that convert gp140 to p80 increased phosphorylation of p80 (Fig. 1, lane 2, and Fig. 3, lane 5). Apparently, detachment affected phosphorylation of p80 differently than gp140. Second, we determined whether deadhesion alters phosphorylation of gp140-GFP distinct from p80-GFP. Trypsin detachment of 293T cells converts gp140-GFP to p80-GFP, and the p80-GFP remained phosphorylated in the detached cells (Fig. 7, B and C, lane 9). Surprisingly, the gp140-GFP that was resistant to trypsin was not phosphorylated. This raised the possibility that the trypsin detachment dephosphorylated gp140 but not p80. This possibility was evaluated as follows. The 293T are easily detached from the substratum by agitation without trypsin digestion or EDTA. Detachment of the 293T cells with agitation dephosphorylated gp140-GFP (Fig. 7B, lane 12, compared with Fig. 7C, lane 12). We conclude that phosphorylation of gp140 and p80 are both altered by adhesion, but they are regulated differently; detachment dephosphorylates gp140 and phosphorylates p80. Proteolytic conversion of gp140 to p80 distinguishes phosphorylation of p80 from phosphorylation of gp140. After conversion, both suramin- and adhesion-dependent phosphorylation of p80 is distinct from gp140.

Conversion of gp140 to p80 by Plasmin—Here, we evaluated possible trypsin-like proteases that might convert gp140 to p80 as a physiological event in wounds. Wounding of epidermis exposes leading keratinocytes to trypsin-like proteases in tissue and serum that regulate coagulation, fibrinolysis, and tissue remodeling and might convert gp140 to p80. Therefore, we determined whether exogenous plasmin, a trypsin-like protease in serum that contacts leading keratinocytes in wounds, could convert gp140 to p80. Digestion of gp140 in HKs (Fig. 10A) or gp140-GFP in 293T cells (Fig. 10B) with purified plasmin was sufficient to convert gp140 to p80. A plasmin titration curve indicated that 1 µg plasmin/ml on adherent HKs for 1–3 h (plasmin/substrate ratio = 1:1000) was sufficient to decrease gp140 and increase p80. This level of plasmin is well below the concentration of plasminogen in plasma (120 µg/ml) (21). Thrombin, a trypsin-like protease involved in clot formation, failed to convert gp140 to p80 under conditions where it converts precursor laminin 5 to mature laminin 5 (results not shown). Digestion with plasmin had no detectable effect on cell-cell or cell-substrate adhesion in contrast to trypsin that rounds and detaches cells. Consistently, plasmin conversion, like trypsin (Fig. 9A), prolonged phosphorylation of p80 relative to gp140 (data not shown).

View larger version (23K): [in this window] [in a new window]   FIG. 10. Plasmin converts gp140 to p80. A, exogenous plasmin converts gp140 to p80 in HKs. HKs, grown in serum-free, defined medium, were incubated with (+) or without (–) exogenous purified plasmin (1 µg of plasmin/ml/mg of cell protein) and/or EDTA (5 mM) for 3 h. The cells were treated with suramin (35 µM, 20 min) and then extracted with 1% Triton X-100 (Buffer A). These soluble Triton extracts (75 µg of protein of each) were immunoblotted with anti-FAK Tyr(P)861 Ab. Plasmin converts gp140 to p80, whereas EDTA added to round the cells had no detectable effect on the cleavage. B, exogenous plasmin converts gp140-GFP to p80-GFP in 293T cells. 293T cells expressing gp140-GFP cDNA as described under "Experimental Procedures" and in the Fig. 7 legend were digested with purified plasmin (1 µg of plasmin/ml/mg of cell protein) for 1 or 3 h as indicated and then extracted with 1% Triton X-100 detergent (buffer A). The soluble Triton extracts (75 µg of protein each) were immunoblotted with anti-GFP Ab. Plasmin converted gp140-GFP to p80-GFP. C, an endogenous protease converts gp140 to p80 in epidermis. Epidermis from a neonatal mouse was treated with suramin (35 µM, 20 min) and then homogenized in 1% Triton X-100 in buffer A. The soluble Triton extract (75 µg of protein) was immunoblotted with anti-FAK Tyr(P)861 Ab. Both P-gp140 and P-p80 bands were detected, indicating the presence of endogenous plasmin or trypsin-like protease that cleaves gp140 to p80. The epidermis was obtained from a neonatal mouse with homozygous null mutations in the LAMA3 gene encoding the 3 chain of laminin 5. This laminin 5 defect generates neonatal lethal blistering, allowing for easy separation of the epidermis from dermis without use of exogenous protease (26).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In cultures of epidermal, prostate, cervical, and esophageal epithelial cells, phosphorylation of p80 is increased by cell detachment, whereas reattachment to the endogenous substrate, laminin 5, dephosphorylates P-p80 (Fig. 1).² Here, we purified P-p80 from trypsin-suspended HKs and P-gp140 from suramin-treated adherent HKs and expressed a recombinant gp140-GFP protein. Conclusions from the findings under "Results" are integrated in a diagram in Fig. 11 and are as follows. (i) gp140 is a transmembrane glycoprotein and the trypsin-sensitive precursor to p80 (Figs. 1, 3, and 7). gp140 and p80 contain three extracellular CUB protein-protein interaction domains of unknown function (Fig. 2). (ii) gp140 is the product of the CDCP1 gene that is overexpressed in human colorectal and lung cancers (9). (iii) Plasmin in serum that contacts leading cells in wounds converts gp140 to p80 (Fig. 10). (iv) Proteolytic conversion increases phosphorylation of p80 relative to gp140 in response to extracellular signals from adhesion or suramin (Figs. 7 and 9). (v) Tyrosine phosphorylation of gp140 on Tyr⁷³⁴ (Fig. 6) and possibly other tyrosine residues is mediated by SFK(s) (Fig. 5). Phosphorylation of gp140 is balanced by dephosphorylation by an unidentified PTP(s), yielding low equilibrium phosphorylation of gp140 (Fig. 5). (vi) Extracellular suramin, plasmin, or adhesion regulates phosphorylation of gp140 and p80 by perturbing the balance of SFK and PTPs (Figs. 9 and 11). We conclude that plasmin conversion of gp140 to p80 alters the phosphorylation balance mediated by SFKs and PTPs. Conversion prolongs phosphorylation of p80 relative to gp140 in response to adhesion or suramin. We suggest that plasmin cleavage and adhesion provide transmembrane signals that regulate phosphorylation of gp140 distinct from p80 in epithelial wounds.

View larger version (22K): [in this window] [in a new window]   FIG. 11. Plasmin conversion of gp140 to p80 prolongs phosphorylation of p80 in response to suramin or changes in adhesion. Results are summarized in a model that integrates outside-in signals from plasmin cleavage, substrate adhesion, and suramin that differentially regulate phosphorylation of gp140/CDCP1 and p80 in keratinocytes. gp140/CDCP1 is a transmembrane glycoprotein containing three extracellular CUB domains. Phosphorylation at cytoplasmic Tyr734 of gp140 is dynamically balanced by SFK and PTP activities, yielding low equilibrium phosphorylation. Plasmin or trypsin converts gp140 to p80 by removal of an extracellular fragment(s). After conversion, signals from extracellular suramin or substrate extracellular matrix through uncharacterized surface receptors (?) unbalance SFKs and PTPs (arrows) to prolong phosphorylation (Pi) of p80 relative to gp140.

gp140/p80 is encoded by the CDCP1 gene, which is elevated in human lung and colon tumors. During the preparation of this manuscript, a novel cell surface glycoprotein, SIMA135, was reported as the product of the CDCP1 gene and a marker for metastasis (11). P-p80 was originally identified in a screen for adhesion-dependent phosphorylation in an epidermal wound model (4). Chronic wound activation associated with inherited blistering diseases is a cancer promoter (25). It will be interesting to determine whether gp140/p80 phosphorylation or expression is altered in mice with adhesion defects generating chronic wounds (26).

Physiological Plasmin Converts gp140 to p80 —Extracts of epidermis prepared without exogenous protease express both P-gp140 and P-p80 (Fig. 10C). This indicates that proteolytic conversion of gp140 to p80 occurs in tissue via an endogenous protease. As a source of epidermis, we utilized mice with adhesion defects in laminin 5, allowing for separation of the epidermis without exogenous protease. This raises the interesting possibility that conversion of gp140 to p80 is elevated in laminin 5 defects relative to wild type epidermis. Plasmin, a trypsin-like protease in serum, converts gp140 to p80 (Fig. 10, A and B). Together, these results suggest that serum plasmin contacts leading keratinocytes in wounds and may convert gp140 to p80.

Studies are under way to identify the location of the trypsin and plasmin cleavage site(s) in gp140. Conversion of gp140 to p80 leaves most of the first CUB domain intact along with the complete second and third CUB domains, the transmembrane, and the cytoplasmic tyrosine phosphorylation sites. Sequencing of tryptic peptides from purified p80 identified peptide 65 (residues 278–296 of gp140) as the most amino-terminal of the five identified p80 peptides (Fig. 2). When trypsin cleaves gp140, the resulting p80 contains peptide 65. Reasonably, trypsin cleaves gp140 at Lys/Arg residues 261, 276, or 277 on the amino-terminal side of peptide 65. The extended optimal substrate specificities for plasmin have been identified as follows: P4-Lys/norleucine/Val/Ile/Phe, P3-Xaa, P2-Tyr/Phe/Trp, P1-Lys/Arg (27). Candidate se