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

Abstract

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 Tyr 734 . 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) 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 ␣ 6 ␤ 4 and ␣ 3 ␤ 1 . 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 ␣ 6 ␤ 4 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 ␣ 2 ␤ 1 and fibronectin via integrin ␣ 5 ␤ 1 . However, leading cells also deposit laminin 5 as a provisional BM and interact with these deposits via integrin ␣ 3 ␤ 1 (5-7). The interaction of leading cells with deposited laminin 5 generates distinct transmembrane signals when compared with interaction with dermal ligands. For example,...

CDCP1 Regulates the Function of MT1-MMP and Invadopodia-Mediated Invasion of Cancer Cells

“…Complement C1r/C1s, Uegf, Bmp1 (CUB) domaincontaining protein 1 (CDCP1), also described as SIMA135 and TRASK (1,2), is a type 1 transmembrane protein with 3 CUB domains as the extracellular domains and several tyrosine residues that can be phosphorylated by Src family kinases (SFK) in the intracellular domain (1)(2)(3)(4)(5)(6). The expression of CDCP1 was reported in several human malignancies, such as colon and breast cancers (3,7).…”

The cell surface glycoprotein CDCP1 in cancer—Insights, opportunities, and challenges

“…For example, trypsin treatment of keratinocytes results in loss of HMW-CDCP1-p-Y734 and appearance of LMW-CDCP1-p-Y734. On balance it appears, as proposed by Brown et al (3), that proteolytic conversion results in increased phosphorylation of LMW-CDCP1 as shown in Fig. 2C.…”

“…2B). For example, trypsin treatment of keratinocytes in vitro generates 80-kDa CDCP1 via cleavage at, or amino terminal of, E278 (3,5). It appears that similarly sized LMW-CDCP1 species can also be generated by endogenous mechanisms, as treatment of adherent keratinocytes with the polysulfonated membrane impermeable naphthylurea, suramin and several of its analogues, results in the appearance of SFK-phosphorylated LMW-and HMW-CDCP1 (3).…”

“…1A). The extracellular domain contains three regions with low homology to complement protein subcomponents C1r/C1s, urchin embryonic growth factor, and bone morphogenetic protein 1 (CUB) domains, as well as 14 consensus N-glycosylation sites and 20 cysteines likely involved in disulfide bond formation (1)(2)(3)(4). Interestingly, 12 of the consensus N-glycosylation sites and 19 of the extracellular cysteines are conserved in human, chimpanzee, dog, cow, mouse, and rat-N39 is not found in dog, cow, mouse, and rat, N339 is not found in mouse, and C423 is not present in rat (Fig.…”

“…CUB domain-containing protein 1 (CDCP1) (1) is a cell surface glycoprotein also known as subtractive immunization associated 135 kDa (SIMA135) (2), gp140 (3), and transmembrane and associated with Src kinases (Trask) (4) and has been assigned the cluster of differentiation (CD) designation CD318. In hindsight, the independent identification of CDCP1, using several different biased genetic and protein based approaches, provided the initial indications of the potential importance of this protein in cancer progression.…”