Proteolysis-induced N-terminal Ectodomain Shedding of the Integral Membrane Glycoprotein CUB Domain-containing Protein 1 (CDCP1) Is Accompanied by Tyrosine Phosphorylation of Its C-terminal Domain and Recruitment of Src and PKCδ

Abstract

CUB-domain-containing protein 1 (CDCP1) is an integral membrane glycoprotein with potential as a marker and therapeutic target for a number of cancers. Here we examine mechanisms regulating cellular processing of CDCP1. By analyzing cell lines exclusively passaged non-enzymatically and through use of a panel of protease inhibitors, we demonstrate that full-length 135 kDa CDCP1 is post-translationally processed in a range of cell lines by a mechanism involving serine protease activity, generating a C-terminal 70-kDa fragment. Immunopurification and N-terminal sequencing of this cell-retained fragment and detailed mutagenesis, show that proteolytic processing of CDCP1 occurs at two sites, Arg-368 and Lys-369. We show that the serine protease matriptase is an efficient, but not essential, cellular processor of CDCP1 at Arg-368. Importantly, we also demonstrate that proteolysis induces tyrosine phosphorylation of 70-kDa CDCP1 and recruitment of Src and PKC␦ to this fragment. In addition, Western blot and mass spectroscopy analyses show that an N-terminal 65-kDa CDCP1 ectodomain is shed intact from the cell surface. These data provide new insights into mechanisms regulating CDCP1 and suggest that the biological role of this protein and, potentially, its function in cancer, may be mediated by both 70-kDa cell retained and 65-kDa shed fragments, as well as the full-length 135-kDa protein. CUB-domain-containing protein 1 (CDCP1)3 is an 836 amino acid integral membrane glycoprotein with a type I orientation at the cell surface (1-4), that is up-regulated in a number of malignancies including breast (1, 5, 6), colon (1, 2, 7), and lung (1) cancers. Of potential clinical significance, CDCP1 expression correlates with recurrence and patient survival rate in renal cell carcinomas (8) and lung adenocarcinomas (9), indicating that it may be suitable as a prognostic marker. Consistent with a role in cancer progression, silencing of CDCP1 reduced the metastatic ability of lung cancer A549 cells (10) and the peritoneal dissemination of gastric cancer 44As3 cells (11) in mice. Although its biological function is not known, the potential of CDCP1 as a therapeutic target for cancer treatment has been highlighted by studies showing that antibody-mediated inhibition of CDCP1 reduced metastasis of prostate cancer PC3 cells in mice (12, 13) and chicken embryos (13). Currently the mechanisms regulating CDCP1 in cancer and normal physiology are not well defined (14).During cellular processing, the 29-residue CDCP1 N-terminal signal peptide is removed generating a protein with molecular mass identified as either 135 kDa (2, 10, 15) or 140 kDa (3, 4) that contains 30 -40 kDa of N-linked glycans (2). In addition to this full-length form, there is evidence that a shorter CDCP1 species is expressed endogenously by a range of cell lines or is generated through the action of exogenous serine proteases. For example, lung cancer A549, PC14, H520, H322, and H157 cells (10) and gastric cancer 44As3 and 58As9 cells (11) resuspended non-enzymatically ...

Trask phosphorylation defines the reverse mode of a phosphotyrosine signaling switch that underlies cell anchorage state <link href="file

“…The 80 kD form is a proteolytic product of the 140 kD form and can be generated by serine proteases including cellular proteases such as MT-SP1 or proteases used in cell culture such as trypsin. 24,25,28 As such, the use of trypsin to induce cell detachment results in the concomitant cleavage and phosphorylation of Trask. But the use of EDTA more clearly reveals the detachment-induced phosphorylation of Trask without the confounding attribute of cleavage.…”

“…26,27 Trask is a 140 kD type I transmembrane glycoprotein that undergoes proteolytic cleavage within its extracellular domain (ECD) by serine proteases including MTSP1, plasmin and trypsin, generating a smaller 80-85 kD product. 24,25,28 Trask is not just a minor component of the phosphotyrosine proteome of unanchored epithelial cells; rather it accounts for the entirety of the abundant 140 and 80 kD signals seen on phosphotyrosine immunoblots. To determine this we depleted Trask from detached MCF10A cell lysates by two methodologies.…”

“…29,31 One group has suggested a link between Trask/CDCP1 cleavage and its phosphorylation, but this was shown in experiments using proteaseinduced Trask cleavage. 28 We believe the use of proteases can confound the study of Trask phosphorylation, since even the …”

Development of an enzyme-linked immunosorbent assay for detection of CDCP1 shed from the cell surface and present in colorectal cancer serum specimens

“…This occurs at two adjacent sites, arginine 368 (R368) and lysine 369 (K369), and generates two 65 kDa amino-terminal fragments that are shed from the cell surface. These are designated ShE-CDCP1 368 and ShE-CDCP1 369 (collectively termed ShE-CDCP1) and differ by a single carboxylterminal amino acid [20]. Western blot analysis has revealed elevated levels of ShE-CDCP1 in the urine of prostate cancer patients at high risk of poor survival [21], and in conditioned media of prostate cancer cell lines [20].…”

“…These are designated ShE-CDCP1 368 and ShE-CDCP1 369 (collectively termed ShE-CDCP1) and differ by a single carboxylterminal amino acid [20]. Western blot analysis has revealed elevated levels of ShE-CDCP1 in the urine of prostate cancer patients at high risk of poor survival [21], and in conditioned media of prostate cancer cell lines [20]. Also, there is indirect evidence from Western blot analyses of tissue lysates, that CDCP1 cleavage occurs in high grade serous ovarian carcinoma patients [22], and in many cell lines derived from epithelial tumors [3,20].…”