Journal of Biological Chemistry
Volume 285, Issue 34, 20 August 2010, Pages 26162-26173
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Cell Biology
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*

https://doi.org/10.1074/jbc.M109.096453Get rights and content
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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 PKCδ 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.

Membrane Proteins
Phosphotyrosine
Protease
Receptors
Shedding
CDCP1
PKCδ
Src
Serine Protease
Tyrosine Phosphorylation

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*

This work was supported by the National Health and Medical Research Council of Australia (Fellowship 339732 to J. D. H.), by a Canadian Institutes of Health Research (CIHR) grant (to R. L.), and by funding supplied by the Cancer & Bowel Research Trust (scholarship to A. W.).

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

1

A Chercheur National of the Fonds de la Recherche en Santé du Québec (FRSQ).