Journal: The Journal of Biological Chemistry

Article Title: 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 PKC?

doi: 10.1074/jbc.M109.096453

Figure Lengend Snippet: Examination of tyrosine phosphorylation of CDCP1 and binding of Src and PKCδ induced by DU145 media. A, anti-Src, -PKCδ, -p-CDCP1-Y734 (performed with an antibody that detects both p-CDCP1-Y734 and p-FAK-Y861), and -CDCP1 (CST 4115) Western blot analysis of anti-CDCP1 immunoprecipitates (CST 4115) obtained from DU145 cells. B, anti-CDCP1 (CST 4115), -p-CDCP1-Y734 (performed with an antibody that detects both p-CDCP1-Y734 and p-FAK-Y861), -PKCδ, and -Src Western blot analysis of anti-Src immunoprecipitates obtained from DU145 cells. C, anti-phosphotyrosine, anti-PKCδ, anti-Src, and anti-CDCP1 (CST 4115) Western blot analysis of anti-CDCP1 immunoprecipitates obtained from 22Rv1 cells either untreated (−) or treated (+) with 3 day serum-free conditioned medium from DU145 cells for 36 h. The media was either untreated (−) or treated (+) with protease inhibitor (PI) mixture before incubation with 22Rv1 cells. Lysates from all experiments were collected in the presence (+) or absence (−) of sodium vanadate and sodium fluoride. All experiments included control immunoprecipitations performed with species matched IgG. NS, nonspecific.

Article Snippet: Antibodies were from the following suppliers: goat polyclonal antibody against the last 13 C-terminal residues of CDCP1 from Abcam (Cambridge, MA; ab1377); rabbit polyclonal antibody against unspecified C-terminal residues of CDCP1 from Cell Signaling Technology (CST; Danvers, MA; 4115); goat antibody against the extracellular domain of CDCP1 from R&D Systems (Bio-Scientific Pty Ltd, Gymea, Australia; AF2666); rabbit anti-matriptase antibody from Bethyl Laboratories (Montgomery, TX); rabbit anti-Src antibody from CST (2108); rabbit anti-PKCδ antibody from Santa Cruz Biotechnology (Santa Cruz, CA; SC-937); rabbit anti-p-FAK-Y861 antibody that detects both p-CDCP1-Y734 and p-FAK-Y861 ( 3 ), from Invitrogen (Mulgrave, Australia); rabbit and mouse monoclonal anti-Flag epitope (DYKDDDDK) antibodies from Sigma; monoclonal anti-phosphotyrosine antibody PY20 from Calbiochem (La Jolla, CA); monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody from Chemicon International (Boronia, Australia); and HRP-conjugated secondary antibodies from Thermo Scientific (Murarrie, Australia).

Techniques: Binding Assay, Western Blot, Protease Inhibitor, Incubation

Journal: The Journal of Biological Chemistry

Article Title: 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 PKC?

doi: 10.1074/jbc.M109.096453

Figure Lengend Snippet: Matriptase proteolysis mediates tyrosine phosphorylation and binding of Src and PKCδ to 70 kDa CDCP1. A, anti-phosphotyrosine Western blot analysis of proteins immunoprecipitated from 22Rv1 cell lysates using a rabbit anti-CDCP1 antibody (CST 4115). Lysates were collected from 22Rv1 cells either untreated (−) or treated (+) with 20 nm matriptase for the indicated times. The blot was reprobed with an anti-rabbit secondary antibody to detect rabbit IgG to assess consistency in the amount of antibody used for immunoprecipitations. B, photographic images of 22Rv1 cells either untreated (left panel) or treated for 2 h with 20 nm matriptase (right panel). Bar, 50 μm. C, anti-phosphotyrosine, -PKCδ, -Src, and -CDCP1 (CST 4115) Western blot analysis of anti-CDCP1 (CST 4115) immunoprecipitates obtained from 22Rv1 cells either untreated (−) or treated (+) with matriptase (20 nm; 0.5 h). Lysates were collected in the presence (+) or absence (−) of sodium vanadate and sodium fluoride. All experiments included control immunoprecipitations performed with species matched IgG. NS, nonspecific.

Article Snippet: Antibodies were from the following suppliers: goat polyclonal antibody against the last 13 C-terminal residues of CDCP1 from Abcam (Cambridge, MA; ab1377); rabbit polyclonal antibody against unspecified C-terminal residues of CDCP1 from Cell Signaling Technology (CST; Danvers, MA; 4115); goat antibody against the extracellular domain of CDCP1 from R&D Systems (Bio-Scientific Pty Ltd, Gymea, Australia; AF2666); rabbit anti-matriptase antibody from Bethyl Laboratories (Montgomery, TX); rabbit anti-Src antibody from CST (2108); rabbit anti-PKCδ antibody from Santa Cruz Biotechnology (Santa Cruz, CA; SC-937); rabbit anti-p-FAK-Y861 antibody that detects both p-CDCP1-Y734 and p-FAK-Y861 ( 3 ), from Invitrogen (Mulgrave, Australia); rabbit and mouse monoclonal anti-Flag epitope (DYKDDDDK) antibodies from Sigma; monoclonal anti-phosphotyrosine antibody PY20 from Calbiochem (La Jolla, CA); monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody from Chemicon International (Boronia, Australia); and HRP-conjugated secondary antibodies from Thermo Scientific (Murarrie, Australia).

Techniques: Binding Assay, Western Blot, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: 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 PKC?

doi: 10.1074/jbc.M109.096453

Figure Lengend Snippet: Protease-mediated processing of CDCP1. Serine proteolysis generates a 65 kDa shed ectodomain and a tyrosine-phosphorylated cell retained 70 kDa CDCP1 fragment. Proteolysis occurs at Arg-368 and Lys-369 between CUB-like domain 1 and 2 of CDCP1 (the 3 CDCP1 CUB-like domains are shown as blue ovals). Proteolysis results in tyrosine phosphorylation of 70 kDa CDCP1 (yellow-filled circles) and recruitment of Src and PKCδ in a phosphorylation-dependent manner. We propose that the CDCP1 ectodomain may function as a ligand or competitive inhibitor for a cell surface receptor (autocrine receptor binding is shown, but paracrine and endocrine signaling may also be relevant) or as a matrix-interacting protein potentially modulating cell:matrix interactions occurring via known CDCP1 interacting proteins such as syndecan 1 and 4 and the tetraspannin CD9 (dotted arrows). Potential signaling downstream of the 70 kDa CDCP1 cell-retained fragment and ectodomain resulting in direct cellular responses and changes in gene expression are represented by curved and straight arrows, respectively. Currently the endogenous serine protease or proteases mediating cleavage of CDCP1 at Arg-368 and Lys-369 are not known. However, we have shown that the cell surface and shed serine protease matriptase efficiently cleaves CDCP1 exclusively at Arg-368.

Article Snippet: Antibodies were from the following suppliers: goat polyclonal antibody against the last 13 C-terminal residues of CDCP1 from Abcam (Cambridge, MA; ab1377); rabbit polyclonal antibody against unspecified C-terminal residues of CDCP1 from Cell Signaling Technology (CST; Danvers, MA; 4115); goat antibody against the extracellular domain of CDCP1 from R&D Systems (Bio-Scientific Pty Ltd, Gymea, Australia; AF2666); rabbit anti-matriptase antibody from Bethyl Laboratories (Montgomery, TX); rabbit anti-Src antibody from CST (2108); rabbit anti-PKCδ antibody from Santa Cruz Biotechnology (Santa Cruz, CA; SC-937); rabbit anti-p-FAK-Y861 antibody that detects both p-CDCP1-Y734 and p-FAK-Y861 ( 3 ), from Invitrogen (Mulgrave, Australia); rabbit and mouse monoclonal anti-Flag epitope (DYKDDDDK) antibodies from Sigma; monoclonal anti-phosphotyrosine antibody PY20 from Calbiochem (La Jolla, CA); monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody from Chemicon International (Boronia, Australia); and HRP-conjugated secondary antibodies from Thermo Scientific (Murarrie, Australia).

Techniques: Cell Surface Receptor Assay, Binding Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: 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 PKC? *

doi: 10.1074/jbc.M109.096453

Figure Lengend Snippet: Examination of tyrosine phosphorylation of CDCP1 and binding of Src and PKCδ induced by DU145 media. A, anti-Src, -PKCδ, -p-CDCP1-Y734 (performed with an antibody that detects both p-CDCP1-Y734 and p-FAK-Y861), and -CDCP1 (CST 4115) Western blot analysis of anti-CDCP1 immunoprecipitates (CST 4115) obtained from DU145 cells. B, anti-CDCP1 (CST 4115), -p-CDCP1-Y734 (performed with an antibody that detects both p-CDCP1-Y734 and p-FAK-Y861), -PKCδ, and -Src Western blot analysis of anti-Src immunoprecipitates obtained from DU145 cells. C, anti-phosphotyrosine, anti-PKCδ, anti-Src, and anti-CDCP1 (CST 4115) Western blot analysis of anti-CDCP1 immunoprecipitates obtained from 22Rv1 cells either untreated (−) or treated (+) with 3 day serum-free conditioned medium from DU145 cells for 36 h. The media was either untreated (−) or treated (+) with protease inhibitor (PI) mixture before incubation with 22Rv1 cells. Lysates from all experiments were collected in the presence (+) or absence (−) of sodium vanadate and sodium fluoride. All experiments included control immunoprecipitations performed with species matched IgG. NS, nonspecific.

Article Snippet: Antibodies were from the following suppliers: goat polyclonal antibody against the last 13 C-terminal residues of CDCP1 from Abcam (Cambridge, MA; ab1377); rabbit polyclonal antibody against unspecified C-terminal residues of CDCP1 from Cell Signaling Technology (CST; Danvers, MA; 4115); goat antibody against the extracellular domain of CDCP1 from R&D Systems (Bio-Scientific Pty Ltd, Gymea, Australia; AF2666); rabbit anti-matriptase antibody from Bethyl Laboratories (Montgomery, TX); rabbit anti-Src antibody from CST (2108); rabbit anti-PKCδ antibody from Santa Cruz Biotechnology (Santa Cruz, CA; SC-937); rabbit anti-p-FAK-Y861 antibody that detects both p-CDCP1-Y734 and p-FAK-Y861 ( 3 ), from Invitrogen (Mulgrave, Australia); rabbit and mouse monoclonal anti-Flag epitope (DYKDDDDK) antibodies from Sigma; monoclonal anti-phosphotyrosine antibody PY20 from Calbiochem (La Jolla, CA); monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody from Chemicon International (Boronia, Australia); and HRP-conjugated secondary antibodies from Thermo Scientific (Murarrie, Australia).

Techniques: Binding Assay, Western Blot, Protease Inhibitor, Incubation