c-Src association with and phosphorylation of p58gag, a membrane- and microfilament-associated retroviral Gag-like protein in a xenotransplantable rat mammary tumor.
The retroviral Gag-like protein p58gag expressed in a highly metastatic ascites rat mammary adenocarcinoma has been implicated in cell surface changes contributing to xenotransplantability. p58gag is present in the cells in a plasma membrane- and microfilament-associated signal transduction particle containing Src and is phosphorylated on tyrosine. Overlay analyses and affinity chromatography with glutathione S-transferase (GST) fusion proteins of Src homology-3 (SH3) domains showed direct binding of the Src but not the Crk SH3 domain to p58gag. This association was confirmed by co-immunoprecipitation of partially purified p58gag from ascites cell lysates with platelet Src. Further, a GST-p58gag fusion protein bound full length c-Src from either platelets or c-Src-expressing insect cells. The GST-p58gag fusion protein, but not GST, was phosphorylated by platelet or insect cell-expressed c-Src, but not by a kinase negative c-Src variant. The binding of GST-p58gag to c-Src was almost completely abolished by a 50-fold excess of the GST-SH3 domain of Src, and a parallel decrease in tyrosine phosphorylation of p58gag was observed. These results demonstrate that p58gag is tyrosine-phosphorylated as a consequence of its specific association with c-Src via its SH3 domain. These observations suggest a mechanism by which Gag proteins may contribute to retroviral maturation or pathogenesis through binding and relocalization of SH3 domain-containing proteins such as Src-like tyrosine kinases to sites of association of microfilaments with the plasma membrane.
Huang
J
J
Department of Biochemistry & Molecular Biology, University of Miami School of Medicine, Florida 33101, USA.
Zhang
B T
BT
Li
Y
Y
Mayer
B
B
Carraway
K L
KL
Carraway
C A
CA
D000230:Adenocarcinoma / Q000378:metabolism*; D000818:Animals; D001201:Ascites / Q000378:metabolism; D001667:Binding, Competitive; D001792:Blood Platelets / Q000201:enzymology; D005260:Female; D008325:Mammary Neoplasms, Experimental / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism*; D008954:Models, Biological; D009363:Neoplasm Proteins / Q000378:metabolism*; D009368:Neoplasm Transplantation; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D050737:Proto-Oncogene Proteins c-crk; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D018411:Spodoptera / Q000166:cytology; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured; D018909:src Homology Domains
C493872:Crk protein, rat; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D011993:Recombinant Fusion Proteins; C500813:membrane and microfilament-associated protein p58, rat; D016392:Proto-Oncogene Proteins pp60(c-src)