c-Src association with and phosphorylation of p58(gag), a membrane- and microfilament-associated retroviral Gag-like protein in a xenotransplantable rat mammary tumor

The retroviral Gag-like protein p58(gag) expressed in a highly metastatic a scites rat mammary adenocarcinoma has been implicated in cell surface chang es contributing to xenotransplantability. p58(gag) is present in the cells in a plasma membrane- and microfilament-associated signal transduction part icle containing Src and is phosphorylated on tyrosine. Overlay analyses and affinity chromatography with glutathione S-transferase (GST) fusion protei ns of Src homology-3 (SH3) domains showed direct binding of the Src but not the Crk SH3 domain to p58(gag), This association was confirmed by coimmuno precipitation of partially purified p58(gag) from ascites cell lysates with platelet Src, Further, a GST-p58(gag) fusion protein bound full length c-S rc from either platelets or c-Src-expressing insect cells. The GST-p58(gag) 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 o f GST-p58(gag) 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 phosphor ylation of p58(gag) was observed. These results demonstrate that p58(gag) i s 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 throug h binding and relocalization of SH3 domain-containing proteins such as Src- like tyrosine kinases to sites of association of microfilaments with the pl asma membrane.