TYROSINE PHOSPHORYLATION AT THE MEMBRANE-MICROFILAMENT INTERFACE - A P185(NEU)-ASSOCIATED SIGNAL-TRANSDUCTION PARTICLE CONTAINING SRC, ABL AND PHOSPHORYLATED P58, A MEMBRANE-ASSOCIATED AND MICROFILAMENT-ASSOCIATED RETROVIRAL GAG-LIKE PROTEIN

Microfilaments are associated with the microvillar membrane in the 137 62 ascites rat mammary carcinoma cells by stable interaction with a la rge, multimeric signal transduction particle (STP) containing the (pro to)oncogene receptor p185(neu). In vitro kinase assays on isolated mic rovilli and microvillar fractions enriched in the putative signal tran sduction particle showed a high specific activity of tyrosine kinase a ctivity compared to that of membranes from EGF receptor-overexpressing A431 cells maximally activated by EGF. Assays of velocity sedimentati on fractions from microvillar lysates in the presence and absence of t he exogenous tyrosine kinase substrate poly-glu-tyr polypeptide (poly- E(4)Y) suggested association of the tyrosine kinase activity with STP- enriched microvillar fractions. The particulate fractions also contain ed discrete endogenous tyrosine-phosphorylated proteins, including pro minent bands of approximate to 42 and 58 kDa. Addition of ATP to these fractions resulted in a rapid increase in tyrosine phosphorylation of these and several other proteins, as detected by antiphosphotyrosine blots. Immunoprecipitation and immunoblotting with anti-phosphotyrosin e antibody of SDS-solubilized ascites cells and microfilament core fra ctions showed nine major bands; the electrophoretic mobilities of most of these in the cell immunoprecipitate and microfilament core were th e same. In vivo and in situ phosphorylation, phosphoamino acid analysi s, immunoprecipitation, 2-dimensional isoelectric focusing/SDS PAGE an d immunoblot analysis showed that one of the prominent substrates is p 58E(gag), a retroviral Gag-like cytoplasmic STP component implicated i n stabilizing microfilament-membrane interactions. Immunoblotting iden tified two peripheral membrane tyrosine kinases, p60(src) and p120(abl ), stably associated with the p185(neu)-containing signal transduction particle. These results provide further evidence for the constitutive activation of this aggressive mammary tumor and suggest a role for ph osphorylation of p58S(gag) in organization of the STP at the membrane- microfilament interface in these cells.