Association of the Ras to mitogen-activated protein kinase signal transduction pathway with microfilaments - Evidence for a p185(neu)-containing cellsurface signal transduction particle linking the mitogenic pathway to a membrane-microfilament association site

Microvilli of the aggressive 13762 ascites mammary adenocarcinoma contain a large, microfilament-associated signal transduction particle whose scaffol ding is a stable glycoprotein complex (Li. Y., Hua. F., Carraway, K. L., an d Carraway, C. A. C. (1999) J. Biol, Chem, 274, 25651-25658) associated wit h the growth factor receptor p185(neu). The receptor is constitutively tyro sine-phosphorylated in the cells and microvilli, predicting that it should recruit mitogenic pathway components to this membrane-microfilament interac tion site. Immunoprecipitation of cell lysates with anti-phosphotyrosine an d immunoblotting showed phosphorylated forms of the mitogenic pathway prote ins Shc and MAPK in addition to p185(neu), Suggesting that the Ras to MAPK mitogenic pathway is activated. Immunoblotting of p185(neu) containing micr ovillar fractions revealed the presence in each of stably associated She, G rb-2, Sos, Ras, Raf, mitogen-activated protein kinase kinase, and mitogen-a ctivated protein kinase/extracellular signal-regulated kinase, as well as t he transcription factor-phosphorylating kinase Rsk, All of these pathway co mponents coimmunoprecipitated with p185(neu) from cleared lysates of microv illi solubilized under microfilament-depolymerizing conditions, The recruit ment of constitutively phosphorylated p185(neu) and the activated mitogenic pathway proteins to this membrane-microfilament interaction site provides a physical model for integrating the assembly of the mitogenic pathway with the transmission of growth factor signal to the cytoskeleton, This linkage is probably a requisite step in the global. cytoskeleton remodeling accomp anying mitogenesis.