Association of the Ras to mitogen-activated protein kinase signal transduction pathway with microfilaments -: Evidence for a p185neu-containing cell surface 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 scaffolding is a stable glycoprotein complex (Li. Y., Hua. F., Carraway, K. L., and Carraway, C. A. C. (1999) J. Biol, Chem, 274, 25651-25658) associated with the growth factor receptor p185(neu). The receptor is constitutively tyrosine-phosphorylated in the cells and microvilli, predicting that it should recruit mitogenic pathway components to this membrane-microfilament interaction site. Immunoprecipitation of cell lysates with anti-phosphotyrosine and immunoblotting showed phosphorylated forms of the mitogenic pathway proteins Shc and MAPK in addition to p185(neu), Suggesting that the Ras to MAPK mitogenic pathway is activated. Immunoblotting of p185(neu) containing microvillar fractions revealed the presence in each of stably associated She, Grb-2, Sos, Ras, Raf, mitogen-activated protein kinase kinase, and mitogen-activated protein kinase/extracellular signal-regulated kinase, as well as the transcription factor-phosphorylating kinase Rsk, All of these pathway components coimmunoprecipitated with p185(neu) from cleared lysates of microvilli solubilized under microfilament-depolymerizing conditions, The recruitment 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 accompanying mitogenesis.