Association of the Ras to Mitogen-activated Protein Kinase Signal Transduction Pathway with Microfilaments

Abstract

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 p185neu . 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 Shc, Grb-2, Sos, Ras, Raf, mitogen-activated protein kinase kinase, and mitogenactivated protein kinase/extracellular signal-regulated kinase, as well as the transcription factor-phosphorylating kinase Rsk. All of these pathway components coimmunoprecipitated with p185neu 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.Growth factors trigger both mitogenesis and changes in cell morphology and microfilament organization (1). Since both of these effects result from the binding of the factors to a single type of receptor, their pathways must somehow be integrated. One suggestion for this integration is that the receptors and components of the signaling pathways are localized to sites where microfilaments interact with membranes (2). The EGF 1 receptor (3) and the EGF receptor family member p185 neu (ErbB2/HER2) (4) have been shown to be associated with microfilaments. EGF receptor binds directly to actin through a motif similar to the actin-binding motif of profilin (5). The mechanism for the association of p185 neu /ErbB2 with microfilaments is presently unclear. Interestingly, p185neu /ErbB2, which has been implicated in tumor cell metastasis, is proposed to play a role in the regulation of cell structure and motility (6). The receptor is concentrated on microvilli in SK-BR-3 breast cancer cells. Activation of its phosphorylation leads to cell spreading, enlargement of microvilli, formation of pseudopodia, and aggregation of the p185 at the plasma membrane protrusions and pseudopodia (6). A specific role for p185 neu /ErbB2 in these microfilament-dependent processes known to enhance metastatic capability of tumor cells was shown in p185 neuoverexpressing transfectants of NCI-H460 lung carcinoma ce...

Sialomucin Complex (Rat Muc4) Is Regulated by Transforming Growth Factor β in Mammary Gland by a Novel Post-translational Mechanism

“…Moreover, SMC has been shown to bind to and modulate phosphorylation of the receptor ErbB2 (21). Supporting the conclusion that ASGP-2 is a ligand is the observation that ErbB2 is constitutively phosphorylated in the 13762 ascites cells and associated with a multimeric complex of signaling components, including Src (22) and all of the components of the Ras to MAP kinase mitogenic pathway (23). Thus, the transmembrane subunit ASGP-2 is proposed to modulate signaling through the epidermal growth factor family of receptors via its interaction with ErbB2 (21,24), the critical receptor for formation of active heterodimeric class I receptor tyrosine kinases (25).…”

Muc4/sialomucin complex, an intramembrane modulator of ErbB2/HER2/Neu, potentiates primary tumor growth and suppresses apoptosis in a xenotransplanted tumor

“…Up-regulation of Muc4/ SMC expression in transfected A375 melanoma cells resulted in potentiation of ErbB2 phosphorylation in response to heregulin ligand (Carraway et al, 1999b). Furthermore, complexes of Muc4/SMC and ErbB2 in 13762 ascites tumor cell membranes are associated with other cellular signal transducers, including elements of the Ras to MAPK signaling pathway (Carraway et al, 1999a).…”

Muc4/Sialomucin Complex, the Intramembrane ErbB2 Ligand, Translocates ErbB2 to the Apical Surface in Polarized Epithelial Cells

“…This interaction induces phosphorylation of ErbB2 in the absence of a soluble ligand and potentiates the phosphorylation of the ErbB2-ErbB3 heterodimer in the presence of the ErbB3-soluble ligand neuregulin. The Muc4/SMC-ErbB2 complex was first observed in highly metastatic rat ascites 13762 mammary adenocarcinoma cells, wherein the receptor and several of its associated intracellular signaling proteins appeared constitutively tyrosine-phosphorylated (15). The Muc4/SMC-ErbB2 in-teraction has been demonstrated in several systems, including normal lactating mammary gland, ascites tumors, isolated rat mammary epithelial cells, Muc4/SMC-transfected MCF-7 breast cancer cells, and a baculovirus-insect cell expression system (16).…”