The tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), acutely stimulates the tyrosine phosphorylation of proteins of approximately 190, 120, and 70 kDa in the well differentiated Fao rat hepatoma cell line. This phosphorylation is dependent on protein kinase C (PKC) and is abolished by down-regulation of PKC or pretreatment with a PKC inhibitor. Purification of the 190-kDa tyrosine-phosphorylated protein revealed that it consists of both ErbB2 and ErbB3. Following PMA-induced tyrosine phosphorylation, ErbB2 and ErbB3 were able to associate with the SH2 domains of several signaling proteins including the p85␣ subunit of phosphatidylinositol 3-kinase, Syp, and Grb2. The 120-kDa protein phosphorylated in response to PMA consists of at least two proteins: focal adhesion kinase that exhibits a minor increase in tyrosine phosphorylation following treatment with PMA, and a major 120-kDa tyrosine-phosphorylated species in PMA-stimulated Fao cells which as yet is unidentified. Similarly, the 70-kDa tyrosine-phosphorylated protein also appears to represent more than one protein, including paxillin and a second protein of similar mobility which appears to be the major tyrosine phosphorylation in response to PMA. Both ErbB2 and paxillin also exhibit reduced migration on SDS-polyacrylamide gel electrophoresis following PMA treatment, suggesting that they are also phosphorylated on serine/threonine residues. The mobility shift of both of these proteins is abolished by treatment with inhibitors of PKC or mitogen-activated protein kinase/ extracellular signal-related kinase kinase. These results suggest a novel mechanism of cross-talk between the serine/threonine kinase PKC and tyrosine phosphorylation pathways. The activation of ErbB2 and ErbB3 that is initiated by PMA may contribute to the tumor promoting activity of these compounds.The ErbB (HER) family of receptor tyrosine kinases consists of the following four members: ErbB1 (the epidermal growth factor receptor; EGFR), 1 ErbB2/Neu, ErbB3, and ErbB4 (1-5).The hallmarks of this family of proteins include the presence of two cysteine-rich regions in the extracellular domain, a single transmembrane domain, and, with the exception of ErbB3 (6), an intrinsic tyrosine kinase activity contained in the large intracellular domain (7). In addition to the role of ErbB1 in EGF action, members of the ErbB family are often overexpressed in various human tumors (8, 9). Many human breast and ovarian carcinomas contain elevated levels of ErbB2 that result in constitutive activation of its intrinsic tyrosine kinase activity (8, 10 -12). This correlates with a poor clinical prognosis (1,7,9,13,14). Similarly, elevated levels of expression and tyrosine phosphorylation of ErbB3 have been detected in several breast cancer cell lines (3,15). It is clear that constitutive activation of the intrinsic kinase activity of these receptors is a crucial step in the oncogenic process (16). Activation of the ErbB family of receptors depends on formation of homodimers or heterodimers wi...