Human cancer cells exhibit protein kinase C-dependent c-erbB-2 transmodulation that correlates with phosphatase sensitivity and kinase activity

The c-erbB-2 receptor tyrosine kinase is often overexpressed in human tumors, but the functional implications of this phenotype remain unclear. We previously used phosphorylation-specific antibodies to define major differences in c-erbB-2 tyrosine kinase activity between overexpressing human tumor cell lines (Epstein, R. J., Druker, B. J., Roberts, T.M., and Stiles, C. D. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 10435-10439). Here we extend this approach to define the relationship between c-erbB-2 tyrosine phosphorylation and protein kinase C (PRC)-dependent transmodulation. Phosphorylation-specific antibodies to the juxtamembrane PKC site Thr(686) recognize tyrosine-dephosphorylated wild-type c-erbB-2 following G8/DHFR 3T3 cell treatment with PKC agonists. B104-1-1 cells transformed by activated c-erbB-2 express a subset of tyrosine-phosphorylated receptors that are homologously phosphorylated on Thr(686), indicating that Thr(686) phosphorylation alone is insufficient to abrogate receptor tyrosine phosphorylation, Similarly, the c-erbB-2-overexpressing human cancer cell lines SK-Ov-3 and BT-474 express constitutively Thr(686)-phosphorylated receptors. SK-Ov-3 cells express predominantly kinase-inactive c-erbB-2 that is heavily Thr(686)-phosphorylated, indicating that Thr(686) phosphorylation in this line is heterologous in origin. In contrast, BT-474 cells express constitutively autophosphorylated c-erbB-2 despite Thr(686) phosphorylation. These results indicate that Thr(686) phosphorylation does not directly abolish c-erbB-2 activity and suggest that such phosphorylation reflects constitutive PKC activity induced by either receptor-activating mutations or heterologous growth factors. The latter possibility suggests in turn that c-erbB-2 interacts in an as yet undefined way with heterologous growth factor receptors in human tumor cells.