The focus of this project is to better elucidate the role of altered transmembrane signaling in such processes as cell growth regulation, tumor promotion, cell differentiation, and cellular resistance to chemotherapeutic drugs. Protein kinase C (PKC) is increased significantly (5-7 fold) in the nuclei of drug resistant MCF-7 cells. A good correlation is found between the increased level of nuclear PKC and the increased degree of drug resistance. Results indicate that MCF7/ADR cells, which are multidrug-resistant, contain a modified form of PKC alpha as determined by Western blot analysis, DEAE cellulose chromatography, and altered sensitivity to PKC inhibitors. These results suggest that elevated levels of a modified form of PKC alpha may act to modulate nuclear events to influence the development of multidrug resistance in MCF-7 cells. A quantitative immunofiltration assay was developed to determine Raf-1 protein kinase (Raf-1 PK) activities in cell extracts. With this method two distinct forms of Raf-like enzyme activity were found to be present in the cytosolic fraction of NIH 3T3 cells. Immunohistochemical studies indicate that changes in cell population density and growth rate correlate with changes in the intracellular level and distribution of Raf-1 PK. Low levels of Raf-1 PK are found diffusely distributed throughout the cytoplasm in confluent, growth arrested cells, while the level of Raf-1 PK is increased in subconfluent, rapidly dividing cells and is found to be markedly elevated in the nuclear fraction. These results support the suggestion that Raf- 1 PK may be involved in mitogenic signaling from the plasma membrane to the nucleus. Evidence from two-dimensional gel analysis, Mono Q column separations, and specific immunoprecipitation studies indicate that the RII regulatory subunit, and to a lesser extent the RI regulatory subunit, of cyclic AMP-dependent protein kinases (PKA) may be covalently modified by retinoylation in HL-60 cells exposed to retinoic acid. The retinoylation of a subpopulation of RII, and perhaps RI, may play a role in the synergistic interrelationship noted between cyclic AMP and retinoic acid in regulating cell growth and differentiation.
