The aim of this project is to better elucidate the role(s) of specific protein kinases in the regulation of cell growth and in malignant transformation. Since subcellular localization is important in determining the functional and regulatory specificity of protein kinases, studies were carried out to characterize the relationship between the domain organization and molecular targeting of protein kinase Ce (PKCe). Studies have shown that PKC can interact with the pleckstrin homology (PH) domain of Btk PH protein tyrosine kinase. Thus, studies were carried out to characterize the PKCe-Btk PH domain interaction. It was found that the C1 zinc finger-like regulatory region of PKCe exhibits strong binding to the Btk PH domain. Phorbol ester tumor promoter (PMA) and phosphatidylinositol 4,5-bisphosphate (PIP2) both compete with PKC for binding to the PH domain. These results indicate that agents which interact with the PH domain of Btk (PIP2) or the C1 region of PKCe (PMA) might act to regulate PKC-PH domain binding. Our previous studies have shown that the generation of oxygen free radicals may be an important regulatory mechanism for PKC and cyclic AMP-dependent protein kinase signaling pathways. It now has been determined that irradiation of MDA-MB 231 human breast cancer cells increased the level of membrane bound, tyrosine phosphorylated Raf protein kinase (Raf PK), and stimulated the catalytic activity of Raf PK. Irradiation also was found to increase the level of Raf PK coimmunoprecipitation with Ras. Other studies showed that treatment of NIH 3T3 cells with hydrogen peroxide also resulted in a significant stimulation of Raf PK catalytic activity. Overexpression of a Raf fragment (which acts as a dominant-negative inhibitor of Ras-Raf interaction) inhibited hydrogen peroxide-induced activation of Raf PK. These results suggest that ionizing radiation and oxygen free radicals may act to enhance Ras-mediated activation of Raf PK.
