PROTEIN KINASES, CA++, AND PI IN STIMULATED LYMPHOCYTES

Lymphocytes, normally non-dividing cells will divide in culture in the presence of a mitogenic lectin such as ConA. This system serves as a useful model to study mammalian cell proliferation and cellular immune regulation. Recent advances in both of these areas suggest that the role of protein kinases as mediators of the proliferative response be re-examined. There are several reasons to suggest that the recently discovered protein kinase C plays an important role. One of the early responses to lectins is an enhanced turnover of phosphatidylinositol. A by-product of this turnover, diacylglycerol, activates a protein kinase C (PKC). In addition it is now known that proliferation requires macrophages or the macrophage product, interleukin 1 (IL. 1). The phorbol ester tumor promoter, 12-0-tetradecanoylphorbol-13-acetate (TPA) can substitute for IL 1 in this response. The receptor for TPA is believed to be a protein kinase C and it can activate this kinase. Furthermore, protein kinase C requires Ca++, and Ca++ flux is an early response of lymphocytes to lectins. In light of the biochemical and cellular information, it is proposed that kinase activities in lymphocytes be examined under conditions where proliferation is limited by lack of macrophages, or by the use of incomplete mitogens. A techniques will be developed to separate and to assay the kinases in situ on non-denaturing gels. It should be possible to compare the major classes of kinases in the same assay. Two early biochemical events in stimulation will also be examined, Ca++ flux and phosphatidylinositol (PI) turnover. The changes in kinase activity particularly PKC will be compared with Ca++ flux, PI turnover and DNA synthesis. These responses will be examined in lymphocytes stimulated with a mitogenic lectin such as Concanavalin A or one that is non-mitogenic alone such as WGA or A23187 but mitogenic in combination with TPA. It is hoped that this approach will allow one to determine which biochemical parameters are required for proliferation and which cells are responsible for the signals.