The calcium/phospholipid-dependent protein kinase (PKC) is an integral part of a major signal transduction pathway for many growth stimuli. Cellular mitogens, including polypeptide growth factors and the tumor promoting phorbol esters, activate PKC and initiate a coordinated cascade of events leading to the entrance of cells into cell cycle. The mechanisms by which such proliferative signals, generated at the cell surface, reach the nucleus are virtually unknown. Recently, a novel protein kinase C-like activity (PKCn) has been identified in our laboratory. PKCn appears to be translocated and activated at the nuclear membrane in a variety of cell types in response to both pharmacological PKC activators and the polypeptide growth factors interleukin-3, platelet- derived growth factor and fibroblast growth factor. Several specific nuclear substrates were also identified for PKCn; these include the nuclear envelope polypeptide lamins and RNA polymerase II. An in-vitro reconstitution system has been devised in which PKCn is selectively translocated to purified nuclear membranes from purified rat brain PKC preparations. Using this as an assay system, the first aim of this proposal is to purify PKCn and characterize its specificity for identified nuclear membrane substrates, requirements for cofactors and sensitivity to pharmacologic activators and inhibitors of PKC. The role for nuclear membrane phospholipid and protein in the specific translocation of PKCn to nuclear membranes will be assessed in related studies. Specific immunological probes will be generated against purified PKCn and used in immunolocalization studies to determine the intracellular distribution of PKCn in several cell types. The effect of various growth t stimuli, including polypeptide growth factors, on the intracellular distribution and expression of PKCn will be investigated. The role of PKCn in the mitogenic responses of both PKC activators and polypeptide growth factors will be assessed by determining the effects of specific activators and inhibitors of PKCn on these responses. Finally, the involvement of PKCn-mediated phosphorylation of the nuclear envelope lamin proteins and RNA polymerase II in mediating growth responses will be investigated.
These aims will be accomplished using a combination of biochemical, immunohistochemical, immunological and pharmacological approaches.
