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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29GM043186-05
Application #
2181853
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1989-12-01
Project End
1995-11-30
Budget Start
1993-12-01
Budget End
1995-11-30
Support Year
5
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Case Western Reserve University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Fields, A P; Thompson, L J (1995) The regulation of mitotic nuclear envelope breakdown: a role for multiple lamin kinases. Prog Cell Cycle Res 1:271-86
Singh, K R; Taylor, L K; Campbell, X Z et al. (1994) A bryostatin-sensitive protein kinase C required for nerve growth factor activity. Biochemistry 33:542-51
Goss, V L; Hocevar, B A; Thompson, L J et al. (1994) Identification of nuclear beta II protein kinase C as a mitotic lamin kinase. J Biol Chem 269:19074-80
Murray, N R; Burns, D J; Fields, A P (1994) Presence of a beta II protein kinase C-selective nuclear membrane activation factor in human leukemia cells. J Biol Chem 269:21385-90
Hocevar, B A; Burns, D J; Fields, A P (1993) Identification of protein kinase C (PKC) phosphorylation sites on human lamin B. Potential role of PKC in nuclear lamina structural dynamics. J Biol Chem 268:7545-52
Murray, N R; Baumgardner, G P; Burns, D J et al. (1993) Protein kinase C isotypes in human erythroleukemia (K562) cell proliferation and differentiation. Evidence that beta II protein kinase C is required for proliferation. J Biol Chem 268:15847-53
Misra-Press, A; Fields, A P; Samols, D et al. (1992) Protein kinase C isoforms in human glioblastoma cells. Glia 6:188-97
Hocevar, B A; Morrow, D M; Tykocinski, M L et al. (1992) Protein kinase C isotypes in human erythroleukemia cell proliferation and differentiation. J Cell Sci 101 ( Pt 3):671-9