MAPPING OF A MOLECULAR DETERMINANT FOR PROTEIN-KINASE-C BETA(II) ISOZYME FUNCTION

In human erythroleukemia (K562) cells, the highly related protein kina se C (PRC) alpha and PKC beta(II) isozymes serve distinct functions in cellular differentiation and proliferation, respectively. Previous st udies using two domain switch PRC chimera revealed that the catalytic domains of PHC alpha and beta(II) contain molecular determinants impor tant for isozyme-specific function (Walker, S. D,, Murray, N, R,, Burn s, D, J,, and Fields, A. P, (1995) Proc. Natl, Acad, Sci. U.S.A. 92, 9 156-9160), We have now analyzed a panel of PKC chimeras to determine t he specific region within the catalytic domain important for PKC beta( II) function. A cellular assay for PKC beta(II) function was devised b ased on the finding that PKC beta(II) selectively translocates to the nucleus and phosphorylates nuclear lamin B in response to the PKC acti vator bryostatin, This response is strictly dependent upon expression of PKC beta(II) or a PKC chimera that functions like PKC beta(II). We demonstrate that a PKC alpha/beta(II) chimera containing only the carb oxyl-terminal 13 amino acids from PRC beta(II) (beta(II) V5) is capabl e of nuclear translocation and lamin B phosphorylation. These results are consistent with our recent observation that the PRC beta(II) V5 re gion binds to phosphatidylglycerol (PG), a potent and selective PRC be ta(II) activator present in the nuclear membrane (Murray, N, R,, and F ields, A. P, (1998) J. Biol. Chem. 273, 11514-11520). Soluble beta(II) V5 peptide selectively inhibits PG-stimulated PKC beta(II) activity i n a dose-dependent fashion, indicating that PG-mediated activation of PKC beta(II) involves interactions with the beta(II) V5 region of the enzyme. We conclude that beta(II) V5 is a major determinant for PKC be ta(II) nuclear function and suggest a model in which binding of PG to the beta(II) V5 region stimulates nuclear PKC beta(II) activity during G(2) phase of the cell cycle.