3 DISTINCT MECHANISMS FOR TRANSLOCATION AND ACTIVATION OF THE DELTA-SUBSPECIES OF PROTEIN-KINASE-C

We expressed delta subspecies of protein kinase C (delta-PKC) fused wi th green fluorescent protein (GFP) in CHO-K1 cells and observed the mo vement of this fusion protein in living cells after three different st imulations. The delta-PKC-GFP fusion protein had enzymological charact eristics very similar to those of the native delta-PKC and was present throughout the cytoplasm in CHO-K1 cells. ATP at 1 mM caused a transi ent translocation of delta-PKC-GFP to the plasma membrane approximatel y 30 s after the stimulation and a sequent retranslocation to the cyto plasm within 3 min. A tumor-promoting phorbol ester, 12-O-tetradecanoy lphorbol 13-acetate (TPA; 1 mu M), induced a slower translocation of d elta-PKC-GFP, and the translocation was unidirectional. Concomitantly, the kinase activity of delta-PKC-GFP was increased by these two stimu lations, when the kinase activity of the immunoprecipitated delta-PKC- GFP was measured in vitro in the absence of PKC activators such as pho sphatidylserine and diacylglycerol. Hydrogen peroxide (H2O2; 5 mM) fai led to translocate delta-PKC-GFP but increased its kinase activity mor e than threefold. delta-PKC-GFP was strongly tyrosine phosphorylated w hen treated with H2O2 but was tyrosine phosphorylated not at all by AT P stimulation and only slightly by TPA treatment. Both TPA and ATP ind uced the translocation of delta-PKC-GFP even after treatment with H2O2 . Simultaneous treatment with TPA and H2O2 further activated delta-PKC -GFP up to more than fivefold. TPA treatment of cells overexpressing d elta-PKC-GFP led to an increase in the number of cells in G(2)/M phase and of dikaryons, while stimulation with H2O2 increased the number of cells in S phase and induced no significant change in cell morphology . These results indicate that at least three different mechanisms are involved in the translocation and activation of delta-PKC.