Human biliverdin reductase, a previously unknown activator of protein kinase C βII

Human biliverdin reductase (hBVR), a dual specificity kinase (Ser/Thr/Tyr) is, as protein kinase C (PKC) beta II, activated by insulin and free radicals (Miralem, T., Hu, Z., Torno, M. D., Lelli, K. M., and Maines, M. D. (2005) J. Biol. Chem. 280, 17084-17092; Lerner-Marmarosh, N., Shen, J., Torno, M. D., Kravets, A., Hu, Z., and Maines, M. D. (2005) Proc. Natl. Acad Sci. U.S.A. 102, 7109-7114). Here, by using 293A cells co-transfected with pcDNA3-hBVR and PKC beta II plasmids, we report the co-immunoprecipitation of the proteins and co-purification in the glutathione S-transferase (GST) pulldown assay. hBVR and PKC beta II, but not the reductase and PKC zeta, transphosphorylated in assay systems supportive of activity of only one of the kinases. PKC beta II K371R mutant protein (kinase-dead) was also a substrate for hBVR. The reductase increased the V-max but not the apparent K-m values of PKC beta II for myelin basic protein; activation was independent of phospholipids and extended to the phosphorylation of S2, a PKC-specific substrate. The increase in substrate phosphorylation was blocked by specific inhibitors of conventional PKCs and attenuated by sihBVR. The effect of the latter could be rescued by subsequent overexpression of hBVR. To a large extent, the activation was a function of the hBVR N-terminal chain of valines and intact ATP-binding site and the cysteine-rich C-terminal segment. The cobalt protoporphyrin-activated hBVR phosphorylated a threonine in a peptide corresponding to the Thr(500) in the human PKC beta II activation loop. Neither serine nor threonine residues in peptides corresponding to other phosphorylation sites of the PKC beta II nor PKC C activation loop-derived peptides were substrates. The phosphorylation of Thr(500) was confirmed by immunoblotting of hBVR center dot PKC beta II immunocomplex. The potential biological relevance of the hBVR activation of PKC beta II was suggested by the finding that in cells transfected with the PKC beta II, hBVR augmented phorbol myristate acetate-mediated c-fos expression, and infection with sihBVR attenuated the response. Also, in cells overexpressing hBVR and PKC beta II, as well as in untransfected cells, upon treatment with phorbol myristate acetate, the PKC translocated to the plasma membrane and co-localized with hBVR. hBVR activation of PKC beta II underscores its potential function in propagation of signals relayed through PKCs.