title: Human biliverdin reductase, a previously unknown activator of protein kinase C betaII.
abstract: Human biliverdin reductase (hBVR), a dual specificity kinase (Ser/Thr/Tyr) is, as protein kinase C (PKC) betaII, 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 betaII plasmids, we report the co-immunoprecipitation of the proteins and co-purification in the glutathione S-transferase (GST) pulldown assay. hBVR and PKC betaII, but not the reductase and PKC zeta, transphosphorylated in assay systems supportive of activity of only one of the kinases. PKC betaII K371R mutant protein ("kinase-dead") was also a substrate for hBVR. The reductase increased the Vmax but not the apparent Km values of PKC betaII 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 Thr500 in the human PKC betaII activation loop. Neither serine nor threonine residues in peptides corresponding to other phosphorylation sites of the PKC betaII nor PKC zeta activation loop-derived peptides were substrates. The phosphorylation of Thr500 was confirmed by immunoblotting of hBVR.PKC betaII immunocomplex. The potential biological relevance of the hBVR activation of PKC betaII was suggested by the finding that in cells transfected with the PKC betaII, hBVR augmented phorbol myristate acetate-mediated c-fos expression, and infection with sihBVR attenuated the response. Also, in cells overexpressing hBVR and PKC betaII, 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 betaII underscores its potential function in propagation of signals relayed through PKCs.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York 14624, USA. mahin_maines@urmc.rochester.edu
authors.forename: Mahin D
authors.initials: MD
authors.lastname: Maines
authors.forename: Tihomir
authors.initials: T
authors.lastname: Miralem
authors.forename: Nicole
authors.initials: N
authors.lastname: Lerner-Marmarosh
authors.forename: Jenny
authors.initials: J
authors.lastname: Shen
authors.forename: Peter E M
authors.initials: PE
authors.lastname: Gibbs
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D047468:Immunoprecipitation; D007700:Kinetics; D018613:Microscopy, Confocal; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000502:physiology*; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D064546:Protein Kinase C beta; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry; D013347:Subcellular Fractions; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
chemical_list: D011994:Recombinant Proteins; D044925:Oxidoreductases Acting on CH-CH Group Donors; C019789:biliverdin reductase; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D013755:Tetradecanoylphorbol Acetate