Human Biliverdin Reductase, a Previously Unknown Activator of Protein Kinase C βII
2007; Elsevier BV; Volume: 282; Issue: 11 Linguagem: Inglês
10.1074/jbc.m513427200
ISSN1083-351X
AutoresMahin D. Maines, Tihomir Miralem, Nicole Lerner‐Marmarosh, Jenny Y. Shen, Peter Gibbs,
Tópico(s)Aldose Reductase and Taurine
ResumoHuman biliverdin reductase (hBVR), a dual specificity kinase (Ser/Thr/Tyr) is, as protein kinase C (PKC) βII, 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 βII plasmids, we report the co-immunoprecipitation of the proteins and co-purification in the glutathione S-transferase (GST) pulldown assay. hBVR and PKC βII, but not the reductase and PKC ζ, transphosphorylated in assay systems supportive of activity of only one of the kinases. PKC βII K371R mutant protein ("kinase-dead") was also a substrate for hBVR. The reductase increased the Vmax but not the apparent Km values of PKC βII 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 βII activation loop. Neither serine nor threonine residues in peptides corresponding to other phosphorylation sites of the PKC βII nor PKC ζ activation loop-derived peptides were substrates. The phosphorylation of Thr500 was confirmed by immunoblotting of hBVR·PKC βII immunocomplex. The potential biological relevance of the hBVR activation of PKC βII was suggested by the finding that in cells transfected with the PKC βII, hBVR augmented phorbol myristate acetate-mediated c-fos expression, and infection with sihBVR attenuated the response. Also, in cells overexpressing hBVR and PKC βII, 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 βII underscores its potential function in propagation of signals relayed through PKCs. Human biliverdin reductase (hBVR), a dual specificity kinase (Ser/Thr/Tyr) is, as protein kinase C (PKC) βII, 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 βII plasmids, we report the co-immunoprecipitation of the proteins and co-purification in the glutathione S-transferase (GST) pulldown assay. hBVR and PKC βII, but not the reductase and PKC ζ, transphosphorylated in assay systems supportive of activity of only one of the kinases. PKC βII K371R mutant protein ("kinase-dead") was also a substrate for hBVR. The reductase increased the Vmax but not the apparent Km values of PKC βII 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 βII activation loop. Neither serine nor threonine residues in peptides corresponding to other phosphorylation sites of the PKC βII nor PKC ζ activation loop-derived peptides were substrates. The phosphorylation of Thr500 was confirmed by immunoblotting of hBVR·PKC βII immunocomplex. The potential biological relevance of the hBVR activation of PKC βII was suggested by the finding that in cells transfected with the PKC βII, hBVR augmented phorbol myristate acetate-mediated c-fos expression, and infection with sihBVR attenuated the response. Also, in cells overexpressing hBVR and PKC βII, 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 βII underscores its potential function in propagation of signals relayed through PKCs. Biliverdin reductase catalyzes the final step in the heme metabolic pathway, the reduction of biliverdin IXα to bilirubin. The enzyme remains unique among all biological catalysts described to date in having a dual pH/cofactor-dependent activity profile (3Kutty R.K. Maines M.D. J. Biol. Chem. 1981; 256: 3956-3962Abstract Full Text PDF PubMed Google Scholar). The protein displays microheterogeneity because of post-translational phosphorylation that is required for its activity (4Salim M. Brown-Kipphut B.A. Maines M.D. J. Biol. Chem. 2001; 276: 10929-10934Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 5Huang T.J. McCoubrey Jr., W.K. Maines M.D. Antioxid. Redox. Signal. 2001; 3: 685-696Crossref PubMed Scopus (42) Google Scholar). Free radical generators such as H2O2 and Na2AsO3, as well as insulin and the metalloporphyrin Co-PP, 3The abbreviations used are: Co-PP, cobalt protoporphyrin; ATF-2, activating transcription factor-2; CREB, cAMP-regulatory element-binding protein; DAG, diacylglycerol; GST, glutathione S-transferase; IRK, insulin receptor kinase; IRS, insulin receptor substrate; HO, heme oxygenase; hBVR, human biliverdin reductase; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; PMA, phorbol myristate acetate; PKCi, PKC inhibiting peptide; PS, phosphatidylserine; RACK, receptor for activated C-kinase; siRNA, small interfering RNA; BVR, biliverdin reductase; WT, wild type; LDH, lactate dehydrogenase; PBS, phosphate-buffered saline; sihBVR, small interference RNA for hBVR. 3The abbreviations used are: Co-PP, cobalt protoporphyrin; ATF-2, activating transcription factor-2; CREB, cAMP-regulatory element-binding protein; DAG, diacylglycerol; GST, glutathione S-transferase; IRK, insulin receptor kinase; IRS, insulin receptor substrate; HO, heme oxygenase; hBVR, human biliverdin reductase; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; PMA, phorbol myristate acetate; PKCi, PKC inhibiting peptide; PS, phosphatidylserine; RACK, receptor for activated C-kinase; siRNA, small interfering RNA; BVR, biliverdin reductase; WT, wild type; LDH, lactate dehydrogenase; PBS, phosphate-buffered saline; sihBVR, small interference RNA for hBVR. activate BVR and increase its phosphorylation (1Miralem T. Hu Z. Torno M.D. Lelli K.M. Maines M.D. J. Biol. Chem. 2005; 280: 17084-17092Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 2Lerner-Marmarosh N. Shen J. Torno M.D. Kravets A. Hu Z. Maines M.D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 7109-7114Crossref PubMed Scopus (119) Google Scholar, 4Salim M. Brown-Kipphut B.A. Maines M.D. J. Biol. Chem. 2001; 276: 10929-10934Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 6Bell J.E. Maines M.D. Arch. Biochem. Biophys. 1988; 263: 1-9Crossref PubMed Scopus (27) Google Scholar). Reduction of biliverdin IXα to lipophilic bilirubin serves several functions that include trafficking of the heme degradation product through the cell membrane, inactivation of a potent kinase inhibitor, biliverdin, and formation of bilirubin, an effective quencher of free radicals (7Stocker R. Yamamoto Y. McDonagh A.F. Glazer A.N. Ames B.N. Science. 1987; 235: 1043-1046Crossref PubMed Scopus (2886) Google Scholar, 8McDonagh A.F. Nat. Struct. Biol. 2001; 8: 198-200Crossref PubMed Scopus (107) Google Scholar). BVR is present across metazoan species, and its homologue is found in unicellular cyanobacteria (9Beale S.I. Cornejo J. Arch. Biochem. Biophys. 1984; 235: 371-384Crossref PubMed Scopus (53) Google Scholar, 10Maines M.D. Physiology (Bethesda). 2005; 20: 382-389Crossref PubMed Scopus (110) Google Scholar, 11Schluchter W.M. Glazer A.N. J. Biol. Chem. 1997; 272: 13562-13569Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Plants use biliverdin IXα produced by ferredoxin-dependent heme oxygenase (HO) to synthesize phytochromes, the sensory photoreceptors (9Beale S.I. Cornejo J. Arch. Biochem. Biophys. 1984; 235: 371-384Crossref PubMed Scopus (53) Google Scholar, 12Kohchi T. Mukougawa K. Frankenberg N. Masuda M. Yokota A. Lagarias J.C. Plant Cell. 2001; 13: 425-436Crossref PubMed Scopus (198) Google Scholar).BVR is a small soluble protein (296 residues) found mainly in the cytoplasm. If activated, the BVR leads to nuclear translocation and association with the nucleolus (13Maines M.D. Ewing J.F. Huang T.J. Panahian N. J. Pharmacol. Exp. Ther. 2001; 296: 1091-1097PubMed Google Scholar). Notably, small proteins, similar in molecular weight to hBVR, have been shown to bind to and activate PKCs (14Ron D. Chen C.H. Caldwell J. Jamieson L. Orr E. Mochly-Rosen D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 839-843Crossref PubMed Scopus (641) Google Scholar). The N terminus of the reductase is composed of hydrophobic and charged residues, which include a chain of four valines flanking the consensus Walker A homology ATP-binding motif, GXGXXG (15Hunter T. Cooper J.A. Annu. Rev. Biochem. 1985; 54: 897-930Crossref PubMed Google Scholar, 16Hanks S.K. Hunter T. FASEB J. 1995; 9: 576-596Crossref PubMed Scopus (2259) Google Scholar), and a notable degree of sequence similarity to IRK and IRS (2Lerner-Marmarosh N. Shen J. Torno M.D. Kravets A. Hu Z. Maines M.D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 7109-7114Crossref PubMed Scopus (119) Google Scholar). In this domain is the AQELWE sequence (amino acids 107–112) that shares identity of sequence and composition with the conserved six-residue RACK1 sequence in PKC β, SVEIWD (pseudo-RACK), and PKC pseudosubstrate AVEIWD. Tryptophan at position 5 and the negatively charged residue at position 3 characterize these sequences (14Ron D. Chen C.H. Caldwell J. Jamieson L. Orr E. Mochly-Rosen D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 839-843Crossref PubMed Scopus (641) Google Scholar, 17Mochly-Rosen D. Miller K.G. Scheller R.H. Khaner H. Lopez J. Smith B.L. Biochemistry. 1992; 31: 8120-8124Crossref PubMed Scopus (82) Google Scholar). A synthetic pseudo-RACK1 has an effect on PKC β, activating the kinase in the absence of activators by inducing structural changes in the protein to expose the catalytic site (14Ron D. Chen C.H. Caldwell J. Jamieson L. Orr E. Mochly-Rosen D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 839-843Crossref PubMed Scopus (641) Google Scholar, 17Mochly-Rosen D. Miller K.G. Scheller R.H. Khaner H. Lopez J. Smith B.L. Biochemistry. 1992; 31: 8120-8124Crossref PubMed Scopus (82) Google Scholar).Traditionally, the presence of 12 motifs characterizes a protein as a kinase (15Hunter T. Cooper J.A. Annu. Rev. Biochem. 1985; 54: 897-930Crossref PubMed Google Scholar); the primary structure of hBVR predicts its sharing several of those motifs, including the aforementioned, but not all 12. The BVR kinase motifs are conserved among mammalian species (2Lerner-Marmarosh N. Shen J. Torno M.D. Kravets A. Hu Z. Maines M.D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 7109-7114Crossref PubMed Scopus (119) Google Scholar, 4Salim M. Brown-Kipphut B.A. Maines M.D. J. Biol. Chem. 2001; 276: 10929-10934Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 10Maines M.D. Physiology (Bethesda). 2005; 20: 382-389Crossref PubMed Scopus (110) Google Scholar, 18Fakhrai H. Maines M.D. J. Biol. Chem. 1992; 267: 4023-4029Abstract Full Text PDF PubMed Google Scholar, 19Maines M.D. Polevoda B.V. Huang T.J. McCoubrey Jr., W.K. Eur. J. Biochem. 1996; 235: 372-381Crossref PubMed Scopus (50) Google Scholar). Notably, not all kinases have a complete set of 12 motifs. Recently, a number of non-conventional protein kinases have been identified. For instance, the Goodpasture antigen-binding protein has only a modified Walker A domain and no other motifs, but nonetheless has serine/threonine kinase activity (20Raya A. Revert F. Navarro S. Saus J. J. Biol. Chem. 1999; 274: 12642-12649Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Similarly, the catalytic domain of myosin heavy chain kinase A does not resemble the catalytic domains of protein kinases (21Cote G.P. Luo X. Murphy M.B. Egelhoff T.T. J. Biol. Chem. 1997; 272: 6846-6849Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar).The carboxyl domain of hBVR, as predicted by the crystal structures of rat BVR, consists of six strands that form a large β-sheet, an ideal interface for protein-protein interaction (22Whitby F.G. Phillips J.D. Hill C.P. McCoubrey W. Maines M.D. J. Mol. Biol. 2002; 319: 1199-1210Crossref PubMed Scopus (57) Google Scholar). The predicted basic leucine zipper protein domain, which links the two termini of the protein, has been shown to bind to the consensus sequences of AP-1 (activator protein-1) and ATF-2/CREB-binding sites (1Miralem T. Hu Z. Torno M.D. Lelli K.M. Maines M.D. J. Biol. Chem. 2005; 280: 17084-17092Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 23Ahmad Z. Salim M. Maines M.D. J. Biol. Chem. 2002; 277: 9226-9232Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 24Kravets A. Hu Z. Miralem T. Torno M.D. Maines M.D. J. Biol. Chem. 2004; 279: 19916-19923Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar).The human enzyme, in strictly Mn2+-dependent assay conditions, phosphorylates known serine/threonine and tyrosine kinase substrates e.g. MBP, casein, IRS-1 (insulin receptor substrate), and Raytide (a PTK substrate). In addition, hBVR autophosphorylates several serines, at least one threonine, and two of its six tyrosine residues (2Lerner-Marmarosh N. Shen J. Torno M.D. Kravets A. Hu Z. Maines M.D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 7109-7114Crossref PubMed Scopus (119) Google Scholar, 4Salim M. Brown-Kipphut B.A. Maines M.D. J. Biol. Chem. 2001; 276: 10929-10934Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The remaining four tyrosine residues are phosphorylated by the insulin receptor kinase (IRK). This includes Tyr198 in the YMXM motif that, in insulin receptor interactive proteins, is the binding site for proteins with Src homology domain, such as phosphatidylinositol 3-kinase (25Songyang Z. Shoelson S.E. McGlade J. Olivier P. Pawson T. Bustelo X.R. Barbacid M. Sabe H. Hanafusa H. Yi T. Mol. Cell. Biol. 1994; 14: 2777-2785Crossref PubMed Scopus (829) Google Scholar). hBVR tyrosine kinase activity was demonstrated by the recombinant human protein expressed in Escherichia coli. Notably the E. coli genome does not encode protein-tyrosine kinases, which are a multigenic family of Mn2+-dependent kinases exclusive to higher organisms (15Hunter T. Cooper J.A. Annu. Rev. Biochem. 1985; 54: 897-930Crossref PubMed Google Scholar). Although there is much information available on identification of IRK function in hBVR tyrosine phosphorylation, to date there is no information available on the identity of the serine/threonine kinase that phosphorylates hBVR.The MAPK and IRS/phosphatidylinositol 3-kinases are considered the major arms of the insulin/insulin-like growth factor-1 signaling pathway; signaling through the two arms is "linked" by the family of PKC isozymes that includes PKC βII, classified as a conventional PKC. hBVR prominently figures in oxidative stress response of the cell by its being a member of the basic leucine zipper protein family of transcription factors that regulate expression of stress-responsive genes, such as ho-1, ATF-2/CREB, and c-jun in the MAPK signaling pathway (1Miralem T. Hu Z. Torno M.D. Lelli K.M. Maines M.D. J. Biol. Chem. 2005; 280: 17084-17092Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 23Ahmad Z. Salim M. Maines M.D. J. Biol. Chem. 2002; 277: 9226-9232Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 24Kravets A. Hu Z. Miralem T. Torno M.D. Maines M.D. J. Biol. Chem. 2004; 279: 19916-19923Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). PKC enzymes are activated by oxidative stress, insulin, and growth factors (26Cacace A.M. Ueffing M. Philipp A. Han E.K. Kolch W. Weinstein I.B. Oncogene. 1996; 13: 2517-2526PubMed Google Scholar, 27Cai H. Smola U. Wixler V. Eisenmann-Tappe I. Diaz-Meco M.T. Moscat J. Rapp U. Cooper G.M. Mol. Cell. Biol. 1997; 17: 732-741Crossref PubMed Scopus (263) Google Scholar, 28Bourbon N.A. Yun J. Berkey D. Wang Y. Kester M. Am. J. Physiol. 2001; 280: C1403-C1411Crossref PubMed Google Scholar, 29Kawakami T. Kawakami Y. Kitaura J. J. Biochem. (Tokyo). 2002; 132: 677-682Crossref PubMed Scopus (57) Google Scholar, 30Chalfant C.E. Ohno S. Konno Y. Fisher A.A. Bisnauth L.D. Watson J.E. Cooper D.R. Mol. Endocrinol. 1996; 10: 1273-1281Crossref PubMed Scopus (62) Google Scholar). PKC β isozymes (I and II) stimulate cell division and differentiation by regulating the expression of several oncogenes, including c-fos (31Messina J.L. Standaert M.L. Ishizuka T. Weinstock R.S. Farese R.V. J. Biol. Chem. 1992; 267: 9223-9228Abstract Full Text PDF PubMed Google Scholar). Spatial localization within the cell is a component of the biological function of many protein kinases, including the PKC enzymes whose catalytic competence and localization are regulated by serine/threonine phosphorylation (14Ron D. Chen C.H. Caldwell J. Jamieson L. Orr E. Mochly-Rosen D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 839-843Crossref PubMed Scopus (641) Google Scholar, 32Shirai Y. Saito N. J. Biochem. (Tokyo). 2002; 132: 663-668Crossref PubMed Scopus (127) Google Scholar, 33Newton A.C. Chem. Rev. 2001; 101: 2353-2364Crossref PubMed Scopus (825) Google Scholar). In the case of PKC βII, when activated, the kinase translocates to the plasma membrane from the cytoplasm (34Farrar W.L. Anderson W.B. Nature. 1985; 315: 233-235Crossref PubMed Scopus (289) Google Scholar). This kinase is a member of the Mg2+- and Ca2+-dependent, phospholipid/phorbol ester-activated family of the conventional PKCs.Presently, we have identified hBVR as a substrate for PKC βII kinase activity. In the course of the study, the reciprocal phosphorylation and activation of PKC βII by hBVR was uncovered. Collectively, the present findings and past reports define hBVR not only as an enzyme with a unique activity profile but also as one with the possibility of having input at multiple stages in cell signaling pathways.EXPERIMENTAL PROCEDURESMaterials and Constructs—Recombinant human PKC βII was purchased from Calbiochem. [γ-32P]ATP and [α-32P]dCTP were from PerkinElmer Life Sciences. Monoclonal and polyclonal anti-PKC βII antibodies were from Zymed Laboratories Inc. and Abgent (San Diego, CA), respectively. Polyclonal anti-phospho-Thr500 PKC βII antibodies were from Abcam (Cambridge, MA). Biotrace polyvinylidene difluoride membrane was a product of Pall Science Corp. (Pensacola, FL). PKC βII-based peptides referred to as Thr500 (MCKENIWDGVTTKTFCG), Thr500mut (MAKENIWDGVTTKAFAG), Thr641 (VLTPPDQEVIRNIDQ), Ser661 (FEGFSFVNSEFLKPEVKS), and the control peptide Smut (FEGFAFVNAEFLKPEVKA) were synthesized by Anaspec Inc. (San Jose, CA). PKC-ζ-based peptides, PKCζ281 (DQIYAMKVVKKE), PKCζ410 (GDTTSTFCGTPN), PKCζ560 (EPVQLTPDDEDA), and PKCζ585 (EFEGFEYINPLLL), were custom-synthesized by Synpep (Dublin, CA). The numbered residues in PKC βII are detrimental to its kinase activity (35Keranen L.M. Dutil E.M. Newton A.C. Curr. Biol. 1995; 5: 1394-1403Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar). The mutant constructs, G17A and V11A/V12A/V13A/V14A, were generated by site-directed mutagenesis of the wild-type hBVR cDNA (19Maines M.D. Polevoda B.V. Huang T.J. McCoubrey Jr., W.K. Eur. J. Biochem. 1996; 235: 372-381Crossref PubMed Scopus (50) Google Scholar) and used to create expression plasmids in pcDNA3 (used for transfection into 293A cells) and pGEX4-T2 (used for transfection into E. coli) hBVR. Expression plasmids for PKC βII and PKC ζ were constructed by subcloning cDNA from pSP65-PKC βII or pCO2-PKC ζ (generous gift from Peter Parker, London Research Institute, London, UK) into pcDNA3 and pGEX4-T2. Site-directed mutagenesis of the pGEX4-T2 construct was used to replace Lys371 in the ATP-binding site with arginine to express a "kinase-dead" protein (36Ohno S. Konno Y. Akita Y. Yano A. Suzuki K. J. Biol. Chem. 1990; 265: 6296-6300Abstract Full Text PDF PubMed Google Scholar, 37Feng X. Hannun Y.A. J. Biol. Chem. 1998; 273: 26870-26874Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). hBVR truncations 1–108, 109–175, 272–296, 272–296 C/A, and 272–296 Y/F were constructed from existing pGEX4-T2-hBVR by appropriate deletion and site-directed mutagenesis of WT-hBVR. pSuper-Retro-siBVR was constructed as described previously (1Miralem T. Hu Z. Torno M.D. Lelli K.M. Maines M.D. J. Biol. Chem. 2005; 280: 17084-17092Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The primers 5′-GATCCCCTCCTCAGTCCGTTCGAACCTGTTCAAGAGACAGGTTGCTGCAACGGACTGAGGATTTTTGGAAA-3′, and 5′-AGCTTTTCCAAAAATCCTCAGTCCGTTCGAACCTGTCTCTTGAACAGGTTGCAACGGACTGAGGAGGG-3′ were designed as a scrambled form of the hBVR siRNA and were used to make the siBVR-sc control construct. The PKC-specific substrate S2 (VRKRTLRRL) was purchased from Anaspec Inc. The PKC β-specific inhibitor LY333531 was obtained from A.G. Scientific, Inc. (San Diego); and the inhibitor of conventional PKCs, Go-6976, was from Calbiochem. PKCi, MBP, and PKC lipid activator (PS: DAG) were purchased from Upstate (Charlottesville, VA). Co-PP was obtained from Porphyrin Products Inc. (Logan, UT).Cell Culture, Transfection, Co-immunoprecipitation, and GST Pulldown—293A cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum and 1% penicillin G/streptomycin for 24 h or until the cells reached 70% confluency. Depending on the experiment, cells were subsequently transfected with up to 5 μg of pcDNA3-hBVR or pcDNA3-PKC βII plasmid using transfectin lipid reagent (Bio-Rad) in 10-cm plates, according to the manufacturer's instructions. Western blotting confirmed overexpression of hBVR or PKC βII. To prepare hBVR siRNA or siBVR-sc, pSuper-Retro-siBVR or siBVR-sc was transfected into 293A cells for packaging, and the siBVR or siBVR-sc retrovirus was then titrated using NIH3T3 cells. 293A cells were infected with 4 plaque-forming units/cell to inhibit hBVR synthesis (1Miralem T. Hu Z. Torno M.D. Lelli K.M. Maines M.D. J. Biol. Chem. 2005; 280: 17084-17092Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). For the protein-protein interaction experiments, cells were seeded into 10-cm dishes and co-transfected with both WT pcDNA3-hBVR and pcDNA3-PKC βII for co-immunoprecipitation, whereas for GST pulldown pcDNA3-PKC βII and pEGFP-HO2 were used. Prior to treatment with 100 nm PMA, the cells were serum-starved in growth medium containing 0.1% fetal bovine serum for 24 h and then lysed in RIPA buffer.For immunoprecipitation experiments, cell lysate (500 μg of protein) was incubated with monoclonal anti-PKC βII antibodies or normal mouse serum overnight at 4 °C. A/G-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) were added to select antibody-bound protein. The agarose beads were washed three times in lysis buffer, and the samples were boiled in Laemmli gel loading buffer, separated by SDS-PAGE, and detected by Western blotting using rabbit polyclonal anti-hBVR antibodies. For the GST pulldown assay, cell lysate was incubated with 10 μg of GST-hBVR fusion protein or GST immobilized on GSH-agarose beads (Amersham Biosciences) at 4 °C for 2 h. The beads were washed three times and boiled in Laemmli buffer to release the bound proteins. Resolved by SDS-PAGE, proteins were detected by immunoblotting using mouse monoclonal anti-PKC βII antibodies.Cell Fractionation—After a cold wash in PBS, cells were scraped and collected by centrifuging at 500 × g for 5 min. Intact cell pellets were resuspended in homogenization medium containing 0.28 m sucrose, 50 mm Tris-HCl (pH 7.5), 25 mm KCl, 5 mm MgCl2, 1 mm EDTA and 1 μg/ml each of the protease inhibitors leupeptin, pepstatin, and aprotinin and 1 mm phenylmethylsulfonyl fluoride and homogenized by passing five times through a 25-gauge needle. Cell homogenates were centrifuged for 25 min at 120 × g at 4 °C to remove large cellular debris (most nuclei and larger), and collected supernatants were centrifuged 15 min at 13,000 × g, 4 °C, to pellet mitochondria. Collected supernatants were centrifuged at 25,000 × g, 4 °C, for 60 min to separate plasma membrane (pellet) from cytoplasm (supernatant). Both fractions were collected, dissolved in RIPA buffer +1% Triton X-100 (membrane) or RIPA buffer (cytoplasm), processed for protein determination, and stored at -20 °C for further examination. To test the purity of extracted cell fractions, aliquots of cell fractions were examined for LDH activity as described earlier (3Kutty R.K. Maines M.D. J. Biol. Chem. 1981; 256: 3956-3962Abstract Full Text PDF PubMed Google Scholar).PKC βII Activity in Vitro—PKC βII kinase activity was assayed in vitro as recommended by the manufacturer (Calbiochem). MBP was used as the substrate, as it is commonly used for PKC isozymes and for BVR serine/threonine kinase activity (4Salim M. Brown-Kipphut B.A. Maines M.D. J. Biol. Chem. 2001; 276: 10929-10934Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Phosphorylation of MBP can be detected by SDS-PAGE or by trapping on P81 phosphocellulose filters. The design of the assay system was modified, depending on whether hBVR or PKC βII was used as the enzyme or substrate. Unless otherwise specified, for PKC βII activity 5 ng of PKC βII was incubated in a 50-μl assay containing 20 mm HEPES (pH 7.2), 15 mm MgCl2, 0.2 mm CaCl2, 12.5 μm MBP, 10 mm β-glycerophosphate, and 1 mm dithiothreitol in the presence of a sonicated lipid activator at final concentrations of 0.05 mg/ml PS and 0.005 mg/ml DAG, or PS alone. The addition of 100 μm ATP containing 5 μCi of [γ-32P]ATP initiated the reaction. To examine the effect of WT or mutant hBVR on PKC βII activity, they were incubated for 10 min with PKC prior to addition of MBP. If WT or mutant hBVR was used as substrate, MBP was omitted. If used, the inhibitor PKCi was added to PKC βII 2 min prior to hBVR or MBP addition. The incubation lasted for 10 min at 30 °C when MBP was the substrate and 20 min with hBVR as the sole substrate, unless otherwise stated. The reaction was terminated on ice, either by the addition of Laemmli buffer for SDS-PAGE followed by transfer to polyvinylidene difluoride membrane and autoradiography, or by the addition of 1 volume of 10% phosphoric acid for the P81 phosphocellulose binding assay. An aliquot of the samples was directly applied to the center of the filter, washed six times in 0.75% phosphoric acid, and dried with acetone prior to measurement for radioactivity.PKC Assay in Situ—The assay was performed by a modification of procedures detailed by Williams and Schrier (38Williams B. Schrier R.W. J. Clin. Investig. 1993; 92: 2889-2896Crossref PubMed Scopus (141) Google Scholar). Cells were seeded into 48-well plates and transfected with 0.5 μg/well pcDNA3-hBVR or with G17A or V11A/V12A/V13A/V14A mutants. 24 h later, the medium was replaced with starvation medium (0.1% serum), and the incubation was continued for another 24 h to synchronize cells. In some cases, cells were pretreated with the PKC inhibitors Go-6976 (200 nm) for conventional PKCs or LY333531 (30 nm) for PKC β (39Jirousek M.R. Gillig J.R. Gonzalez C.M. Heath W.F. McDonald 3rd, J.H. Neel D.A. Rito C.J. Singh U. Stramm L.E. Melikian-Badalian A. Baevsky M. Ballas L.M. Hall S.E. Winneroski L.L. Faul M.M. J. Med. Chem. 1996; 39: 2664-2671Crossref PubMed Scopus (325) Google Scholar, 40Nakamura J. Kasuya Y. Hamada Y. Nakashima E. Naruse K. Yasuda Y. Kato K. Hotta N. Diabetologia. 2001; 44: 480-487Crossref PubMed Scopus (67) Google Scholar, 41Watterson J.M. Watson D.G. Meyer E.M. Lenox R.H. Brain Res. 2002; 934: 69-80Crossref PubMed Scopus (32) Google Scholar) for 30 min before addition of PMA (100 nm, 15 min). Cells were washed with medium and incubated for 10 min at 30 °C in 50 μl of kinase assay buffer (137 mm NaCl, 5.4 mm KCl, 10 mm MgCl2, 0.3 mm Na2HPO4, 0.4 mm KH2PO4, 25 mm β-glycerophosphate, 5.5 mm d-glucose, 5 mm EGTA, 1 mm CaCl2, 20 mm HEPES (pH 7.2), 50 μg/ml digitonin, 120 μg/ml S2 substrate, and 100 μm ATP labeled with 10 μCi/ml [γ-32P]ATP). The reaction stopped with the addition of 25 μl of ice-cold 30% (w/v) trichloroacetic acid on ice. The trichloroacetic acid-soluble fraction samples were transferred to P81 phosphocellulose filters. After 15 min at room temperature, the filters were washed three times in 75 mm phosphoric acid, once in 2.75 mm sodium phosphate (pH 7.5), and once with acetone before liquid scintillation counting. Kinase activity was normalized to protein content.hBVR Kinase Activity—The activity was measured as described recently (2Lerner-Marmarosh N. Shen J. Torno M.D. Kravets A. Hu Z. Maines M.D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 7109-7114Crossref PubMed Scopus (119) Google Scholar). For routine assays, purified hBVR, at concentrations noted in the appropriate figure legends, was incubated at 30 °C in a 50-μl reaction mixture cont
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