Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Activation of Protein Kinase Cζ by Peroxynitrite Regulates LKB1-dependent AMP-activated Protein Kinase in Cultured Endothelial Cells

2006; Elsevier BV; Volume: 281; Issue: 10 Linguagem: Inglês

10.1074/jbc.m511178200

ISSN

1083-351X

Autores

Zhonglin Xie, Yunzhou Dong, Miao Zhang, Mei‐Zhen Cui, Richard A. Cohen, Uwe Riek, Dietbert Neumann, Uwe Schlattner, Ming-Hui Zou,

Tópico(s)

Cancer, Hypoxia, and Metabolism

Resumo

We previously reported the phosphoinositide 3-kinase-dependent activation of the 5′-AMP-activated kinase (AMPK) by peroxynitrite (ONOO-) and hypoxia-reoxygenation in cultured endothelial cells. Here we show the molecular mechanism of activation of this pathway. Exposure of bovine aortic endothelial cells to ONOO- significantly increased the phosphorylation of both Thr172 of AMPK and Ser1179 of endothelial nitric-oxide synthase, a known downstream enzyme of AMPK. In addition, activation of AMPK by ONOO- was accompanied by increased phosphorylation of protein kinase Cζ (PKCζ) (Thr410/403) and translocation of cytosolic PKCζ into the membrane. Further, inhibition of PKCζ abrogated ONOO--induced AMPK-Thr172 phosphorylation as that of endothelial nitric-oxide synthase. Furthermore, overexpression of a constitutively active PKCζ mutant enhanced the phosphorylation of AMPK-Thr172, suggesting that PKCζ is upstream of AMPK activation. In contrast, ONOO- activated PKCζ in LKB1-deficient HeLa-S3 but affected neither AMPK-Thr172 nor AMPK activity. These data suggest that LKB1 is required for PKCζ-enhanced AMPK activation. In vitro, recombinant PKCζ phosphorylated LKB1 at Ser428, resulting in phosphorylation of AMPK at Thr172. Further, direct mutation of Ser428 of LKB1 into alanine, like the kinase-inactive LKB1 mutant, abolished ONOO--induced AMPK activation. In several cell types originating from human, rat, and mouse, inhibition of PKCζ significantly attenuated the phosphorylation of both LKB1-Ser428 and AMPK-Thr172 that were enhanced by ONOO-. Taken together, we conclude that PKCζ can regulate AMPK activity by increasing the Ser428 phosphorylation of LKB1, resulting in association of LKB1 with AMPK and consequent AMPK Thr172 phosphorylation by LKB1. We previously reported the phosphoinositide 3-kinase-dependent activation of the 5′-AMP-activated kinase (AMPK) by peroxynitrite (ONOO-) and hypoxia-reoxygenation in cultured endothelial cells. Here we show the molecular mechanism of activation of this pathway. Exposure of bovine aortic endothelial cells to ONOO- significantly increased the phosphorylation of both Thr172 of AMPK and Ser1179 of endothelial nitric-oxide synthase, a known downstream enzyme of AMPK. In addition, activation of AMPK by ONOO- was accompanied by increased phosphorylation of protein kinase Cζ (PKCζ) (Thr410/403) and translocation of cytosolic PKCζ into the membrane. Further, inhibition of PKCζ abrogated ONOO--induced AMPK-Thr172 phosphorylation as that of endothelial nitric-oxide synthase. Furthermore, overexpression of a constitutively active PKCζ mutant enhanced the phosphorylation of AMPK-Thr172, suggesting that PKCζ is upstream of AMPK activation. In contrast, ONOO- activated PKCζ in LKB1-deficient HeLa-S3 but affected neither AMPK-Thr172 nor AMPK activity. These data suggest that LKB1 is required for PKCζ-enhanced AMPK activation. In vitro, recombinant PKCζ phosphorylated LKB1 at Ser428, resulting in phosphorylation of AMPK at Thr172. Further, direct mutation of Ser428 of LKB1 into alanine, like the kinase-inactive LKB1 mutant, abolished ONOO--induced AMPK activation. In several cell types originating from human, rat, and mouse, inhibition of PKCζ significantly attenuated the phosphorylation of both LKB1-Ser428 and AMPK-Thr172 that were enhanced by ONOO-. Taken together, we conclude that PKCζ can regulate AMPK activity by increasing the Ser428 phosphorylation of LKB1, resulting in association of LKB1 with AMPK and consequent AMPK Thr172 phosphorylation by LKB1. The AMP-activated protein kinase (AMPK) 2The abbreviations used are: AMPK, 5′-AMP activated-kinase; BAEC, bovine aortic endothelial cell; CA, constitutively active mutant; DN, dominant negative mutants; eNOS, endothelial nitric-oxide synthase; GFP, green fluorescent protein; H/R, hypoxia-reoxygenation; ONOO-, peroxynitrite; PDK1, phosphoinositide-dependent kinase 1; PI, phosphoinositide; PKA, protein kinase A; PKG, protein kinase G; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PS, pseudosubstrate; AMPKK, AMPK upstream kinase; ACC, acetyl-CoA carboxygenase; AICAR, aminoimidazole-4-carboxamide-1-β-d-riboside. is a serine/threonine kinase and a member of the Snf1/AMPK protein kinase family (1Hardie D.G. Carling D. Carlson M. Ann. Rev. Biochem. 1998; 67: 821-855Crossref PubMed Scopus (1281) Google Scholar, 2Kemp B.E. Stapleton D. Campbell D.J. Chen Z.P. Murthy S. Walter M. Gupta A. Adams J.J. Katsis F. Van Denderen B. Jennings I.G. Iseli T. Michell B.J. Witters L.A. Biochem. Soc. Trans. 2003; 31: 162-168Crossref PubMed Google Scholar, 3Winder W.W. Hardie D.G. Am. J. Physiol. 1999; 277: E1-E10PubMed Google Scholar). Its activity is stimulated by an increase in intracellular AMP-to-ATP ratio in response to stresses such as exercise (4Winder W.W. Hardie D.G. Am. J. Physiol. 1996; 270: E299-E304Crossref PubMed Google Scholar, 5Hutber C.A. Hardie D.G. Winder W.W. Am. J. Physiol. 1996; 272: E262-E266Google Scholar, 6Vavvas D. Apazidis A. Saha A.K. Gamble J. Patel A. Kemp B.E. Witters L.A. Ruderman N.B. J. Biol. Chem. 1997; 272: 13255-13261Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar), hypoxia (7Kudo N. Barr A.J. Barr R.L. Desai S. Lopaschuk G.D. J. Biol. Chem. 1995; 270: 17513-17520Abstract Full Text Full Text PDF PubMed Scopus (510) Google Scholar, 8Marsin A.S. Bertrand L. Rider M.H. Deprez J. Beauloye C. Vincent M.F. van den Berghe G. Carling D. Hue L. Curr. Biol. 2000; 10: 1247-1255Abstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar), oxidant stress (9Zou M.H. Hou X. Shi M. Nagata D. Walsh K. Cohen R.A. J. Biol. Chem. 2002; 277: 32552-32557Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 10Choi S.L. Kim S.J. Lee K.T. Kim J. Mu J. Birnbaum M.J. Soo-Kim S. Ha J. Biochem. Biophys. Res. Commun. 2001; 287: 92-97Crossref PubMed Scopus (259) Google Scholar), and glucose deprivation (11Salt I.P. Johnson G. Ashcroft S.J.H. Hardie D.G. Biochem. J. 1998; 335: 533-539Crossref PubMed Scopus (339) Google Scholar). AMPK activation switches on catabolic pathways that produce ATP and switches off anabolic pathways that consume ATP. The activation of AMPK leads to phosphorylation of a number of proteins that result in increased glucose uptake and metabolism as well as fatty acid oxidation and simultaneously in inhibition of hepatic lipogenesis, cholesterol synthesis, and glucose production (reviewed in Refs. 12Hardie D. J. Cell Sci. 2004; 117: 5479-5487Crossref PubMed Scopus (965) Google Scholar, 13Kahn B.B. Alquier T. Carling D. Hardie D.G. Cell Metab. 2005; 1: 15-25Abstract Full Text Full Text PDF PubMed Scopus (2347) Google Scholar, 14Hardie D.G. Curr. Opin. Cell Biol. 2005; 17: 167-173Crossref PubMed Scopus (225) Google Scholar). AMPK is also responsible for increased fatty acid oxidation in response to the adipocyte-derived hormones leptin (15Minokoshi Y. Kim Y.B. Peroni O.D. Fryer L.G. Muller C. Carling D. Kahn B.B. Nature. 2002; 415: 339-343Crossref PubMed Scopus (1691) Google Scholar) and adiponectin (16Yamauchi T. Kamon J. Minokoshi Y. Ito Y. Waki H. Uchida S. Yamashita S. Noda M. Kita S. Ueki K. Eto K. Akanuma Y. Froguel P. Foufelle F. Ferre P. Carling D. Kimura S. Nagai R. Kahn B.B. Kadowaki T. Nat. Med. 2002; 8: 1288-1295Crossref PubMed Scopus (3483) Google Scholar). Because AMPK activation could have beneficial metabolic consequences for diabetic patients, AMPK has emerged as a potential target for the treatment of obesity and type II diabetes (reviewed in Refs. 3Winder W.W. Hardie D.G. Am. J. Physiol. 1999; 277: E1-E10PubMed Google Scholar and 17Winder W.W. Hardie D.G. Am. J. Physiol. 1999; 277: E1-E10PubMed Google Scholar). It has been demonstrated that two classes of anti-diabetic drugs, metformin (18Zhou G. Myers R. Li Y. Chen Y. Shen X. Fenyk-Melody J. Wu M. Ventre J. Doebber T. Fujii N. Musi N. Hirshman M.F. Goodyear L.J. Moller D.E. J. Clin. Investig. 2001; 108: 1167-1174Crossref PubMed Scopus (4472) Google Scholar, 19Zou M.H. Kirkpatrick S.S. Davis B.J. Nelson J.S. Wiles 4th, W.G Schlattner U. Neumann D. Brownlee M. Freeman M.B. Goldman M.H. J. Biol. Chem. 2004; 279: 43940-43951Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar) and thiazolidinediones (20Fryer L.G. Parbu-Patel A. Carling D. J. Biol. Chem. 2002; 277: 25226-25232Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar), can act at least in part through activation of AMPK in liver and muscle. AMPK is an obligatory heterotrimer containing catalytic α subunit and regulatory β and γ subunits, each of which occur in at least two isoforms. Activation of AMPK absolutely requires its phosphorylation at Thr172 in the activation loop of α1 and α2 subunits by one or more upstream kinases (AMPKKs) (21Dyck J.R. Gao G. Widmer J. Stapleton D. Fernandez C.S. Kemp B.E. Witters L.A. J. Biol. Chem. 1996; 271: 17798-17803Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 22Hawley S.A. Davison M. Woods A. Davies S.P. Beri R.K. Carling D. Hardie D.G. J. Biol. Chem. 1996; 271: 27879-27887Abstract Full Text Full Text PDF PubMed Scopus (1015) Google Scholar). The major breakthrough in identifying the first AMPKK came from research on the regulation of the AMPK ortholog Snf-1 in Saccharomyces cerevisiae (23Hong S.P. Leiper F.C. Woods A. Carling D. Carlson M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8839-8843Crossref PubMed Scopus (481) Google Scholar, 24Hardie D.G. Carling D. Halford N. Semin. Cell Biol. 1994; 5: 409-416Crossref PubMed Scopus (80) Google Scholar). The T-loop residue of Snf-1 was phosphorylated by a group of three related protein kinases bearing homology to mammalian LKB1, which was subsequently identified by several laboratories as being the major upstream kinase for AMPK (25Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Makela T.P. Alessi D.R. Hardie D.G. J. Biol. 2003; 2: 28Crossref PubMed Google Scholar, 26Shaw R.J. Kosmatka M. Bardeesy N. Hurley R.L. Witters L.A. DePinho R.A. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3329-3335Crossref PubMed Scopus (1453) Google Scholar, 27Woods A. Johnstone S.R. Dickerson K. Leiper F.C. Fryer L.G. Neumann D. Schlattner U. Wallimann T. Carlson M. Carling D. Curr. Biol. 2003; 13: 2004-2008Abstract Full Text Full Text PDF PubMed Scopus (1352) Google Scholar). LKB1 was found to co-purify with liver AMPK and to phosphorylate recombinant AMPK complexes. In addition, AMPK could not be activated in mammalian cells that lacked LKB1 expression or in cells that were treated with Hsp90 inhibitors, which decrease LKB1 expression (28Boudeau J. Sapkota G. Alessi D.R. FEBS Lett. 2003; 546: 159-165Crossref PubMed Scopus (149) Google Scholar, 29Boudeau J. Deak M. Lawlor M.A. Morrice N.A. Alessi D.R. Biochem. J. 2003; 370: 849-857Crossref PubMed Scopus (74) Google Scholar). Finally, LKB1 turned out to phosphorylate the T-loop of all the 12 human kinases that are phylogenetically related to AMPK (AMPK subfamily) (30Lizcano J.M. Goransso N.O. Toth R. Deak M. Morrice N.A. Boudeau J. Hawley S.A. Udd L. Makela T.P. Hardie D.G. Alessi D.R. EMBO J. 2004; 23: 833-843Crossref PubMed Scopus (1068) Google Scholar). However, paradoxically, neither the activity of LKB1 itself nor that of AMPK-related kinases was regulated directly by the stimuli known to activate AMPK, like e.g. AMP, AICAR, or muscle contraction (31Sakamoto K. Goransson O. Hardie D.G. Alessi D.R. Am. J. Physiol. 2004; 287: 310-317Crossref PubMed Scopus (27) Google Scholar, 32Sakamoto K. McCarthy A. Smith D. Green K.A. Hardie G.D. Ashworth A. Alessi D.R. EMBO J. 2005; 24: 1810-1820Crossref PubMed Scopus (446) Google Scholar). Thus, the question remained regarding how AMPK stimuli can lead to LKB1-dependent AMPK activation. We had previously reported that peroxynitrite (ONOO-), a potent oxidant formed by the combination of superoxide anions (O2·¯) and NO at a diffusion-controlled rate, activated AMPK independent of the cellular AMP/ATP ratios (19Zou M.H. Kirkpatrick S.S. Davis B.J. Nelson J.S. Wiles 4th, W.G Schlattner U. Neumann D. Brownlee M. Freeman M.B. Goldman M.H. J. Biol. Chem. 2004; 279: 43940-43951Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 33Davis B. Xie Z. Viollet B. Zou M.H. Diabetes. 2006; (in press)Google Scholar, 34Zou M.H. Hou X.Y. Shi C.M. Kirkpatick S. Liu F. Goldman M.H. Cohen R.A. J. Biol. Chem. 2003; 278: 34003-34010Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). We further demonstrated that short periods of hypoxia-reoxygenation (H/R)-activated AMPK in a ONOO --dependent manner, which was also independent of the AMP/ATP ratios, but was sensitive to PI 3-kinase inhibition with either pharmacological inhibitors or overexpression of PDK1 dominant negative mutants (PDK1-DN) (34Zou M.H. Hou X.Y. Shi C.M. Kirkpatick S. Liu F. Goldman M.H. Cohen R.A. J. Biol. Chem. 2003; 278: 34003-34010Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). PI 3-kinase-dependent AMPK activation has since been observed by others in insulin-stimulated platelets (35Fleming I. Schulz C. Fichtlscherer B. Kemp B.E. Fisslthaler B. Busse R. Thromb. Haemostasis. 2003; 90: 863-871Crossref PubMed Google Scholar) and adiponectin-stimulated endothelial cells (36Chen H. Montagnani M. Funahashi T. Shimomura I. Quon M.J. J. Biol. Chem. 2003; 278: 45021-45026Abstract Full Text Full Text PDF PubMed Scopus (888) Google Scholar). Paradoxically, activation of PI 3-kinase with insulin or growth factors that stimulate the PI 3-kinase/PDK1 pathway either did not affect AMPK in most cell types (37Williams M.R. Arthur J.S. Balendran A. van der Kaay J. Poli V. Cohen P. Alessi D.R. Curr. Biol. 2000; 10: 439-448Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar) or rather caused AMPK inhibition in some cases (34Zou M.H. Hou X.Y. Shi C.M. Kirkpatick S. Liu F. Goldman M.H. Cohen R.A. J. Biol. Chem. 2003; 278: 34003-34010Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 38Beauloye C. Marsin A.S. Bertrand L. Vanoverschelde J.L. Rider M.H. Hue L. FEBS Lett. 2002; 531: 324-328Crossref PubMed Scopus (38) Google Scholar). Therefore, PI 3-kinase/PDK1 will very likely not serve as a direct upstream kinase for LKB1, and the mechanism for AMPK activation remains to be established. In the present study, we have established a central role of atypical protein kinase Cζ (PKCζ), a protein kinase of the AGC family, as a key regulator in LKB1-dependent AMPK activation. We found that inhibition of PKCζ with pharmacological and genetic inhibitors effectively blocked AMPK activation caused by ONOO-. This AMPK activation pathway was LKB1-dependent, involved LKB1 phosphorylation at Ser428 within the C-terminal part of LKB1, and led to the association of LKB1 with AMPK. The central role of PKCζ in the LKB1-AMPK axis is further supported by the fact that inhibition of PKCζ with either pharmacological (PKCζ-PS) or genetic (PKCζ-dominant negative mutants, PKCζ-DN) inhibitors blunted AMPK activation caused by (ONOO-). Finally, in vitro, recombinant PKCζ phosphorylated both LKB1 at Ser428. We conclude that PKCζ-dependent and LKB1-mediated AMPK activation might play important roles in regulating not only cellular energy metabolism but also signaling pathways that control cell growth, differentiation, and survival. Bovine aortic endothelial cells (BAEC) and cell culture media were obtained from Clonetics Inc. (Walkersville, MD). BAEC were maintained in endothelial basal medium with 2% serum and growth factors before use. HeLa-S3 cells were obtained from ATCC (Manassas, VA) and were grown in Dulbecco's modified Eagle's medium supplemented with 10% serum. All culture media were added with penicillin (100 units/ml) and streptomycin (100 μg/ml) (32Sakamoto K. McCarthy A. Smith D. Green K.A. Hardie G.D. Ashworth A. Alessi D.R. EMBO J. 2005; 24: 1810-1820Crossref PubMed Scopus (446) Google Scholar). [32P]ATP was obtained from PerkinElmer Life Sciences. The SAMS peptide and recombinant LKB1 were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Antibodies against phospho-AMPK (Thr172), AMPK-α, phospho-eNOS (Ser1177), phosphor-LKB1 (Ser428, Ser334, and Thr189), LKB1 (for Western blot), PKCζ, phospho-PKCζ (Thr410-403), PKCα/β, and phospho-PKCα/β (Thr638/641) were obtained from Cell Signaling Inc. (Beverly, MA). Antibody against eNOS was obtained from the BD Bioscience. Protein A/G-agarose beads, antibodies against PKCζ and LKB1 (used for immunoprecipitation assay, catalogue number Sc-5638, D-19) were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). ONOO-, SIN-1, recombinant PKCζ, PKC βII, Go6983, H89, KT5823, calphostin C, wortmanin, LY294002, and arcyriarubin A (2,3-bis(1H-Indol-3-yl)maleimide) were obtained from Calbiochem. Cell-permeable myristoylated both PKC-ζ-PS and PKC-βII-PS were from BIOSOURCE International (Camarillo, CA). Other chemicals and organic solvents of highest grade were obtained from Sigma. Cell Culture and Adenoviral Infection—To generate the adenovial vector expressing a constitutively active mutant of AMPK α1 (AMPK-CA), a rat cDNA encoding residues 1-312 of AMPKα1 and bearing a mutation of Thr172 into aspartic acid (T172D) was subcloned into a shuttle vector (p-shuttle CMV). The c-Myc epitope tag was fused in frame to the 5′-terminus of the coding sequence. The resulting plasmid was linearized by digesting with PmeI and co-transfected into Escherichia coli BJ5183 with the adenoviral backbone plasmid, pAdEasy-1. Homogenous recombinants were selected with kanamycin. The linearized recombinant plasmid was infected into transformed human embryonic kidney 293 cells. Recombinant adenoviruses were amplified on 293 cells and purified by two ultracentrifugation steps on cesium chloride gradients. The number of viral particles was assessed by measurement of the optical density at 260 nm. BAEC were infected with adenovirus expressing a constitutively active PKCζ mutant (PKCζ-CA), a dominant negative mutant PKCζ (PKCζ-DN) (39Tan M. Xu X. Ohba M. Ogawa W. Cui M.Z. J. Biol. Chem. 2003; 278: 2824-2828Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 40Minami T. Abid M.R. Zhang J. King G. Kodama T. Aird W.C. J. Biol. Chem. 2003; 278: 6976-6984Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), or adenovirus coding constitutively active AMPK (AMPK-CA) (33Davis B. Xie Z. Viollet B. Zou M.H. Diabetes. 2006; (in press)Google Scholar, 34Zou M.H. Hou X.Y. Shi C.M. Kirkpatick S. Liu F. Goldman M.H. Cohen R.A. J. Biol. Chem. 2003; 278: 34003-34010Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). A replication-defective adenoviral vector expressing green fluorescence protein (Ad-GFP) was used as control. BAEC were infected with the adenoviruses with a multiplicity of infection of at least 10 in medium deprived of serum overnight. The cells were then washed and incubated in fresh endothelial base medium without serum for an additional 18-24 h prior to experimentation. Under these conditions, infection efficiency was typically >80% as determined by GFP expression. Hypoxia-Reoxygenation of BAEC—H/R was performed as described previously (34Zou M.H. Hou X.Y. Shi C.M. Kirkpatick S. Liu F. Goldman M.H. Cohen R.A. J. Biol. Chem. 2003; 278: 34003-34010Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Briefly, BAEC were cultured in 6-well plates. The cells were first infected with indicated adenoviruses for 2 days if required. The cells were placed in a water bath (37 °C, total volume of 2 liters) filled with 1 liter of prewarmed Kreb-Ringer's buffer, gassed with 95% O2,5%CO2. After 30 min of incubation, the oxygen tension was reduced abruptly from 95% O2, 5% CO2 to 95% N2, 5% CO2 and was maintained for 15 min as indicated. After this phase of hypoxia, 95% O2,5%CO2 was resumed (reoxygenation) for 15 min. After that the cells were washed with phosphate-buffered saline buffer twice and collected for Western blot and immunoprecipitation assays. The control BAEC was gassed only with 95% O2, 5% CO2 for equivalent periods. Assay of AMPK Activity—AMPK activity was assayed by using the SAMS peptide, as previously described (19Zou M.H. Kirkpatrick S.S. Davis B.J. Nelson J.S. Wiles 4th, W.G Schlattner U. Neumann D. Brownlee M. Freeman M.B. Goldman M.H. J. Biol. Chem. 2004; 279: 43940-43951Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 33Davis B. Xie Z. Viollet B. Zou M.H. Diabetes. 2006; (in press)Google Scholar, 34Zou M.H. Hou X.Y. Shi C.M. Kirkpatick S. Liu F. Goldman M.H. Cohen R.A. J. Biol. Chem. 2003; 278: 34003-34010Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). The difference between the presence and absence of AMP (200 μm) was calculated as the AMPK activity. LKB1 Activity Assay—LKB1 was immunoprecipitated from untreated (control) or treated cells with an antibody against LKB1 (Santa Cruz, catalogue number Sc-5638, D-19) overnight at 4 °C in the presence of protein A/G-agarose. LKB1 activity present in the immunoprecipitates was determined by its ability to activate recombinant AMPK as described previously (20Fryer L.G. Parbu-Patel A. Carling D. J. Biol. Chem. 2002; 277: 25226-25232Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar). In Vitro Kinase Assays—To determine the effects of PKCζ or PKCβII on AMPK or LKB1 or both, recombinant LKB1 or recombinant AMPK α1β1r1 (41Neumann D. Woods A. Carling D. Wallimann T. Schlattner U. Protein Expr. Purif. 2003; 30: 230-237Crossref PubMed Scopus (119) Google Scholar) were incubated with PKCζ at concentrations indicated for 15 min at 37 °C in the presence of [32P]ATP (1 μCi) with or without AMP (200 μm). The SAMS peptides (final concentration, 200 μm) were added if needed. AMPK activity was calculated by counting the phosphorylated SAMS peptides in the supernatants (25 μl) as described previously (33Davis B. Xie Z. Viollet B. Zou M.H. Diabetes. 2006; (in press)Google Scholar, 34Zou M.H. Hou X.Y. Shi C.M. Kirkpatick S. Liu F. Goldman M.H. Cohen R.A. J. Biol. Chem. 2003; 278: 34003-34010Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). The beads were added 20 μlof3× sample buffer and boiled for 5 min at 95 °C. The proteins were separated with 12% SDS-PAGE, and the dried gels were subjected to radioautography. Immunoprecipitation and Western Blotting—The proteins were immunoprecipitated with specific antibodies and Western blotted into nitrocellulose membranes, and the proteins were detected by specific antibodies, as described previously (33Davis B. Xie Z. Viollet B. Zou M.H. Diabetes. 2006; (in press)Google Scholar, 34Zou M.H. Hou X.Y. Shi C.M. Kirkpatick S. Liu F. Goldman M.H. Cohen R.A. J. Biol. Chem. 2003; 278: 34003-34010Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Preparation of Membrane Fractions—Cellular cytosolic and membrane fractions were prepared as described previously (42Xie Z. Singh M. Singh K. J. Biol. Chem. 2004; 279: 39513-39519Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). The membranes were then incubated with appropriate secondary antibodies and analyzed in Western blots. Site-directed Mutagenesis of Ser428 and Asp194 of Human LKB1 and Plasmid Transfection—Human cDNA clone was purchased from Invitrogen (clone 3689780). Wild type LKB1 gene coding region was amplified by PCR. The PCR product was ligated into TA cloning vector pGEM-T easy (Promega). LKB1 gene was released with enzymes of EcoRI/NotI from TA cloning vector and was cloned into pCI-neo mammalian expression vector (Promega; catalogue number E184). Ser428 of LKB1 was mutated into either alanine or aspartic acid. Aspartic acid 194 of LKB1, which is essential for maintaining LKB1 activity, was mutated into alanine. All of the site-directed mutagenesis was done by using the QuikChange kits from Stratagene according to the manufacturer's instructions. All of the mutation vectors were confirmed by DNA sequencing. Plasmid DNA was extracted in large scale by using Qiagen EndoFree plasmid maxi kit (catalogue number 12362) and were transfected to HeLa-S3 by using Lipofectamine 2000 kit from Invitrogen (catalogue number 11668-019), according to the instruction provided by the supplier. Twenty four hours after transfection, the cells were treated as indicated. In this experiment, both LacZ expression vector and untreated cells are used as control. Quantification of Western Blot—The intensity (area × density) of the individual bands on Western blots was measured by densitometry (model GS-700, Imaging Densitometer; Bio-Rad). The background was subtracted from the calculated area. Statistical Analysis—The results were analyzed by using two-way analysis of variance. The values are expressed as the means ± S.E. of the mean for n assays. A p value of < 0.05 is considered statistically significant. Inhibition of Protein Kinase C Attenuates ONOO--enhanced AMPK Activity and eNOS Phosphorylation—We had previously shown that ONOO-, either given exogenously or generated endogenously during H/R, activated AMPK in cultured endothelial cells (19Zou M.H. Kirkpatrick S.S. Davis B.J. Nelson J.S. Wiles 4th, W.G Schlattner U. Neumann D. Brownlee M. Freeman M.B. Goldman M.H. J. Biol. Chem. 2004; 279: 43940-43951Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 33Davis B. Xie Z. Viollet B. Zou M.H. Diabetes. 2006; (in press)Google Scholar, 34Zou M.H. Hou X.Y. Shi C.M. Kirkpatick S. Liu F. Goldman M.H. Cohen R.A. J. Biol. Chem. 2003; 278: 34003-34010Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). In addition, inhibition of PI 3-kinase with pharmacological inhibitors or overexpression of dominant negative PDK1 abolished AMPK activation caused by either ONOO-or metformin (31Sakamoto K. Goransson O. Hardie D.G. Alessi D.R. Am. J. Physiol. 2004; 287: 310-317Crossref PubMed Scopus (27) Google Scholar, 32Sakamoto K. McCarthy A. Smith D. Green K.A. Hardie G.D. Ashworth A. Alessi D.R. EMBO J. 2005; 24: 1810-1820Crossref PubMed Scopus (446) Google Scholar, 33Davis B. Xie Z. Viollet B. Zou M.H. Diabetes. 2006; (in press)Google Scholar, 34Zou M.H. Hou X.Y. Shi C.M. Kirkpatick S. Liu F. Goldman M.H. Cohen R.A. J. Biol. Chem. 2003; 278: 34003-34010Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). PDK1 serves as an important link between PI 3-kinase and several other kinases in the so-called AGC family, consisting of protein kinase A (PKA), protein kinase G (PKG), and atypical PKC (43Farese R.V. Am. J. Physiol. 2002; 283: E1-E11Crossref PubMed Scopus (147) Google Scholar). To identify which of these kinases leads to AMPK activation, various selective protein kinase inhibitors were preincubated with BAEC before exposure to ONOO-. After treatment, AMPK activation was determined by monitoring both Thr172 phosphorylation of AMPK using a specific antiphospho-antibody and AMPK activity by using [32P]ATP phosphorylation of the SAMS peptide (19Zou M.H. Kirkpatrick S.S. Davis B.J. Nelson J.S. Wiles 4th, W.G Schlattner U. Neumann D. Brownlee M. Freeman M.B. Goldman M.H. J. Biol. Chem. 2004; 279: 43940-43951Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 34Zou M.H. Hou X.Y. Shi C.M. Kirkpatick S. Liu F. Goldman M.H. Cohen R.A. J. Biol. Chem. 2003; 278: 34003-34010Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). As expected, ONOO- (50 μm) significantly increased AMPK-Thr172 phosphorylation and AMPK activity (Fig. 1, a and b). Interestingly, inhibition of PKC with either GO6983 (1 μm) or arcyriarubin A (1 μm) significantly attenuated ONOO--enhanced AMPK Thr172 phosphorylation and AMPK activity (Fig. 1b). In contrast, inhibition of either PKA with H89 (10 μm) or PKG with KT5823 (10 μm) had no effect (Fig. 1, a and b). These results suggest that ONOO- might activate AMPK via selective PKC activation. eNOS residue Ser1179 (homologous to 1177 in the human sequence) is a substrate for several kinases including both AMPK and protein kinase B/Akt. We had previously reported that ONOO- induced AMPK-dependent phosphorylation of eNOS-Ser1179 in BAEC (33Davis B. Xie Z. Viollet B. Zou M.H. Diabetes. 2006; (in press)Google Scholar, 34Zou M.H. Hou X.Y. Shi C.M. Kirkpatick S. Liu F. Goldman M.H. Cohen R.A. J. Biol. Chem. 2003; 278: 34003-34010Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Thus, we examined the effects of protein kinase inhibitors on eNOS-Ser1179 in BAEC. As expected, ONOO- (50 μm) significantly increased the phosphorylation of eNOS-Ser1179. Neither H89 nor KT5823 altered ONOO--enhanced eNOS-Ser1179 phosphorylation, whereas GO6983 and arcyriarubin A, both of which were shown to inhibit AMPK (Fig. 1, a and b), significantly attenuated ONOO--enhanced eNOS-Ser1179 phosphorylation. These results again suggest that ONOO- might up-regulate AMPK and its downstream enzyme, eNOS, by activating PKC. Activation of Protein Kinase Cζ by ONOO-—Because the employed PKC inhibitors cannot distinguish between these isoforms, we next determined which PKC isoforms are activated by ONOO-. Specific antibodies revealed that BAEC expressed several major isoforms of PKC such as PKCα, β, and atypical PKCζ (data not shown). Atypical PKCζ belongs to the PI 3-kinase/PDK1 family, and its activation is linked to phosphorylation at Thr410/403 that can be monitored by antibodies (43Farese R.V. Am. J. Physiol. 2002; 283: E1-E11Crossref PubMed Scopus (147) Google Scholar, 44Puceat M. Vassort G. Mol. Cell Biochem. 1996; 157: 65-72Crossref PubMed Google Scholar, 45Shizukuda Y. Buttrick P.M. Am. J. Physiol. 2002; 282: H320-H327Crossref PubMed Scopus (67) Google Scholar). Exposure of BAEC to either ONOO- or ONOO- donor SIN-1 significantly increased Thr410/403 phosphorylation without altering PKCζ expression (Fig. 2a). In contrast, ONOO- decreased phosphorylated PKCα/β (Thr638/641), an activated form o

Referência(s)