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W1979037899 abstract "We previously demonstrated that lysophosphatidylcholine up-regulated endothelial nitric-oxide synthase promoter activity by increasing Sp1 binding via the action of protein serine/threonine phosphatase 2A (Cieslik, K., Zembowicz, A., Tang, J.-L., and Wu, K.K. (1998) J. Biol. Chem. 273, 14885–14890). To characterize the regulation of basal endothelial nitric-oxide synthase promoter activity and the signaling pathway through which lysophosphatidylcholine augments endothelial nitric-oxide synthase transcription, we used a casein kinase 2 inhibitor coupled with immunoprecipitation to demonstrate that basal Sp1 binding and endothelial nitric-oxide synthase promoter activity were controlled by casein kinase 2 complexed with protein serine/threonine phosphatase 2A. Casein kinase 2 catalyzed protein serine/threonine phosphatase 2A phosphorylation thereby inhibiting its activity. Lysophosphatidylcholine selectively activated p42/p44 mitogen-activated protein kinase. Purified extracellular regulated kinase 2 blocked casein kinase 2 activity and increased protein serine/threonine phosphatase 2A activity, resulting in an increased Sp1 binding and endothelial nitric-oxide synthase promoter activity. These results indicate that Sp1 binding to its cognate site on the endothelial nitric-oxide synthase promoter and its transactivation of endothelial nitric-oxide synthase is regulated by post-translational Sp1 phosphorylation and dephosphorylation through a dynamic interaction between casein kinase 2 and protein serine/threonine phosphatase 2A. We previously demonstrated that lysophosphatidylcholine up-regulated endothelial nitric-oxide synthase promoter activity by increasing Sp1 binding via the action of protein serine/threonine phosphatase 2A (Cieslik, K., Zembowicz, A., Tang, J.-L., and Wu, K.K. (1998) J. Biol. Chem. 273, 14885–14890). To characterize the regulation of basal endothelial nitric-oxide synthase promoter activity and the signaling pathway through which lysophosphatidylcholine augments endothelial nitric-oxide synthase transcription, we used a casein kinase 2 inhibitor coupled with immunoprecipitation to demonstrate that basal Sp1 binding and endothelial nitric-oxide synthase promoter activity were controlled by casein kinase 2 complexed with protein serine/threonine phosphatase 2A. Casein kinase 2 catalyzed protein serine/threonine phosphatase 2A phosphorylation thereby inhibiting its activity. Lysophosphatidylcholine selectively activated p42/p44 mitogen-activated protein kinase. Purified extracellular regulated kinase 2 blocked casein kinase 2 activity and increased protein serine/threonine phosphatase 2A activity, resulting in an increased Sp1 binding and endothelial nitric-oxide synthase promoter activity. These results indicate that Sp1 binding to its cognate site on the endothelial nitric-oxide synthase promoter and its transactivation of endothelial nitric-oxide synthase is regulated by post-translational Sp1 phosphorylation and dephosphorylation through a dynamic interaction between casein kinase 2 and protein serine/threonine phosphatase 2A. endothelial nitric-oxide synthase lysophosphatidylcholine casein kinase 2 protein serine/threonine phosphatase 2A mitogen-activated protein kinase extracellular regulated kinase 1 (p44) and 2 (p42) nuclear extracts enhanced chemiluminescence immunoprecipitation bovine serum albumin dithiothreitol phenylmethylsulfonyl fluoride Tris-buffered saline 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole c-Jun N-terminal kinase polyacrylamide gel electrophoresis Endothelial nitric-oxide synthase (eNOS)1 is a member of the NOS family, which catalyzes the oxidation of l-arginine to generate nitric oxide (NO) and l-citrulline (1Lowenstein C.J. Snyder S.H. Cell. 1992; 70: 705-707Abstract Full Text PDF PubMed Scopus (736) Google Scholar, 2Nathan C. FASEB J. 1992; 6: 3051-3064Crossref PubMed Scopus (4133) Google Scholar, 3Marletta M.A. J. Biol. Chem. 1993; 268: 12231-12234Abstract Full Text PDF PubMed Google Scholar, 4Wu K.K. Adv. Pharmacol. 1995; 33: 179-207Crossref PubMed Scopus (193) Google Scholar). Nitric oxide induces smooth muscle cell relaxation, inhibits platelet and leukocyte activation, and plays a key role in maintaining vascular integrity and tone (5Moncada S. Palmer R.J. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142PubMed Google Scholar). Altered endothelial NO production has been implicated in several important cardiovascular diseases: hypertension, coronary heart disease, diabetes, and ischemic stroke. eNOS is constitutively expressed in endothelial cells and has features of a housekeeping gene (4Wu K.K. Adv. Pharmacol. 1995; 33: 179-207Crossref PubMed Scopus (193) Google Scholar). However, it is inducible by various vasoactive agents including fluid shear stress (6Nadaud S. Philippe M. Arnal JF. Michel J.B. Soubrier F. Circ. Res. 1996; 79: 857-863Crossref PubMed Scopus (206) Google Scholar), physical exercise (7Sessa W.C. Pritchard K. Seyedi N. Wang J. Hintze T.H. Circ. Res. 1994; 74: 349-353Crossref PubMed Scopus (818) Google Scholar), hypoxia (8Arnet U.A. McMillan A. Dinerman J.L. Ballermann B. Lowenstein C.J. J. Biol. Chem. 1996; 271: 15069-15073Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar), estrogen (9Weiner C.P. Lizasoain I. Baylis S.A. Knowles R.G. Charles I.G. Moncada S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5212-5216Crossref PubMed Scopus (1072) Google Scholar), low levels of oxidized low density lipoprotein (10Hirata K. Miki N. Kuroda Y. Sakoda T. Kanashima S. Yokoyama M. Circ. Res. 1995; 76: 958-962Crossref PubMed Scopus (144) Google Scholar), and lysophosphatidylcholine (lysoPC or LPC) (11Zembowicz A. Tang J.-L. Wu K.K. J. Biol. Chem. 1995; 270: 17006-17010Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). eNOS induction is considered to be important in fortifying the vasoprotective role of NO (12Wu K.K. Proc. Assoc. Am. Physicians. 1998; 110: 163-170PubMed Google Scholar).The mechanism by which eNOS is induced by diverse physical and chemical factors remains unclear. We have used lysoPC as a model system to elucidate the mechanism by which eNOS gene transcription is regulated. Basal eNOS transcription requires binding of Sp1 to its cognate site (−104 to −90) at the 5′-flanking region of human eNOS gene (13Tang J.-L. Zembowicz A. Xu X.-M. Wu K.K. Biochem. Biophys. Res. Commun. 1995; 213: 673-680Crossref PubMed Scopus (36) Google Scholar, 14Zhang R. Min W. Sessa W.C. J. Biol. Chem. 1995; 270: 15320-15326Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Recent work from our laboratory indicated that lysoPC increases Sp1 binding activity, thereby augmenting eNOS promoter activity (15Cieslik K. Zembowicz A. Tang J.-L. Wu K.K. J. Biol. Chem. 1998; 273: 14885-14890Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Our work demonstrated that lysoPC-induced increase in Sp1 binding activity was blocked by okadaic acid and correlated with an elevated PP2A activity (15Cieslik K. Zembowicz A. Tang J.-L. Wu K.K. J. Biol. Chem. 1998; 273: 14885-14890Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), suggesting that eNOS transcription is regulated by post-translational modification of Sp1 via the actions of PP2A and a nuclear kinase, which was recently identified as casein kinase 2 (CK2) (16Armstrong S. Barry D.A. Leggett R.W. Mueller C.R. J. Biol. Chem. 1997; 272: 13489-13495Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). The purpose of this study was to determine how Sp1 binding and eNOS promoter activities are controlled by CK2 and PP2A and to elucidate the signal pathway through which lysoPC alters this control mechanism. Our results indicate that CK2 complexes with PP2A and suppresses PP2A activity at the basal state, resulting in a low level of Sp1 binding and eNOS promoter activity. LysoPC activates selectively the extracellular signal-regulated kinase (ERK-1 and ERK-2), which suppresses the CK2 activity and unleashes its inhibition of PP2A, leading to a higher Sp1 binding and eNOS promoter activity.DISCUSSIONOur findings shed light on how basal and lysoPC-induced eNOS transcriptions are controlled and regulated. Previous studies have shown that basal eNOS promoter activity depends almost entirely on binding of Sp1 to a cognate site at the proximal region of eNOS promoter (13Tang J.-L. Zembowicz A. Xu X.-M. Wu K.K. Biochem. Biophys. Res. Commun. 1995; 213: 673-680Crossref PubMed Scopus (36) Google Scholar, 14Zhang R. Min W. Sessa W.C. J. Biol. Chem. 1995; 270: 15320-15326Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Results from this study indicate that basal Sp1 binding activity is controlled by CK2, and lysoPC treatment of endothelial cells leads to suppression of the basal CK2 activity (Fig.4) with an increase in PP2A activity (Fig. 2). These results implied inhibition of PP2A by CK2 at the basal state. This notion was supported by two pieces of evidence from our experimental data. 1) Inhibition of CK2 activity with DRB raised the PP2A activity (Fig. 2), and 2) coincubation of PP2A with CK2 suppressed PP2A activity (Fig.8 B). LysoPC enhances Sp1 binding and eNOS promoter activities by about 2-fold, while it reduces the CK2 activity in the CK2-PP2A complex to half of the basal level, consistent with the notion that reduction of CK2 activity accounts for increased eNOS promoter activity in lysoPC-treated cells. Our results further implied that lysoPC unleashes CK2's control of PP2A by an ERK1/2-mediated inhibition of CK2. This is supported by the following evidence. 1) Coincubation of ERK2 with purified CK2 or CK2 + PP2A resulted in a reduction of CK2 activity (Fig. 8 A) and an increase in PP2A activity (Fig. 8 B), and 2) lysoPC selectively activates the ERK1/2 pathway (Fig. 6, A–C) through which PP2A activity is enhanced in cells (Fig. 2). From these experimental results, a model for basal and lysoPC-induced eNOS transcription regulation is proposed as depicted in Fig. 9. According to this model, Sp1 binding to its DNA motif which has been suggested to be governed by a balance between Sp1 phosphorylation and dephosphorylation (16Armstrong S. Barry D.A. Leggett R.W. Mueller C.R. J. Biol. Chem. 1997; 272: 13489-13495Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 22Daniel S. Zhang S. DePach-Roach A.A. Kim K.-H. J. Biol. Chem. 1996; 271: 14692-14697Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar) is controlled by CK2 interacting with PP2A. At the basal state, CK2 dominates over PP2A thereby maintaining a high level of Sp1 phosphorylation with a controlled basal Sp1 binding activity and eNOS promoter activity. Stimulation with lysoPC leads to a reduction of CK2 activity and increased PP2A activity via ERK activation, tilting the balance to favor Sp1 dephosphorylation and consequently unleashing the control accompanied by increased Sp1 binding and gene transcription.PP2A occupies a pivotal position in eNOS transcription. Its activity is regulated by phosphorylation through its complex formation with CK2. This mode of PP2A regulation is in keeping with the PP2A properties. PP2A is a heterotrimer composed of a 36-kDa catalytic C subunit and a 65-kDa regulatory A subunit that form the core enzyme and a regulatory B subunit that binds to the core enzyme to form the holoenzyme (for a review, see Ref. 23Mumby M.C. Walter G. Physiol. Rev. 1993; 73: 673-699Crossref PubMed Scopus (624) Google Scholar). Phosphorylation of the catalytic or regu-latory subunit has been reported to influence its catalytic activity (24Chen J. Martin B.L. Brautigan D.L. Science. 1992; 257: 1261-1264Crossref PubMed Scopus (532) Google Scholar, 25Chen J. Parsons S. Brautigan D.L. J. Biol. Chem. 1994; 269: 7957-7962Abstract Full Text PDF PubMed Google Scholar, 26Guo H. Damuni Z. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2500-2504Crossref PubMed Scopus (145) Google Scholar, 27Guo H. Reddy S.A. Damuni Z. J. Biol. Chem. 1993; 268: 11193-11198Abstract Full Text PDF PubMed Google Scholar). Our results are consistent with a previous report that CK2 catalyzes the phosphorylation of the PP2A catalytic subunit (21Heriche J.-K. Lebrin F. Rabilloud T. Leroy D. Chambaz E.M. Goldberg Y. Science. 1997; 276: 952-954Crossref PubMed Scopus (246) Google Scholar). Regulation of PP2A activity is facilitated by its binding to diverse proteins including several kinases (20Westphal R.S. Anderson K.A. Means A.R. Wadzinski B.E. Science. 1998; 280: 1258-1261Crossref PubMed Scopus (222) Google Scholar, 21Heriche J.-K. Lebrin F. Rabilloud T. Leroy D. Chambaz E.M. Goldberg Y. Science. 1997; 276: 952-954Crossref PubMed Scopus (246) Google Scholar, 28Fuhrer D.K. Yang Y.-C. Biochem. Biophys. Res. Commun. 1996; 224: 289-296Crossref PubMed Scopus (58) Google Scholar, 29Westphal R.S. Coffee R.L. Marotta A. Pelech S.L. Wadzinski B.E. J. Biol. Chem. 1999; 274: 687-692Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). A recent report indicates that it binds to CK2α but not CK2 holoenzyme and its activity is enhanced by complex with CK2α (21Heriche J.-K. Lebrin F. Rabilloud T. Leroy D. Chambaz E.M. Goldberg Y. Science. 1997; 276: 952-954Crossref PubMed Scopus (246) Google Scholar). This is contrary to our results, which show that it complexes with either CK2 holoenzyme or CK2α and its activity is suppressed by either form of CK2 in the complex. When ATP was omitted from the reaction mixture, the suppressing effect of CK2 on PP2A was abrogated. In our experimental system, CK2-catalyzed phosphorylation of PP2A is responsible for its inhibition of PP2A activity.Our experimental data support the notion that Sp1 is a substrate for CK2. 2K. Cieslik and K. K. Wu, unpublished data. Recent reports showed that Sp1 is phosphorylated by CK2 and phosphorylated Sp1 has reduced DNA binding activity during terminal differentiation of the liver (16Armstrong S. Barry D.A. Leggett R.W. Mueller C.R. J. Biol. Chem. 1997; 272: 13489-13495Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar,30Leggett R.W. Armstrong S. Barry D.A. Mueller C.R. J. Biol. Chem. 1995; 270: 25879-25884Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). It was subsequently shown that Thr-579 located within the second zinc finger at the C-terminal region of Sp1 is actively phosphorylated by CK2 and mutation of this residue eliminated Sp1 phosphorylation and CK2-mediated inhibition of Sp1 binding activity (16Armstrong S. Barry D.A. Leggett R.W. Mueller C.R. J. Biol. Chem. 1997; 272: 13489-13495Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Other serine residues in this region are also phosphorylated, but their identities and their functional roles remain unclear. Two classes of protein serine/threonine phosphatases have been implicated to catalyze the dephosphorylation of CK2-mediated Sp1 phosphorylation. In the liver differentiation study, protein phosphatase 1 (PP1) was implicated based on okadaic acid inhibition of Sp1 binding activity (16Armstrong S. Barry D.A. Leggett R.W. Mueller C.R. J. Biol. Chem. 1997; 272: 13489-13495Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). However, okadaic acid is a nonselective inhibitor for PP1 and PP2A (31Favre B. Jurowski P. Hemmings B.A. J. Biol. Chem. 1997; 272: 13586-13863Abstract Full Text Full Text PDF Scopus (278) Google Scholar). Our results provide direct evidence for the involvement of PP2A in regulating Sp1 binding activity.LysoPC, a lipid mediator, is generated from phosphatidylcholine via the action of phospholipase A2 (32Parthasarathy S. Streinbrecher U.P. Barnett J. Witztum J.L. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3000-3004Crossref PubMed Scopus (306) Google Scholar). It induces transcription of a series of endothelial genes (33Kume N. Gimbrone M.A. J. Clin. Invest. 1994; 93: 907-911Crossref PubMed Scopus (294) Google Scholar, 34Kume N. Cybulski M.I. Gimbrone M.A. J. Clin. Invest. 1992; 90: 1138-1144Crossref PubMed Scopus (720) Google Scholar, 35Nakamo T. Raines E.W. Abaraham J.A. Klagsbrun M. Ross R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1069-1073Crossref PubMed Scopus (123) Google Scholar, 36Zembowicz A. Jones S.L. Wu K.K. J. Clin. Invest. 1995; 96: 1688-1692Crossref PubMed Scopus (88) Google Scholar). The signaling pathway through which lysoPC activates gene transcription has not been clearly defined. Several studies have shown that it activates protein kinase C (37Kugiyama K. Ohgushi M. Sugiyama S. Murohura T. Fukunaga K. Miyamoto E. Yasue H. Circ. Res. 1992; 71: 1422-1428Crossref PubMed Scopus (125) Google Scholar, 38Ohgushi M. Kugiyama K. Fukunaga K. Murohura T. Sugiyama S. Miyamoto E. Yasue H. Arterioscler. Thromb. 1993; 13: 1525-1533Crossref PubMed Scopus (73) Google Scholar). Others have shown that it activates MAP kinases (39Bassa B.V. Roh D.D. Kirschenbaum M.A. Kamanna V.S. J. Am. Soc. Nephrol. 1998; 9: 488-496PubMed Google Scholar, 40Fang X. Gibson S. Bast R. Mills G.B. J. Biol. Chem. 1997; 272: 13683-13689Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Our results indicate that lysoPC selectively activates ERK1/2. Our results demonstrate for the first time that ERK2 exerts a direct inhibitory effect on CK2. LysoPC induces MEK-1 and ERK1/2 activation probably by at least two upstream pathways: 1) through PKC activation, which may lead to Ras or Raf activation with subsequent MEK-1 and ERK1/2 activation, and 2) through Ras activation that results in Raf activation followed by MEK-1 and ERK activation. Endothelial nitric-oxide synthase (eNOS)1 is a member of the NOS family, which catalyzes the oxidation of l-arginine to generate nitric oxide (NO) and l-citrulline (1Lowenstein C.J. Snyder S.H. Cell. 1992; 70: 705-707Abstract Full Text PDF PubMed Scopus (736) Google Scholar, 2Nathan C. FASEB J. 1992; 6: 3051-3064Crossref PubMed Scopus (4133) Google Scholar, 3Marletta M.A. J. Biol. Chem. 1993; 268: 12231-12234Abstract Full Text PDF PubMed Google Scholar, 4Wu K.K. Adv. Pharmacol. 1995; 33: 179-207Crossref PubMed Scopus (193) Google Scholar). Nitric oxide induces smooth muscle cell relaxation, inhibits platelet and leukocyte activation, and plays a key role in maintaining vascular integrity and tone (5Moncada S. Palmer R.J. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142PubMed Google Scholar). Altered endothelial NO production has been implicated in several important cardiovascular diseases: hypertension, coronary heart disease, diabetes, and ischemic stroke. eNOS is constitutively expressed in endothelial cells and has features of a housekeeping gene (4Wu K.K. Adv. Pharmacol. 1995; 33: 179-207Crossref PubMed Scopus (193) Google Scholar). However, it is inducible by various vasoactive agents including fluid shear stress (6Nadaud S. Philippe M. Arnal JF. Michel J.B. Soubrier F. Circ. Res. 1996; 79: 857-863Crossref PubMed Scopus (206) Google Scholar), physical exercise (7Sessa W.C. Pritchard K. Seyedi N. Wang J. Hintze T.H. Circ. Res. 1994; 74: 349-353Crossref PubMed Scopus (818) Google Scholar), hypoxia (8Arnet U.A. McMillan A. Dinerman J.L. Ballermann B. Lowenstein C.J. J. Biol. Chem. 1996; 271: 15069-15073Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar), estrogen (9Weiner C.P. Lizasoain I. Baylis S.A. Knowles R.G. Charles I.G. Moncada S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5212-5216Crossref PubMed Scopus (1072) Google Scholar), low levels of oxidized low density lipoprotein (10Hirata K. Miki N. Kuroda Y. Sakoda T. Kanashima S. Yokoyama M. Circ. Res. 1995; 76: 958-962Crossref PubMed Scopus (144) Google Scholar), and lysophosphatidylcholine (lysoPC or LPC) (11Zembowicz A. Tang J.-L. Wu K.K. J. Biol. Chem. 1995; 270: 17006-17010Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). eNOS induction is considered to be important in fortifying the vasoprotective role of NO (12Wu K.K. Proc. Assoc. Am. Physicians. 1998; 110: 163-170PubMed Google Scholar). The mechanism by which eNOS is induced by diverse physical and chemical factors remains unclear. We have used lysoPC as a model system to elucidate the mechanism by which eNOS gene transcription is regulated. Basal eNOS transcription requires binding of Sp1 to its cognate site (−104 to −90) at the 5′-flanking region of human eNOS gene (13Tang J.-L. Zembowicz A. Xu X.-M. Wu K.K. Biochem. Biophys. Res. Commun. 1995; 213: 673-680Crossref PubMed Scopus (36) Google Scholar, 14Zhang R. Min W. Sessa W.C. J. Biol. Chem. 1995; 270: 15320-15326Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Recent work from our laboratory indicated that lysoPC increases Sp1 binding activity, thereby augmenting eNOS promoter activity (15Cieslik K. Zembowicz A. Tang J.-L. Wu K.K. J. Biol. Chem. 1998; 273: 14885-14890Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Our work demonstrated that lysoPC-induced increase in Sp1 binding activity was blocked by okadaic acid and correlated with an elevated PP2A activity (15Cieslik K. Zembowicz A. Tang J.-L. Wu K.K. J. Biol. Chem. 1998; 273: 14885-14890Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), suggesting that eNOS transcription is regulated by post-translational modification of Sp1 via the actions of PP2A and a nuclear kinase, which was recently identified as casein kinase 2 (CK2) (16Armstrong S. Barry D.A. Leggett R.W. Mueller C.R. J. Biol. Chem. 1997; 272: 13489-13495Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). The purpose of this study was to determine how Sp1 binding and eNOS promoter activities are controlled by CK2 and PP2A and to elucidate the signal pathway through which lysoPC alters this control mechanism. Our results indicate that CK2 complexes with PP2A and suppresses PP2A activity at the basal state, resulting in a low level of Sp1 binding and eNOS promoter activity. LysoPC activates selectively the extracellular signal-regulated kinase (ERK-1 and ERK-2), which suppresses the CK2 activity and unleashes its inhibition of PP2A, leading to a higher Sp1 binding and eNOS promoter activity. DISCUSSIONOur findings shed light on how basal and lysoPC-induced eNOS transcriptions are controlled and regulated. Previous studies have shown that basal eNOS promoter activity depends almost entirely on binding of Sp1 to a cognate site at the proximal region of eNOS promoter (13Tang J.-L. Zembowicz A. Xu X.-M. Wu K.K. Biochem. Biophys. Res. Commun. 1995; 213: 673-680Crossref PubMed Scopus (36) Google Scholar, 14Zhang R. Min W. Sessa W.C. J. Biol. Chem. 1995; 270: 15320-15326Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Results from this study indicate that basal Sp1 binding activity is controlled by CK2, and lysoPC treatment of endothelial cells leads to suppression of the basal CK2 activity (Fig.4) with an increase in PP2A activity (Fig. 2). These results implied inhibition of PP2A by CK2 at the basal state. This notion was supported by two pieces of evidence from our experimental data. 1) Inhibition of CK2 activity with DRB raised the PP2A activity (Fig. 2), and 2) coincubation of PP2A with CK2 suppressed PP2A activity (Fig.8 B). LysoPC enhances Sp1 binding and eNOS promoter activities by about 2-fold, while it reduces the CK2 activity in the CK2-PP2A complex to half of the basal level, consistent with the notion that reduction of CK2 activity accounts for increased eNOS promoter activity in lysoPC-treated cells. Our results further implied that lysoPC unleashes CK2's control of PP2A by an ERK1/2-mediated inhibition of CK2. This is supported by the following evidence. 1) Coincubation of ERK2 with purified CK2 or CK2 + PP2A resulted in a reduction of CK2 activity (Fig. 8 A) and an increase in PP2A activity (Fig. 8 B), and 2) lysoPC selectively activates the ERK1/2 pathway (Fig. 6, A–C) through which PP2A activity is enhanced in cells (Fig. 2). From these experimental results, a model for basal and lysoPC-induced eNOS transcription regulation is proposed as depicted in Fig. 9. According to this model, Sp1 binding to its DNA motif which has been suggested to be governed by a balance between Sp1 phosphorylation and dephosphorylation (16Armstrong S. Barry D.A. Leggett R.W. Mueller C.R. J. Biol. Chem. 1997; 272: 13489-13495Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 22Daniel S. Zhang S. DePach-Roach A.A. Kim K.-H. J. Biol. Chem. 1996; 271: 14692-14697Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar) is controlled by CK2 interacting with PP2A. At the basal state, CK2 dominates over PP2A thereby maintaining a high level of Sp1 phosphorylation with a controlled basal Sp1 binding activity and eNOS promoter activity. Stimulation with lysoPC leads to a reduction of CK2 activity and increased PP2A activity via ERK activation, tilting the balance to favor Sp1 dephosphorylation and consequently unleashing the control accompanied by increased Sp1 binding and gene transcription.PP2A occupies a pivotal position in eNOS transcription. Its activity is regulated by phosphorylation through its complex formation with CK2. This mode of PP2A regulation is in keeping with the PP2A properties. PP2A is a heterotrimer composed of a 36-kDa catalytic C subunit and a 65-kDa regulatory A subunit that form the core enzyme and a regulatory B subunit that binds to the core enzyme to form the holoenzyme (for a review, see Ref. 23Mumby M.C. Walter G. Physiol. Rev. 1993; 73: 673-699Crossref PubMed Scopus (624) Google Scholar). Phosphorylation of the catalytic or regu-latory subunit has been reported to influence its catalytic activity (24Chen J. Martin B.L. Brautigan D.L. Science. 1992; 257: 1261-1264Crossref PubMed Scopus (532) Google Scholar, 25Chen J. Parsons S. Brautigan D.L. J. Biol. Chem. 1994; 269: 7957-7962Abstract Full Text PDF PubMed Google Scholar, 26Guo H. Damuni Z. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2500-2504Crossref PubMed Scopus (145) Google Scholar, 27Guo H. Reddy S.A. Damuni Z. J. Biol. Chem. 1993; 268: 11193-11198Abstract Full Text PDF PubMed Google Scholar). Our results are consistent with a previous report that CK2 catalyzes the phosphorylation of the PP2A catalytic subunit (21Heriche J.-K. Lebrin F. Rabilloud T. Leroy D. Chambaz E.M. Goldberg Y. Science. 1997; 276: 952-954Crossref PubMed Scopus (246) Google Scholar). Regulation of PP2A activity is facilitated by its binding to diverse proteins including several kinases (20Westphal R.S. Anderson K.A. Means A.R. Wadzinski B.E. Science. 1998; 280: 1258-1261Crossref PubMed Scopus (222) Google Scholar, 21Heriche J.-K. Lebrin F. Rabilloud T. Leroy D. Chambaz E.M. Goldberg Y. Science. 1997; 276: 952-954Crossref PubMed Scopus (246) Google Scholar, 28Fuhrer D.K. Yang Y.-C. Biochem. Biophys. Res. Commun. 1996; 224: 289-296Crossref PubMed Scopus (58) Google Scholar, 29Westphal R.S. Coffee R.L. Marotta A. Pelech S.L. Wadzinski B.E. J. Biol. Chem. 1999; 274: 687-692Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). A recent report indicates that it binds to CK2α but not CK2 holoenzyme and its activity is enhanced by complex with CK2α (21Heriche J.-K. Lebrin F. Rabilloud T. Leroy D. Chambaz E.M. Goldberg Y. Science. 1997; 276: 952-954Crossref PubMed Scopus (246) Google Scholar). This is contrary to our results, which show that it complexes with either CK2 holoenzyme or CK2α and its activity is suppressed by either form of CK2 in the complex. When ATP was omitted from the reaction mixture, the suppressing effect of CK2 on PP2A was abrogated. In our experimental system, CK2-catalyzed phosphorylation of PP2A is responsible for its inhibition of PP2A activity.Our experimental data support the notion that Sp1 is a substrate for CK2. 2K. Cieslik and K. K. Wu, unpublished data. Recent reports showed that Sp1 is phosphorylated by CK2 and phosphorylated Sp1 has reduced DNA binding activity during terminal differentiation of the liver (16Armstrong S. Barry D.A. Leggett R.W. Mueller C.R. J. Biol. Chem. 1997; 272: 13489-13495Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar,30Leggett R.W. Armstrong S. Barry D.A. Mueller C.R. J. Biol. Chem. 1995; 270: 25879-25884Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). It was subsequently shown that Thr-579 located within the second zinc finger at the C-terminal region of Sp1 is actively phosphorylated by CK2 and mutation of this residue eliminated Sp1 phosphorylation and CK2-mediated inhibition of Sp1 binding activity (16Armstrong S. Barry D.A. Leggett R.W. Mueller C.R. J. Biol. Chem. 1997; 272: 13489-13495Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Other serine residues in this region are also phosphorylated, but their identities and their functional roles remain unclear. Two classes of protein serine/threonine phosphatases have been implicated to catalyze the dephosphorylation of CK2-mediated Sp1 phosphorylation. In the liver differentiation study, protein phosphatase 1 (PP1) was implicated based on okadaic acid inhibition of Sp1 binding activity (16Armstrong S. Barry D.A. Leggett R.W. Mueller C.R. J. Biol. Chem. 1997; 272: 13489-13495Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). However, okadaic acid is a nonselective inhibitor for PP1 and PP2A (31Favre B. Jurowski P. Hemmings B.A. J. Biol. Chem. 1997; 272: 13586-13863Abstract Full Text Full Text PDF Scopus (278) Google Scholar). Our results provide direct evidence for the involvement of PP2A in regulating Sp1 binding activity.LysoPC, a lipid mediator, is generated from phosphatidylcholine via the action of phospholipase A2 (32Parthasarathy S. Streinbrecher U.P. Barnett J. Witztum J.L. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3000-3004Crossref PubMed Scopus (306) Google Scholar). It induces transcription of a series of endothelial genes (33Kume N. Gimbrone M.A. J. Clin. Invest. 1994; 93: 907-911Crossref PubMed Scopus (294) Google Scholar, 34Kume N. Cybulski M.I. Gimbrone M.A. J. Clin. Invest. 1992; 90: 1138-1144Crossref PubMed Scopus (720) Google Scholar, 35Nakamo T. Raines E.W. Abaraham J.A. Klagsbrun M. Ross R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1069-1073Crossref PubMed Scopus (123) Google Scholar, 36Zembowicz A. Jones S.L. Wu K.K. J. Clin. Invest. 1995; 96: 1688-1692Crossref PubMed Scopus (88) Google Scholar). The signaling pathway through which lysoPC activates gene transcription has not been clearly defined. Several studies have shown that it activates protein kinase C (37Kugiyama K. Ohgushi M. Sugiyama S. Murohura T. Fukunaga K. Miyamoto E. Yasue H. Circ. Res. 1992; 71: 1422-1428Crossref PubMed Scopus (125) Google Scholar, 38Ohgushi M. Kugiyama K. Fukunaga K. Murohura T. Sugiyama S. Miyamoto E. Yasue H. Arterioscler. Thromb. 1993; 13: 1525-1533Crossref PubMed Scopus (73) Google Scholar). Others have shown that it activates MAP kinases (39Bassa B.V. Roh D.D. Kirschenbaum M.A. Kamanna V.S. J. Am. Soc. Nephrol. 1998; 9: 488-496PubMed Google Scholar, 40Fang X. Gibson S. Bast R. Mills G.B. J. Biol. Chem. 1997; 272: 13683-13689Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Our results indicate that lysoPC selectively activates ERK1/2. Our results demonstrate for the first time that ERK2 exerts a direct inhibitory effect on CK2. LysoPC induces MEK-1 and ERK1/2 activation probably by at least two upstream pathways: 1) through PKC activation, which may lead to Ras or Raf activation with subsequent MEK-1 and ERK1/2 activation, and 2) through Ras activation that results in Raf activation followed by MEK-1 and ERK activation. Our findings shed light on how basal and lysoPC-induced eNOS transcriptions are controlled and regulated. Previous studies have shown that basal eNOS promoter activity depends almost entirely on binding of Sp1 to a cognate site at the proximal region of eNOS promoter (13Tang J.-L. Zembowicz A. Xu X.-M. Wu K.K. Biochem. Biophys. Res. Commun. 1995; 213: 673-680Crossref PubMed Scopus (36) Google Scholar, 14Zhang R. Min W. Sessa W.C. J. Biol. Chem. 1995; 270: 15320-15326Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Results from this study indicate that basal Sp1 binding activity is controlled by CK2, and lysoPC treatment of endothelial cells leads to suppression of the basal CK2 activity (Fig.4) with an increase in PP2A activity (Fig. 2). These results implied inhibition of PP2A by CK2 at the basal state. This notion was supported by two pieces of evidence from our experimental data. 1) Inhibition of CK2 activity with DRB raised the PP2A activity (Fig. 2), and 2) coincubation of PP2A with CK2 suppressed PP2A activity (Fig.8 B). LysoPC enhances Sp1 binding and eNOS promoter activities by about 2-fold, while it reduces the CK2 activity in the CK2-PP2A complex to half of the basal level, consistent with the notion that reduction of CK2 activity accounts for increased eNOS promoter activity in lysoPC-treated cells. Our results further implied that lysoPC unleashes CK2's control of PP2A by an ERK1/2-mediated inhibition of CK2. This is supported by the following evidence. 1) Coincubation of ERK2 with purified CK2 or CK2 + PP2A resulted in a reduction of CK2 activity (Fig. 8 A) and an increase in PP2A activity (Fig. 8 B), and 2) lysoPC selectively activates the ERK1/2 pathway (Fig. 6, A–C) through which PP2A activity is enhanced in cells (Fig. 2). From these experimental results, a model for basal and lysoPC-induced eNOS transcription regulation is proposed as depicted in Fig. 9. According to this model, Sp1 binding to its DNA motif which has been suggested to be governed by a balance between Sp1 phosphorylation and dephosphorylation (16Armstrong S. Barry D.A. Leggett R.W. Mueller C.R. J. Biol. Chem. 1997; 272: 13489-13495Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 22Daniel S. Zhang S. DePach-Roach A.A. Kim K.-H. J. Biol. Chem. 1996; 271: 14692-14697Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar) is controlled by CK2 interacting with PP2A. At the basal state, CK2 dominates over PP2A thereby maintaining a high level of Sp1 phosphorylation with a controlled basal Sp1 binding activity and eNOS promoter activity. Stimulation with lysoPC leads to a reduction of CK2 activity and increased PP2A activity via ERK activation, tilting the balance to favor Sp1 dephosphorylation and consequently unleashing the control accompanied by increased Sp1 binding and gene transcription. PP2A occupies a pivotal position in eNOS transcription. Its activity is regulated by phosphorylation through its complex formation with CK2. This mode of PP2A regulation is in keeping with the PP2A properties. PP2A is a heterotrimer composed of a 36-kDa catalytic C subunit and a 65-kDa regulatory A subunit that form the core enzyme and a regulatory B subunit that binds to the core enzyme to form the holoenzyme (for a review, see Ref. 23Mumby M.C. Walter G. Physiol. Rev. 1993; 73: 673-699Crossref PubMed Scopus (624) Google Scholar). Phosphorylation of the catalytic or regu-latory subunit has been reported to influence its catalytic activity (24Chen J. Martin B.L. Brautigan D.L. Science. 1992; 257: 1261-1264Crossref PubMed Scopus (532) Google Scholar, 25Chen J. Parsons S. Brautigan D.L. J. Biol. Chem. 1994; 269: 7957-7962Abstract Full Text PDF PubMed Google Scholar, 26Guo H. Damuni Z. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2500-2504Crossref PubMed Scopus (145) Google Scholar, 27Guo H. Reddy S.A. Damuni Z. J. Biol. Chem. 1993; 268: 11193-11198Abstract Full Text PDF PubMed Google Scholar). Our results are consistent with a previous report that CK2 catalyzes the phosphorylation of the PP2A catalytic subunit (21Heriche J.-K. Lebrin F. Rabilloud T. Leroy D. Chambaz E.M. Goldberg Y. Science. 1997; 276: 952-954Crossref PubMed Scopus (246) Google Scholar). Regulation of PP2A activity is facilitated by its binding to diverse proteins including several kinases (20Westphal R.S. Anderson K.A. Means A.R. Wadzinski B.E. Science. 1998; 280: 1258-1261Crossref PubMed Scopus (222) Google Scholar, 21Heriche J.-K. Lebrin F. Rabilloud T. Leroy D. Chambaz E.M. Goldberg Y. Science. 1997; 276: 952-954Crossref PubMed Scopus (246) Google Scholar, 28Fuhrer D.K. Yang Y.-C. Biochem. Biophys. Res. Commun. 1996; 224: 289-296Crossref PubMed Scopus (58) Google Scholar, 29Westphal R.S. Coffee R.L. Marotta A. Pelech S.L. Wadzinski B.E. J. Biol. Chem. 1999; 274: 687-692Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). A recent report indicates that it binds to CK2α but not CK2 holoenzyme and its activity is enhanced by complex with CK2α (21Heriche J.-K. Lebrin F. Rabilloud T. Leroy D. Chambaz E.M. Goldberg Y. Science. 1997; 276: 952-954Crossref PubMed Scopus (246) Google Scholar). This is contrary to our results, which show that it complexes with either CK2 holoenzyme or CK2α and its activity is suppressed by either form of CK2 in the complex. When ATP was omitted from the reaction mixture, the suppressing effect of CK2 on PP2A was abrogated. In our experimental system, CK2-catalyzed phosphorylation of PP2A is responsible for its inhibition of PP2A activity. Our experimental data support the notion that Sp1 is a substrate for CK2. 2K. Cieslik and K. K. Wu, unpublished data. Recent reports showed that Sp1 is phosphorylated by CK2 and phosphorylated Sp1 has reduced DNA binding activity during terminal differentiation of the liver (16Armstrong S. Barry D.A. Leggett R.W. Mueller C.R. J. Biol. Chem. 1997; 272: 13489-13495Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar,30Leggett R.W. Armstrong S. Barry D.A. Mueller C.R. J. Biol. Chem. 1995; 270: 25879-25884Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). It was subsequently shown that Thr-579 located within the second zinc finger at the C-terminal region of Sp1 is actively phosphorylated by CK2 and mutation of this residue eliminated Sp1 phosphorylation and CK2-mediated inhibition of Sp1 binding activity (16Armstrong S. Barry D.A. Leggett R.W. Mueller C.R. J. Biol. Chem. 1997; 272: 13489-13495Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Other serine residues in this region are also phosphorylated, but their identities and their functional roles remain unclear. Two classes of protein serine/threonine phosphatases have been implicated to catalyze the dephosphorylation of CK2-mediated Sp1 phosphorylation. In the liver differentiation study, protein phosphatase 1 (PP1) was implicated based on okadaic acid inhibition of Sp1 binding activity (16Armstrong S. Barry D.A. Leggett R.W. Mueller C.R. J. Biol. Chem. 1997; 272: 13489-13495Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). However, okadaic acid is a nonselective inhibitor for PP1 and PP2A (31Favre B. Jurowski P. Hemmings B.A. J. Biol. Chem. 1997; 272: 13586-13863Abstract Full Text Full Text PDF Scopus (278) Google Scholar). Our results provide direct evidence for the involvement of PP2A in regulating Sp1 binding activity. LysoPC, a lipid mediator, is generated from phosphatidylcholine via the action of phospholipase A2 (32Parthasarathy S. Streinbrecher U.P. Barnett J. Witztum J.L. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3000-3004Crossref PubMed Scopus (306) Google Scholar). It induces transcription of a series of endothelial genes (33Kume N. Gimbrone M.A. J. Clin. Invest. 1994; 93: 907-911Crossref PubMed Scopus (294) Google Scholar, 34Kume N. Cybulski M.I. Gimbrone M.A. J. Clin. Invest. 1992; 90: 1138-1144Crossref PubMed Scopus (720) Google Scholar, 35Nakamo T. Raines E.W. Abaraham J.A. Klagsbrun M. Ross R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1069-1073Crossref PubMed Scopus (123) Google Scholar, 36Zembowicz A. Jones S.L. Wu K.K. J. Clin. Invest. 1995; 96: 1688-1692Crossref PubMed Scopus (88) Google Scholar). The signaling pathway through which lysoPC activates gene transcription has not been clearly defined. Several studies have shown that it activates protein kinase C (37Kugiyama K. Ohgushi M. Sugiyama S. Murohura T. Fukunaga K. Miyamoto E. Yasue H. Circ. Res. 1992; 71: 1422-1428Crossref PubMed Scopus (125) Google Scholar, 38Ohgushi M. Kugiyama K. Fukunaga K. Murohura T. Sugiyama S. Miyamoto E. Yasue H. Arterioscler. Thromb. 1993; 13: 1525-1533Crossref PubMed Scopus (73) Google Scholar). Others have shown that it activates MAP kinases (39Bassa B.V. Roh D.D. Kirschenbaum M.A. Kamanna V.S. J. Am. Soc. Nephrol. 1998; 9: 488-496PubMed Google Scholar, 40Fang X. Gibson S. Bast R. Mills G.B. J. Biol. Chem. 1997; 272: 13683-13689Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Our results indicate that lysoPC selectively activates ERK1/2. Our results demonstrate for the first time that ERK2 exerts a direct inhibitory effect on CK2. LysoPC induces MEK-1 and ERK1/2 activation probably by at least two upstream pathways: 1) through PKC activation, which may lead to Ras or Raf activation with subsequent MEK-1 and ERK1/2 activation, and 2) through Ras activation that results in Raf activation followed by MEK-1 and ERK activation. We thank Susan Mitterling for editorial assistance." @default.
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W1979037899 title "Transcriptional Regulation of Endothelial Nitric-oxide Synthase by an Interaction between Casein Kinase 2 and Protein Phosphatase 2A" @default.
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