To help define essential interactions of cGMP with the catalytic site, we tested a series of cGMP analogs as competitive inhibitors of each cyclic nucleotide phosphodiesterase (PDE) family known to hydrolyze cGMP (PDE1, PDE2, PDE3, PDE5, and PDE6). IC50 values, relative to cGMP, were used to predict which functional groups of cGMP contribute to binding by the catalytic sites of each isozyme. The results indicate that the N1-nitrogen of cGMP contributes to binding at the catalytic site of all PDEs, probably as a hydrogen donor. All PDEs tested, with the exception of PDE2, also use the 6-oxo group, probably as a hydrogen acceptor. In contrast to other cGMP-binding enzymes, the 2-amino and 2'-hydroxyl groups of cGMP are not major requirements for binding to any PDE. The 8-bromo- and 8-p-chlorophenylthio-substituted analogs inhibit PDE1, PDE2, and PDE6 activity with high relative affinities, suggesting that these PDEs are not sterically hindered with bulky 8-position substitutions and that they do not preferentially bind the anti-conformation of cGMP. PDE3 and PDE5 have reduced apparent affinity for these analogs and therefore either are sterically hindered with these substitutions or bind cGMP in the anti-conformation. Overall, the data show substantial differences in structural requirements for cGMP binding to the catalytic sites of the different PDE families. Comparisons with published data show different structural requirements for binding to the catalytic, compared with noncatalytic, binding domains of PDEs. Even larger differences are seen between the requirements for binding to PDE catalytic sites and those for the cGMP-dependent protein kinase and the cGMP-gated cation channel.
Lysophosphatidic acid (LPA) stimulates the c-Fos serum response element (SRE) by activating two distinct signal pathways regulated by the small GTPases, Ras and RhoA. Ras activates the ERK cascade leading to phosphorylation of the transcription factors Elk-1 and Sap1a at the Ets/TCF site. RhoA regulates an undefined pathway required for the activation of the SRF/CArG site. Here we have examined the role of the Ras and RhoA pathways in activation of the SRE and c-Fos expression in Rat-1 cells. Pertussis toxin and PD98059 strongly inhibited LPA-stimulated c-Fos expression and activation of a SRE:Luc reporter. C3 toxin completely inhibited RhoA function, partially inhibited SRE:Luc activity, but had no effect on LPA-stimulated c-Fos expression. Thus, in a physiological context the Ras-Raf-MEK-ERK pathway, but not RhoA, is required for LPA-stimulated c-Fos expression in Rat-1 cells. C3 toxin stimulated the stress-activated protein kinases JNK and p38 and potentiated c-Jun expression and phosphorylation; these properties were shared by another cellular stress agonist the protein kinase C inhibitor Ro-31-8220. However, C3 toxin alone or in combination with growth factors did not stimulate AP-1:Luc activity and actually antagonized the synergistic activation of AP-1:Luc observed in response to co-stimulation with growth factors and Ro-31-8220. These data indicate that C3 toxin is a cellular stress which antagonizes activation of AP-1 at a point downstream of stress-activated kinase activation or immediate-early gene induction.
A newly designed cyclic AMP (cAMP) analogue, Sp-5,6-dichloro-1-beta-D- ribofuranosylbenzimidazole-3′,5′-monophosphorothioate (Sp-5,6-DCl-cBiMPS), and 8-(p-chlorophenylthio)-cAMP (8-pCPT-cAMP) were compared with respect to their chemical and biological properties in order to assess their potential as activators of the cAMP-dependent protein kinases (cAMP-PK) in intact cells. Sp-5,6-DCl-cBiMPS was shown to be both a potent and specific activator of purified cAMP-PK and of cAMP-PK in platelet membranes, whereas 8-pCPT-cAMP proved to be a potent activator of cAMP-PK and cyclic-GMP-dependent protein kinase (cGMP-PK) both as purified enzymes and in platelet membranes. Sp-5,6-DCl-cBiMPS was not significantly hydrolysed by three types of cyclic nucleotide phosphodiesterases, whereas 8-pCPT-cAMP (and 8-bromo-cAMP) was hydrolysed to a significant extent by the Ca2+/calmodulin-dependent phosphodiesterase and by the cGMP-inhibited phosphodiesterase. The apparent lipophilicity, a measure of potential cell-membrane permeability, of Sp-5,6-DCl-cBiMPS was higher than that of 8-pCPT-cAMP. Extracellular application of Sp-5,6-DCl-cBiMPS to intact human platelets reproduced the pattern of protein phosphorylation induced by prostaglandin E1, a cAMP-increasing inhibitor of platelet activation. In intact platelets, Sp-5,6- DCl-cBiMPS was also more effective than 8-pCPT-cAMP in inducing quantitative phosphorylation of the 46/50 kDa vasodilator-stimulated phosphoprotein (VASP), a major substrate of cAMP-PK in platelets. As observed with prostaglandin E1, pretreatment of human platelets with Sp-5,6-DCl-cBiMPS prevented the aggregation induced by thrombin. The results suggest that Sp-5,6-DCl-cBiMPS is a very potent and specific activator of cAMP-PK in cell extracts and intact cells and, in this respect, is superior to any other cAMP analogue used for intact-cell studies. In contrast with 8-pCPT-cAMP, Sp-5,6-DCl-cBiMPS can be used to distinguish the signal-transduction pathways mediated by cAMP-PK and cGMP-PK.
Stimulation of Rat-1 cells with lysophosphatidic acid (LPA) or epidermal growth factor (EGF) results in a biphasic, sustained activation of extracellular signal-regulated kinase 1 (ERK1). Pretreatment of Rat-1 cells with either cycloheximide or sodium orthovanadate had little effect on the early peak of ERK1 activity but potentiated the sustained phase. Cycloheximide also potentiated ERK1 activation in Rat-1 cells expressing ΔRaf-1:ER, an estradiol-regulated form of the oncogenic, human Raf-1. Since cycloheximide did not potentiate MEK activity but abrogated the expression of mitogen-activated protein kinase phosphatase (MKP-1) normally seen in response to EGF and LPA, we speculated that the level of MKP-1 expression may be an important regulator of ERK1 activity in Rat-1 cells. Inhibition of LPA-stimulated MEK and ERK activation with PD98059 and pertussis toxin, a selective inhibitor of Gi-protein-coupled signaling pathways, reduced LPA-stimulated MKP-1 expression by only 50%, suggesting the presence of additional MEK- and ERK-independent pathways for MKP-1 expression. Specific activation of the MEK/ERK pathway by ΔRaf-1:ER had little or no effect on MKP-1 expression, suggesting that activation of the Raf/MEK/ERK pathway is necessary but not sufficient for MKP-1 expression in Rat-1 cells. Activation of PKC played little part in growth factor-stimulated MKP-1 expression, but LPA- and EGF-induced MKP-1 expression was blocked by buffering [Ca2+]i, leading to a potentiation of the sustained phase of ERK1 activation without potentiating MEK activity. In Rat-1ΔRaf-1:ER cells, we observed a strong synergy of MKP-1 expression when cells were stimulated with estradiol in the presence of ionomycin, phorbol 12-myristate 13-acetate, or okadaic acid under conditions where these agents did not synergize for ERK activation. These results suggest that activation of the Raf/MEK/ERK pathway is insufficient to induce expression of MKP-1 but instead requires other signals, such as Ca2+, to fully reconstitute the response seen with growth factors. In this way, ERK-dependent and -independent signals may regulate MKP-1 expression, the magnitude of sustained ERK1 activity, and therefore gene expression. Stimulation of Rat-1 cells with lysophosphatidic acid (LPA) or epidermal growth factor (EGF) results in a biphasic, sustained activation of extracellular signal-regulated kinase 1 (ERK1). Pretreatment of Rat-1 cells with either cycloheximide or sodium orthovanadate had little effect on the early peak of ERK1 activity but potentiated the sustained phase. Cycloheximide also potentiated ERK1 activation in Rat-1 cells expressing ΔRaf-1:ER, an estradiol-regulated form of the oncogenic, human Raf-1. Since cycloheximide did not potentiate MEK activity but abrogated the expression of mitogen-activated protein kinase phosphatase (MKP-1) normally seen in response to EGF and LPA, we speculated that the level of MKP-1 expression may be an important regulator of ERK1 activity in Rat-1 cells. Inhibition of LPA-stimulated MEK and ERK activation with PD98059 and pertussis toxin, a selective inhibitor of Gi-protein-coupled signaling pathways, reduced LPA-stimulated MKP-1 expression by only 50%, suggesting the presence of additional MEK- and ERK-independent pathways for MKP-1 expression. Specific activation of the MEK/ERK pathway by ΔRaf-1:ER had little or no effect on MKP-1 expression, suggesting that activation of the Raf/MEK/ERK pathway is necessary but not sufficient for MKP-1 expression in Rat-1 cells. Activation of PKC played little part in growth factor-stimulated MKP-1 expression, but LPA- and EGF-induced MKP-1 expression was blocked by buffering [Ca2+]i, leading to a potentiation of the sustained phase of ERK1 activation without potentiating MEK activity. In Rat-1ΔRaf-1:ER cells, we observed a strong synergy of MKP-1 expression when cells were stimulated with estradiol in the presence of ionomycin, phorbol 12-myristate 13-acetate, or okadaic acid under conditions where these agents did not synergize for ERK activation. These results suggest that activation of the Raf/MEK/ERK pathway is insufficient to induce expression of MKP-1 but instead requires other signals, such as Ca2+, to fully reconstitute the response seen with growth factors. In this way, ERK-dependent and -independent signals may regulate MKP-1 expression, the magnitude of sustained ERK1 activity, and therefore gene expression. One of the major signal pathways responsible for regulating reentry into the cell cycle leads to activation of the extracellular signal-regulated kinases (ERKs) 1The abbreviations used are: ERK, extracellular signal-regulated kinase (also called mitogen-activated protein kinase or MAP kinase); BAPTA-AM, (1,2-bis(2)aminophenoxy)ethaneN,N,N′,N′-tetraacetic acid tetra(acetoxymethyl ester); EGF, epidermal growth factor; JNK, JUN N-terminal kinase; LPA, lysophosphatidic acid; MBP, myelin basic protein MEK, MAP kinase, or ERK kinase; MKP, MAP kinase phosphatase; PMA, phorbol 12-myristate 13-acetate; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; PBS, phosphate-buffered saline; β-E2, β-estradiol; PKC, protein kinase C; FBS, fetal bovine serum. 1The abbreviations used are: ERK, extracellular signal-regulated kinase (also called mitogen-activated protein kinase or MAP kinase); BAPTA-AM, (1,2-bis(2)aminophenoxy)ethaneN,N,N′,N′-tetraacetic acid tetra(acetoxymethyl ester); EGF, epidermal growth factor; JNK, JUN N-terminal kinase; LPA, lysophosphatidic acid; MBP, myelin basic protein MEK, MAP kinase, or ERK kinase; MKP, MAP kinase phosphatase; PMA, phorbol 12-myristate 13-acetate; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; PBS, phosphate-buffered saline; β-E2, β-estradiol; PKC, protein kinase C; FBS, fetal bovine serum. p44ERK1 and p42ERK2 (also called mitogen-activated protein kinases or MAP kinases) (1Cobb M.H. Hepler J.E. Cheng M. Robbins D. Semin. Cancer Biol. 1994; 5: 261-268PubMed Google Scholar, 2Johnson G.L. Vaillancourt R.R. Curr. Opin. Cell Biol. 1994; 6: 230-238Crossref PubMed Scopus (307) Google Scholar, 3Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4213) Google Scholar, 4Treisman R. Curr. Opin. Genet. Devel. 1994; 4: 96-101Crossref PubMed Scopus (618) Google Scholar). Growth factor-induced activation of p74Raf-1 leads to the phosphorylation and activation of the dual specificity protein kinase MEK, which in turn activates the ERK/MAP kinases by phosphorylation of threonine and tyrosine residues in the motif TEY (5Boulton T.G. Nye S.H. Robbins D.J. Ip E. Radziejewska S.D. Morgenbesser S.D. DePinho N. Cobb M.H. Yancopoulos G.D. Cell. 1991; 65: 663-675Abstract Full Text PDF PubMed Scopus (1473) Google Scholar, 6Anderson N.G. Maller J.L. Tonks N.K. Sturgill T.W. Nature. 1990; 343: 651-653Crossref PubMed Scopus (791) Google Scholar, 7Crews C.M. Alessandrini A. Erickson R.L. Science. 1992; 258: 478-480Crossref PubMed Scopus (733) Google Scholar, 8Kyriakis J.M. App H. Zhang X.F. Banerjee P. Brautigan D.L. Rapp U.R. Avruch J. Nature. 1992; 358: 417-421Crossref PubMed Scopus (964) Google Scholar, 9Howe L.R. Leevers S.J. Gomez N. Nakielny S. Cohen P. Marshall C.J. Cell. 1992; 71: 335-342Abstract Full Text PDF PubMed Scopus (627) Google Scholar). p74Raf-1 is recruited to the plasma membrane and regulated by the Ras GTPase proteins (10Traverse S. Cohen P. Paterson H. Marshall C. Rapp U. Grand R.J.A. Oncogene. 1993; 8: 3175-3181PubMed Google Scholar, 11Leevers S.J. Paterson H.F. Marshall C.J. Nature. 1994; 369: 411-414Crossref PubMed Scopus (875) Google Scholar, 12Stokoe D. Macdonald S.G. Cadwallader K. Symons M. Hancock J.F. Science. 1994; 264: 1463-1467Crossref PubMed Scopus (832) Google Scholar). This cascade is activated by receptor tyrosine kinases such as the EGF receptor (reviewed in Ref. 13Egan S.E. Weinberg R.A. Nature. 1993; 365: 781-783Crossref PubMed Scopus (523) Google Scholar), cytokine receptors such as the GMCSF-receptor (14Kishimoto T. Taga T. Akira S. Cell. 1994; 76: 253-262Abstract Full Text PDF PubMed Scopus (1239) Google Scholar), and G-protein-coupled receptors such as the lysophosphatidic acid (LPA) or thrombin receptors (15Cook S.J. Rubinfeld B. Albert I. McCormick F. EMBO J. 1993; 12: 3475-3485Crossref PubMed Scopus (331) Google Scholar, 16van Corven E.J. Hordijk P.L. Medema R.H. Bos J.L. Moolenaar W.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1257-1261Crossref PubMed Scopus (334) Google Scholar, 20Vouret-Craviari V. Van Obberghen-Schilling E. Scimeca J.C. Van Obberghen E. Pouysségur J. Biochem. J. 1993; 289: 209-214Crossref PubMed Scopus (143) Google Scholar). Several observations demonstrate the importance of this signaling pathway in cell growth. First, activating mutations in Ras (17Leevers S.J. Marshall C.J. EMBO J. 1992; 11: 569-574Crossref PubMed Scopus (381) Google Scholar), Raf (9Howe L.R. Leevers S.J. Gomez N. Nakielny S. Cohen P. Marshall C.J. Cell. 1992; 71: 335-342Abstract Full Text PDF PubMed Scopus (627) Google Scholar), or MEK (18Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1844) Google Scholar) are sufficient to activate ERK, induce gene expression, or cause oncogenic transformation. Second, inhibition in this signaling pathway is associated with inhibition of ERK activation and cell growth (19Pagés G. Lenormand P. L'Allemain G. Chambard J.C. Méloche S. Pouysségur J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8319-8323Crossref PubMed Scopus (918) Google Scholar). Third, among the substrates for ERK1 and ERK2 are transcription factors of the Ets family, Elk-1 and Sap1a (4Treisman R. Curr. Opin. Genet. Devel. 1994; 4: 96-101Crossref PubMed Scopus (618) Google Scholar, 21Marais R. Wynne J. Treisman R. Cell. 1993; 73: 381-393Abstract Full Text PDF PubMed Scopus (1102) Google Scholar, 22Gille H. Kortenjann M. Thomae O. Moomaw C. Slaughter C. Cobb M.H. Shaw P.E. EMBO J. 1995; 14: 951-962Crossref PubMed Scopus (578) Google Scholar), which regulate transcription of c-Fos (4Treisman R. Curr. Opin. Genet. Devel. 1994; 4: 96-101Crossref PubMed Scopus (618) Google Scholar). Finally, many growth factors stimulate the sustained activation of ERKs (20Vouret-Craviari V. Van Obberghen-Schilling E. Scimeca J.C. Van Obberghen E. Pouysségur J. Biochem. J. 1993; 289: 209-214Crossref PubMed Scopus (143) Google Scholar, 23Kahan C. Seuwen K. Meloche S. Pouysségur J. J. Biol. Chem. 1992; 267: 13369-13375Abstract Full Text PDF PubMed Google Scholar, 24Cook S.J. McCormick F. Biochem J. 1996; 320: 237-245Crossref PubMed Scopus (63) Google Scholar), which is temporally associated with their accumulation in the nucleus (25Chen R.-H. Sarnecki C. Blenis J. Mol. Cell. Biol. 1992; 12: 915-927Crossref PubMed Google Scholar, 26Lenormand P. Sardet C. Pagés G. L'Allemain G. Brunet A. Pouysségur J. J. Cell. Biol. 1993; 122: 1079-1088Crossref PubMed Scopus (577) Google Scholar, 27Traverse S. Seedorf K. Paterson H. Marshall C.J. Cohen P. Ullrich A. Curr. Biol. 1994; 4: 694-701Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). In this way, the ERK cascade provides a link between receptor signaling events at the plasma membrane and regulated gene expression in the nucleus. Whereas much attention has focused on the mechanisms by which this cascade is activated by growth factors and oncoproteins, several studies have turned to the question of which protein phosphatases are responsible for dephosphorylating and thereby inactivating each kinase in the cascade. In the case of ERK, dephosphorylation and inactivation is of considerable interest because the activating phosphorylations are on both threonine and tyrosine (6Anderson N.G. Maller J.L. Tonks N.K. Sturgill T.W. Nature. 1990; 343: 651-653Crossref PubMed Scopus (791) Google Scholar) and because sustained ERK activation, which seems to be important for proliferative signaling (20Vouret-Craviari V. Van Obberghen-Schilling E. Scimeca J.C. Van Obberghen E. Pouysségur J. Biochem. J. 1993; 289: 209-214Crossref PubMed Scopus (143) Google Scholar, 23Kahan C. Seuwen K. Meloche S. Pouysségur J. J. Biol. Chem. 1992; 267: 13369-13375Abstract Full Text PDF PubMed Google Scholar, 24Cook S.J. McCormick F. Biochem J. 1996; 320: 237-245Crossref PubMed Scopus (63) Google Scholar), is compartmentalized within the nucleus (25Chen R.-H. Sarnecki C. Blenis J. Mol. Cell. Biol. 1992; 12: 915-927Crossref PubMed Google Scholar, 26Lenormand P. Sardet C. Pagés G. L'Allemain G. Brunet A. Pouysségur J. J. Cell. Biol. 1993; 122: 1079-1088Crossref PubMed Scopus (577) Google Scholar, 27Traverse S. Seedorf K. Paterson H. Marshall C.J. Cohen P. Ullrich A. Curr. Biol. 1994; 4: 694-701Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). Recent work has identified a family of dual specificity protein phosphatases that dephosphorylate both the tyrosine (Tyr183) and threonine (Thr185) residues on ERK (28Keyse S.M. Emslie E.A. Nature. 1992; 359: 644-646Crossref PubMed Scopus (568) Google Scholar, 29Charles C.H. Abler A.S. Lau L.F. Oncogene. 1992; 7: 187-190PubMed Google Scholar, 30Charles C.H. Sun H. Lau L.F. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5292-5296Crossref PubMed Scopus (183) Google Scholar, 31Sun H. Charles C.H. Lau L.F. Tonks N.K. Cell. 1993; 75: 487-493Abstract Full Text PDF PubMed Scopus (1020) Google Scholar, 32Noguchi T. Metz R. Chen L. Mattei M.-G. Carrasco D. Bravo R. Mol. Cell. Biol. 1993; 13: 5195-5205Crossref PubMed Scopus (168) Google Scholar). The prototype of this family is MAP kinase phosphatase-1 (MKP-1) (which is encoded by human CL100 and mouse3CH134 or erp genes, which are immediate early genes induced by oxidative stress and mitogenic stimulation (28Keyse S.M. Emslie E.A. Nature. 1992; 359: 644-646Crossref PubMed Scopus (568) Google Scholar, 29Charles C.H. Abler A.S. Lau L.F. Oncogene. 1992; 7: 187-190PubMed Google Scholar,32Noguchi T. Metz R. Chen L. Mattei M.-G. Carrasco D. Bravo R. Mol. Cell. Biol. 1993; 13: 5195-5205Crossref PubMed Scopus (168) Google Scholar)). Overexpression of MKP-1 blocks activation of ERK (31Sun H. Charles C.H. Lau L.F. Tonks N.K. Cell. 1993; 75: 487-493Abstract Full Text PDF PubMed Scopus (1020) Google Scholar) and cell cycle reentry (33Sun H. Tonks N.K. Bar-Sagi D. Science. 1994; 266: 285-288Crossref PubMed Scopus (205) Google Scholar, 34Brondello J.M. McKenzie F.R. Sun H. Tonks N.K. Pouysségur J. Oncogene. 1995; 10: 1895-1904PubMed Google Scholar). Furthermore, pretreatment of cells with cycloheximide, to block de novo expression of MKP-1, is able to potentiate sustained ERK activation by serum in NIH3T3 cells (31Sun H. Charles C.H. Lau L.F. Tonks N.K. Cell. 1993; 75: 487-493Abstract Full Text PDF PubMed Scopus (1020) Google Scholar). Since MKP-1 appears to be expressed exclusively in the nucleus, a simple model envisages growth factor-stimulated activation and nuclear accumulation of ERK leading to increased MKP-1 expression, which in turn accumulates in the nucleus and inactivates ERK, affording exquisite fine tuning of the sustained phase of ERK activation (35Clarke P.R. Curr. Biol. 1994; 4: 647-650Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). This model has been challenged by the observation that in PC12 cells and endothelial cells MKP-1 is induced but may not be the phosphatase that regulates ERK activity; it was suggested that PP2A and an unidentified PTPase were responsible for dephosphorylation of ERK (36Alessi D.R. Gomez N. Moorhead G. Lewis T. Keyse S.M. Cohen P. Curr. Biol. 1995; 5: 283-295Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). In Rat-1 fibroblasts, there is a strong correlation between sustained ERK activation and DNA synthesis in response to mitogenic stimulation with LPA (24Cook S.J. McCormick F. Biochem J. 1996; 320: 237-245Crossref PubMed Scopus (63) Google Scholar); factors that regulate this sustained ERK activation are therefore of great interest. In this report, we demonstrate a role for MKP-1 or a closely related molecule in regulating sustained ERK activity in Rat-1 cells and characterize the pathways regulating expression of MKP-1 in response to growth factor stimulation. Induction of MKP-1 clearly reflects activation of the MEK/ERK cascade, but this pathway alone is insufficient; maximal expression of MKP-1 requires synergistic activation of the ERK pathway and additional ERK-independent signals including Ca2+. In addition, we show that preventing Ca2+-dependent MKP-1 expression potentiates sustained ERK activation, suggesting that MKP-1 is a focus for regulatory cross-talk to the ERK pathway from other signal pathways. Cell culture reagents were from Irvine Scientific. Prepoured SDS-PAGE reagents were from Novex Gel Systems. LPA was obtained from Avanti Polar Lipids. EGF was from Boehringer Mannheim. Okadaic acid was from LC Laboratories. [γ-32P]ATP was from DuPont NEN. Goat-anti-rabbit horseradish peroxidase-conjugated secondary antibodies were from Bio-Rad. All other reagents including myelin basic protein, cycloheximide, and sodium orthovanadate were from Sigma. Antibodies to ERK1 (E1.2) have been described previously (15Cook S.J. Rubinfeld B. Albert I. McCormick F. EMBO J. 1993; 12: 3475-3485Crossref PubMed Scopus (331) Google Scholar). Antibodies to MKP-1 and MKP-2 (Alb-1), generated using a peptide derived from the C-terminal 12 amino acids of mouse CL100 (YLKSPITTSPSC) were the very generous gift of Dr. Fergus McKenzie and Prof. Jacques Pouysségur (Center de Biochimie, Université de Nice, Nice, France). Monoclonal antibodies to ERK and MEK were from Pharmingen and Zymed, respectively. Phosphospecific antibodies for ERK and MEK were from New England Biolabs. The MEK inhibitor PD98059 was prepared by Cheri Blume and Dr. Dan Rogers in the Chemistry Group at ONYX Pharmaceuticals and confirmed by NMR analysis. The Rat-1 cells used in this and previous studies (15Cook S.J. Rubinfeld B. Albert I. McCormick F. EMBO J. 1993; 12: 3475-3485Crossref PubMed Scopus (331) Google Scholar, 24Cook S.J. McCormick F. Biochem J. 1996; 320: 237-245Crossref PubMed Scopus (63) Google Scholar, 37Cook S.J. McCormick F. Science. 1993; 262: 1069-1072Crossref PubMed Scopus (860) Google Scholar) were originally provided by Dr. J. L. Bos (Department of Physiological Chemistry, University of Utrecht, The Netherlands). Rat-1 cells were cultured in Dulbecco's modified Eagle's medium containing penicillin/streptomycin, glutamine, and 10% fetal bovine serum. Cells were washed once in serum-free medium and then placed in fresh serum-free medium for at least 24 h prior to the experiments described herein. Pretreatments with various agents were as follows: 50 μg/ml cycloheximide for 45 min prior to growth factor addition, 100–1000 μm sodium orthovanadate for 30 min prior to growth factor addition, 40 μm PD98059 for 30 min prior to growth factor addition, and 100 μg/ml pertussis toxin for 18 h prior to growth factor addition. The derivation and characterization of R1ΔRaf-1:ER-4 cells will be described elsewhere. 2S. J. Cook, M. McMahon, and F. McCormick, manuscript in preparation. These are a clone of Rat-1 cells expressing the conditional form of oncogenic human Raf-1 in which the catalytic domain of Raf-1 is fused to the hormone-binding domain of the human estrogen receptor (38Samuels M.H. Weber M.J. Bishop J.M. McMahon M. Mol. Cell. Biol. 1993; 13: 6241-6252Crossref PubMed Scopus (322) Google Scholar), allowing estrogen-dependent activation of MEK and ERK independently of Ras. R1ΔRaf-1:ER-4 cells were maintained in the same medium as Rat-1 cells but supplemented with 400 μg/ml G418. G418 selection was maintained throughout and only removed during serum deprivation for the last 24 h. For ERK1 assays, experiments were performed upon six-well plates of confluent, quiescent cells that had been serum-starved for 24–36 h. Following the addition of the indicated drug or inhibitor, cells were stimulated by the addition of 10 × solutions of growth factors, and stimulations proceeded at 37 °C for the time indicated. Incubations were terminated by aspiration and the addition of ice-cold TG lysis buffer (20 mm Tris/HCl (pH 8), 1% Triton X-100, 10% glycerol, 137 mm NaCl, 1.5 mm MgCl2, 1 mm EGTA, 50 mm NaF, 1 mmNa3VO4, 1 mm Pefabloc, 20 μm leupeptin, 10 μg/ml aprotinin). Clarified cell lysates were prepared as described previously (15Cook S.J. Rubinfeld B. Albert I. McCormick F. EMBO J. 1993; 12: 3475-3485Crossref PubMed Scopus (331) Google Scholar, 24Cook S.J. McCormick F. Biochem J. 1996; 320: 237-245Crossref PubMed Scopus (63) Google Scholar, 39Beltman J. McCormick F. Cook S.J. J. Biol. Chem. 1996; 271: 27018-27024Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Anti-peptide antibodies directed to the extreme C termini of ERK1 (E1.2) and the assay of immunoprecipitated ERK1 were described previously (15Cook S.J. Rubinfeld B. Albert I. McCormick F. EMBO J. 1993; 12: 3475-3485Crossref PubMed Scopus (331) Google Scholar, 24Cook S.J. McCormick F. Biochem J. 1996; 320: 237-245Crossref PubMed Scopus (63) Google Scholar, 37Cook S.J. McCormick F. Science. 1993; 262: 1069-1072Crossref PubMed Scopus (860) Google Scholar,39Beltman J. McCormick F. Cook S.J. J. Biol. Chem. 1996; 271: 27018-27024Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). We have been unable to derive immunoprecipitating antiserum for ERK2. However, we have derived a Rat-1 cell line that stably expresses physiological levels of a Myc epitope-tagged version of ERK2. In all experiments performed, we have noted that ERK1 and MycERK2 are regulated identically in response to the agents used in this study. 3K. A. Cadwallader and S. J. Cook, unpublished results. Confluent 10-cm dishes of Rat-1 cells were serum-starved in 5 ml of serum-free Dulbecco's modified Eagle's medium for 24 h before being treated and stimulated as indicated. Cells were then washed briefly in ice-cold PBS before the addition of ice-cold TG lysis buffer. Following removal of detergent-insoluble material, the clarified supernatant was boiled in sample buffer, and equal amounts of cell lysate were resolved on 10 or 12% SDS-PAGE gels until the 30-kDa marker approached the bottom. Gels were transferred to PVDF using a Bio-Rad transblot apparatus, and the filter was stained with Coomassie Brilliant Blue to confirm equal loading of lanes. Filters were washed thoroughly in 0.1% (v/v) Tween 20 in PBS (TPBS) and then "blocked" overnight in TPBS, 5% (w/v) Carnation powdered milk (TPBS/milk). Filters were then probed at room temperature for 1 h in TPBS/milk with anti-peptide antiserum that specifically recognizes the phosphorylated, activated versions of ERK1 and ERK2 or MEK1 and MEK2. Following five 5-min washes with TPBS, the second antibody, goat anti-rabbit horseradish peroxidase, was used at a 1:5000 dilution in TPBS/milk for 1 h at room temperature. Following five washes in TPBS, the filter was dried and incubated with Amersham ECL reagents according to the manufacturer's instructions; exposures to hyperfilm were typically 1–2 min. In addition, duplicate blots were probed with conventional, nonphosphospecific monoclonal antibodies to ERK1/2 and MEK1/2 to confirm that equal amounts of these proteins were present in each sample. Cell samples were fractionated, transferred to PVDF and "blocked" in TPBS/milk as described above. Filters were then probed at room temperature for 1 h in TPBS/milk with a 1:500 dilution of crude antiserum Alb-1 raised against the C-terminal peptide of mouse3CH134 (40Brondello J.M. Brunet A. Pouysségur J. McKenzie F.R. J. Biol. Chem. 1997; 272: 1368-1376Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). Following five 5-min washes with TPBS, the second antibody, goat anti-rabbit horseradish peroxidase, was used at a 1:4000 dilution in TPBS/milk for 1 h at room temperature. Following five washes in TPBS, the filter was dried and incubated with Amersham ECL reagents according to the manufacturer's instructions; exposures to hyperfilm were typically 10–30 s. Results are from single experiments representative of between three and six experiments giving similar results. For ERK1 MBP kinase assays, results are expressed as raw cpm of 32P incorporated into MBP from single point assays. This is a sensitive and highly reproducible assay (15Cook S.J. Rubinfeld B. Albert I. McCormick F. EMBO J. 1993; 12: 3475-3485Crossref PubMed Scopus (331) Google Scholar, 20Vouret-Craviari V. Van Obberghen-Schilling E. Scimeca J.C. Van Obberghen E. Pouysségur J. Biochem. J. 1993; 289: 209-214Crossref PubMed Scopus (143) Google Scholar, 23Kahan C. Seuwen K. Meloche S. Pouysségur J. J. Biol. Chem. 1992; 267: 13369-13375Abstract Full Text PDF PubMed Google Scholar). In addition, some cells were stimulated and assayed in duplicate and gave identical results with errors generally less than 10%. Data were pooled for statistical analysis by student's t test. Experiments involving Western blotting were performed at least three or four times with identical results; a representative experiment is shown in each case. Stimulation of Rat-1 cells with 100 μm LPA resulted in a biphasic increase in ERK1 activity, which peaked at 5–10 min before declining rapidly until 30 min, after which a smaller second phase persisted above basal level for up to 3 h (Fig.1 A). Pretreatment of cells with 50 μg/ml cycloheximide for 45 min followed by challenge with LPA had no effect on the magnitude of peak ERK1 activity or the rapid decline after 10 min of stimulation but did potentiate the sustained phase of ERK1 activity from 60 min onward (Fig. 1 A). For example, the magnitude of LPA-stimulated ERK1 activity, measured as a percentage of the maximum response at 10 min, was 27 ± 15% after 120 min in control cells but 135 ± 28% after 120 min in cycloheximide-treated cells; the difference between the control and cycloheximide-treated values was statistically significant (p < 0.01). Similar results were obtained when EGF was the stimulus (Fig. 1 B). Prolonged treatment with cycloheximide alone had a small, poorly reproducible effect on ERK1 activity (see Fig. 1 C). We also examined the effect of cycloheximide on ERK1 activation by ΔRaf-1:ER (38Samuels M.H. Weber M.J. Bishop J.M. McMahon M. Mol. Cell. Biol. 1993; 13: 6241-6252Crossref PubMed Scopus (322) Google Scholar) in the R1Raf-1:ER-4 cell line. Activation of ΔRaf-1:ER by β-estradiol (β-E2) results in the rapid activation of MEK and ERK in these cells, thereby circumventing the other pathways activated by receptor signaling. The ability of ΔRaf-1:ER to activate ERK1 was greatly potentiated by pretreatment with cycloheximide (Fig.1 C). These results suggest that a labile protein acts as a negative regulator of sustained ERK1 activity, whether stimulated by a receptor tyrosine kinase, a G-protein-coupled receptor, or the conditional form of the human c-Raf-1 protooncogene. This effect was not confined to ERK1. Western blotting with a phosphospecific antibody that only recognizes the activated versions of ERK1 and ERK2 (phosphorylated at Tyr185 in the TEY motif) confirmed that both ERK1 and ERK2 isoforms were activated in a strongly sustained manner in the presence of cycloheximide (Fig. 1 D). Furthermore, duplicate blots probed with a phosphospecific antibody that only recognizes activated MEK1 and MEK2 confirmed that cycloheximide did not amplify or prolong LPA-stimulated MEK activation under these conditions (Fig. 1 D). These results demonstrate that cycloheximide exerts its effect at the level of ERK, not MEK, by preserving the activating phosphorylation sites of ERK1 and ERK2. Since these sites are substrates for MKP family phosphatases, we were interested in seeing if MKPs were expressed in response to growth factors in these cells. We investigated the expression of MKP-1 in response to LPA, EGF, and serum in Rat-1 cells by Western blotting whole cell lysates with an antibody raised to the C terminus of human 3CH134 (Alb-1) (40Brondello J.M. Brunet A. Pouysségur J. McKenzie F.R. J. Biol. Chem. 1997; 272: 1368-1376Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). This antibody recognizes MKP-1 (CL100/3CH134) and MKP-2 (hVH2) (41Guan K.-L. Butch E. J. Biol. Chem. 1995; 270: 7197-7203Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 42King A.G. Ozanne B.W. Smythe C. Ashworth A. Oncogene. 1995; 11: 2553-2563PubMed Google Scholar) but not MKP-3 (rVH6, Pyst1) (43Muda M. Boschert U. Dickinson R. Martinou J.-C. Martinou I. Camps M. Schlegel W. Arkinstall S. J. Biol. Chem. 1996; 271: 4319-4326Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 44271, 3795–3802Mourey, R. J., Vega, Q. C., Campbell, J. S., Wenderoth, M. P., Hauschka, S. D., Krebs, E. G. & Dixon, J. E. (1996) 271,3795–3802.Google Scholar, 45Groom L.A. Sneddon A.A. Alessi D.R. Dowd S. Keyse S.M. EMBO J. 1996; 15: 3621-3632Crossref PubMed Scopus (365) Google Scholar). As a control for the MKP-1 antiserum, we immunoblotted samples from COS cells transfected with empty vector (EXV) or EXV-CL100. Transfected cells were lysed and Western blotted as described under "Experimental Procedures." We detected a strong immunoreactive band of 39–40 kDa in lysates from CL100-transfected cells that was absent in the cells transfected with empty vector (Fig.2 A). A band of similar molecular weight was strongly induced in quiescent Rat-1 cells stimulated with LPA (Fig.2 A). This protein is consistent with the molecular weight of MKP-1 and indicates that antiserum Alb-1 recognizes both recombinant MKP-1 and endogenous MKP-1 induced by growth factor stimulation. The ability of LPA, EGF, or FBS to induce MKP-1 protein expression was completely blocked by pretreatment of cells with cycloheximide (Fig.2 B), indicating that MKP-1 expression is a growth factor-stimulated event that requires de
The role of protein kinase C (PKC) in inflammation, mitogenesis, and differentiation has been deduced in part through the use of a variety of PKC inhibitors. Two widely used inhibitors are the structurally related compounds GF109203X and Ro-31-8220, both of which potently inhibit PKC activity and are believed to be highly selective. While using GF109203X and Ro-31-8220 to address the role of PKC in immediate early gene expression, we observed striking differential effects by each of these two compounds. Growth factors induce the expression of the immediate early gene products MAP kinase phosphatase-1 (MKP-1), c-Fos and c-Jun. Ro-31-8220 inhibits growth factor-stimulated expression of MKP-1 and c-Fos but strongly stimulated c-Jun expression, even in the absence of growth factors. GF109203X displays none of these properties.These data suggest that Ro-31-8220 may have other pharmacological actions in addition to PKC inhibition. Indeed, Ro-31-8220 strongly stimulates the stress-activated protein kinase, JNK1. Furthermore, Ro-31-8220 apparently activates JNK in a PKC-independent manner. Neither the down-regulation of PKC by phorbol esters nor the inhibition of PKC by GF109203X affected the ability of Ro-31-8220 to activate JNK1. These data suggest that, in addition to potently inhibiting PKC, Ro-31-8220 exhibits novel pharmacological properties which are independent of its ability to inhibit PKC. The role of protein kinase C (PKC) in inflammation, mitogenesis, and differentiation has been deduced in part through the use of a variety of PKC inhibitors. Two widely used inhibitors are the structurally related compounds GF109203X and Ro-31-8220, both of which potently inhibit PKC activity and are believed to be highly selective. While using GF109203X and Ro-31-8220 to address the role of PKC in immediate early gene expression, we observed striking differential effects by each of these two compounds. Growth factors induce the expression of the immediate early gene products MAP kinase phosphatase-1 (MKP-1), c-Fos and c-Jun. Ro-31-8220 inhibits growth factor-stimulated expression of MKP-1 and c-Fos but strongly stimulated c-Jun expression, even in the absence of growth factors. GF109203X displays none of these properties. These data suggest that Ro-31-8220 may have other pharmacological actions in addition to PKC inhibition. Indeed, Ro-31-8220 strongly stimulates the stress-activated protein kinase, JNK1. Furthermore, Ro-31-8220 apparently activates JNK in a PKC-independent manner. Neither the down-regulation of PKC by phorbol esters nor the inhibition of PKC by GF109203X affected the ability of Ro-31-8220 to activate JNK1. These data suggest that, in addition to potently inhibiting PKC, Ro-31-8220 exhibits novel pharmacological properties which are independent of its ability to inhibit PKC.
