cAMP-dependent Protein Kinase and Ca2+Influx through L-type Voltage-gated Calcium Channels Mediate Raf-independent Activation of Extracellular Regulated Kinase in Response to Glucagon-like Peptide-1 in Pancreatic β-Cells

Glucagon like peptide-1 (GLP1) is a Gs-coupled receptor agonist that exerts multiple effects on pancreatic β-cells, including the stimulation of insulin gene expression and secretion. In this report, we show that treatment of the mouse pancreatic β-cell line MIN6 with GLP1 leads to the glucose-dependent activation of Erk. These effects are mimicked by forskolin, a direct activator of adenylate cyclase, and blocked by H89, an inhibitor of cAMP-dependent protein kinase. Additionally, we provide evidence that GLP1-stimulated activation of Erk requires an influx of calcium through L-type voltage-gated calcium channels and the activation of calcium/calmodulin-dependent protein kinase II. GLP1-stimulated activation of Erk is blocked by inhibitors of MEK, but GLP1 does not induce the activation of A-Raf, B-Raf, C-Raf, or Ras. Additionally, dominant negative forms of Ras(N17) and Rap1(N17) fail to block GLP1-stimulated activation of Erk. In conclusion, our results indicate that, in the presence of stimulatory concentrations of glucose, GLP1 stimulates the activation of Erk through a mechanism dependent on MEK but independent of both Raf and Ras. This requires 1) the activation of cAMP-dependent protein kinase, 2) an influx of extracellular Ca2+ through L-type voltage-gated calcium channels, and 3) the activation of CaM kinase II. Glucagon like peptide-1 (GLP1) is a Gs-coupled receptor agonist that exerts multiple effects on pancreatic β-cells, including the stimulation of insulin gene expression and secretion. In this report, we show that treatment of the mouse pancreatic β-cell line MIN6 with GLP1 leads to the glucose-dependent activation of Erk. These effects are mimicked by forskolin, a direct activator of adenylate cyclase, and blocked by H89, an inhibitor of cAMP-dependent protein kinase. Additionally, we provide evidence that GLP1-stimulated activation of Erk requires an influx of calcium through L-type voltage-gated calcium channels and the activation of calcium/calmodulin-dependent protein kinase II. GLP1-stimulated activation of Erk is blocked by inhibitors of MEK, but GLP1 does not induce the activation of A-Raf, B-Raf, C-Raf, or Ras. Additionally, dominant negative forms of Ras(N17) and Rap1(N17) fail to block GLP1-stimulated activation of Erk. In conclusion, our results indicate that, in the presence of stimulatory concentrations of glucose, GLP1 stimulates the activation of Erk through a mechanism dependent on MEK but independent of both Raf and Ras. This requires 1) the activation of cAMP-dependent protein kinase, 2) an influx of extracellular Ca2+ through L-type voltage-gated calcium channels, and 3) the activation of CaM kinase II. The peptide hormone GLP1 1The abbreviations used are: GLP1, glucagon-like peptide-1; GLP1-R, GLP1 receptor; MIN6, mouse insulinoma cell line 6; PKA, cAMP-dependent protein kinase; TPA, 12-O-tetradecanoylphorbol 13-acetate; EGF, epidermal growth factor; Erk, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/Erk kinase; PKC, protein kinase C; VGCC, voltage-gated calcium channel; KRB, Krebs-Ringer bicarbonate buffer; MEF, mouse embryonic fibroblast; GST, glutathioneS-transferase; PI, phosphatidylinositol; PTP, protein-tyrosine phosphatases; MBP, myelin basic protein; EGFP, enhanced green fluorescent protein; CaM kinase II, calcium/calmodulin-dependent protein kinase II. is secreted from intestinal L-cells in response to oral ingestion of nutrients such as carbohydrates and fats (1Perfetti R. Merkel P. Eur. J. Endocrinol. 2000; 143: 717-725Google Scholar). A major target for GLP1 action is the pancreatic β-cell, where it exerts multiple effects including the stimulation of β-cell proliferation, differentiation, insulin gene transcription, and the potentiation of glucose dependent insulin secretion (1Perfetti R. Merkel P. Eur. J. Endocrinol. 2000; 143: 717-725Google Scholar). These effects are mediated through the binding of GLP1 to its receptor (GLP1-R), a member of the secretin/glucagon/vasoactive intestinal peptide G-protein-coupled receptor superfamily, that can couple to Gαs-containing heterotrimeric G-proteins leading to the activation of adenylate cyclase and the increase in the production of cAMP (1Perfetti R. Merkel P. Eur. J. Endocrinol. 2000; 143: 717-725Google Scholar). However, it has also been reported that GLP1-R can also signal through Gαi and Gαqproteins (2Montrose-Rafizadeh C. Avdonin P. Garant M.J. Rodgers B.D. Kole S. Yang H. Levine M.A. Schwindinger W. Bernier M. Endocrinology. 1999; 140: 1132-1140Google Scholar). In pancreatic β-cells, the binding of GLP1 to its receptor not only increases cAMP but also induces a rise in intracellular free Ca2+ levels through the closure of ATP sensitive K+ channels, an increase in Ca2+ influx through L-type voltage-gated calcium channels (L-type VGCC), and Ca2+-dependent calcium release from intracellular stores (3Holz G.G. Leech C.A. Habener J.F. J. Biol. Chem. 1995; 270: 17749-17757Google Scholar, 4Holz G.G. Leech C.A. Heller R.S. Castonguay M. Habener J.F. J. Biol. Chem. 1999; 274: 14147-14156Google Scholar, 5Bode H.P. Moormann B. Dabew R. Goke B. Endocrinology. 1999; 140: 3919-3927Google Scholar). Additionally, a number of kinases are activated upon GLP1 binding to their receptor, including phosphatidylinositol 3-kinase (PI 3-kinase) and the extracellular regulated kinase, Erk (2Montrose-Rafizadeh C. Avdonin P. Garant M.J. Rodgers B.D. Kole S. Yang H. Levine M.A. Schwindinger W. Bernier M. Endocrinology. 1999; 140: 1132-1140Google Scholar, 6Buteau J. Foisy S. Rhodes C.J. Carpenter L. Biden T.J. Prentki M. Diabetes. 2001; 50: 2237-2243Google Scholar, 7Buteau J. Roduit R. Susini S. Prentki M. Diabetologia. 1999; 42: 856-864Google Scholar). In a number of cell types, Erk activation has been shown to be important in many cellular processes including mitosis, cell differentiation, apoptosis, and the modulation of gene expression. Therefore, Erk activation is likely to be important in mediating a number of the effects induced by GLP1 in pancreatic β-cells. However, the mechanism by which GLP1 activates Erk in pancreatic β-cells is poorly understood. Typically, Erk1 and Erk2 are activated upon phosphorylation by the dual specificity tyrosine serine kinases, MEK1 and MEK2, which are themselves activated by one of several Raf isoforms, Raf1 (C-Raf), B-Raf, and A-Raf (8Kolch W. Biochem. J. 2000; 351: 289-305Google Scholar). The mechanism of Raf activation is complex and not fully understood; however, its activation is known to require the binding of the small G-proteins Ras or Rap1 in their GTP ligated forms (8Kolch W. Biochem. J. 2000; 351: 289-305Google Scholar). In many cell types, drugs or hormones that increase intracellular cAMP inhibit Erk signaling (9Chen J. Iyengar R. Science. 1994; 263: 1278-1281Google Scholar, 10Wu J. Dent P. Jelinek T. Wolfman A. Weber M.J. Sturgill T.W. Science. 1993; 262: 1065-1069Google Scholar, 11Cook S.J. McCormick F. Science. 1993; 262: 1069-1072Google Scholar, 12Qiu W. Zhuang S. von Lintig F.C. Boss G.R. Pilz R.B. J. Biol. Chem. 2000; 275: 31921-31929Google Scholar). However, in other cell types, such as neuronal cells, increased intracellular cAMP leads to an increase in Erk activation (13Vossler M.R. Yao H. York R.D. Pan M.G. Rim C.S. Stork P.J. Cell. 1997; 89: 73-82Google Scholar, 14Dugan L.L. Kim J.S. Zhang Y. Bart R.D. Sun Y. Holtzman D.M. Gutmann D.H. J. Biol. Chem. 1999; 274: 25842-25848Google Scholar). cAMP activation or inhibition of Erk is thought to occur through the activation of either cAMP-dependent protein kinase (PKA) or the cAMP-responsive Ras guanine nucleotide exchange factor, Epac (15Stork P.J. Schmitt J.M. Trends Cell Biol. 2002; 12: 258-266Google Scholar, 16de Rooij J. Zwartkruis F.J. Verheijen M.H. Cool R.H. Nijman S.M. Wittinghofer A. Bos J.L. Nature. 1998; 396: 474-477Google Scholar). In cells that mainly express Raf1, the activation of Rap1 is thought to inhibit Erk activation (17Schmitt J.M. Stork P.J. Mol. Cell. Biol. 2001; 21: 3671-3683Google Scholar, 18Naor Z. Benard O. Seger R. Trends Endocrinol. Metab. 2000; 11: 91-99Google Scholar). However, in other cells types, the activation of Rap1 leads to the activation of B-Raf, which in turn activates Erk (12Qiu W. Zhuang S. von Lintig F.C. Boss G.R. Pilz R.B. J. Biol. Chem. 2000; 275: 31921-31929Google Scholar, 13Vossler M.R. Yao H. York R.D. Pan M.G. Rim C.S. Stork P.J. Cell. 1997; 89: 73-82Google Scholar, 14Dugan L.L. Kim J.S. Zhang Y. Bart R.D. Sun Y. Holtzman D.M. Gutmann D.H. J. Biol. Chem. 1999; 274: 25842-25848Google Scholar). In this report, we provide evidence that, in the mouse pancreatic β-cell line MIN6, GLP1 stimulates the activation of Erk through a mechanism dependent of MEK but independent of both Raf and Ras. This requires 1) the activation of PKA, 2) an influx of extracellular Ca2+ through L-type voltage-gated calcium channels, and 3) the activation of CaM kinase II. PD098059, KN-93, KN-62, nifedipine, and Bay K8644 were all purchased from Calbiochem. U0126 was purchased from Promega. All other chemicals (unless stated) were obtained from Sigma. In this study, MIN6 cells were used between passages 25 and 35 at ∼80% confluence. MIN6 cells were grown in Dulbecco's modified Eagle's medium containing 25 mm glucose supplemented with 15% heat-inactivated fetal calf serum, 100 μg/ml streptomycin, 100 units/ml penicillin sulfate, and 75 μm β-mercaptoethanol, equilibrated with 5% CO2, 95% air at 37 °C. Prior to treatment, the medium was removed and the cells washed twice with HEPES-balanced Krebs-Ringer bicarbonate buffer (115 mm NaCl, 5 mm KCl, 10 mm NaHCO3, 2.5 mmMgCl2, 2.5 mm CaCl2, 20 mm HEPES, pH 7.4) containing 0.5% bovine serum albumin (KRB buffer). The cells were then incubated for 1 h at 37 °C in KRB buffer containing 1 mm glucose prior to incubation in KRB buffer containing 2.8 or 16.7 mm glucose and the test substances for the times indicated in the figure legends (details of treatments are provided in the figure legends). When cells were treated with elevated extracellular K+concentration, the K+ concentration in the KRB was increased to 50 mm and the Na+ concentration decreased to 70 mm to maintain isotonicity. In the calcium-free experiments, after preincubation the cells were incubated for 15 min in a calcium-free KRB buffer containing 1 mmEGTA and stimulated in the same buffer. All treatments were stopped by the addition of ice-cold lysis buffer containing 1% Triton, 10 mm β-glycerophosphate, 50 mm Tris-HCl, pH 7.5, 1 mm EDTA, 1 mm EGTA, 1 mmsodium orthovanadate, 1 mm benzamidine HCl, 0.2 mm phenylmethylsulfonyl fluoride, 1 μg/ml each of leupeptin and pepstatin, 0.1% β-mercaptoethanol, and 50 mm sodium fluoride. The lysates were then centrifuged for 10 min at 16,000 × g. The supernatants were kept, and total protein concentrations were determined by the Bradford assay (Bio-Rad) using bovine serum albumin as standard. The protein lysates were stored at −80 °C until further analysis. and were as G.R. J. Biol. Chem. 2000; 275: Scholar). only the activated forms of Erk was purchased from was purchased from was by and enhanced embryonic or for were used as and negative for this were in Dulbecco's modified Eagle's medium containing 0.5% fetal calf serum for h prior to stimulation by 10 for 5 of the MIN6 cell treatments are provided in the to and lysates were as by R. Y. C.S. C.J. J. Biol. Chem. 1997; Scholar). Raf were for h at °C from to 1 and of cell with of of C.S. C.J. C.J. R. J. 1999; and of mouse The of each Raf protein was by using the Raf kinase assay using and as with R. Y. C.S. C.J. J. Biol. Chem. 1997; Scholar). incubation at °C for min the kinase were stopped by addition of buffer and 5 min at 100 °C. The were on a 15% The was and the phosphorylation of MBP, the activation of the kinase by binding was in using and then used to Ras activation in MIN6 cells and to the in the or MIN6 cells and for were stimulated with for 5 min and of were incubated with binding at °C for The were on a 15% and the activated Ras was then by using mouse containing was using the S. J. B. S. A. 1998; Scholar). The and were provided by and and the was provided by J. L. the was by from the as a and ligated a the of the and of the with the in a The was then cells, and was obtained by the cells after expression was used to the production of the several of The a from J. of and the containing from B. and C. were in a MIN6 cells, or were with and cells were to the with of for h prior to the of cells were as determined by expression. the mechanism by which GLP1 activates Erk, we the activation of Erk in response to GLP1 in the pancreatic β-cell line MIN6, a cell line that and insulin in response to glucose concentrations H. T. H. M. M. Y. Y. Diabetologia. 1993; Scholar, R.H. H. Y. Rhodes C.J. Diabetes. Scholar). MIN6 cells were for 1 h in KRB supplemented with 1 mm The cells were then incubated in 2.8 or 16.7 mm glucose in the presence of of either GLP1 or forskolin, an activator of adenylate were a and the activation of Erk using a the activated forms of Erk. activation of Erk was MIN6 cells were incubated in KRB supplemented with 2.8 However, MIN6 cells were incubated in KRB supplemented with 2.8 in the presence of GLP1, a increase in the activation of Erk was at 5 min of cells in KRB supplemented with 16.7 mm glucose to an activation of Erk with cells incubated at 2.8 mm glucose This is in with that glucose can Erk in MIN6 cells C. H. C. J. R. J. Biol. Chem. 1998; Scholar, C. J. R. Biochem. J. 1999; Scholar). However, GLP1 treatment in the presence of 16.7 mm glucose to a and increase in Erk with activation between 5 and 15 GLP1 stimulation of Erk was mimicked by at either 2.8 or 16.7 mm glucose GLP1 or can adenylate cyclase, and both Erk a glucose-dependent the activation of Erk by GLP1 or is likely to be mediated through GLP1 or glucose-dependent insulin secretion (1Perfetti R. Merkel P. Eur. J. Endocrinol. 2000; 143: 717-725Google glucose-dependent GLP1 activation of Erk be mediated by an of this MIN6 cells were incubated in KRB supplemented with 2.8 or 16.7 mm glucose and treated with 1 μm insulin in the presence or of treatment not to Erk activation and stimulatory on GLP1 induced Erk This that GLP1 activation of Erk is not mediated by insulin not glucose-dependent GLP1 activation of Erk is mimicked by forskolin, it is likely to be mediated through the activation of adenylate the of in GLP1 activation of Erk, MIN6 cells were treated with GLP1 or as a forskolin, in KRB supplemented with 16.7 mm glucose in either the presence or of the H89, at a concentration shown to block stimulation of Erk. both and Erk activation GLP1-stimulated activation of Erk is mimicked by forskolin, an activator of adenylate cyclase, and by H89, an inhibitor of PKA, it is that GLP1-stimulated activation of Erk is mediated by the activation of adenylate cyclase, leading to an increase in intracellular cAMP and the activation of in pancreatic β-cells leads to an increase in intracellular calcium through an influx of calcium through L-type VGCC, which is by hormones such as GLP1 G.G. Habener J.F. Trends Biochem. Scholar, G.G. Habener J.F. Nature. 1993; Scholar). This rise in intracellular Ca2+ a number of important pancreatic β-cell including insulin secretion (1Perfetti R. Merkel P. Eur. J. Endocrinol. 2000; 143: 717-725Google Scholar). Ca2+ influx is an important in GLP1-stimulated Erk MIN6 cells were incubated at 16.7 mm glucose in the presence or of to extracellular calcium GLP1 treatment to the activation of Erk, which was by the presence Therefore, GLP1 requires an influx of extracellular calcium to Erk. an increase in intracellular calcium is for GLP1-stimulated Erk MIN6 cells were treated with the Ca2+ to intracellular calcium in the presence or of GLP1 at 2.8 or 16.7 mm glucose of MIN6 cells with in the presence of 2.8 or 16.7 mm glucose on Erk of MIN6 with and GLP1 at 2.8 mm glucose also on Erk activation Additionally, GLP1-stimulated Erk activation in the presence of 16.7 mm glucose was by the presence of These results indicate that an influx of extracellular calcium is not for GLP1-stimulated Erk However, it is that the of calcium is important in GLP1-stimulated Erk activation and that through L-type to be in signaling to Erk. this MIN6 cells were treated with GLP1 at 16.7 mm glucose in the presence or of nifedipine, an L-type GLP1-stimulated activation of Erk was by the presence of Therefore, GLP1 stimulation of Erk is mediated through an influx of extracellular calcium through L-type through L-type was for Erk cells were incubated in the presence of 1) increased extracellular K+ which leads to the of the the of L-type VGCC, and the influx of extracellular Ca2+ or 2) Bay an L-type agonist of MIN6 cells in 50 mm extracellular K+ to a stimulation of Erk that this was mediated through Ca2+ influx through L-type VGCC, the was also in the presence of or treatments Erk activation This is in with that Erk activation in MIN6 cells requires the influx of Ca2+ through L-type C. H. C. J. R. J. Biol. Chem. 1998; Scholar, C. J. R. Biochem. J. 1999; Scholar). Additionally, treatment of MIN6 cells with Bay K8644 was to induce Erk activation results indicate that an influx of is for Erk activation and is for GLP1-stimulated Erk However, it is the influx of Ca2+ through L-type is both and for GLP1-stimulated Erk GLP1 be which are important for GLP1-stimulated Erk GLP1 stimulation of Erk requires an influx of extracellular calcium through L-type of the effects of calcium on intracellular signaling are mediated through such as Therefore, we the of in GLP1-stimulated Erk MIN6 cells were for min with a inhibitor of prior to incubation in KRB containing 16.7 mm glucose in the presence of GLP1 of cells with to a inhibition of GLP1-stimulated Erk activation that is likely to be for GLP1-stimulated Erk activation the important in the activation of calcium/calmodulin-dependent kinases and the CaM kinase in insulin secretion in pancreatic β-cells, we the of CaM kinase in Erk activation using inhibitors of CaM kinase II, and Biochem. Scholar, L. Mol. 1999; Scholar). MIN6 cells were incubated with either or for min or 1 prior to incubation in KRB supplemented with 16.7 mm glucose and GLP1 and of cells with either or a inhibition of GLP1-stimulated Erk activation and These results indicate that and the activation of CaM kinase be for glucose-dependent GLP1 activation of Erk. further the signaling by which GLP1 stimulates Erk we the of MEK, the kinase of Erk, using inhibitors of MEK, and U0126 MIN6 cells were with either or U0126 prior to treatment with either GLP1 or in the presence of 16.7 mm glucose, or 1 μm as The activation of Erk was using a to Erk and U0126 and Erk that the activation of MEK is for and Erk activation MEK is and activated by several of Raf including Raf1 (C-Raf), B-Raf, and Therefore, we the activation of all of Raf upon treatment with GLP1 using the kinase assay R. Y. C.S. C.J. J. Biol. Chem. 1997; Scholar). MIN6 cells were treated for the times indicated in with 10 GLP1 or 10 μm in the presence of 16.7 mm glucose or 1 μm as lysates were and Raf were using to the Raf B-Raf, and The were incubated with MBP, and and the of Raf by the phosphorylation of mouse embryonic or for treated or not with 10 EGF, were used as and negative for this M. J. A. M. S. Sun J. R. C. J. 2001; Scholar). In with M. J. A. M. S. Sun J. R. C. J. 2001; elevated in cells, both and A-Raf were not was in the stimulated the of the Raf A-Raf, and in the and the of A-Raf and in the In the MIN6 cell stimulation to the activation of both A-Raf and kinases In that to the activation of Erk, glucose or glucose forskolin, not to the activation of of the Raf isoforms, A-Raf, B-Raf, or further we the of dominant negative Rap1(N17) or Ras(N17) on GLP1 stimulation of Erk. MIN6 cells were or with either or h the cells were treated for 5 min with 16.7 mm glucose or 16.7 mm glucose GLP1 or and the activation of Erk determined The of dominant negative Rap1 on the of GLP1 or to Erk activation The of dominant negative Ras on the of GLP1 to Erk activation but Erk activation Therefore, GLP1-stimulated Erk activation to be independent of both Ras and an to the we also the activation of Ras in response to agonist Ras activation was with a binding which with the of but not with the of Ras in of MIN6 cells with GLP1 not to an increase in the binding of Ras to binding with cells incubated in 16.7 mm glucose This that Ras is not activated by GLP1 In or treatment of MIN6 cells and treatment of a increase in the activation of Ras as shown by the increased of the of Ras to the binding indicate that glucose-dependent GLP1 stimulation of Erk is independent of the activation of A-Raf, B-Raf, C-Raf, or Ras. In pancreatic β-cells, GLP1 activates the extracellular regulated kinase, Erk (2Montrose-Rafizadeh C. Avdonin P. Garant M.J. Rodgers B.D. Kole S. Yang H. Levine M.A. Schwindinger W. Bernier M. Endocrinology. 1999; 140: 1132-1140Google Scholar, 6Buteau J. Foisy S. Rhodes C.J. Carpenter L. Biden T.J. Prentki M. Diabetes. 2001; 50: 2237-2243Google Scholar, 7Buteau J. Roduit R. Susini S. Prentki M. Diabetologia. 1999; 42: 856-864Google a poorly In this report, we provide evidence that, in the pancreatic β-cell line MIN6, glucose-dependent GLP1 activation of Erk is mediated by an influx of calcium through L-type and requires both the activation of and calcium/calmodulin-dependent protein kinase kinase we show that GLP1 stimulates the activation of Erk a mechanism independent of both Raf and Ras but dependent on the activation of MEK in pancreatic β-cells leads to an influx of calcium through L-type VGCC, which is by hormones such as GLP1 G.G. Habener J.F. Trends Biochem. Scholar, G.G. Habener J.F. Nature. 1993; Scholar). The rise in intracellular Ca2+ a number of important pancreatic β-cell including insulin secretion (1Perfetti R. Merkel P. Eur. J. Endocrinol. 2000; 143: 717-725Google Scholar). GLP1 stimulation of Erk also requires Ca2+ through L-type The of Ca2+ through L-type is also for or activation of Erk in MIN6 cells C. H. C. J. R. J. Biol. Chem. 1998; Scholar, C. J. R. Biochem. J. 1999; Scholar). it is both and for Erk as Bay an L-type activates Erk and nifedipine, an L-type Erk activation induced by an increase in extracellular K+ concentration and the influx of L-type is for the activation of Erk, and GLP1 Ca2+ influx through L-type G.G. Leech C.A. Heller R.S. Castonguay M. Habener J.F. J. Biol. Chem. 1999; 274: 14147-14156Google it is that the increase influx of calcium is the signal by which GLP1 induces glucose-dependent Erk However, it is that GLP1 activates signaling that are important in the activation of Erk. In the influx of calcium through L-type also leads to the activation of Erk. This of Ca2+ is in which signaling is activated and is thought to be mediated through the increase in the of the L-type in the binding of to the L-type and the activation of Erk J.M. Science. 2001; Scholar). the agonist GLP1-stimulated Erk activation it is that GLP1 stimulates Erk in pancreatic β-cells by a mechanism to that in inhibitors of CaM kinase II, and inhibit GLP1-stimulated activation of Erk but inhibitors specificity and can with both K+ and Ca2+ T. Biochem. 2000; Scholar). However, it has been that CaM kinase is activated by glucose in pancreatic β-cells J. M. Endocrinology. 1997; and that the activation of CaM kinase can Erk activation in other cell such as and cells M.R. J. Biol. Chem. Scholar, R. J. 2002; Scholar). Therefore, it is that CaM kinase GLP1-stimulated Erk activation in MIN6 GLP1 activation of CaM kinase and that this is both and for GLP1 activation of Erk or glucose activation of CaM kinase is but GLP1 an for the activation of Erk is In cells, Ca2+-dependent activation of Erk requires the activation of CaM kinase II. In this Erk activation is dependent on the tyrosine kinase the of the receptor, and the activation of Raf R. J. 2002; Scholar). However, in our we show that GLP1-stimulated Erk activation is independent of as activation of all of Raf was in kinase assay and dominant negative forms of Ras or Rap1 on GLP1-stimulated Erk activation GLP1-stimulated Erk activation not to the activation of as increase in the of Ras to Raf was upon GLP1 treatment with This activation of Erk does not require the activation of the or as the inhibitor and inhibitors or or to block GLP1-stimulated activation of Erk not GLP1-stimulated Erk activation does require the activation of MEK as it is blocked by inhibitors of MEK, and U0126 is that CaM kinase MEK at either or important in MEK as of the known for CaM kinase M. J. Biol. Chem. 1998; Scholar). However, CaM kinase an kinase for MEK mechanism by which Erk be activated by cAMP in a mechanism is through the of binding of the protein-tyrosine and to Erk M. S. T. Cell Biol. 1999; Scholar, C. J. R. J. Cell Biol. 1999; Scholar). When protein-tyrosine are to Erk, Erk is in an however, upon their activation by PKA, Erk is and activated M. S. T. Cell Biol. 1999; Scholar, C. J. R. J. Cell Biol. 1999; Scholar). and for the and J. L. Bos for a also like to of for and the used in the Raf kinase