FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2079238835> ?p ?o ?g. }

• W2079238835 endingPage "32497" @default.
• W2079238835 startingPage "32490" @default.
• W2079238835 abstract "The small G protein RAP1 and the kinase B-RAF have been proposed to link elevations of cAMP to activation of ERK/mitogen-activated protein (MAP) kinase. In order to delineate signaling pathways that link receptor-generated cAMP to the activation of MAP kinase, the human A2A-adenosine receptor, a prototypical Gs-coupled receptor, was heterologously expressed in Chinese hamster ovary cells (referred as CHO-A2A cells). In CHO-A2A cells, the stimulation of the A2A-receptor resulted in an activation of RAP1 and formation of RAP1-B-RAF complexes. However, overexpression of a RAP1 GTPase-activating protein (RAP1GAP), which efficiently clamped cellular RAP1 in the inactive GDP-bound form, did not affect A2A-agonist-mediated MAP kinase stimulation. In contrast, the inhibitor of protein kinase A H89 efficiently suppressed A2A-agonist-mediated MAP kinase stimulation. Neither dynamin-dependent receptor internalization nor receptor-promoted shedding of matrix-bound growth factors accounted for A2A-receptor-dependent MAP kinase activation. PP1, an inhibitor of SRC family kinases, blunted both the A2A-receptor- and the forskolin-induced MAP kinase stimulation (IC50 = 50 nm); this was also seen in PC12 cells, which express the A2A-receptor endogenously, and in NIH3T3 fibroblasts, in which cAMP causes MAP kinase stimulation. In the corresponding murine fibroblast cell line SYF, which lacks the ubiquitously expressed SRC family kinases SRC, YES, and FYN, forskolin barely stimulated MAP kinase; this reduction was reversed in cells in which c-SRC had been reintroduced. These findings show that activation of MAP kinase by cAMP requires a SRC family kinase that lies downstream of protein kinase A. A role for RAP1, as documented for the β2-adrenergic receptor, is apparently contingent on receptor endocytosis. The small G protein RAP1 and the kinase B-RAF have been proposed to link elevations of cAMP to activation of ERK/mitogen-activated protein (MAP) kinase. In order to delineate signaling pathways that link receptor-generated cAMP to the activation of MAP kinase, the human A2A-adenosine receptor, a prototypical Gs-coupled receptor, was heterologously expressed in Chinese hamster ovary cells (referred as CHO-A2A cells). In CHO-A2A cells, the stimulation of the A2A-receptor resulted in an activation of RAP1 and formation of RAP1-B-RAF complexes. However, overexpression of a RAP1 GTPase-activating protein (RAP1GAP), which efficiently clamped cellular RAP1 in the inactive GDP-bound form, did not affect A2A-agonist-mediated MAP kinase stimulation. In contrast, the inhibitor of protein kinase A H89 efficiently suppressed A2A-agonist-mediated MAP kinase stimulation. Neither dynamin-dependent receptor internalization nor receptor-promoted shedding of matrix-bound growth factors accounted for A2A-receptor-dependent MAP kinase activation. PP1, an inhibitor of SRC family kinases, blunted both the A2A-receptor- and the forskolin-induced MAP kinase stimulation (IC50 = 50 nm); this was also seen in PC12 cells, which express the A2A-receptor endogenously, and in NIH3T3 fibroblasts, in which cAMP causes MAP kinase stimulation. In the corresponding murine fibroblast cell line SYF, which lacks the ubiquitously expressed SRC family kinases SRC, YES, and FYN, forskolin barely stimulated MAP kinase; this reduction was reversed in cells in which c-SRC had been reintroduced. These findings show that activation of MAP kinase by cAMP requires a SRC family kinase that lies downstream of protein kinase A. A role for RAP1, as documented for the β2-adrenergic receptor, is apparently contingent on receptor endocytosis. protein kinase A/cAMP-dependent protein kinase mitogen-activated protein kinase extracellular signal-regulated protein kinase influenza hemagglutinin-epitope tag MAP kinase kinase glutathione S-transferase N-ethylcarboxamido-2-[4-(2-carboxyethyl)phenylethyl]adenosine phorbol 12,13-dibutyrate N- [2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide phenylmethylsulfonyl fluoride [4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine] [4-amino-7-phenylpyrazol[3,4-d]pyrimidine] Chinese hamster ovary 8-bromo-cAMP green fluorescent protein epidermal growth factor GTPase-activating protein Elevation of cyclic AMP inhibits the growth of many cells. This effect is thought to reflect, at least in part, the ability of protein kinase A (PKA)1-dependent phosphorylation to disrupt the interaction between p21ras and c-RAF; this results in cAMP-mediated suppression of mitogen-activated protein kinase pathway (1Wu J. Dent P. Jelinek T. Wolfmann A. Weber M.J Sturgill T.W. Science. 1993; 262: 1066-1069Crossref Scopus (817) Google Scholar, 2Cook S.J. McCormick Science. 1993; 262: 1069-1072Crossref PubMed Scopus (860) Google Scholar, 3Burgering B.M. Pronk G.J. van Weeren P.C. Chardin P. Bos J.L. EMBO J. 1993; 12: 4211-4220Crossref PubMed Scopus (313) Google Scholar, 4Graves L.M. Bornfeldt K.E. Raines E.W. Potts B.C. Macdonald S.G. Ross R. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10300-10304Crossref PubMed Scopus (400) Google Scholar). However, in some cells, agonist occupancy of Gs-coupled receptors is associated with both increases in cellular cAMP and with stimulation of MAP kinase. It has been argued that stimulation of MAP kinase is dependent on cAMP and that effectors other than adenylyl cyclase generate the signal that link Gs-coupled receptor to MAP kinase activation. Furthermore, in each of the proposed models, the signal to MAP kinase diverges at a different level from the signaling cascade composed of receptor, Gs, and adenylyl cyclase/cAMP. (i) The cAMP-dependent signaling mechanism is mediated by the p21ras-related, monomeric G protein RAP1 that preferentially activates B-RAF (5Ohtsuka T. Shimizu K. Yamamori B. Kuroda S. Takai Y. J. Biol. Chem. 1996; 271: 1258-1261Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 6Vossler M.R. Yao H. York R.D. Pan M.G. Rim C.S. Stork P.J. Cell. 1997; 89: 73-82Abstract Full Text Full Text PDF PubMed Scopus (940) Google Scholar). Loading of RAP1A with GTP requires guanine nucleotide exchange factors, a class of which (Epac) is directly activated by cAMP, i.e. in a manner independent of PKA (7de Rooij J. Zwartkruis F.J. Verheijen M.H. Cool R.H. Nijman S.M. Wittinghofer A. Bos J.L. Nature. 1998; 396: 474-477Crossref PubMed Scopus (1591) Google Scholar,8Kawasaki H. Springett G.M. Mochizuki N. Toki S. Nakaya M. Matsuda M. Housman D.E. Graybiel A.M. Science. 1998; 282: 2275-2279Crossref PubMed Scopus (1155) Google Scholar). (ii) Alternatively, a role has been invoked for the non-receptor tyrosine kinase SRC, because Gαs directly binds to and activates SRC in vitro (9Ma Y.C. Huang J. Ali S. Lowry W. Huang X.Y. Cell. 2000; 102: 635-646Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar). In this case, stimulation of MAP kinase by Gs-coupled receptors is independent of cAMP but sensitive to inhibition or genetic ablation of SRC. (iii) A large set of experiments support a third model of MAP kinase activation in which the receptor is endocytosed in a dynamin-dependent fashion. Here β-arrestin functions as adapter protein for the recruitment of SRC and for the assembly of a large signaling complex. This model has been primarily developed with the β2-adrenergic receptor (10McDonald P.H. Lefkowitz R.J. Cell. Signal. 2001; 13: 683-689Crossref PubMed Scopus (111) Google Scholar) and predicts that stimulation of MAP kinase by the receptor is blocked by abrogating dynamin and SRC. (iv) Finally, G protein-coupled receptors may promote transactivation of tyrosine kinase receptors by causing the shedding of matrix-bound growth factors; this effect depends on the activation of matrix metalloproteases (11Prenzel N. Zwick E. Daub H. Leserer M. Abraham R. Wallasch C. Ullrich A. Nature. 1999; 402: 884-888Crossref PubMed Scopus (1481) Google Scholar). Although G protein-coupled receptors may recruit multiple and redundant pathways to stimulate MAP kinase (12Marinissen M.J. Gutkind J.S. Trends Pharmacol. Sci. 2001; 22: 368-376Abstract Full Text Full Text PDF PubMed Scopus (823) Google Scholar), it is evident that some of the proposed mechanisms are mutually exclusive. It is also difficult to understand why protein kinase A is required for cAMP-dependent stimulation of MAP kinase (13Yao H. York R.D. Misra-Press A. Carr D.W. Stork P.J. J. Biol. Chem. 1998; 273: 8240-8247Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) if GTP-liganded RAP1 is formed by the action of Epac (7de Rooij J. Zwartkruis F.J. Verheijen M.H. Cool R.H. Nijman S.M. Wittinghofer A. Bos J.L. Nature. 1998; 396: 474-477Crossref PubMed Scopus (1591) Google Scholar, 8Kawasaki H. Springett G.M. Mochizuki N. Toki S. Nakaya M. Matsuda M. Housman D.E. Graybiel A.M. Science. 1998; 282: 2275-2279Crossref PubMed Scopus (1155) Google Scholar). In the present study, we have therefore tested the predictions of the four models; we compared the action of receptor-independent elevations of cAMP (by membrane-permeable analogues and forskolin, the direct activator of adenylyl cyclase) with the effect of the A2A-adenosine receptor. This Gs-coupled receptor activates MAP kinase in CHO cells in a manner dependent on cAMP (14Seidel M.G. Klinger M. Freissmuth M. Holler C. J. Biol. Chem. 1999; 274: 25833-25841Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Our experiments show that RAP1 and dynamin-dependent receptor endocytosis are dispensable for receptor and cAMP-dependent activation of MAP kinase; similarly, MAP kinase stimulation cannot be accounted for by transactivation of a receptor tyrosine kinase due to release of a matrix-bound release growth factor. In contrast, SRC (or an SRC-like kinase) plays an essential role, but it is downstream of protein kinase A. Adenosine deaminase, basic fibroblast growth factor, 12CA5 anti-hemagglutinin mouse monoclonal antibody, and enzymes for DNA manipulation were from Roche Molecular Biochemicals. CGS21680 was from Tocris Cookson Ltd. (Bristol, UK). Hepes was from Biomol(Munich, Germany). The materials required for SDS-PAGE were from Bio-Rad. Fetal calf serum was from PAA Laboratories (Linz, Austria); Dulbecco’s modified Eagle’s medium, Opti-MEM medium, horse serum, non-essential amino acids, β-mercaptoethanol, and G418 (geneticin) were obtained from Invitrogen. Ham’s F-12 medium was from BioConcept (Allschwil, Switzerland). Collagen was from Biomedical Technologies Inc. (Stoughton, MA), Centrifuge tubes and tissue culture plates were from Greiner (Vienna, Austria) and from Corning Costar (Acton, MA). Forskolin, 8-Br-cAMP, l-glutamine, penicillin G, streptomycin, Triton X-100, PMSF, leupeptin, and thrombin were purchased from Sigma. Aprotinin, PP3, and PDBu were from Calbiochem. PP1 was from Alexis Biochemicals (San Diego, CA). The inhibitor of protein kinase A H89 was from Alexis Corp. (Laeufelfingen, Switzerland). The Micro BCA® protein assay reagent kit was from Pierce. Buffers and salts were from Merck. Glutathione-Sepharose and protein G-Sepharose was from Amersham Biosciences. Polyclonal rabbit antisera recognizing the diphosphorylated sequence of ERK1 and ERK2 was from New England Biolabs (Beverly, MA). The monoclonal mouse antibody directed against RAP1 was from Transduction Laboratories (Lexington, KY). Polyclonal rabbit antisera recognizing the carboxyl terminus of ERK1/ERK2 and B-RAF were purchased from and from Santa Cruz Biotechnology (Santa Cruz, CA). The 16B12 anti-hemagglutinin mouse monoclonal antibody was generous gift of E. Ogris (Vienna Biocenter). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit immunoglobulin antibodies were from Amersham Biosciences. The immunoreactive bands on nitrocellulose blots were detected by chemiluminescence using SuperSignal chemiluminescence substrate from Pierce. SuperFect® polycationic transfection reagent and plasmid preparation kits were from Qiagen (Hilden, Germany). PC12 were from ECACC (Salisbury, UK). SYF and SYF cells, in which c-SRC had been reintroduced via retroviral infection (referred as SYF + c-SRC), were purchased from ATCC (Manassas, VA). The plasmid driving the mammalian expression of RAP1GAP was a gift from P. Polakis and J. L. Bos, which encoding mutated c-SRC was generously provided by R. J. Resnick and D. Shalloway, those encoding wild type and mutated versions of dynamin were kindly provided by C. van Koppen. In order to generate an alternative reporter MAP kinase, we amplified the cDNA encoding a hemagglutinin-tagged version of ERK1/p44 MAP kinase by PCR using the following primer (where underlines indicate the restriction sites for the enzymes shown in parentheses): ERK-5′-GCA GGAGCTCCG ATG TAC CCA TAC GAT GTT CCA (SacI); ERK-3′ GCA TGAATTCTC GGG GCC TCT GGT GCC CCT GG (EcoRI). The purified PCR fragment was ligated via theSacI-EcoRI restriction site into pGFP-N1 (CLONTECH, BD PharMingen) linking the HA-ERK in-frame to the amino terminus of the GFP protein. The sources for the other plasmids employed has been listed previously (14Seidel M.G. Klinger M. Freissmuth M. Holler C. J. Biol. Chem. 1999; 274: 25833-25841Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). CHO and HEK293 cells were propagated and transfected as outlined earlier (14Seidel M.G. Klinger M. Freissmuth M. Holler C. J. Biol. Chem. 1999; 274: 25833-25841Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). SYF and SYF + c-SRC cells were maintained in Dulbecco’s modified Eagle’s medium at 5% CO2 and 37 °C supplemented with 10% fetal calf serum, 2 mml-glutamine, β-mercaptoethanol, non-essential amino acids, 100 units/ml penicillin G, and 100 μg/ml streptomycin. PC12 cells were plated onto collagen-coated culture dishes and propagated in Opti-MEM medium containing 10% horse serum, 5% fetal calf serum, 2 mml-glutamine, 100 units/ml penicillin G, and 100 μg/ml streptomycin. For co-culture of CHO or CHO-A2A with reporter CHO cells, expressing reporter MAP kinase, CHO cells were transiently transfected with a plasmid expressing HA-tagged reporter MAP kinase. After 24 h, the transfected reporter CHO cells were seeded with CHO-A2A or control CHO cells (ratio 1:1) and allowed to adhere for 12 h. Thereafter, the cells were rendered quiescent by withdrawing serum for 12 h, and MAP kinase assays were subsequently performed as described below. Confluent cell layers (in 6-cm dishes) were rendered quiescent by serum starvation for 12–24 h. The starving medium was supplemented with 1 unit/ml adenosine deaminase to remove any endogenously produced adenosine; 30 min prior to MAP kinase assays the medium was again changed against prewarmed (37 °C) medium in order to minimize basal activity. If not otherwise indicated, cells were subsequently stimulated by addition of medium containing or lacking agonists and maintained at 37 °C for 5 min. Control incubations were carried out in order to verify that the carry-over of dimethyl sulfoxide, which resulted in final concentrations of ≤0.1%, neither affected the basal levels of MAP kinase phosphorylation nor the response to agonists. The exposure to agonists or vehicle was terminated by rapidly rinsing with ice-cold phosphate-buffered saline; thereafter, the dish was immediately immersed in liquid nitrogen; after rapid thawing, cells were lysed by addition of 80 μl of lysis buffer (in mm: 50 Tris, 40 β-glycerophosphate, 100 NaCl, 10 EDTA, 10 p-nitrophenol phosphate, 1 PMSF, 1 Na3VO4, 10 NaF, pH adjusted to 7.4 with HCl), 1% Nonidet P-40, 0.1% SDS, 250 units/ml aprotinin, 40 μg/μl leupeptin. The cellular debris was removed by centrifugation at 10,000 × g for 10 min, and the total protein content was measured photometrically using bicinchoninic acid (Micro-BCA kit, Pierce). Aliquots corresponding to 2.5–5·104 cells (10–30 μg of protein) were dissolved in Laemmli sample buffer containing 30 mm dithiothreitol and applied to SDS-polyacrylamide gels (monomer concentration 10–15% acrylamide, 0.26–0.4% bisacrylamide). MAP kinase phosphorylation was assayed by incubating nitrocellulose blots with an antiserum that recognizes only the dually phosphorylated forms of p42 and p44 MAP kinase; in order to rule out that the differences observed were due to the application of unequal amounts of lysates, control blots were also probed with an antiserum recognizing both the unphosphorylated (inactive) and phosphorylated (active form). The immunoreactive bands were visualized by enhanced chemiluminescence using horseradish peroxidase-linked secondary antibodies. Immunodetection of the other proteins was performed in an analogous manner, using the appropriate antibodies or antisera. In several instances the reporter MAP kinase was first immunoprecipitated using the monoclonal 12CA5 antibody directed against the HA epitope and protein G-Sepharose that had been pre-equilibrated in lysis buffer; HA-tagged or HA-GFP-tagged ERK1 were used interchangeably with equivalent results. Co-immunoprecipitations of HA-tagged RAP1 with B-RAF were done in a similar manner. GST fusion protein of the minimal RAP1-binding domain of RalGDS (ral-RBD, see Ref. 15Franke B. Akkerman J.W. Bos J.L. EMBO J. 1997; 16: 252-259Crossref PubMed Scopus (362) Google Scholar) were expressed in Escherichia coli (strain BL21DE3); following induction by isopropyl-1-thio-β-d-galactopyranoside, bacterial lysates were prepared as described. GST fusion proteins were immobilized by incubating bacterial lysates for 1 h at 4 °C with GSH-Sepharose pre-equilibrated in RIPA buffer (50 mm Tris·HCl, pH 7.5, 150 mm NaCl, 0.5% deoxycholate, 1% Nonidet P-40, 0.1% SDS) supplemented with 2 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μm PMSF. The Sepharose beads were washed 3 times in order to remove excess GST fusion protein. Cells were prepared for the assay in a similar way as outlined above for MAP kinase assays if not otherwise stated; incubation with agonists was carried out for 5 min followed by rapidly rinsing with ice-cold phosphate-buffered saline and addition of RIPA buffer to achieve cell lysis. Cell lysates were cleared by centrifugation (20,000 × g for 1 min). The resulting supernatants were incubated together with the GSH-Sepharose beads (50 μl of a 1:1 slurry containing about 10 μg of immobilized GST fusion protein) for 1 h to allow for the association of activated RAP1 with the RalGDS-GST fusion protein. Samples were washed 3 times in RIPA buffer, resuspended in Laemmli sample buffer, and applied to SDS-polyacrylamide gels; RAP1 was visualized using specific antibodies in a dilution of 1:250. If HA-tagged RAP1 was co-transfected, the assay was carried out in a similar way as described except that the transfection procedure preceded the assay and that the immunoblot was done with the 16B12 monoclonal antibody directed against HA tag sequence. Each experiment was at least carried out three times. Receptor-dependent stimulation of MAP kinase can be transient (i.e. a monophasic activation that fades after a few minutes) or result in sustained activation. If RAP1 were important for regulation of MAP kinase by the A2A-adenosine receptor, the kinetics of activation should be reasonably similar. To test this prediction, the time course of MAP kinase activation (Fig.1A) and of RAP1 activation (Fig. 1B) was determined in CHO cells that heterologously expressed the human A2A-adenosine receptor (CHO-A2A cells). Addition of the A2A-selective agonist CGS21680 resulted in biphasic stimulation of MAP kinase phosphorylation. An early increment in the phosphorylation of the endogenous p42 and p44 (ERK2 and ERK1) isoforms of MAP kinase was seen at 5–10 min and was followed by a sustained phase of activation after 60–90 min (Fig. 1A). It is evident from Fig. 1B(left panel) that GTP-liganded active RAP1 was also formed with biphasic kinetics. We also verified by co-immunoprecipitation that receptor-dependent activation of RAP1 (by CGS21680 or by thrombin as a positive control) resulted in formation of a complex between of RAP1 with its putative effector B-RAF (Fig. 1B, right panel). RAP1 is under the control of cAMP-dependent exchange factors (7de Rooij J. Zwartkruis F.J. Verheijen M.H. Cool R.H. Nijman S.M. Wittinghofer A. Bos J.L. Nature. 1998; 396: 474-477Crossref PubMed Scopus (1591) Google Scholar, 8Kawasaki H. Springett G.M. Mochizuki N. Toki S. Nakaya M. Matsuda M. Housman D.E. Graybiel A.M. Science. 1998; 282: 2275-2279Crossref PubMed Scopus (1155) Google Scholar). Thus, protein kinase A should be dispensable for both activation of RAP1 and stimulation of MAP kinase. This was clearly not the case. Formation of GTP-bound RAP1 was insensitive to the PKA-inhibitor H89 (Fig. 1C, lower panel); in contrast, activation of MAP kinase was suppressed by H89 (Fig.1C, upper panel). In order to directly activate cAMP-dependent effectors, we also performed a similar set of experiments with the membrane-permeable cAMP analogue 8-Br-cAMP; the results were equivalent to those shown in Fig. 1, A—C, for the A2A-agonist CGS21680 (data not shown). The time course of RAP1 activation and the kinetics of MAP kinase phosphorylation were compatible with a cause and effect relationship. Similarly, the fact that following stimulation by an A2A-agonist, RAP1 associated with B-RAF, which is a MAP kinase kinase kinase and thus, by definition, upstream of ERK1/2. These observations clearly argued for a role of RAP1 in mediating the stimulatory effect of the A2A-receptor on MAP kinase. However, the protein kinase A inhibitor H89 discriminated between stimulation of MAP kinase phosphorylation and GTP loading of RAP1. This indicated a requirement for protein kinase A rather than for an exchange factor of the Epac family. It has been argued that protein kinase A acted upstream of RAP1 activation, possibly on the RAP1 exchange factor C3G (16Schmitt J.M. Stork P.J. J. Biol. Chem. 2000; 275: 25342-25350Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). In order to address this discrepancy, we employed RAP1GAP, a member of the GAP family that regulates RAP1 (17Polakis P. Rubinfeld B. McCormick F. J. Biol. Chem. 1992; 267: 10780-10785Abstract Full Text PDF PubMed Google Scholar). The intrinsic GTPase activity of small G proteins is very low, and it is, in most cases, accelerated by a GTPase-activating protein (GAP) that provides one or more residues required for catalysis. Thus, GAPs switch off the active, GTP-bound state. We transiently expressed an HA-tagged version of RAP1GAP together with epitope versions of reporter RAP1 (Fig.2A) and ERK1 (Fig.2B). Overexpression of HA-tagged RAP1GAP efficiently prevented the accumulation of GTP-bound RAP1 after stimulation of CHO-A2A cells by the A2A-agonist (Fig.2A, top row). Cells that expressed HA-RAP1GAP (middle row in Fig.2A) synthesized less HA-RAP1 (Fig. 2A, bottom row). However, GTP-bound RAP1 was below the detection limit, even if HA-RAP1GAP-transfected cells had been stimulated by the A2A-agonist. In contrast, GTP-loaded RAP1A was detected under basal conditions in vector-transfected control cells; it is evident from a comparison of the level of total (bottom rowin Fig. 2A) and GTP-bound RAP1 (top row, Fig.2A) that the lower level of reporter HA-RAP1 in HA-RAP1GAP-transfected cells clearly still would have sufficed to allow for the detection of receptor-dependent activation. Although the accumulation of GTP-bound RAP1 was completely abrogated by the overexpression of RAP1GAP, it did not have any appreciable effect on the phosphorylation of a co-transfected reporter MAP kinase in response to stimulation by CGS21680 (Fig. 2B, cf. lanes C in vector and HA-RAP1GAP-transfected cells). Similar observations were also made if cells were stimulated with the membrane-permeable cAMP analogue 8-Br-cAMP (Fig. 2B, lanes labeled8Br). These results were difficult to reconcile with published data, namely that RAP1GAP attenuated MAP kinase activation by the β2-adrenergic receptor (16Schmitt J.M. Stork P.J. J. Biol. Chem. 2000; 275: 25342-25350Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). We therefore also transiently co-expressed the β2-adrenergic receptor in CHO-A2A cells together with the reporter MAP kinase in the absence and presence of HA-RAP1-GAP. By contrast with MAP kinase stimulation with the A2A-agonist (and 8-Br-cAMP), RAP1GAP did diminish isoproterenol-stimulated phosphorylation of the reporter MAP kinase construct (Fig. 2C, cf. lanes labeledI and C for isoproterenol and CGS21680, respectively). Finally, we ruled out that any differences observed are due to a variation in the amount of reporter MAP kinase in the immunoprecipitates because comparable levels were detected with an antiserum that recognizes holo-ERK1/2 (bottom rows in Fig.2, B and C). Activation of MAP kinase by β2-adrenoceptors is dependent on receptor internalization (18Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar). In order to test if this was also true for the A2A-receptor, CHO-A2A cells were transiently co-transfected with dynamin K44A, a dominant negative suppressor of dynamin-dependent formation of endocytotic vesicles (19Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1029) Google Scholar), the reporter MAP kinase, and the β2-adrenergic receptor. As a control, we employed the plasmid encoding wild type dynamin (which did not alter the response to the agonists CGS21680 and isoproterenol, not shown). Fig.3A shows that the response to the β2-agonist isoproterenol was blunted in cells that expressed dynamin K44A; in contrast, dynamin K44A did not have any appreciable effect on the stimulation of reporter MAP kinase by the A2A-adenosine receptor (Fig. 3A, lanes C). Two additional manipulations highlighted the fundamental difference between activation of ERK1/2 by the β2-adrenergic receptor and A2A-adenosine receptor; neither overexpression of the carboxyl terminus of the β-adrenergic receptor kinase nor of phosducin, which both act as scavengers for free βγ, impaired A2A-receptor-stimulated MAP kinase activation (data not shown). In contrast, receptor-generated Gβγ is important for MAP kinase activation by the β2-adrenergic receptor (20Daaka Y. Luttrell L.M. Lefkowitz R.J. Nature. 1997; 390: 88-91Crossref PubMed Scopus (1052) Google Scholar). Taken together the observations indicated that there was a fundamental difference in the mechanism by which the A2A-adenosine receptor and the β2-adrenergic receptor impinged on MAP kinase. We have therefore explored if the A2A-adenosine receptor signaled to MAP kinase via transactivation. Originally, receptors with tyrosine kinase activity were proposed to act as scaffolds for the assembly of signaling complexes and to thereby support MAP kinase activation by G protein-coupled receptors, and this was referred to as transactivation; more recently, the emphasis has shifted to the ability of G protein-coupled receptors to promote the release of cell surface-bound growth factors, e.g.heparin-binding EGF, via activation of a matrix metalloprotease (11Prenzel N. Zwick E. Daub H. Leserer M. Abraham R. Wallasch C. Ullrich A. Nature. 1999; 402: 884-888Crossref PubMed Scopus (1481) Google Scholar). By definition, this type of stimulation is paracrine stimulation; we have therefore used a co-culture system that was analogous to the one originally employed by Prenzel et al. (11; see also ref.21Pierce K.L. Tohgo A. Ahn S. Field M.E. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 2001; 276: 23155-23160Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar); CHO-A2A or control CHO cells were mixed at 1:1 ratio with reporter CHO cells that harbored an epitope-tagged MAP kinase and seeded at high density. Growth factor release and the ensuing paracrine stimulation ought to be detected as A2A-receptor-dependent stimulation of MAP kinase in the reporter cells. This was clearly not observed; on the contrary, the reporter MAP kinase was phosphorylated to a negligible extent irrespective of whether the co-culture contained cells endowed with or lacking the A2A-adenosine receptor (Fig.3B, top row). In contrast, we readily detected the phosphorylation of endogenous MAP kinase isoforms in the cellular lysates if these were prepared from co-cultures containing CHO-A2A cells; as expected, MAP kinase was irresponsive to the A2A-agonist in the control co-cultures that lacked A2A-receptor-bearing cells (Fig. 3B, bottom row). We therefore conclude that the A2A-adenosine receptor relies on components that are intrinsically present within the stimulated cell to activate MAP kinase and that there is no evidence for the involvement of an additional, extracellular signal. The observations presented so far suggested that cAMP-dependent activation of PKA played an essential role in linking MAP kinase to the A2A-adenosine receptor. However, Gαs may have effectors other than adenylyl cyclase isoforms; the non-receptor tyrosine kinase SRC has, in particular, been reported as direct target of Gαs (9Ma Y.C. Huang J. Ali S. Lowry W. Huang X.Y. Cell. 2000; 102: 635-646Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar). If the signaling pathway A2A-adenosine receptor/Gs/SRC was upstream of MAP kinase activation, inhibition of SRC (or related kinases) ought to discriminate between receptor- and cAMP-dependent MAP kinase stimulation. We therefore tested incubated CHO-A2A cell in the presence of 1 μm PP1, an inhibitor of SRC family kinases. As can be seen in Fig. 4A, preincubation with PP1 specifically abrogated the ability of the A2A-agonist CGS21680 (lanes C) and of forskolin (lanes F) to stimulate MAP kinase phosphorylation. In control, PP1 did not blunt the effect of basic fibroblast growth factor (lanes FGF in" @default.
• W2079238835 created "2016-06-24" @default.
• W2079238835 creator A5027980182 @default.
• W2079238835 creator A5050428979 @default.
• W2079238835 creator A5060360807 @default.
• W2079238835 creator A5073132442 @default.
• W2079238835 creator A5079620940 @default.
• W2079238835 date "2002-09-01" @default.
• W2079238835 modified "2023-10-14" @default.
• W2079238835 title "MAP Kinase Stimulation by cAMP Does Not Require RAP1 but SRC Family Kinases" @default.
• W2079238835 cites W1497642271 @default.
• W2079238835 cites W1516555591 @default.
• W2079238835 cites W1525183361 @default.
• W2079238835 cites W1533171633 @default.
• W2079238835 cites W1573823819 @default.
• W2079238835 cites W1769585641 @default.
• W2079238835 cites W1885709862 @default.
• W2079238835 cites W1963817582 @default.
• W2079238835 cites W1967861082 @default.
• W2079238835 cites W1967965496 @default.
• W2079238835 cites W1968625794 @default.
• W2079238835 cites W1976849281 @default.
• W2079238835 cites W1976874658 @default.
• W2079238835 cites W1984362855 @default.
• W2079238835 cites W1993876642 @default.
• W2079238835 cites W1994816216 @default.
• W2079238835 cites W2004169102 @default.
• W2079238835 cites W2010767308 @default.
• W2079238835 cites W2012415301 @default.
• W2079238835 cites W2014211428 @default.
• W2079238835 cites W2024068070 @default.
• W2079238835 cites W2032817955 @default.
• W2079238835 cites W2041687747 @default.
• W2079238835 cites W2045876058 @default.
• W2079238835 cites W2046467234 @default.
• W2079238835 cites W2047057752 @default.
• W2079238835 cites W2051152907 @default.
• W2079238835 cites W2064973618 @default.
• W2079238835 cites W2070564840 @default.
• W2079238835 cites W2073039482 @default.
• W2079238835 cites W2078037416 @default.
• W2079238835 cites W2078721878 @default.
• W2079238835 cites W2082889823 @default.
• W2079238835 cites W2083220635 @default.
• W2079238835 cites W2090429239 @default.
• W2079238835 cites W2101257013 @default.
• W2079238835 cites W2118399632 @default.
• W2079238835 cites W2157776145 @default.
• W2079238835 cites W2158615338 @default.
• W2079238835 cites W4232995978 @default.
• W2079238835 cites W4297584855 @default.
• W2079238835 doi "https://doi.org/10.1074/jbc.m200556200" @default.
• W2079238835 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12082090" @default.
• W2079238835 hasPublicationYear "2002" @default.
• W2079238835 type Work @default.
• W2079238835 sameAs 2079238835 @default.
• W2079238835 citedByCount "74" @default.
• W2079238835 countsByYear W20792388352012 @default.
• W2079238835 countsByYear W20792388352013 @default.
• W2079238835 countsByYear W20792388352014 @default.
• W2079238835 countsByYear W20792388352015 @default.
• W2079238835 countsByYear W20792388352016 @default.
• W2079238835 countsByYear W20792388352017 @default.
• W2079238835 countsByYear W20792388352021 @default.
• W2079238835 countsByYear W20792388352022 @default.
• W2079238835 crossrefType "journal-article" @default.
• W2079238835 hasAuthorship W2079238835A5027980182 @default.
• W2079238835 hasAuthorship W2079238835A5050428979 @default.
• W2079238835 hasAuthorship W2079238835A5060360807 @default.
• W2079238835 hasAuthorship W2079238835A5073132442 @default.
• W2079238835 hasAuthorship W2079238835A5079620940 @default.
• W2079238835 hasBestOaLocation W20792388351 @default.
• W2079238835 hasConcept C108636557 @default.
• W2079238835 hasConcept C169760540 @default.
• W2079238835 hasConcept C183141693 @default.
• W2079238835 hasConcept C184235292 @default.
• W2079238835 hasConcept C185592680 @default.
• W2079238835 hasConcept C196347352 @default.
• W2079238835 hasConcept C24998067 @default.
• W2079238835 hasConcept C2776179587 @default.
• W2079238835 hasConcept C2779976819 @default.
• W2079238835 hasConcept C62478195 @default.
• W2079238835 hasConcept C86803240 @default.
• W2079238835 hasConcept C95444343 @default.
• W2079238835 hasConceptScore W2079238835C108636557 @default.
• W2079238835 hasConceptScore W2079238835C169760540 @default.
• W2079238835 hasConceptScore W2079238835C183141693 @default.
• W2079238835 hasConceptScore W2079238835C184235292 @default.
• W2079238835 hasConceptScore W2079238835C185592680 @default.
• W2079238835 hasConceptScore W2079238835C196347352 @default.
• W2079238835 hasConceptScore W2079238835C24998067 @default.
• W2079238835 hasConceptScore W2079238835C2776179587 @default.
• W2079238835 hasConceptScore W2079238835C2779976819 @default.
• W2079238835 hasConceptScore W2079238835C62478195 @default.
• W2079238835 hasConceptScore W2079238835C86803240 @default.
• W2079238835 hasConceptScore W2079238835C95444343 @default.
• W2079238835 hasIssue "36" @default.
• W2079238835 hasLocation W20792388351 @default.

• next