MAP kinase stimulation by cAMP does not require RAP1 but SRC family kinases.
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 A(2A)-adenosine receptor, a prototypical G(s)-coupled receptor, was heterologously expressed in Chinese hamster ovary cells (referred as CHO-A(2A) cells). In CHO-A(2A) cells, the stimulation of the A(2A)-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 A(2A)-agonist-mediated MAP kinase stimulation. In contrast, the inhibitor of protein kinase A H89 efficiently suppressed A(2A)-agonist-mediated MAP kinase stimulation. Neither dynamin-dependent receptor internalization nor receptor-promoted shedding of matrix-bound growth factors accounted for A(2A)-receptor-dependent MAP kinase activation. PP1, an inhibitor of SRC family kinases, blunted both the A(2A)-receptor- and the forskolin-induced MAP kinase stimulation (IC(50) = 50 nm); this was also seen in PC12 cells, which express the A(2A)-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 beta(2)-adrenergic receptor, is apparently contingent on receptor endocytosis.
Klinger
Markus
M
Institute of Pharmacology, University of Vienna, Währinger Strasse 13a, A-1090 Vienna, Austria.
Kudlacek
Oliver
O
Seidel
Markus G
MG
Freissmuth
Michael
M
Sexl
Veronika
V
D016475:3T3 Cells; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D034281:Dynamins; D004705:Endocytosis; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000378:metabolism; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D020128:Inhibitory Concentration 50; D007545:Isoproterenol / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D020935:MAP Kinase Signaling System; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016716:PC12 Cells; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D051381:Rats; D013449:Sulfonamides; D013997:Time Factors; D015533:Transcriptional Activation; D014162:Transfection; D020827:rap1 GTP-Binding Proteins / Q000502:physiology*; D019061:src-Family Kinases / Q000502:physiology*
D004791:Enzyme Inhibitors; D020690:GTPase-Activating Proteins; D007546:Isoquinolines; C484658:RAP1GAP protein, human; D013449:Sulfonamides; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000242:Cyclic AMP; D005982:Glutathione Transferase; D019061:src-Family Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D020558:GTP Phosphohydrolases; D020827:rap1 GTP-Binding Proteins; D034281:Dynamins; D007545:Isoproterenol; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide