DeltaRaf-1:ER* bypasses the cyclic AMP block of extracellular signal-regulated kinase 1 and 2 activation but not CDK2 activation or cell cycle reentry.
Elevation of cellular cyclic AMP (cAMP) levels inhibits cell cycle reentry in a variety of cell types. While cAMP can prevent the activation of Raf-1 and extracellular signal-regulated kinases 1 and 2 (ERK1/2) by growth factors, we now show that activation of ERK1/2 by DeltaRaf-1:ER is insensitive to cAMP. Despite this, DeltaRaf-1:ER-stimulated DNA synthesis is still inhibited by cAMP, indicating a cAMP-sensitive step downstream of ERK1/2. Although cyclin D1 expression has been proposed as an alternative target for cAMP, we found that cAMP could inhibit DeltaRaf-1:ER-induced cyclin D1 expression only in Rat-1 cells, not in CCl39 or NIH 3T3 cells. DeltaRaf-1:ER-stimulated activation of CDK2 was strongly inhibited by cAMP in all three cell lines, but cAMP had no effect on the induction of p21(CIP1). cAMP blocked the fetal bovine serum (FBS)-induced degradation of p27(KIP1); however, loss of p27(KIP1) in response to DeltaRaf-1:ER was less sensitive in CCl39 and Rat-1 cells and was completely independent of cAMP in NIH 3T3 cells. The most consistent effect of cAMP was to block both FBS- and DeltaRaf-1:ER-induced expression of Cdc25A and cyclin A, two important activators of CDK2. When CDK2 activity was bypassed by activation of the ER-E2F1 fusion protein, cAMP no longer inhibited expression of Cdc25A or cyclin A but still inhibited DNA synthesis. These studies reveal multiple points of cAMP sensitivity during cell cycle reentry. Inhibition of Raf-1 and ERK1/2 activation may operate early in G(1), but when this early block is bypassed by DeltaRaf-1:ER, cells still fail to enter S phase due to inhibition of CDK2 or targets downstream of E2F1.
Balmanno
Kathryn
K
Signalling Programme, The Babraham Institute, Babraham Hall, Cambridge CB2 4AT, England, UK.
Millar
Tracy
T
McMahon
Martin
M
Cook
Simon J
SJ
D000818:Animals; D042846:CDC2-CDC28 Kinases / Q000378:metabolism*; D002453:Cell Cycle; D002460:Cell Line; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology*; D019925:Cyclin A / Q000378:metabolism; D019938:Cyclin D1 / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D004247:DNA / Q000096:biosynthesis; D004789:Enzyme Activation / Q000187:drug effects; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008954:Models, Biological; D041681:NIH 3T3 Cells; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D018808:Transcription Factor AP-1 / Q000378:metabolism; D020687:cdc25 Phosphatases / Q000378:metabolism
D019925:Cyclin A; D011993:Recombinant Fusion Proteins; D018808:Transcription Factor AP-1; D019938:Cyclin D1; D004247:DNA; D000242:Cyclic AMP; D019908:Proto-Oncogene Proteins c-raf; D042846:CDC2-CDC28 Kinases; C495893:Cdk2 protein, mouse; C495892:Cdk2 protein, rat; D051357:Cyclin-Dependent Kinase 2; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; C517005:Cdc25a protein, mouse; C517007:Cdc25a protein, rat; D020687:cdc25 Phosphatases