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Raf-1:ER* Bypasses the Cyclic AMP Block of Extracellular Signal-Regulated Kinase 1 and 2 Activation but Not CDK2 Activation or Cell Cycle Reentry

Signalling Programme, The Babraham Institute, Babraham Hall, Cambridge, CB2 4AT, England,¹ Cancer Research Institute and Department of Cellular and Molecular Pharmacology, University of California—San Francisco, San Francisco, California 94115²

Received 8 September 2003/ Accepted 11 September 2003

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 Raf-1:ER is insensitive to cAMP. Despite this, Raf-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 Raf-1:ER-induced cyclin D1 expression only in Rat-1 cells, not in CCl39 or NIH 3T3 cells. Raf-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 Raf-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 Raf-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 G₁, but when this early block is bypassed by Raf-1:ER, cells still fail to enter S phase due to inhibition of CDK2 or targets downstream of E2F1.

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