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

Abstract

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.

FIG. 1.

cAMP does not inhibit ΔRaf-1:ER in RR1 cells. Quiescent, serum-starved RR1 cells expressing ΔRaf-1:ER were treated with 100 or 500 μM IBMX or vehicle control (solid bars) for 30 min prior to stimulation with E₂ for 1 h. (A) Activation of ΔRaf-1:ER, assayed by its ability to phosphorylate catalytically inactive MEK1, was monitored after immunoprecipitation with anti-estrogen receptor antibodies as described in Materials and Methods. (B) The same lysates as in panel A were also used to immunoprecipitate endogenous ERK1, which was assayed by its ability to phosphorylate myelin basic protein. In both cases, the results are means plus ranges from duplicate cell cultures taken from a single experiment. Similar results were obtained in two other experiments. PI, phosphorimager.

FIG. 2.

cAMP does not inhibit ΔRaf-1:ER-mediated ERK1 activation in RR1, CR1-11, or NIH 3T3/ΔRaf-1:ER cells. All experiments were performed on quiescent, serum-starved cells. (A) RR1 cells were pretreated with increasing concentrations of IBMX for 30 min prior to stimulation with EGF or E₂(left) or treated with 1 μM E2 in the absence (solid bars) or presence (hatched bars) of 1 mM db-cAMP (right). ERK1 activity was determined by immune complex kinase assay, and the results are from a single representative experiment. (B) CR1-11 cells were treated with control vehicle (4-HT) or 1 mM db-cAMP (4-HT+cAMP) for 5 min before stimulation with increasing concentrations of 4-HT as indicated for 20 h. ERK1 activity was determined by immune complex kinase assay and quantified on a phosphorimager. The results are expressed as phosphorimager (PI) units and are taken from a single experiment representative of three that had similar results. For reference, the same lysates were used to assay CDK2 activity, which was completely inhibited (Balmanno and Cook, unpublished). (C) NIH 3T3/ΔRaf-1:ER cells were treated with 1 mM db-cAMP (cAMP) or vehicle control (Con) for 30 min prior to being stimulated for 2 h with 10 or 100 nM 4-HT. Activation of ERK1 was determined by immune complex kinase assay. The data are means plus ranges of duplicate cell dishes and are taken from a single experiment representative of three.

FIG. 3.

cAMP fails to inhibit the expression of AP-1 proteins by ΔRaf-1:ER. All experiments were performed on quiescent serum-starved cells. (A) RR1 cells were stimulated with 100 μM lysophosphatidic acid (LPA) in the absence or presence of 1 mM db-cAMP for the indicated times. Phosphorylation of ERK1 and ERK2 (P-ERK) and expression of c-Fos, Fra-1, and JunB were assayed by performing Western immunoblotting on whole-cell lysates using the appropriate antibodies. (B and C) RR1 cells (B) and CR1-11 cells (C) were stimulated with 100 nM 4-HT in the absence or presence of 1 mM db-cAMP for the indicated times. Expression of Fra-1 and JunB was assayed by performing Western immunoblotting on whole-cell lysates using the appropriate antibodies. (D) NIH 3T3/ΔRaf-1:ER cells were stimulated with 1, 10, or 100 nM 4-HT in the absence or presence of 1 mM db-cAMP for 5 h. Expression of Fra-1, Fra-2, and JunB was assayed by performing Western immunoblotting on whole-cell lysates using the appropriate antibodies. In all cases, the results are from single experiments representative of between three and seven others that gave similar results.

FIG. 4.

cAMP can still inhibit ΔRaf-1:ER-stimulated DNA synthesis. (A) NIH 3T3/ΔRaf-1:ER cells were stimulated with increasing concentrations of 4-HT in the absence (4-HT) or presence (4-HT+cAMP) of 1 mM db-cAMP, and cell cycle reentry was assayed by [³H]thymidine incorporation ([3H]Thy Inc) (left), or the cells were stimulated with 10 nM 4-HT or 10% (vol/vol) FBS in the absence (Con) or presence (cAMP) of 1 mM db-cAMP, and cell cycle reentry was assayed by [³H]thymidine incorporation (right). The results are means ± ranges of duplicate cell cultures. (B) CR1-11 cells were pretreated for 30 min with increasing concentrations of IBMX and then stimulated for 24 h with either 100 nM thrombin (Thr) or 100 nM 4-HT. Cell cycle reentry was assayed by [³H]thymidine incorporation, and the results are the means ± ranges of duplicate cell cultures. (C) RR1 cells were pretreated with 1 mM db-cAMP or vehicle control and then stimulated for 20 h with either 10% (vol/vol) FBS (left) or 100 nM 4-HT (right). Cell cycle reentry was assayed by [³H]thymidine incorporation, and the results are the means ± ranges of duplicate cell cultures. SF, serum free; cA, cAMP. In all cases, the results are from a single experiment representative of three that had the same result.

FIG. 5.

Effects of cAMP on activation of CDK2 and expression of cyclin D1, cyclin A, Cdc25A, and CDKIs in CR1-11 cells. Quiescent CR1-11 cells were stimulated for the indicated times with 10% (vol/vol) FBS (A and B) or 100 nM 4-HT (C and D) in the absence (solid symbols) or presence (open symbols) of 1 mM db-cAMP. Whole-cell lysates were used to assay CDK2 activity, quantified as phosphorimager (PI) units (A and C), or were resolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with antibodies specific for cyclin (cyc) D1, phospho (P)-Rb-Ser⁷⁹⁵, p27^KIP1, p21^CIP1, cyclin A, Cdc25A, phospho-CDK2-Tyr¹⁵, and CDK2 (B and D). Phospho-CDK2-Tyr¹⁵ phosphorylation was assessed by immunoblotting following immunoprecipitation of CDK2. In all cases, the results are from a single experiment representative of three that had similar results.

FIG. 6.

Effects of cAMP on activation of CDK2 and expression of cyclin D1, cyclin A, Cdc25A, and CDKIs in RR1 cells. Quiescent RR1 cells were stimulated for the indicated times with 10% (vol/vol) FBS (A and B) or 100 nM 4-HT (C and D) in the absence (▪) or presence (□) of 1 mM db-cAMP. Whole-cell lysates were used to assay CDK2 activity, quantified as phosphorimager (PI) units (A and C), or were resolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with antibodies specific for cyclin (cyc) D1, p27^KIP1, cyclin A, Cdc25A, phospho (P)-CDK2-Tyr¹⁵, and CDK2 (B and D). Phospho-CDK2-Tyr¹⁵ phosphorylation was assessed by immunoblotting following immunoprecipitation of CDK2. The results are from a single experiment representative of three that had similar results.

FIG. 7.

Effects of cAMP on activation of CDK2 and expression of cyclin D1, cyclin A, Cdc25A, and CDKIs in NIH 3T3/ΔRaf-1:ER cells. Quiescent NIH 3T3/ΔRaf-1:ER cells were stimulated with increasing concentrations of 4-HT in the absence (4-HT) or presence (4-HT+cAMP) of 1 mM db-cAMP. Whole-cell lysates were used to assay CDK2 activity, quantified as phosphorimager (PI) units (A), or were resolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with antibodies specific for cyclin (cyc) D1, p21^CIP1, or p27^KIP1 (B) or Cdc25A, cyclin E, cyclin A, and CDK2 (C). The results are taken from a single experiment representative of three that had similar results.

FIG. 8.

Ectopic expression of Cdc25A fails to overcome cAMP growth arrest. (A) CR1-11 cells were transfected with pCMVNeo-HA-Cdc25A, and positive clones were identified by Western immunoblotting with anti-HA antibody (WB:HA). The expression of HA-Cdc25A is shown in one of the positive clones (lane 2) but not in the control CR1-11/Neo cells (lane 1). The asterisk indicates the position of proteolytic breakdown products of the full-length HA-CDc25A. (B and C) Duplicate dishes of CR1-11/Neo or CR1-11/Cdc25A cells were serum starved and then restimulated with 4-HT in the presence or absence of 0.5 mM db-cAMP for 24 h. The cells were assayed for CDK2 activity (B) or [³H]thymidine incorporation ([³H]Thy Inc) (C), and the data are shown as means plus ranges of duplicate dishes from a single experiment representative of three. PI, phosphorimager; cA, cAMP.

FIG. 9.

Activation of ER-E2F1 overcomes the cAMP block of Cdc25A and cyclin A expression but not inhibition of DNA synthesis. (A) Rat-1 cells were infected with pBabePuro HA-ER-E2F1 retrovirus, and positive clones were detected by Western immunoblotting with anti-HA antibodies (WB:HA). The blot shows the expression of HA-ER-E2F1 in one representative positive clone (lane 2) compared to Rat-1 Puro control cells (lane 1). (B) R1-ER-E2F1 cells were treated with 100 nM 4-HT for the indicated times in the absence or presence of 0.5 mM db-cAMP, and whole-cell lysates were assayed for expression of Cdc25A and cyclin A (cyc A) by Western immunoblotting. The asterisk indicates the position of a nonspecific reactive band which served as a useful loading control. (C and D) R1-ER-E2F1 cells were serum starved and then stimulated with 10% FBS (C) or 100 nM 4-HT (D) for 24 h in the absence (solid bars) or presence (hatched bars) of 0.5 mM db-cAMP. The cells were assayed for [³H]thymidine incorporation ([³H]Thy Inc), and the data are means plus ranges of duplicate dishes from a single representative experiment. cA, cAMP.

FIG. 10.

Model depicting sites of cAMP sensitivity during cell cycle reentry. (A) cAMP can promote phosphorylation of Raf-1 at S⁴³, S²³³, and S²⁵⁹ (indicated by PPP), thereby blocking activation of Raf-1 by growth factors and preventing ERK1/2 activation. As a result, most ERK1/2-dependent events downstream of Raf-1 will be blocked by cAMP (indicated by shading). (B) ΔRaf-1:ER is insensitive to cAMP. As a result, many ERK1/2-dependent events downstream, such as expression of AP-1 proteins and cyclin (cyc) D1, are no longer blocked by cAMP. However, cAMP can still prevent the activation of CDK2 by inhibiting the expression of cyclin A, Cdc25A, and perhaps p27^KIP1 (indicated by shading). The extent to which the early cAMP block at Raf-1 (A) operates will depend on the expression of cAMP-insensitive Raf isoforms, such as B-Raf. In such cases, where cAMP fails to inhibit ERK1/2 activation, the later checkpoint at CDK2 may be more important for cAMP-mediated growth arrest. (C) Once CDK2 has been activated, cAMP can still inhibit E2F1-stimulated DNA synthesis, presumably by inhibiting the expression of select E2F1 target genes (indicated by shading), but not by inhibiting E2F1 per se, since Cdc25A and cyclin A expression proceed normally. For details, see Discussion.