Guanine nucleotide exchange factor Epac2-dependent activation of the GTP-binding protein Rap2A mediates cAMP-dependent growth arrest in neuroendocrine cells

Abstract

First messenger-dependent activation of MAP kinases in neuronal and endocrine cells is critical for cell differentiation and function and requires guanine nucleotide exchange factor (GEF)-mediated activation of downstream Ras family small GTPases, which ultimately lead to ERK, JNK, and p38 phosphorylation. Because there are numerous GEFs and also a host of Ras family small GTPases, it is important to know which specific GEF-small GTPase dyad functions in a given cellular process. Here we investigated the upstream activators and downstream effectors of signaling via the GEF Epac2 in the neuroendocrine NS-1 cell line. Three cAMP sensors, Epac2, PKA, and neuritogenic cAMP sensor-Rapgef2, mediate distinct cellular outputs: p38-dependent growth arrest, cAMP response element-binding protein-dependent cell survival, and ERK-dependent neuritogenesis, respectively, in these cells. Previously, we found that cAMP-induced growth arrest of PC12 and NS-1 cells requires Epac2-dependent activation of p38 MAP kinase, which posed the important question of how Epac2 engages p38 without simultaneously activating other MAP kinases in neuronal and endocrine cells. We now show that the small GTP-binding protein Rap2A is the obligate effector for, and GEF substrate of, Epac2 in mediating growth arrest through p38 activation in NS-1 cells. This new pathway is distinctly parcellated from the G protein-coupled receptor → G_s → adenylate cyclase → cAMP → PKA → cAMP response element-binding protein pathway mediating cell survival and the G protein-coupled receptor → G_s → adenylate cyclase → cAMP → neuritogenic cAMP sensor-Rapgef2 → B-Raf → MEK → ERK pathway mediating neuritogenesis in NS-1 cells.

Keywords: ERK; cAMP; guanine nucleotide exchange factor (GEF); p38; p38 MAPK; small GTPase.

Figure 1.

Differential farnesylation requirement for PACAP-initiated signaling to ERK and p38 MAP kinases. A, measurements of p38 phosphorylation by a phospho-specific antibody cell-based ELISA in NS-1 cells pretreated with 0.02% DMSO or 10 μm FTS-A, followed by treatment with either 100 nm PACAP-38 or 100 ng/ml NGF. B, measurements of phosphorylated ERK in NS-1 cells following identical treatments to those shown in A. Bars represent means from three experiments, and error bars correspond to standard deviations. Data were analyzed by two-way ANOVA and Bonferroni-corrected post hoc tests: ***, p < 0.001; **, p < 0.01; comparing the effects of each condition with its respective untreated control. Statistical significance of inhibitory effects of FTS-A on either PACAP or NGF are denoted by ##, p < 0.01.

Figure 2.

Epac-dependent p38 phosphorylation does not require farnesylation. A, representative Western blots of phosphorylated and total p38 in NS-1 cells pretreated for 30 min with vehicle (0.02% DMSO), FTS-A (10 μm), or ESI-09 (10 μm), followed by addition of 8-CPT-2′-O-Me-cAMP (100 μm) for 30 min. MW, molecular weight; IB, immunoblot. B, quantifications of three independent experiments. Bars represent means, and error bars correspond to the standard deviations. Data were analyzed by ANOVA and Bonferroni-corrected t tests. ***, p < 0.001; **, p < 0.01; comparing the effect of 8-CPT-2′-O-Me-cAMP with vehicle-treated controls.

Figure 3.

Rit1 is not involved in NS-1 cell growth arrest. A, NS-1 cells were transduced with a lentiviral shRNA targeting RIT1 mRNA (shRIT1) or a control construct expressing scrambled control shRNA (Ctl shRNA). After stable cell lines were established, Rit1 protein was compared in lysates from cells either expressing scrambled shRNA (lane 1) or shRNA targeting RIT1 mRNA (lane 2). Bottom panel, the membrane was stripped and reprobed for GAPDH to confirm equivalent protein loading. MW, molecular weight; IB, immunoblot. B, growth curves from NS-1 sublines expressing scrambled shRNA or RIT1 shRNA grown in 96-well plates and treated with 100 μm 8-CPT-2′-O-Me-cAMP for 5 days. C, representative images of NS-1 cells expressing scrambled shRNA or shRNA targeting RIT1 obtained after treatment for 5 days with 100 μm 8-CPT-2′-O-Me-cAMP or 100 μm 8-CPT-cAMP.

Figure 4.

Cyclic AMP/Epac-dependent p38 activation requires geranylgeranylation. A and B, NS-1 cells were pretreated with the farnesylation inhibitor FTS-A or the geranylgeranyltransferase I inhibitor GGTI-298, followed by stimulation with either 10 μm forskolin (A) or 100 μm 8-CPT-2′-O-Me-cAMP (B). p38 phosphorylation was determined by phospho-specific antibody cell-based ELISA. Data points are means from six determinations, with error bars corresponding to the standard errors of the mean.

Figure 5.

Activation of Rap2A requires geranylgeranylation. A, profiling of Rap2 isoform expression levels in three cultures of NS-1 cells using a Rap2A/B antibody. B, measurements of active Rap2 in NS-1 cells pretreated with 10 μm FTS-A for 30 min, followed by treatment with 100 μm 8-CPT-2′-O-Me-cAMP (8-CPT-2′-O-Me) for 10 min. Active Rap GTPases were purified from lysates with a recombinant fusion protein comprising the Ras binding domain of the Ras/Ral GEF RalGDS. C, detection of active Rap2A using the treatments and method described for B. D, representative Western blot depicting GTP-bound and total Rap2A following pretreatment for 30 min with 10 μm GGTI-298 or 10 μm FTS-A, followed by treatment with 8-CPT-2′-O-Me-cAMP (100 μm) for 10 min. Total levels of Rap2 protein prior to affinity purification of samples in B and C are shown in A. E, quantification of three independent experiments. Levels of active Rap are expressed as the ratio of total Rap, and data were analyzed by ANOVA and Bonferroni-corrected t tests. ***, p < 0.001 compared with controls. F, measurements of active and total Rap1 following the same treatments as used in D and E. G, quantification of three independent experiments.

Figure 6.

Blockade of PACAP-dependent growth arrest in NS-1 cells by inhibition of Epac or p38. A, growth curves of NS-1 cells grown for 5 days in 96-well plates in the absence or presence of 100 nm PACAP-38 with or without the Epac inhibitor ESI-09 (10 μm) or the p38 inhibitor SB239063 (10 μm). Five images per well were acquired every 24 h by automated microscopy. Data points represent means, and error bars correspond to the standard error of the mean from quadruplicate determinations. B, representative images of phase-contrast photomicrographs (×20 objective) obtained after 5 days of treatment.

Figure 7.

Blockade of cyclic AMP–dependent growth arrest in NS-1 cells by inhibition of Epac or p38. A, growth curves of NS-1 cells grown for 5 days in 96-well plates in the absence or presence of 100 μm 8-CPT-cAMP (8-CPT) in the absence or presence of either the Epac inhibitor ESI-09 (10 μm) or the p38 inhibitor SB239063 (10 μm). Five images per well were acquired every 24 h by automated microscopy. Data points represent means, and error bars correspond to the standard error of the mean from eight independent determinations. B, representative phase-contrast photomicrographs (×20 objective) obtained after 5 days of treatment.

Figure 8.

Epac activation promotes growth arrest in NS-1 cells. A, NS-1 cells growing in 96-well plates were treated with either 100 μm 8-CPT-cAMP (pan-specific) or 100 μm 8-CPT-2′-O-Me-cAMP (Epac-specific). Cells were imaged every 24 h, and cell number was determined for each set of images. Data points represent means from quadruplicate determinations, and error bars show standard errors of the mean. B, representative phase-contrast ×20 photomicrographs acquired after treatment for 5 days.

Figure 9.

Rap2A is necessary for Epac-dependent p38 activation. A, measurements of Rap2A protein levels in four NS-1 cell lines generated to stably express either scrambled shRNA or shRNAs targeting various regions of RAP2A-encoding mRNA. Lane 1, NS-1 cells expressing RAP2A-targeting shRNA. Lane 2, NS-1 cells expressing shRAP2A-1. Lane 3, NS-1 cells expressing shRAP2A-2. Lane 4, NS-1 cells stably expressing shRAP2A-3. MW, molecular weight. B, levels of Rap1 protein in samples from A. C, measurements of p38 phosphorylation in NS-1 cells stably expressing scrambled shRNA or shRNA targeting RAP2A mRNA. Cells were treated with varying concentrations of 8-CPT-2′-O-Me-cAMP for 30 min. Data points are means from three experiments performed in duplicate, with error bars representing standard error. D, p38 phosphorylation in NS-1 cells expressing either scrambled shRNA or shRNA targeting RAP2A following treatment with NGF (100 ng/ml) for 30 min. Data were analyzed by ANOVA and Bonferroni-corrected t tests. ***, p < 0.001 relative to comparable untreated control cells.

Figure 10.

Rap2A mediates Epac-dependent growth arrest. A and B, growth properties of NS-1 cells stably expressing either (A) scrambled shRNA or (B) shRNA targeting RAP2A mRNA grown in 96-well plates and treated with 8-CPT-2′-O-Me-cAMP (100 μm) or NGF (100 ng/ml) for 5 days. C, representative images obtained on the final measurement shown in A and B.

Figure 11.

Parcellation of neuropeptide- and neurotrophin-initiated signaling to growth arrest and neuritogenesis in NS-1 cells. Rap1 denotes Rap1A or B (not yet determined); MEK-ERK denotes MEK1/2 and ERK1/2 or both (not yet determined); p38 is most likely p38α, although minor contributions of p38β or others cannot be ruled out.