Rap1-mediated activation of extracellular signal-regulated kinases by cyclic AMP is dependent on the mode of Rap1 activation

Abstract

Like other small G proteins of the Ras superfamily, Rap1 is activated by distinct guanine nucleotide exchange factors (GEFs) in response to different signals to elicit cellular responses. Activation of Rap1 by cyclic AMP (cAMP) can occur via cAMP-dependent protein kinase A (PKA)-independent and PKA-dependent mechanisms. PKA-independent activation of Rap1 by cAMP is mediated by direct binding of cAMP to Rap1-guanine nucleotide exchange factors (Rap1-GEFs) Epac1 (exchange protein directly activated by cAMP 1) and Epac2 (Epac1 and Epac2 are also called cAMP-GEFI and -GEFII). The availability of cAMP analogues that selectively activate Epacs, but not PKA, provides a specific tool to activate Rap1. It has been argued that the inability of these analogues to regulate extracellular signal-regulated kinases (ERKs) signaling despite activating Rap1 provides evidence that Rap1 is incapable of regulating ERKs. We confirm that the PKA-independent activation of Rap1 by Epac1 activates a perinuclear pool of Rap1 and that this does not result in ERK activation. However, we demonstrate that this inability to regulate ERKs is not a property of Rap1 but is rather a property of Epacs themselves. The addition of a membrane-targeting motif to Epac1 (Epac-CAAX) relocalizes Epac1 from its normal perinuclear locale to the plasma membrane. In this new locale it is capable of activating ERKs in a Rap1- and cAMP-dependent manner. Rap1 activation by Epac-CAAX, but not wild-type Epac, triggers its association with B-Raf. Therefore, we propose that its intracellular localization prevents Epac1 from activating ERKs. C3G (Crk SH3 domain Guanine nucleotide exchanger) is a Rap1 exchanger that is targeted to the plasma membrane upon activation. We show that C3G can be localized to the plasma membrane by cAMP/PKA, as can Rap1 when activated by cAMP/PKA. Using a small interfering RNA approach, we demonstrate that C3G is required for the activation of ERKs and Rap1 by cAMP/PKA. This activation requires the GTP-dependent association of Rap1 with B-Raf. These data demonstrate that B-Raf is a physiological target of Rap1, but its utilization as a Rap1 effector is GEF specific. We propose a model that specific GEFs activate distinct pools of Rap1 that are differentially coupled to downstream effectors.

FIG. 1.

Activation of Rap1 in a PKA-dependent and PKA-independent manner and its role in ERK activation. (A) PC12 cells were left untreated (U) or were treated with F/I and were pretreated with H89 (1 μM) or myr-PKI or left without pretreatment (−). Cell lysates were examined for activated Rap1 (Rap1-GTP) by GST-Ral-GDS assay as described previously. Total Rap1 protein levels are shown in the bottom panel. (B) AtT20 cells were left untreated (−) or treated with F/I (+) and were pretreated with H89 (1 μM) or myr-PKI or left without pretreatment (U). Cell lysates were examined for activated Flag-Rap1 (Flag-Rap1-GTP). Total Flag-Rap1 in cell extracts is indicated as a loading control. (C) PC12 cells were transfected with Flag-Rap1 and either Epac1, vector, or C3G/CrkL along with Myc-Rap1-AGE at 4 μg/ml (4) or 6 μg/ml (6) or no Myc-Rap1-AGE (0). Epac-transfected cells were stimulated with 8-CPT-2Me-cAMP (8-CPT-OMe). Cell lysates were examined for activated Flag-Rap1 (Flag-Rap1-GTP). Total Flag-Rap1 in cell extracts is indicated as a loading control. (D) PC12 cells were transfected with Flag-Rap1 with (+) or without (−) Myc-Rap1-AGE (6 μg/ml) and were left untreated (U) or stimulated with F/I, as indicated. In addition, cells were cotransfected with C3G and CrkL (C3G/CrkL) with (+) or without (−) Myc-Rap1-AGE. Cell lysates were examined for activated Flag-Rap1 (Flag-Rap1-GTP). Total Flag-Rap1 in cell extracts is indicated as a loading control. (E) AtT20 cells were transfected with Flag-Rap1, with (+) or without (−) Myc-Rap1-AGE, and were left untreated (U) or stimulated with F/I, as indicated. In addition, cells were cotransfected with C3G and CrkL (C3G/CrkL) with (+) or without (−) Myc-Rap1-AGE. Cell lysates were examined for activated Flag-Rap1 (Flag-Rap1-GTP). Total Flag-Rap1 in cell extracts is indicated as a loading control.

FIG. 2.

Activation of PKA, Rap1, and C3G is required for the phosphorylation of ERK by Forskolin in PC12 cells. (A) Cells were left untreated (Un), treated with F/I, or pretreated with H89 prior to F/I treatment (F/I + H89). Cell lysates were examined for activated Rap1 (Rap1-GTP). Phosphorylation of ERK1/2 was visualized by Western blotting utilizing a phospho-ERK antibody (pERK1/2). Phosphorylation of MEK was visualized by Western blotting utilizing a phospho-MEK antibody (pMEK). Total Rap1 protein levels are shown in the bottom panel. (B) PC12 cells were transfected with Flag-ERK2 and vector, Rap1GAP1, or RasN17 as indicated and left untreated (−) or treated with F/I (+). Flag-ERK2 was immunoprecipitated from cell lysates and analyzed by immunoblotting with phospho-ERK antibodies (pFlag-ERK2). The levels of total Flag-ERK2 are indicated as a loading control. (C) Rap1 is required for the phosphorylation of ERK by C3G/CrkL in PC12 cells. PC12 cells were transfected with Flag-ERK2 and vector plus C3G/CrkL, Rap1GAP1 plus C3G/CrkL, or RasN17 plus C3G/CrkL, as indicated. Flag-ERK2 was immunoprecipitated from cell lysates and analyzed by immunoblotting with phospho-ERK antibodies (pFlag-ERK2). The levels of total Flag-ERK2 are indicated as a loading control. (D) PC12 cells were transfected with Flag-ERK2, with (+) or without (−) Myc-Rap1-AGE, and left untreated or stimulated with F/I, as indicated. In addition, cells were cotransfected with C3G and CrkL (C3G/CrkL) with (+) or without (−) Myc-Rap1-AGE. Flag-ERK2 was immunoprecipitated from cell lysates and analyzed by immunoblotting with phospho-ERK antibodies (pFlag-ERK2). The levels of total Flag-ERK2 are indicated as a loading control.

FIG. 3.

Transfection of C3G siRNA blocks activation of Rap1 and ERKs by Forskolin in PC12 cells. (A) PC12 cells were transfected with C3G siRNA or mock transfected (mock) for 72 h and stimulated with F/I for 0, 10, or 20 min, as indicated. The expression of C3G was analyzed by immunoblotting with anti-C3G antibodies (C3G). Active Rap1 within cell lysates is shown (Rap1-GTP). Total Rap1 in cell extracts is indicated as a loading control. Activation of ERKs was analyzed by immunoblotting with phospho-ERK specific antibodies (pERK1/2). Total ERK1/2 from cell lysates was used as a loading control. (B) Quantitation of the efficacy of siRNA knockdown is presented as percentage of control (n = 3 [mean ± standard error of the mean]). (C) PC12 cells were transfected with C3G siRNA or control siRNA for 72 h or left untransfected (mock) and were stimulated with F/I or left untreated, as indicated. Activation of ERKs was analyzed by immunoblotting with phospho-ERK-specific antibodies (pERK1/2). Active Rap1 within cell lysates is shown (Rap1-GTP). Total Rap1 in cell extracts is indicated as a loading control.

FIG. 4.

Localization of C3G and activation of Rap1 at the PM by Forskolin. (A) C3G localization at the PM is triggered by PKA in COS cells. COS cells were transfected with GFP-C3G with CrkL and were left untreated or were treated with Forskolin or Forskolin plus H89, as indicated. (B) C3G localization at the PM is triggered by PKA in PC12 cells. PC12 cells were transfected with GFP-C3G with CrkL and were left untreated or were treated with Forskolin or Forskolin plus H89. (C) Activated Rap1-GTP can be detected at the PM upon cAMP treatment. COS cells were transfected with GFP-Ral-GDS and Rap1 or RapV12 and were left untreated or treated with Forskolin, as indicated. Arrows identify membrane patches that represent areas of GFP-Ral-GDS binding to Rap1-GTP (middle panel) and RapV12-GTP (right panel).

FIG. 5.

Epac and C3G localize to distinct cellular compartments and correlate with distinct pools of Rap1 activation. (A) Epac proteins are present in AtT20 cells. Protein levels for Epac1 and Epac2 were examined in PC12 (P) and AtT20 (A) cell lysates by use of antibodies to Epac1 (upper left panel) and Epac2 (upper right panel). The levels of total ERK2 are indicated as a loading control. (B) C3G expression in PC12 cells and AtT20 cells. Endogenous C3G expression levels for PC12 cells (P) and AtT20 cells (A) were examined using antibodies to C3G. AtT20 cells that were transfected with C3G were used as a positive control. (C) 8-CPT-2Me-cAMP (8-CPT-OMe) activates Rap1 in AtT20 cells. AtT20 cells were left untreated (−) or treated with H89, F/I, F/I plus H89, or 8-CPT-2Me-cAMP (8-CPT-OMe), as indicated. Cell lysates were examined for activated Rap1 (Rap1-GTP). Total Rap1 within cell extracts is indicated as a loading control. (D) Rap activity and regulation within different cellular fractions. AtT20 cells were stimulated with either 8-CPT-2Me-cAMP (8-CPT-OMe) or F/I, with or without pretreatment with H89, as indicated. Cells were lysed, and cytosolic (Cyto.), membrane (Mem.), and perinuclear (Nuc.) fractions were isolated as described in Materials and Methods. Subcellular fractions (derived from equivalent numbers of cells) were assayed for activation of Rap1 (Rap-1-GTP). Total Rap1 is shown in the second panel. Endogenous Epac1, C3G, and B-Raf levels within each fraction are also shown (lower panels).

FIG. 6.

PKA-independent Rap1 activation is mediated by Epacs in AtT20 cells and does not lead to ERK activation. (A) 8-CPT-2Me-cAMP does not activate ERKs in AtT20 cells. AtT20 cells were left untreated (−) or treated with 8-CPT-2Me-cAMP (8-CPT-OMe) (+), as indicated. Fibroblast growth factor (FGF) served as a positive control. Total cell lysates were examined for the phosphorylation of ERK1/2 by Western blotting (pERK1/2, upper panel). Total ERK1/2 is shown as a loading control. (B) Expression of Epac1 is required for 8-CPT-2Me-cAMP to activate Rap1 in PC12 cells. PC12 cells were transfected with Flag-Rap1 and either WT Epac1, vector, or C3G/CrkL and treated with 8-CPT-2Me-cAMP (8-CPT-OMe) (+) or left untreated (−), as indicated. Cell lysates were examined for activated Flag-Rap1 (Flag-Rap1-GTP). Total Flag-Rap1 is shown as a loading control. (C) Activation of transfected Epac1 in PC12 cells does not result in ERK activation. PC12 cells were transfected with Flag-ERK2 and either WT Epac1, vector, or C3G/CrkL and treated with 8-CPT-2Me-cAMP (8-CPT-OMe) (+) or left untreated (−) as indicated. Flag-ERK2 was immunoprecipitated from cell lysates and analyzed by Western blotting using phospho-ERK antibodies (pFlag-ERK2). The levels of total Flag-ERK2 are indicated as a loading control. (D) ERK is activated by PKA in AtT20 cells and participates in cAMP activation of ERKs. (A) AtT20 cells were left untreated (Un), treated with F/I alone for 20 min, or pretreated with H89 (F/I + H89). Cell lysates were examined for activated Rap1 (Rap1-GTP). Phosphorylation of ERK1/2 was visualized by Western blotting utilizing a phospho-ERK antibody (pERK1/2). Phosphorylation of MEK was visualized by Western blotting utilizing a phospho-MEK antibody (pMEK). Total Rap1 protein levels are shown in the bottom panel as a loading control. (E) Ras is required for the phosphorylation of ERK by F/I in AtT20 cells. AtT20 cells transfected with Myc-ERK2 were cotransfected with vector, Rap1GAP, or RasN17 as indicated. Cells were left untreated or treated with F/I and lysed. Myc-ERK2 was immunoprecipitated from cell lysates and analyzed by Western blotting using phospho-ERK antibodies (pMyc-ERK2). Total Myc-ERK2 levels are shown.

FIG. 7.

Transfection of C3G/CrkL triggers Rap1/B-Raf association and ERK activation in AtT20 cells. (A) C3G/CrkL-mediated ERK activation was blocked by Rap1GAP1. Cells were transfected Myc-ERK2, and with vector alone or with C3G/CrkL, in the presence of vector, Rap1GAP1, or RasN17, as indicated. Myc-ERK2 was immunoprecipitated from cell lysates and analyzed by Western blotting using phospho-ERK antibodies (pMyc-ERK2). Total Myc-ERK2 levels are shown. (B) Forskolin/IBMX, but not 8-CPT-2Me-cAMP, promotes Rap1/B-Raf association in AtT20 cells. AtT20 cells were transfected with Myc-Rap1, and either cotransfected with C3G/CrkL or treated with F/I or 8-CPT-2Me-cAMP (8-CPT-OMe), as indicated. Cell lysates were immunoprecipitated (IP) with Myc antibodies to recover Myc-Rap1. Association of Myc-Rap1 with endogenous B-Raf was examined by Western blotting following Myc IP (B-Raf). Total B-Raf is shown as a loading control.

FIG. 8.

Epac1 is localized to the perinuclear region. PC12 cells were transfected with GFP-Epac-WT. (A) The localization of GFP-Epac-WT is shown using epifluorescence. (B) The localization of the nuclear pore complex is shown using immunofluorescence using the antibody Mab414. (C) A merged image of GFP-Epac-WT and the nuclear pore complex shown in panels A and B is shown. (D) The addition of the Ki-Ras CAAX domain relocalizes Epac1 to the PM. PC12 cells were transfected with GFP-Epac-CAAX, and the localization of GFP-Epac-CAAX is shown using epifluorescence. (E) PC12 cells were transfected with GFP-Epac-SAAX and the localization of GFP-Epac-SAAX is shown using epifluorescence. Fixed cells were stained with Hoechst 33258 and analyzed using Image Restoration microscopy.

FIG. 9.

Epac-CAAX, but not Epac WT or Epac-SAAX, promotes ERK activation and Rap1/B-Raf association. (A and B) Parallel experiments in PC12 cells were conducted to examine the selectivity of Rap1-GEFs for Rap1 activation and association with B-Raf, shown in panel A, and activation of ERKs, shown in panel B. For the experiments depicted in both panels A and B, cells were transfected with Myc-Epac WT (lanes 5 and 6), or Myc-Epac-CAAX (lanes 7 and 8), or Myc-Epac-SAAX (lanes 9 and 10). All cells transfected with Epac constructs were either treated with 8-CPT-2Me-cAMP (OMe; lanes 6, 8, and 10) or left untreated (lanes 5, 7, and 9). Cells were transfected with C3G/CrkL (lanes 11 and 12). Cells were treated with F/I in lanes 4 and 12. (A) All Epac constructs activate Rap1, but only Epac-CAAX can trigger Rap1 to associate with B-Raf. In addition to use of the cDNAs described above, cells were cotransfected with vector (lane 1), Flag-RapV12 (lane 2), or WT Flag-Rap1 (lanes 3 to 12) and treated as described above. Cell lysates were split and one fraction examined for Rap1 activation (Flag-Rap1-GTP). Total Flag-Rap1 is shown to indicate equal transfection and loading. The second fraction was immunoprecipitated (IP) with Flag antibody to recover Flag-Rap1, and association with endogenous B-Raf was examined by Western blotting [B-Raf (within IP)]. The expression levels of Myc-Epacs (WT, Epac-CAAX, and Epac-SAAX) are shown in the lower panel (Total Myc-Epac). (B) Only Epac-CAAX, but neither Epac WT nor Epac-SAAX, can activate ERKs. In addition to the cDNAs described above, cells were transfected with vector (lane 1), or Flag-ERK2 (lanes 2 to 12), and treated as described above. Flag-RapV12 (lane 2) was used a positive control. Flag-ERK2 was immunoprecipitated from cell lysates and analyzed by Western blotting using phospho-ERK antibodies (pFlag-ERK2). The levels of total Flag-ERK2 and total Myc-Epac proteins are shown. (C) Neither Epac nor Epac-CAAX can activate Ras. PC12 cells were transfected with Flag-Ras and either Epac-WT, Epac-CAAX, or vector. Cells were left untreated (−) or treated with 8-CPT-2Me-cAMP (8-CPT-OMe) (+), as indicated. NGF was used as a positive control. Cell lysates were examined for activated Flag-Ras (Flag-Ras-GTP). Total Flag Ras levels are shown to indicate equal transfection and loading (lower panel). (D) ERK activation by Epac-CAAX is Rap1 dependent. PC12 cells were transfected with Flag-ERK2 and Epac-CAAX in the presence of vector, Rap1GAP1, or RasN17, as indicated. Cells were left untreated (−) or treated with 8-CPT-2Me-cAMP (8-CPT-OMe) (+), as indicated. Flag-ERK2 was immunoprecipitated from cell lysates and analyzed by Western blotting using phospho-ERK antibodies (pFlag-ERK2). The levels of total Flag-ERK2 protein are shown.

FIG. 10.

Stable expression of Epac-CAAX in PC12 cells permits ERK activation by 8-CPT-2Me-cAMP. (A) Stably selected populations of Flag-Epac-WT, Flag-Epac-CAAX and Flag-Epac-SAAX-expressing PC12 cells show Rap1 activation by 8-CPT-2Me-cAMP. Cells were treated in the presence (+) or absence (−) of 8-CPT-2Me-cAMP (8-CPT-OMe), and lysates were examined for activation of Rap1 (Rap1-GTP). Total Rap1 levels are shown as a loading control. Flag-Epac-WT-, Flag-Epac-SAAX-, and Flag-Epac-CAAX-expressing PC12 cells are shown in the left, middle, and right panels, respectively. (B) PC12 cells stably expressing Flag-Epac-CAAX show ERK activation in response to 8-CPT-2Me-cAMP. Cells were treated with 8-CPT-2Me-cAMP (8-CPT-OMe) for the indicated times (min). F/I (1 μM) was used as a normalization control. Phosphorylation of ERK1/2 was visualized by Western blotting utilizing a phospho-ERK antibody (pERK1/2). Total ERK2 levels are shown as a loading control. The bottom panels show Flag-Epac expression for each transfectant. Flag-Epac-WT-, Flag-Epac-SAAX-, and Flag-Epac-CAAX-expressing PC12 cells are shown in the left, middle, and right panels, respectively. (C) Rap1 activation by cAMP in stable populations of Epac-expressing PC12 cells is not inhibited by PKA. Stable populations of Epac-CAAX- and Epac-WT-expressing PC12 cells were stimulated with 8-CPT-2Me-cAMP (8-CPT-OMe) or F/I with (+) or without (−) H89 pretreatment. Cell lysates were examined for activated Rap1 (Rap1-GTP, top panel). Total Rap1 was visualized by Western blotting (middle panel). Total cell lysates were examined for Flag-Epac expression (bottom panel).