Phosphorylation of Rap1 by cAMP-dependent Protein Kinase (PKA) Creates a Binding Site for KSR to Sustain ERK Activation by cAMP

Abstract

Cyclic adenosine monophosphate (cAMP) is an important mediator of hormonal stimulation of cell growth and differentiation through its activation of the extracellular signal-regulated kinase (ERK) cascade. Two small G proteins, Ras and Rap1 have been proposed to mediate this activation. Using HEK293 cells as a model system, we have recently shown that both Ras and Rap1 are required for cAMP signaling to ERKs. However, cAMP-dependent Ras signaling to ERKs is transient and rapidly terminated by PKA phosphorylation of the Raf isoforms C-Raf and B-Raf. In contrast, cAMP-dependent Rap1 signaling to ERKs and Rap1 is potentiated by PKA. We show that this is due to sustained binding of B-Raf to Rap1. One of the targets of PKA is Rap1 itself, directly phosphorylating Rap1a on serine 180 and Rap1b on serine 179. We show that these phosphorylations create potential binding sites for the adaptor protein 14-3-3 that links Rap1 to the scaffold protein KSR. These results suggest that Rap1 activation of ERKs requires PKA phosphorylation and KSR binding. Because KSR and B-Raf exist as heterodimers within the cell, this binding also brings B-Raf to Rap1, allowing Rap1 to couple to ERKs through B-Raf binding to Rap1 independently of its Ras-binding domain.

Keywords: 14-3-3 protein; Raf kinase; Ras-related protein 1 (Rap1); extracellular-signal-regulated kinase (ERK); protein kinase A (PKA).

FIGURE 1.

B-Raf binding to Rap1 following cAMP stimulation is indirect. A, HEK293 cells were transfected with scrambled shRNA, or shRNA against B-Raf or C-Raf, as indicated. A, HEK293 cells were treated with F/I for 0, 10, 20, and 30 min. The endogenous levels of pERK and total ERK2, and the efficacies of the shRNAs are shown. B, cells transfected with wild type GFP-B-Raf (WT) or GFP-B-Raf R188L (R188L) and either FLAG-RasV12 or FLAG-RapE63 were treated with F/I for the indicated times. The presence of GFP-B-Raf following FLAG immunoprecipitation (IP) was detected by Western blotting (upper panel). The levels of FLAG-RasV12/RapE63 and GFP-B-Raf in the TCL are shown in the lower two panels. C, cells transfected with FLAG-Rap1 and wild type GFP-B-Raf (WT) or GFP-B-Raf R188L (R188L) were treated with F/I, as indicated. The presence of GFP-B-Raf following FLAG IP was examined. The levels of transfected proteins in the TCL are shown. D, wild type B-Raf, but not B-Raf R509H, can rescue cAMP activation of ERKs in cells depleted of B-Raf. HEK293 cells that were stably transfected with shRNA against B-Raf were transiently transfected with FLAG-ERK2 and either GFP-tagged vector control, shRNA-resistant (*) wild type B-Raf (WT*), or the R509H mutant (RH*) as indicated or vector (not shown), and treated with F/I as indicated. Phosphorylated FLAG-ERK2 was detected within the FLAG IPs (pFlag-ERK2). The levels of FLAG-ERK2 and endogenous B-Raf within total cell lysates (TCL) are shown. E, cAMP activation of ERKs by Rap1 requires B-Raf dimerization and KSR. HEK293 cells transfected with wild type GFP-B-Raf (WT) or GFP-B-Raf R509H (R509H), and either FLAG-RasV12 or FLAG-RapE63 were treated with F/I, as indicated. The presence of GFP-B-Raf following FLAG IP and the levels of transfected proteins in the TCL are shown.

FIGURE 2.

KSR is required for ERK activation by cAMP. A, cAMP does not increase the levels of B-Raf/KSR dimers. HEK293 cells were transfected with FLAG-tagged KSR (WT), or the mutant KSR R615H, as indicated, and treated with F/I for 20 min (20) or left untreated (0). The association of B-Raf with FLAG-KSR or FLAG-KSR R615H was determined by examining the level of endogenous (endo) B-Raf within each FLAG immunoprecipitation (IP). The levels of FLAG KSR within each IP are shown as a control. Levels of endogenous B-Raf are shown. B, cells transfected with scrambled short hairpin RNA (scrambled) or shRNA against KSR1 were treated with F/I, as indicated. The levels of endogenous pERK and ERK2 are shown. C, the efficacy of the KSR1 shRNA is shown. HEK293 cells were transfected with scrambled shRNA (scram), or shRNA against human KSR, as indicated. Cells were treated with F/I as indicated. The levels of endogenous (endo) KSR were examined following KSR IP, using an anti-KSR Ab. The endogenous levels of activated phospho-ERK1/2 (pERK), ERK2, B-Raf, and C-Raf were detected by Western blotting. D, KSR^−/− MEF cells transfected with GFP-ERK2 and vector (not shown), wild type KSR (WT), or the R615H mutant, were treated with F/I for 20 min (20) or left untreated (0). The levels of phosphorylated GFP-ERK2 (pGFP-ERK2) following GFP IP and the levels of GFP-ERK2 and FLAG-KSR in the TCL are shown.

FIGURE 3.

KSR/B-Raf dimerization correlates with ERK activation by cAMP. A, HEK293 cells transfected with GFP-RasV12, GFP-RapE63 (E63), or GFP-RapE63AA (E63AA) and either FLAG-tagged WT KSR or KSR R615H (R615H) were treated with F/I, as indicated. The levels of GFP-Ras/Rap following FLAG immunoprecipitation (IP) are shown. The levels of FLAG-KSR (WT/RH) and the levels of GFP-RasV12, GFP-RapE63, and GFP-RapE63AA (GFP-Ras/Rap) in the TCL are shown. B, cells transfected with FLAG-KSR, and GFP-Rap1 (WT) or GFP-RapAA (AA), were treated with F/I, as indicated. The levels of GFP-Rap1 (WT/AA) following FLAG IP, and the levels of GFP-Rap1 (WT/AA) and FLAG-KSR in the TCL are shown.

FIGURE 4.

Rap1 activation of ERKs requires PKA phosphorylation of Rap1. A, HEK293 cells transfected with wild type GFP-B-Raf and FLAG-RasV12, FLAG-RapE63, or FLAG-RapE63AA were treated with F/I for the indicated times. The presence of GFP-B-Raf following FLAG immunoprecipitation (IP) was detected by Western blotting (upper panel). The levels of GFP-B-Raf and FLAG-Ras/Rap in the TCL are shown in the lower two panels. B, cells transfected with FLAG-ERK2, and either vector or GFP-Rap1AA were treated with F/I for the times indicated. The levels of phosphorylated FLAG-ERK2 (pFlag-ERK2) following FLAG IP are shown in the first panel. The levels of FLAG ERK2 and GFP-Rap1AA in the TCL are also shown. C, cells transfected with FLAG-ERK2, scrambled shRNA (scram), or shRNA against Rap1a/b, and GFP-tagged shRNA-resistant (*) wild type Rap1b (GFP-Rap1*), or the corresponding phosphorylation-deficient mutant (GFP-Rap1AA*) were treated with F/I, as indicated. The levels of pFLAG-ERK2 following FLAG IP and the levels of FLAG ERK2, GFP-Rap (WT/AA), and the endogenous Rap1 (endo Rap1) in the TCL are also shown.

FIGURE 5.

Both Rap1a and Rap1b contain putative 14-3-3 binding sites. A, HEK293 cells transfected with Myc-14-3-3 and either GFP-Rap1b (WT) or the phosphorylation mutant Rap1bAA (AA), constitutively active mutant Rap1bE63 (E63), or Rap1bE63AA (E63AA), were treated with F/I, as indicated. The levels of Myc-14-3-3 and GFP-Rap1 following GFP immunoprecipitation (IP) are shown. The levels of Myc-14-3-3 in the TCL are shown in the bottom panel. B, cells transfected with the constitutively active mutants Rap1bE63 or Rap1bE63AA were treated with F/I, as indicated. The levels of endogenous 14-3-3 and FLAG-Rap1 following FLAG IP are shown. The levels of endogenous 14-3-3 in the TCL are shown in the bottom panel. C, KSR^−/− MEF cells transfected with FLAG-Rap1b or vector and GFP-KSR were treated with F/I, as indicated. Endogenous 14-3-3 and FLAG-Rap1b following FLAG IP are shown. The levels of endogenous 14-3-3 and FLAG-RapE63, and GFP-KSR in the TCL are shown. D, HEK293 cells transfected with FLAG-RapE63 and vector and HA-14-3-3γ, HA-14-3-3β, and HA-14-3-3σ, and were treated with F/I, as indicated. HA-14-3-3 and GFP-Rap1b following FLAG IP are shown. The levels of HA-14-3-3 and GFP-RapE63 in the TCL are shown.

FIGURE 6.

KSR 14-3-3 sites differentially interact with Rap1. A, HEK293 cells transfected with FLAG-KSR were treated with F/I as indicated. Lysates were subjected to FLAG immunoprecipitation (IP) and probed with a phospho-specific antibody recognizing phosphorylated Ser-392 of KSR. The levels of Ser(P)-392 are shown in the upper panel. The levels of FLAG-KSR within the IP are shown in the lower panel. B, HEK293 cells transfected with GFP-Rap1bE63 (RapE63) and FLAG-KSR (WT), FLAG-KSR S297A (S297A), FLAG-KSR S392A (S392A), FLAG-KSR S297A/S392A (S297A/S392A), or FLAG-KSR S838A (S838A). Cells were treated with F/I for 20 min (20) or left untreated (0). The levels of GFP-RapE63 following FLAG IP are shown. The levels of GFP-RapE63 and FLAG-KSR WT/mutants within the TCL are shown. C, KSR S838A cannot dimerize with B-Raf. HEK293 cells were transfected with FLAG-tagged KSR (WT), or mutants KSR S392A, KSR S838A, or KSR R615H, as indicated, and treated with F/I for 20 min (20) or left untreated (0). The association of B-Raf with KSR and/or KSR mutants was determined by examining the level of endogenous B-Raf within each FLAG IP. The levels of FLAG KSR within each IP are shown as a control. Levels of endogenous B-Raf in the TCL are shown. D, KSR S297A can dimerize with B-Raf. HEK293 cells were transfected with FLAG-tagged KSR (WT) or KSR S297A, and treated with F/I for 20 min (20) or left untreated (0). The association of B-Raf with KSR and/or KSR mutants was determined by examining the level of endogenous B-Raf within each FLAG IP. The levels of FLAG KSR within each IP are shown as a control. Levels of endogenous B-Raf in the TCL are shown.

FIGURE 7.

Model of cAMP activation of ERK. A, Ras activation recruits B-Raf/KSR to trigger ERK activation. This occurs through the direct binding of Ras-GTP to the B-Raf RBD. The binding of B-Raf to Ras is regulated by PKA phosphorylation of B-Raf on Ser-365 (pS365). This phosphorylated site interrupts B-Raf binding to Ras-GTP, terminating signals to ERK. This occurs through the binding of 14-3-3 (not shown). The release of B-Raf also releases KSR, as part of the B-Raf/KSR dimer. Ras-GTP also recruits C-Raf and a similar mechanism involving PKA phosphorylation of C-Raf S295 releases C-Raf from Ras (not shown). B, Rap1 recruitment of B-Raf/KSR is regulated by two phosphorylations induced by cAMP/PKA, one on B-Raf and one on Rap1. Activated Rap1 binds to a B-Raf/KSR dimer through the B-Raf RBD. This direct binding is inhibited by the PKA-dependent phosphorylation on B-Raf (pS365). At the same time, the indirect binding of B-Raf/KSR to Rap1 is enhanced by the PKA-dependent phosphorylations of Rap1. (Here, Rap1b and pS179 are shown.) The inhibitory PKA phosphorylation of B-Raf on Ser-365 functions to release the B-Raf/KSR dimer from Rap1-GTP in a similar fashion to that shown above for Ras. Although we have not definitively proved that 14-3-3 is required for KSR to bind Rap1, the association of both KSR and 14-3-3 with Rap1 requires the same phosphorylated site within Rap1. This PKA phosphorylation of Rap1 on Ser-179 creates a novel 14-3-3 binding site at the carboxyl terminus of Rap1. 14-3-3 binding accommodates this phosphorylation in conjunction with the adjacent the C-terminal geranylgeranyl lipid. We propose that the binding of 14-3-3 to phosphorylated Rap1 occurs as part of a 14-3-3γ dimer that also binds KSR. Therefore, we have drawn 14-3-3 as a bridge between Rap1 and KSR in this model. Rap1 binds to KSR via established 14-3-3 binding sites. Mutational analysis of potential 14-3-3 sites in KSR suggested Ser-297 as a candidate site, although Ser-392 could not be ruled out. We propose that whereas the B-Raf/KSR dimer is released from direct binding to Rap1, the dimer remains associated with phosphorylated Rap1 through a phospho-specific 14-3-3 binding site that links KSR and its dimer partner B-Raf to Rap1. This model explains how KSR and B-Raf can bind to phosphorylated Rap1 even after direct binding of Rap1 to B-Raf is blocked.