Identification of the RGC in adipocytes
Previous studies have demonstrated that GAPs preferentially interact with their target GTPases during the transition state of guanosine triphosphate (GTP) hydrolysis (Bos et al
; Scheffzek et al
). This interaction is transient and occurs with lower affinity when wild-type GTPases are locked in either guanosine diphosphate (GDP)– or GTP-bound states. However, binding of GTPases to GDP in complex with aluminum fluoride (GDP/AlFx) mimics the GTP hydrolysis transition state, thus permitting stabilized GTPase–GAP interaction (Scheffzek et al
; Wittinghofer, 1997
; see A). We took advantage of this property in a proteomic approach to search for potential GAPs that may interact with immobilized Ral loaded with GDP/AlFx in 3T3-L1 adipocytes since wild-type RalA displayed a much lower GTP loading profile than did its mutant form that fails to catalyze GTP hydrolysis in these cells (Supplemental Figure S1). Interestingly, several proteins were found to preferentially associate with glutathione S
-transferase (GST)–RalA loaded with GDP/AlFx but not with GDP-loaded GST-RalA or GST alone, and the most prominent of these proteins migrated at ~200 kDa (p200; B). When subjected to tandem mass spectrometry, four unique peptides were recovered with the protein; each sequence matched that of a protein named AS250 (B). AS250, or Akt substrate of 250 kDa, was originally identified as a phosphoprotein of 1984 amino acids that was purified from adipocytes by virtue of its interaction with a phospho-Akt substrate antibody (Gridley et al
). The protein contains a putative GAP domain homologous to TSC2 and RapGAP and was found to associate with the protein KIAA1219 (Gridley et al
). In agreement with the mass spectrometry data, Western blotting (WB) revealed that p200 GAP (AS250) and its associating protein KIAA1219 specifically interacted with RalA bound to GDP/AlFx, but not with RalA bound to GDP or GTPγS, or GST alone (C). Meanwhile, subunits of the exocyst proteins preferentially interacted with RalA loaded with GTPγS (C), further confirming the specificity of the transition state–dependent binding between KIAA1219/p200 GAP and RalA that is characteristic of a GAP–substrate GTPase interaction. We thus named these two proteins RGC1 and RGC2, to reflect their function and homology with TSC1/2, as will be further described. RGC1 represents the regulatory subunit of the complex, while RGC2 contains the GAP domain and represents the catalytic subunit of the complex.
FIGURE 1: RGC2/AS250 preferentially interacts with RalA in the transition state. (A) Schematic view of the transition state interaction between GAPs and their cognate small GTPase targets. Top: GAPs interact preferentially with their cognate targets during the (more ...)
We sought to explore the importance of the RGC in the regulation of Ral activity by insulin. Consistent with the characteristic GAP–substrate GTPase interaction, siRNA-mediated depletion of the GAP RGC2 in 3T3-L1 adipocytes led to increased RalA activity in both the basal and insulin-stimulated states, as determined by a pulldown assay using the immobilized effector domain of Sec5 (, A and B). This gain-of-function phenotype was specific to the loss of RGC2, as knockdown of other GAPs including p120 Ras GAP, RapGAP, AS160, or TSC2 had little effect on RalA activity (Supplemental Figure S2). When immunoprecipitated from cell lysates of 3T3-L1 adipocytes by an anti-RGC2 antibody, RGC proteins efficiently enhanced GTP hydrolysis of recombinant RalA in vitro, compared with RalA alone or RalA incubated with various control immunoprecipitates (C). The immunoprecipitated RGC proteins also catalyzed GTP hydrolysis of the Ral family member RalB (Figure S3A). These results are in contrast to a previous report that failed to detect RGC GAP activity toward RalA or other G proteins (Gridley et al
), perhaps due to differences in the assay used to detect activity. Immunoprecipitated RGC did not stimulate GTP hydrolysis of other GTPases of close homology to Ral, such as Rheb (Supplemental Figure S3B) or H-Ras (Supplemental Figure S3C), demonstrating the specificity of the GAP activity of the RGC proteins for Ral GTPases.
FIGURE 2: RGC2 negatively regulates RalA activity in adipocytes. (A) 3T3-L1 adipocytes were transfected with control siRNA or siRNA oligos that deplete RGC2. Four days after transfection, cell lysates were subjected to a pulldown assay to detect RalA activation. (more ...)
A protein named GARNL1 was recently isolated in complex with RalAQ72
L and RGC1 in brain lysates (Shirakawa et al
). GARNL1 contains a GAP domain with 83% identity to the GAP domain of RGC2 (Supplemental Figure S4A) and has in vitro activity toward Ral GTPases (Shirakawa et al
). SiRNA-mediated depletion of GARNL1 did not affect the activity of RalA in 3T3-L1 adipocytes (, D and E), despite efficient knockdown of GARNL1 protein (~82% knockdown), whereas knockdown of RGC2 (~95% knockdown) increased both basal and insulin-stimulated RalA activity. Surprisingly, we observed a ~245% increase in GARNL1 protein levels when cells were transfected with siRNA to deplete RGC2 (D). Analysis of the expression of GARNL1 and RGC2 during adipocyte differentiation revealed the up-regulation of GARNL1 protein levels during the clonal expansion stage of 3T3-L1 adipogenesis, followed by a marked down-regulation during later stages of differentiation, with little GARNL1 protein present in mature 3T3-L1 adipocytes (Supplemental Figure S3B). In contrast, RGC2 expression was induced during insulin treatment of adipocytes and remained high in mature adipocytes (Supplemental Figure S4B). Furthermore, RGC2 was the predominant Ral GAP expressed in primary white adipocytes (Supplemental Figure S4C). Taken together, these data indicate that RGC2 is the primary Ral GAP expressed in adipocytes and is responsible for regulating RalA activity in these cells. The identification of a specific Ral GAP in adipocytes points to a novel regulatory process targeting this G protein in insulin action.
RGC1 and RGC2 form a physical and functional complex to inactivate RalA
The precipitation of both RGC1 and 2 with the RalA-GDP/AlFx affinity matrix led us to test whether RGC1/2 forms a functional complex to inactivate RalA in vivo. We assayed RalA activity after depletion of RGC1 from 3T3-L1 adipocytes by siRNA-mediated knockdown. While proximal insulin signaling remained unaffected, as determined by p-Akt blotting, knockdown of RGC1 led to increased RalA activity in both the basal and insulin-stimulated states (, A and B), indicating that both RGC proteins are required to form a functional GAP complex that inactivates RalA. The nature of this complex is reminiscent of the Rheb GAP TSC1/2 complex (Inoki et al
; Li et al
), with which RGC1/2 shares a similar overall structure (C). Furthermore, alignment of the GAP domains revealed that RGC2 shares a conserved catalytic asparagine residue with TSC2 and RapGAP (C), indicating a similar catalytic mechanism among these GAPs (Li et al
). Interestingly, siRNA-mediated knockdown of RGC1 in adipocytes led to decreased levels of RGC2 (D), suggesting that physical interaction is required for maximal stability of the subunits. Consistent with this notion, RGC2 protein was stabilized when coexpressed with RGC1, which interacts with the former protein (E), further indicating that these proteins form a physical complex for stability.
FIGURE 3: RGC1 and RGC2/AS250 form a physical and functional complex to inactivate RalA. (A) 3T3-L1 adipocytes were transfected with control siRNA or siRNA oligos that deplete RGC1 and were subjected to a pulldown assay to detect RalA activity. Pulldowns represent (more ...)
We tested whether overexpression of RGC1/2 could inactivate RalA in vivo via its GAP activity. Consistent with the in vitro GAP assay, coexpression of RGC1 and 2 in cells significantly decreased the activity of the G protein, as determined by the pulldown assay (F). This inhibitory effect was absent in cells expressing RGC1 and RGC2 N/K, in which the critical asparagine residue for GAP activity was substituted to lysine to abolish the catalytic activity (F). Consistent with these data, overexpression of wild-type RGC1/2 decreased the interaction between wild-type RalA and the exocyst subunits Sec8 and Exo70, as assayed by coimmunoprecipitation of endogenous exocyst subunits (G). The RGC2 N/K mutant had a minimal effect on the interaction between wild-type RalA and exocyst subunits, likely due to residual GAP activity of the mutant (G). Taken together, these data demonstrate that RGC1 and RGC2 form a physical and functional GAP complex to inactivate RalA and prevent its interaction with the exocyst in a manner that resembles inactivation of Rheb by the TSC1/2 proteins.
RGC2 is an endogenous substrate for Akt2 downstream of PI 3-kinase
The similarity between the RGC1/2 complex and the Rheb GAP complex TSC1/2 (), as well as the PI 3-kinase dependence of the activation of RalA by insulin, prompted us to investigate whether the GAP RGC2 may also be regulated by Akt-dependent phosphorylation downstream of PI 3-kinase activity. Previous mass spectrometry studies showed that insulin treatment increased the phosphorylation of Ser486, Ser696, and Thr715 on RGC2/AS250 in adipocytes (Gridley et al
), although the protein kinase catalyzing these phosphorylation events was not defined. To elaborate these signaling events, we generated phosphospecific antibodies to each of the three sites. Insulin stimulated the phosphorylation of all three sites on RGC2 in 3T3-L1 adipocytes, as determined by blotting an anti-RGC2 immune complex or total cell lysates with the phosphospecific antibodies (A). Phosphorylation of RGC2 in total cell lysates was depleted upon knockdown of the protein, further demonstrating the specificity of the phospho antibodies (B). Insulin stimulated the phosphorylation of RGC2 in a dose-dependent manner that correlated well with the phosphorylation of Akt (C). A time course of insulin treatment of adipocytes revealed that phosphorylation of the Ser696 and Thr715 sites was sustained for up to 90 min (D). Phosphorylation at the Ser486 site peaked at 45 min and decreased by 90 min, perhaps indicating faster turnover at this site (D). Consistent with the data from 3T3-L1 adipocytes, insulin also stimulated phosphorylation of RGC2 in white adipose tissue for up to 30 min (E), providing in vivo evidence for this phosphorylation event.
FIGURE 4: Insulin stimulates phosphorylation of RGC2 in adipocytes. (A) Serum-starved 3T3-L1 adipocytes were mock treated or stimulated with 100 nM insulin for 10 min, and lysates were immunoprecipitated using control rabbit IgG or anti-RGC2 antibody. The immune (more ...)
To test whether Akt catalyzed the phosphorylation of RGC2 on these sites, immunoprecipitated RGC1/2 complex was subjected to an in vitro phosphorylation assay in the presence of recombinant Akt2. Akt2 directly phosphorylated all three sites on RGC2 (A). To ascertain the Akt isoform responsible for RGC2 phosphorylation in cells, we depleted Akt1, Akt2, or both isoforms in adipocytes by siRNA-mediated knockdown (B, top). Knockdown of Akt1 produced minimal inhibition of Akt signaling, as determined by blotting with the phospho-Akt antibody, while knockdown of Akt2 alone or in combination with Akt1 dramatically attenuated Akt activation (B, middle). SiRNA-mediated reduction in Akt2 activity led to inhibition of RGC2 phosphorylation on all three sites (B, bottom), supporting the notion that the GAP RGC2 is an endogenous substrate of Akt2 in adipocytes. Interestingly, acute inhibition of Akt activity by incubation of cells with wortmannin blunted insulin-stimulated phosphorylation on Ser696 of RGC2, while phosphorylation of Ser486 and Thr715 was less sensitive, indicating that these sites may require lower Akt activity for phosphorylation (C). Alternatively, it remains possible that Ser696 phosphorylation undergoes more efficient turnover. Collectively, the data indicate that activation of Akt2 via the PI 3-kinase pathway regulates RGC2 phosphorylation.
FIGURE 5: RGC2 is an endogenous substrate of Akt2 in adipocytes. (A) COS-1 cells were transfected with HA-RGC1 and FLAG-RGC2 constructs as indicated. The RGC proteins were isolated by IP with FLAG antibody and subjected to an in vitro kinase assay with 100 ng recombinant (more ...)
To further test this hypothesis, we used an active form of Akt2 in which the lipid-binding PH domain is replaced with a myristoylation sequence (Myr-Akt ΔPH). RGC2 phosphorylation was readily detectable when expressed in 293T cells that possess high endogenous Akt activity and was inhibited after blockade of PI 3-kinase/Akt activity by wortmannin, as confirmed by phospho-Akt blotting (D). However, although wortmannin blocked signaling events upstream of Akt, inhibition of RGC phosphorylation with this compound could be bypassed by the expression of Myr-Akt ΔPH, demonstrating that this phosphorylation event is catalyzed directly by Akt (D). To further confirm that phosphorylation of RGC2 is dependent on Akt activity, we also used Akti-1/2, an inhibitor with high specificity for Akt kinases by targeting the PH domain (Lindsley et al., 2005; Logie et al., 2007). Treating adipocytes with Akti-1/2 led to inhibition of Akt-dependent phosphorylation events, as detected by phospho-FKHR and phospho-TSC2 WB, whereas Akt-independent phosphorylation events remained intact, as visualized by phosphotyrosine and phospho-Erk blotting (E). Interestingly, treatment of 3T3-L1 adipocytes with Akti-1/2 blunted insulin-stimulated phosphorylation of RGC2 at Ser486, Ser696, and Thr715 (E). These data suggest that insulin, via activation of Akt2, differentially regulates RGC2 phosphorylation on different sites.
RalA is activated through inhibition of the Ral GAP complex
It has been suggested that the GAP activity of TSC2 and AS160 against their target G proteins is blocked by Akt-catalyzed phosphorylation (Inoki et al
; Manning et al
; Sano et al
; Cai et al
), although the molecular mechanism responsible remains unknown. We postulated that, like TSC2 and AS160, Akt might negatively regulate the inhibitory effect of RGC proteins on RalA. Consistent with this idea, expression of myristoylated, constitutively active Akt (Myr-Akt) produced an increase in wild-type RalA activity (A). To determine whether the stimulation of RalA by Akt occurred through regulation of the GAP, we coexpressed Myr-Akt with RalAF39
L, a fast-exchanging RalA that is activated independently of GEFs but is still responsive to GAPs (A). Because RalAF39
L displays high activity, low concentrations of the mutant were expressed with Myr-Akt so that the amount of RalA in the pulldown would remain within the linear range of the assay. Interestingly, expression of Myr-Akt activated RalAF39
L, suggesting that Akt can activate RalA by inhibiting its GAPs (A). Moreover, coexpression of activated Akt with RGC1/2 alleviated the inhibition of RalA by the GAP complex (B). In addition, inhibition of Akt activity by treatment of adipocytes with Akti-1/2 blunted RalA activation by insulin (, C and D). Taken together, these data suggest that the RGC1/2 proteins provide a direct link for the activation of the small GTPase RalA by Akt.
FIGURE 6: Insulin activates RalA by inhibiting the Ral GAP complex. (A) COS-1 cells were transfected with FLAG-RalA, FLAG-RalAF39L, and Myr-Akt-HA as indicated. Cell lysates were subjected to a pulldown assay to detect RalA activation. Pulldowns represent the amount (more ...)
Previous investigations of GAPs downstream of Akt such as TSC2 or AS160 have suggested that phosphorylation may directly inhibit catalytic activity, though there is little data to definitively evaluate this model. Alternatively, phosphorylation could restrict substrate accessibility of the GAP, therefore leading to the activation of the downstream GTPases. To evaluate whether phosphorylation of RGC2 might directly attenuate its GAP activity, we phosphorylated the immunoprecipitated protein in vitro by incubating with Akt2 kinase and assayed its activity as a GAP for RalA. Akt-catalyzed phosphorylation produced little change in the GAP activity of the RGC immune complex toward recombinant RalA (Figure S5). Because it was possible that our failure to observe a change in GAP activity reflected a low degree of phosphorylation of the complex, we attempted to isolate stoichiometrically phosphorylated GAP complex by immunoprecipitation with the phosphospecific antibody toward the 715 site (p715) on RGC2, with the nonphospho antibody generated from the same epitope (pan-715) as a control (E), followed by assay of GAP activity. RGC2 isolated from basal or insulin-stimulated adipocytes by the pan-715 antibody showed little difference in GAP activity, and phosphorylated RGC2 purified by the p715 antibody from insulin-stimulated adipocytes displayed essentially the same GAP activity, offering evidence that phosphorylation may not inhibit RGC2 catalytic capacity per se (E).
Despite the fact that phosphorylation did not appear to modulate the catalytic activity of RGC2 in vitro, we noticed that the recognition of the native protein by the RGC1 antibody was increased in cells treated with insulin, as visualized by an increased amount of RGC1 in the immunoprecipitates (F, top). This was not due to an increase in the amount of antibody or lysate that was included in the immunoprecipitation reaction (F, Ponceau S staining). These data suggest that insulin augments exposure of the antigenic sequence to the RGC1 antibody, raising the possibility that phosphorylation produces a conformational change in the protein complex involving the regulatory subunit. These data prompted us to evaluate the interaction between RalA and the phosphorylated complex. When expressed in 293T cells with high endogenous Akt activity, the RGC had little interaction with RalAQ72L, a mutant capable of stabilizing GAP interactions (G). Treating the cells with wortmannin lowered Akt activity and consequently RGC2 phosphorylation but increased the amount of RalA in complex with immunoprecipitated RGC1/2 (G), suggesting that phosphorylation led to reduced access of RGC2 GAP to its substrate. Together, these data suggest that insulin activates the protein kinase Akt to directly phosphorylate the RGC, which produces a conformational change in the complex that decreases its access to RalA, resulting in increased activity of the G protein.
RGC proteins regulate GLUT4 trafficking in 3T3-L1 adipocytes
Activation of Akt kinases downstream of PI 3-kinase plays an essential role in insulin-stimulated GLUT4 exocytosis (Whiteman et al
; Watson and Pessin, 2006
), and expression of an activated version of the kinase can promote glucose uptake in adipocytes, although to an extent less than that seen with insulin (Kohn et al
; Ng et al
). We and others have reported that the exocyst complex is required for targeting of GLUT4 vesicles to the plasma membrane in 3T3-L1 adipocytes (Inoue et al
; Ewart et al
), a process that requires activation of RalA in a PI 3-kinase–dependent manner (Chen et al
). This led us to speculate that the RGC proteins, which inhibit RalA activity, may function as negative regulators of GLUT4 exocytosis. Consistent with this idea, Lienhard and colleagues found that adipocytes differentiated from fibroblasts stably expressing AS250/RGC2 knockdown shRNA displayed increased GLUT4 exocytosis (Gridley et al
). However, it remains possible that stable knockdown of RGC2 achieved in these experiments may have affected the differentiation states of adipocytes or led to adaptation of cells to the increased activity of Ral GTPases. To avoid these caveats, we acutely depleted RGC1 or RGC2 by siRNA-mediated knockdown in mature 3T3-L1 adipocytes. Loss of RGC2 in adipocytes led to a decrease of RGC1 (A), a phenomenon not evident in cells in which RGC2 was stably knocked down (Gridley et al
), further implying a potential compensatory process in these cells. Depletion of RGC1 or RGC2 in mature adipocytes did not affect proximal signaling events, including phosphorylation of insulin receptor, Akt, or Erk, nor were there changes in the expression of adipocyte-specific proteins such as GLUT4 or PPARγ, as determined by WB with the specific antibodies (A). Knockdown of RGC1 or RGC2 had little effect on basal glucose uptake despite an increase in basal RalA activity (D, 3A, and 7B), perhaps because RalA activation alone is not sufficient for glucose uptake or because the RalA activity observed in an RGC knockdown is not sufficient to increase basal glucose uptake. However, loss of RGC1 or RGC2 caused an increase in glucose uptake after stimulation with submaximal (1 nM) and maximal (100 nM) insulin concentrations when compared with cells transfected with control oligos (B). Considering that GLUT1 transporters also contribute to glucose uptake in cultured adipocytes (Liao et al
), and the fact that siRNA cannot completely abolish gene expression, these data suggest that the RGC regulates insulin-stimulated GLUT4 trafficking in adipocytes. Previous studies have shown that knockdown of the Rab GAP AS160 increases basal glucose uptake with little effect on insulin-stimulated glucose transport (Eguez et al
), indicating that AS160 and the RGC proteins may engage different mechanisms to regulate GLUT4 trafficking.
FIGURE 7: The Ral GAP complex regulates glucose uptake and GLUT4 trafficking in adipocytes. (A) 3T3-L1 adipocytes were transfected with control siRNA or siRNA to deplete RGC1 or RGC2. After 7 d, cells were serum starved for 6 h and then mock treated or stimulated (more ...)
To examine the effect of RGC knockdown on GLUT4 trafficking, 3T3-L1 adipocytes were infected with a lentivirus expressing a Myc-GLUT4-eGFP construct that allows for quantification of fused GLUT4 in nonpermeabilized cells. Cells that were transfected with siRNA to deplete both RGC1 and RGC2 demonstrated a −70–80% knockdown of each protein (D). Consistent with the glucose uptake data, cells that were depleted of RGC1/2 by siRNA exhibited a 25% increase of cells with exofacial Myc staining when stimulated with 1 nM insulin and a trend of increased GLUT4 insertion when stimulated with 100 nM insulin (, C and E). To quantitatively assess the effect of RGC1/2 knockdown on GLUT4 insertion into the plasma membrane, the amount of Myc rim staining versus total eGFP fluorescence was determined. Cells depleted of RGC1/2 displayed an increase in Myc rim staining versus total eGFP fluorescence when stimulated with 1 nM insulin (F). Together these data indicate that loss of the RGC results in increased plasma membrane GLUT4 levels, either by increased exocytosis, decreased endocytosis, or perhaps regulating both endo- and exocytosis of the glucose transporter. Thus, the RGC is a novel component of the insulin signaling pathway that regulates GLUT4 trafficking and glucose uptake in adipocytes.