Tuberous sclerosis tumor suppressor complex-like complexes act as GTPase-activating proteins for Ral GTPases

Abstract

The small GTPases RalA and RalB are multifunctional proteins regulating a variety of cellular processes. Like other GTPases, the activity of Ral is regulated by the opposing effects of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Although several RalGEFs have been identified and characterized, the molecular identity of RalGAP remains unknown. Here, we report the first molecular identification of RalGAPs, which we have named RalGAP1 and RalGAP2. They are large heterodimeric complexes, each consisting of a catalytic alpha1 or alpha2 subunit and a common beta subunit. These RalGAP complexes share structural and catalytic similarities with the tuberous sclerosis tumor suppressor complex, which acts as a GAP for Rheb. In vitro GTPase assays revealed that recombinant RalGAP1 accelerates the GTP hydrolysis rate of RalA by 280,000-fold. Heterodimerization was required for this GAP activity. In PC12 cells, knockdown of the beta subunit led to sustained Ral activation upon epidermal growth factor stimulation, indicating that the RalGAPs identified here are critical for efficient termination of Ral activation induced by extracellular stimuli. Our identification of RalGAPs will enable further understanding of Ral signaling in many biological and pathological processes.

FIGURE 1.

The p240-p170 complex purified from porcine brain has RalGAP activity. A, RalA^Q72L affinity column eluate was analyzed by SDS-PAGE and Coomassie Blue staining. Components of the exocyst complex (Sec8, Sec5, and Sec6) and GST-RalA^Q72L that had leaked from the column are indicated, as identified by Western blotting (not shown). B, upper panel, the mixture of RalA^Q72L-binding proteins was separated on a Superose 6 gel filtration column, and the resulting fractions were analyzed by SDS-PAGE and Coomassie blue staining. Lower panel, each fraction was tested for RalGAP activity by a filter binding assay using [γ-³²P]GTP-loaded RalA as described under “Experimental Procedures.” C, various Ras family GTPases preloaded with [γ-³²P]GTP were incubated at 30 °C for the indicated periods with (open circles) or without (closed circles) fraction 18, and the [γ-³²P]GTP remaining bound to GTPases was measured by the filter binding assay (mean ± S.E., n = 2). D, Ras subfamily GTPases preloaded with [α-³²P]GTP were incubated at 30 °C for 10 min with (+) or without (−) fraction 18, and bound nucleotides were analyzed by thin layer chromatography and autoradiography. E, co-immunoprecipitation of p240 and p170 from porcine brain cytosol. IP, immunoprecipitation. F, immunodepletion of p240 from fraction 18 results in a concomitant depletion of p170 (left panel) and abolishes the GAP activity for RalA (right graph) (mean ± S.E., n = 3). ID, immunodepletion. G, schematic comparison of the domain structure of the p240-p170 complex, the TSC2-TSC1 complex, and Rap1GAP. aa, amino acids.

FIGURE 2.

Biochemical and catalytic properties of the recombinant p240-p170 complex. A, chromatographic comparison of the recombinant p240-p170 complex (top), p240 alone (middle), and p170 alone (bottom) on Superose 6 gel filtration chromatography. Gels were Coomassie Blue-stained. B, autoradiograph showing that the recombinant complex stimulates Ral GTPase activity. [α-³²P]GTP-loaded RalA or RalB were incubated at 30 °C for 10 min with (+) or without (−) the recombinant complex (5 nm). C, kinetic analysis of the recombinant p240-p170 complex. Initial rates of GTP hydrolysis on RalA were determined at 30 °C at increasing concentrations of GTP-bound RalA by the filter binding assay. The recombinant p240-p170 complex was used at 1 nm ([E₀]). GTPase reaction rates were fitted to the Michaelis-Menten equation to give K_m (12.1 μm) and k_cat (46.8 s⁻¹) values of the reaction. D, p240 requires p170 for GAP activity. [γ-³²P]GTP-loaded RalA or RalB were incubated at 30 °C for 10 min with the recombinant complex, p240 alone, p170 alone, or a mixture of separately purified p240 and p170 at the indicated concentrations (mean ± S.E., n = 3). E, the N1950K mutation abrogates p240 GAP activity. [γ-³²P]GTP-loaded RalA or RalB were incubated at 30 °C for the indicated periods with the wild-type complex (WT; 2 nm or 10 nm) or the mutant complex (N1950K; 10 nm) (mean ± S.E., n = 3).

FIGURE 3.

The p220-p170 complex; a second RalGAP identified from rat lung. A, comparison of RalA^Q72L-binding proteins from rat brain and lung cytosol. RalA^Q72L affinity column eluates were fractionated by gel filtration, and fractions containing p170 (fractions 17–22) were pooled and analyzed by SDS-PAGE and Coomassie Blue staining. p240, p220, p170, and the components of the exocyst complex are indicated, as identified by Western blotting and mass spectrometry. B, co-immunoprecipitation of p220 and p170 from rat testis cytosol. Note that p220 is not co-purified with p240. IP, immunoprecipitation. C, the pooled lung fraction depleted of p240 retains RalGAP activity. Left panel, the pooled lung fraction was treated with anti-p240 IgG- or preimmune IgG-coated beads, and the resulting supernatants were analyzed for the specified proteins. Asterisks denote degradation products of p170. Right graphs, RalGAP activity of the immunodepleted fractions was measured by the filter-binding assay (mean ± S.E., n = 2). ID, immunodepletion. D, autoradiograph showing that the p240-depleted lung fraction has Ral-specific GAP activity. Ras subfamily GTPases preloaded with [α-³²P]GTP were incubated at 30 °C for 10 min with (+) or without (−) the p240-depleted lung fraction. E, immunoprecipitates prepared from rat lung cytosol with preimmune IgG (IP beads 1), anti-p220 IgG (IP beads 2), or anti-p220 IgG absorbed with p220 antigen peptide (IP beads 3) or p240 antigen peptide (IP beads 4) were used in GTPase assays with the indicated GTPases preloaded with [α-³²P]GTP. After 10 min of incubation at 30 °C, bound radiolabeled nucleotides were analyzed by thin layer chromatography and autoradiography. The right panel shows Western blot analysis of the immunoprecipitates with the indicated antibodies. F, Western blot analysis of p240, p220, and p170 in rat tissues.

FIGURE 4.

p170 knockdown leads to sustained activation of RalA. A, PC12 or NIH3T3 cells transfected with the indicated siRNAs were lysed, and the levels of the GTP-bound form of RalA (RalA-GTP) were determined by a pulldown assay using Sec5 Ral binding domain as described under “Experimental Procedures.” Lysates were also analyzed for the levels of the specified proteins. B, PC12 cells transfected with the indicated siRNAs were serum-starved and then stimulated with EGF (20 ng ml⁻¹). At the indicated time points, cells were lysed, and the amounts of RalA-GTP and Ras-GTP in the same lysate were determined. Lysates were also probed for phosphorylated ERK1/2. The lower graphs show quantification of Ras-GTP and RalA-GTP levels (means ± S.E., n = 3).

FIGURE 5.

The RalGAP complexes. Schematic representation of RalGAP1 and RalGAP2. RalGAP1 is composed of the catalytic subunit RalGAPα1 (p240) and the common subunit RalGAPβ (p170). RalGAP2 is composed of the catalytic subunit RalGAPα2 (p220) and the common subunit RalGAPβ (p170).