Structural basis for the allosteric inhibitory mechanism of human kidney-type glutaminase (KGA) and its regulation by Raf-Mek-Erk signaling in cancer cell metabolism

Abstract

Besides thriving on altered glucose metabolism, cancer cells undergo glutaminolysis to meet their energy demands. As the first enzyme in catalyzing glutaminolysis, human kidney-type glutaminase isoform (KGA) is becoming an attractive target for small molecules such as BPTES [bis-2-(5 phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide], although the regulatory mechanism of KGA remains unknown. On the basis of crystal structures, we reveal that BPTES binds to an allosteric pocket at the dimer interface of KGA, triggering a dramatic conformational change of the key loop (Glu312-Pro329) near the catalytic site and rendering it inactive. The binding mode of BPTES on the hydrophobic pocket explains its specificity to KGA. Interestingly, KGA activity in cells is stimulated by EGF, and KGA associates with all three kinase components of the Raf-1/Mek2/Erk signaling module. However, the enhanced activity is abrogated by kinase-dead, dominant negative mutants of Raf-1 (Raf-1-K375M) and Mek2 (Mek2-K101A), protein phosphatase PP2A, and Mek-inhibitor U0126, indicative of phosphorylation-dependent regulation. Furthermore, treating cells that coexpressed Mek2-K101A and KGA with suboptimal level of BPTES leads to synergistic inhibition on cell proliferation. Consequently, mutating the crucial hydrophobic residues at this key loop abrogates KGA activity and cell proliferation, despite the binding of constitutive active Mek2-S222/226D. These studies therefore offer insights into (i) allosteric inhibition of KGA by BPTES, revealing the dynamic nature of KGA's active and inhibitory sites, and (ii) cross-talk and regulation of KGA activities by EGF-mediated Raf-Mek-Erk signaling. These findings will help in the design of better inhibitors and strategies for the treatment of cancers addicted with glutamine metabolism.

Fig. 1.

Schematic view and structure of the cKGA-l-glutamine complex. (A) Human KGA domains and signature motifs (refer to Fig. S1A for details). (B) Structure of the of cKGA and bound substrate (l-glutamine) is shown as a cyan stick. (C) Fourier 2F_o-F_c electron density map (contoured at 1 σ) for l-glutamine, that makes hydrogen bonds with active site residues are shown.

Fig. 2.

Structure of cKGA: BPTES complex and the allosteric binding mode of BPTES. (A) Structure of cKGA dimer and BPTES is shown as a cyan stick. (B) A close-up view of the interactions of BPTES in the cKGA allosteric inhibitor binding pocket. (C) Electron density map (2F_o \x{2013} F_c map, contoured at 1.0σ) for BPTES is shown. (D) A close-up view of the BPTES binding pocket on the surface exposed region of the loop Glu312-Pro329 at the dimer interface. (E) Perpendicular view of dimer interface formed by the sulphate ion, hydrogen bonding, salt bridge, and hydrophobic interactions between residues from each monomer. (F) Conformational changes on cKGA induced by binding of the BPTES. For clarity only half of the BPTES is shown. Structure superposition of monomeric BPTES complex (magenta) and apo cKGA (green), showing conformational changes of key residues on the loop Glu312-Pro329. The BPTES binding site is located ∼18 Å away from the active site (Ser286).

Fig. 3.

Mutations at allosteric loop and BPTES binding pocket abrogate KGA activity and BPTES sensitivity. (A) 293T cells were transfected with vector control or plasmids expressing wild-type, single, or multiple point mutants of HA-tagged KGA for 24 h before cell lysates were prepared for glutaminase assays. For clarity, the dotted line was included to indicate the basal level. Equal expression levels of the wild-type and mutants KGA were verified with Western blots. Each value represents the mean ± SD of three independent experiments. (B) Mutational analyses of cKGA residues in the BPTES binding pocket. BPTES sensitivity for the wild-type and cKGA mutants indicated were measured and their IC₅₀ values calculated. Each value represents the mean ± SD of three independent experiments performed in duplicate.

Fig. 4.

EGFR-Raf-Mek-Erk signaling stimulates KGA activity. (A) 293T cells expressing HA-tagged KGA were starved for 24 h and then stimulated with EGF (100 ng/mL) for the times indicated. Cells were lysed and assayed for their glutaminase activities. The expression levels of KGA and the Erk activation profile (as indicated by levels of phosphorylated Erk) were verified by Western blot analyses. (B) Cells expressing Flag-tagged KGA with or without the HA-tagged wild-type, dominant negative mutants (Raf-1-K375M; Mek2-K101A) or constitutive active mutants (Raf-1-Y340D; Mek2-S222, 226D) were lysed and assayed for glutaminase activity. (C) Same batch of cell lysates prepared for the glutaminase assay in B were subjected to immunoprecipitation (IP) with anti-Flag M2 beads. Bound proteins and their expression in whole-cell lysates (WCL) were analyzed with Western blot. (D) Cells expressing Flag-tagged KGA were lysed for immunoprecipitation using anti-Flag M2 beads and analyzed for the presence of endogenous Raf-1 or Erk1/2 by Western blot analyses. Arrow denotes band for Raf-1. (E) 293T cells were transfected with vector control or plasmids expressing wild-type KGA or the KGA triple mutant (L321A/F322A/L323A), in the absence or presence of Mek2-K101A or Mek2-S222,226D for 24 h before lysates were prepared for glutaminase assays. Expression levels of these proteins were verified by Western blot analyses. All values are mean ± SD of three independent experiments, each with multiple replicates. Data sharing different letters are statistically significant at P values as indicated, tested by ANOVA or t test.

Fig. 5.

KGA activity is regulated by phosphorylation. (A) Lysates were prepared from cells expressing Flag-tagged KGA in the presence or absence of myc-tagged catalytic subunit of the protein phosphatase PP2A and assayed for the glutaminase activity. Values are means ± SD of three independent experiments. Data sharing different letters are statistically significant at P < 0.02, as tested by ANOVA. (B) Separate aliquots from the same batch of cell lysates prepared for the glutaminase assay in A were subjected to immunoprecipitation (IP). Bound myc-PP2A and their expression levels in whole-cell lysates (WCL) were analyzed by Western blots. (C) Schematic model depicting the synergistic cross-talk between KGA-mediated glutaminolysis and EGF-activated Raf-Mek-Erk signaling. Exogenous glutamine can be transported across the membrane and converted to glutamate by glutaminase (KGA), thus feeding the metabolite to the ATP-producing tricarboxylic acid (TCA) cycle. This process can be stimulated by EGF receptor-mediated Raf-Mek-Erk signaling via their phosphorylation-dependent pathway, as evidenced by the inhibition of KGA activity by the kinase-dead and dominant negative mutants of Raf-1 (Raf-1-K375M) and Mek2 (Mek2-K101A), protein phosphatase PP2A, and Mek-specific inhibitor U0126. Consequently, inhibiting KGA with BPTES and blocking Raf-Mek pathway with Mek2-K101A provide a synergistic inhibition on cell proliferation. Refer to the text for more details.