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. 2017 Feb 3;292(5):2032-2045.
doi: 10.1074/jbc.M116.753277. Epub 2016 Dec 12.

Signal Integration at Elongation Factor 2 Kinase: THE ROLES OF CALCIUM, CALMODULIN, AND SER-500 PHOSPHORYLATION

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Free PMC article

Signal Integration at Elongation Factor 2 Kinase: THE ROLES OF CALCIUM, CALMODULIN, AND SER-500 PHOSPHORYLATION

Clint D J Tavares et al. J Biol Chem. .
Free PMC article

Abstract

Eukaryotic elongation factor 2 kinase (eEF-2K), the only calmodulin (CaM)-dependent member of the unique α-kinase family, impedes protein synthesis by phosphorylating eEF-2. We recently identified Thr-348 and Ser-500 as two key autophosphorylation sites within eEF-2K that regulate its activity. eEF-2K is regulated by Ca2+ ions and multiple upstream signaling pathways, but how it integrates these signals into a coherent output, i.e. phosphorylation of eEF-2, is unclear. This study focuses on understanding how the post-translational phosphorylation of Ser-500 integrates with Ca2+ and CaM to regulate eEF-2K. CaM is shown to be absolutely necessary for efficient activity of eEF-2K, and Ca2+ is shown to enhance the affinity of CaM toward eEF-2K. Ser-500 is found to undergo autophosphorylation in cells treated with ionomycin and is likely also targeted by PKA. In vitro, autophosphorylation of Ser-500 is found to require Ca2+ and CaM and is inhibited by mutations that compromise binding of phosphorylated Thr-348 to an allosteric binding pocket on the kinase domain. A phosphomimetic Ser-500 to aspartic acid mutation (eEF-2K S500D) enhances the rate of activation (Thr-348 autophosphorylation) by 6-fold and lowers the EC50 for Ca2+/CaM binding to activated eEF-2K (Thr-348 phosphorylated) by 20-fold. This is predicted to result in an elevation of the cellular fraction of active eEF-2K. In support of this mechanism, eEF-2K knock-out MCF10A cells reconstituted with eEF-2K S500D display relatively high levels of phospho-eEF-2 under basal conditions. This study reports how phosphorylation of a regulatory site (Ser-500) integrates with Ca2+ and CaM to influence eEF-2K activity.

Keywords: CaMK-III; S500D; Ser-500; Thr-348; calcium; calmodulin (CaM); eEF-2K; phosphorylation; translation; translation elongation factor.

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

FIGURE 1.
FIGURE 1.
Proposed organization of eEF-2K and location of regulatory phosphorylation sites. The illustration shows the proposed organization of full-length eEF-2K (725 residues). The N terminus of eEF-2K contains a CBD and an atypical catalytic kinase domain. The C terminus is composed of predicted SLRs. A disordered regulatory loop (R-loop) (located between the kinase domain and SLRs) contains a number of regulatory phosphorylation sites. The primary autophosphorylation site Thr-348 and the regulatory site Ser-500 are shown. Ser-500 is also phosphorylated by PKA.
FIGURE 2.
FIGURE 2.
Two-step mechanism for the Ca2+/CaM-mediated activation of eEF-2K. Ca2+/CaM binds to eEF-2K (red and blue schematic; NTD, N-terminal domain; CTD, C-terminal domain; black loop represents the R-loop; see Fig. 1), and induces autophosphorylation at Thr-348.
FIGURE 3.
FIGURE 3.
Ca2+/CaM-stimulated autophosphorylation sensitizes eEF-2K to CaM through phosphorylation at Ser-500. A, eEF-2K Ca2+-independent activity and the extent of Ser-500 phosphorylation against the time of autophosphorylation. eEF-2K (500 nm) was allowed to autophosphorylate in the presence of CaM (5 μm) and free Ca2+ (50 μm). At various times, the reaction was quenched for 1 min by diluting an aliquot of autophosphorylated eEF-2K 5-fold in a buffer containing EGTA (final concentration of 5 mm), following which kinase activity was assessed against a peptide substrate. Western blotting was used to detect phosphorylation on Ser-500, and both eEF-2K activity and phosphate incorporation at Ser-500 were recorded as the percentage of their maximal values (at 180 min). Inset, expansion of the data for 0–20 min. B, eEF-2K activity following autophosphorylation for 1 h in the presence of CaM (5 μm) and free Ca2+ (50 μm), and a 1-min quench by EGTA (5 mm). Kinase activity in the absence or presence of CaM, Ca2+, or EGTA, as indicated, is reported as a percentage of the maximal activity of eEF-2K obtained in the presence of saturating Ca2+ and CaM. C, Ca2+-independent kinase activity of autophosphorylated eEF-2K against 10 μm wheat germ eEF-2. Upper panel, autoradiograph. Lower panel, Coomassie-stained gel. D, scheme for the analysis of Ca2+-independent activity of eEF-2K with or without separation of CaM. 1, autophosphorylation of eEF-2K in the presence of Ca2+ and CaM. 2, autophosphorylation is quenched in a buffer containing EGTA (final concentration of 5 mm). 3a, without separation of CaM, the effect of autophosphorylation on Ca2+-independent eEF-2K activity against 150 μm peptide substrate is determined by assaying the autophosphorylated enzyme (50 nm) in a buffer containing 5 mm EGTA (lacking CaCl2 and CaM), resulting in a calculated [Ca2+]free of ∼0.2 nm, 500 nm CaM, and 7.5 mm EGTA. 3b, alternatively, to separate CaM, the autophosphorylated quenched sample is run over a HiPrepTM 26/60 SephacrylTM S-200 HR gel filtration column with a buffer containing 5 mm EGTA. Inset, upper panel, chromatogram (peak (i), eEF-2K; and peak (ii), CaM). Lower panel, corresponding peaks from chromatogram resolved by SDS-PAGE. 4b, autophosphorylated eEF-2K separated from CaM is assayed for Ca2+-independent activity against 150 μm peptide substrate in a buffer containing 5 mm EGTA (lacking CaCl2 and CaM). E, activity of autophosphorylated eEF-2K following separation from CaM (separation scheme depicted in D and described under “Experimental Procedures”). Where indicated, CaM, Ca2+, or EGTA was added to the assay. F, kobs versus [CaM] for eEF-2K WT (solid line) and eEF-2K S500D (dashed line) (monophosphorylated on Thr-348) in the absence of Ca2+. The lines correspond to the best fit through the data according to Equation 2 with the following parameters: kcatapp = 11 ± 0.13 s−1 and EC50 = 37 ± 1.1 μm (WT); and kcatapp = 25.7 ± 0.14 s−1 and EC50 = 1.5 ± 0.03 μm (S500D). G, eEF-2K (2 nm) (WT, S500A, and S500D) were assayed using 1 mm [γ-32P]ATP and 4 μm wheat germ eEF-2 in the presence and absence of CaM or Ca2+ as indicated. EGTA (1 mm) was added to all assays conducted in the absence of Ca2+. Upper panel, autoradiograph. Lower panel, Coomassie-stained gel.
FIGURE 4.
FIGURE 4.
Autophosphorylation of eEF-2K on Ser-500 is intramolecular, occurs in mammalian cells, and requires Ca2+, CaM, and prior phosphorylation of Thr-348. A, MDA-MB-231 cells were transfected with vectors encoding either FLAG-tagged eEF-2K WT or S500A. After 48 h, cells were treated with ionomycin (5 μm, 5 min). Lysates or eEF-2K immunoprecipitated using a FLAG antibody were analyzed by Western blotting using a phospho-specific antibody for Ser-500. B, MDA-MB-231 cells were transfected with vectors encoding eEF-2K WT, S500A or D284A. After 48 h, cells were treated with ionomycin and calyculin A, and lysates then analyzed by Western blotting for phosphorylation of Ser-500. C, HEK 293T cells were transfected with vectors encoding eEF-2K WT, S500A, or D284A. After 48 h, cells were treated with forskolin for 30 min, and the media were then supplemented with calyculin A for an additional 5 min. Lysates were analyzed by Western blotting with a phospho-specific antibody for Ser-500. D, various eEF-2K mutants were allowed to autophosphorylate in the presence of saturating CaM and Ca2+. The reactions were quenched after 1 h, and the samples were analyzed by Western blotting with a phospho-specific antibody for Ser-500. E, eEF-2K (500 nm) was allowed to autophosphorylate in the presence or absence of Ca2+ and/or CaM as indicated. Reactions were quenched after 1 h and analyzed by Western blotting for the various phosphorylated forms of eEF-2K. F, comparison between the rate of autophosphorylation at Ser-500 on eEF-2K expressed in mammalian cells and the recombinant kinase expressed in bacteria. MDA-MB-231 cells were transfected with a vector encoding eEF-2K WT and lysed after 48 h, and the lysate was incubated with Ca2+, CaM, and MgATP to allow for eEF-2K autophosphorylation. For comparison, recombinant human eEF-2K expressed in bacteria was allowed to autophosphorylate in the presence of Ca2+, CaM, and MgATP. Phosphorylation at Ser-500 was monitored by Western blotting. Con, control.
FIGURE 5.
FIGURE 5.
S500D promotes phosphorylation of eEF-2K on Thr-348. A, phosphorylation of Thr-348 versus time for eEF-2K WT and S500D in the presence of varying non-saturating concentrations of CaM in the absence of Ca2+. B, phosphorylation of Thr-348 versus time for eEF-2K WT and S500D in the presence of saturating concentrations of Ca2+ and CaM. A solution containing eEF-2K, Ca2+, and CaM was mixed with saturating MgATP. The reaction was quenched at various time points, and the samples were analyzed by Western blotting with a phospho-specific antibody for Thr-348. Blots were quantified and data plotted as the percent of Thr-348 phosphorylation as a function of autophosphorylation time and fit to Equation 1 to give kauto = 2.1 ± 0.4 s−1 (t½ = 330 ms) and kauto = 12 ± 0.5 s−1 (t½ = 58 ms) for eEF-2K WT and S500D, respectively. Results are the average of three independent experiments, and error bars represent S.D. C, binding of eEF-2K S500D to dansyl-CaM. The fraction of fluorescent dansyl-labeled CaM (dansyl-CaM) bound (FB) to eEF-2K was measured and used to determine the affinity of eEF-2K for CaM (KD), as described previously (35). Error bars represent S.D. of duplicate (WT) and quadruplicate (S500D) measurements. Data shown for eEF-2K WT were previously reported (35). The calculated KD values of eEF-2K WT and S500D are 23 ± 7 and 16 ± 7 nm, respectively. D and E, dephosphorylation of Thr-348 on recombinant eEF-2K WT or S500D (pre-autophosphorylated at Thr-348) in MDA-MB-231 cell lysates in the absence (D) or presence (E) of supplemented Ca2+, CaM, and MgATP. Blots were quantified, and data were plotted as the percent of Thr-348 phosphorylation versus time. Results are the average of three independent experiments, and error bars represent S.E. F, kobs versus [CaM] for eEF-2K WT (solid line) and eEF-2K S500D (dashed line) (monophosphorylated on Thr-348) in the presence of 50 μm Ca2+. The lines correspond to the best fit through the data according to Equation 2, and kcatapp = 24.5 ± 0.5 s−1, EC50 = 42 ± 1.7 nm (WT); and kcatapp = 24.9 ± 0.04 s−1, EC50 = 2.3 ± 0.02 nm (S500D).
FIGURE 6.
FIGURE 6.
Phosphorylation of Ser-500 stabilizes active conformations of eEF-2K. A, activation. Ca2+/CaM binds to eEF-2K, (Kd(I)) to induce a conformational transition from an inactive state (I · CaM) to an active state (A · CaM), Keq(IA). Phosphorylation of Ser-500 stabilizes the active state A · CaM relative to the inactive state I · CaM. For activation, we propose that Keq(IA) <1, giving EC50 = Kd(I) and kcat = kcautoKeq(IA). Thus, S500D enhances kcat but has no effect on EC50. B, substrate phosphorylation. Ca2+/CaM binds to eEF-2K, (Kd(I* · S)) to induce a conformational transition from an inactive state (I* · CaM · S) to an active state (F* · CaM · S), Keq(I*F*). Phosphorylation of Ser-500 stabilizes the active state F* · CaM · S relative to the inactive state I* · CaM · S. For activation, we propose that Keq(I*F*) >1, giving EC50 = Kd(I* · S)/Keq(I*F*) and kcat = kcauto. Thus, S500D enhances EC50 but has no effect on kcat.
FIGURE 7.
FIGURE 7.
eEF-2K S500D exhibits high activity in unstimulated cells. A, eEF-2K−/− MCF10A cells were transfected with various mutants of eEF-2K or a control (Cont) vector, and their activity was estimated by assessing eEF-2 phosphorylation by Western blotting. B, comparison of WT and mutant eEF-2K activity (normalized phospho-eEF-2Thr-56) calculated as described under “Experimental Procedures.”
FIGURE 8.
FIGURE 8.
Regulation of eEF-2K. The scheme shows a proposed mechanism for how Ca2+, CaM, and phosphorylation of Ser-500 regulate eEF-2K. Nucleotide binding (ATP) is assumed. Steps predicted to be influenced by Ca2+ or Ser-500 phosphorylation are indicated as circles containing Ca or 500, respectively. According to the mechanism, CaM binds eEF-2K, KCaM, to induce its autophosphorylation on Thr-348, kcauto, to give the fully activated complex, F* · CaM. This complex binds the substrate eEF-2 (S) to give F* · CaM · S, which catalyzes the phosphorylation of eEF-2, which then dissociates, kcsub. Dissociation of CaM from the fully activated complexes, F* · CaM and F* · CaM · S, leads to the inactive complexes I* and I* · S, respectively. Ca2+ enhances the fraction of active eEF-2K by promoting the binding of Ca2+/CaM to I, I*, and I* · S as indicated. Phosphorylation of Ser-500 promotes the fraction of active eEF-2K, as indicated, by enhancing the rate of Thr-348 autophosphorylation, kcauto, to elevate the concentration of activated complexes (e.g. F* · CaM and F* · CaM · S), and by enhancing the association of Ca2+/CaM to I* · S.

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