eEF2 is phosphorylated on serine 595 by cyclin A-CDK2 in vitro.
We initially identified eEF2 in a proteomic screen for CDK2 substrates in fractionated cell lysates (10
). This approach employed recombinant cyclin A-CDK2 containing a CDK2 mutant that utilizes bulky ATP analogs and 2-phenylethyl-ATP-γ-S to thiophosphorylate substrates. This strategy allowed substrate enrichment based upon thiophosphate chemistry, and candidate substrates were subsequently identified by mass spectroscopy (27
). eEF2 was among the initial group of substrates that we used to validated the mass spectrometry results (22
We first confirmed that myc-tagged cyclin A-CDK2 immunoprecipitated from transfected 293A cells robustly phosphorylates eEF2 in vitro (). Our initial screen was not saturated with respect to phosphorylation site identifications, and we used site-directed mutants to determine which residue(s) is phosphorylated by cyclin A-CDK2. eEF2 contains 6 potential proline-directed phosphorylations, two of which, T435 and S595, are CDK consensus sites (S/T-P-X-K/R) (). We mutated most of these sites to alanines, either individually or in combinations, and phosphorylated these mutants with cyclin A-CDK2 ( and not shown). The S595A mutation, either as a single mutation or when combined with other mutations, resulted in greatly reduced eEF2 phosphorylation by cyclin A-CDK2 in vitro (, lanes 5 and 9). In contrast, the remaining mutations had minimal impact on bulk eEF2 phosphorylation ( and not shown).
Cyclin-CDKs often redundantly phosphorylate substrates, and we next examined the ability of other cyclin-CDKs to phosphorylate eEF2. Both cyclin B-CDC2 and cyclin E-CDK2 phosphorylated eEF2 to roughly the same extent as cyclin A-CDK2, when normalized to histone H1 kinase activity ( and ). Thus, like most CDK substrates, eEF2 is phosphorylated by multiple cyclin-CDKs. We also assessed the specificity of eEF2 phosphorylation by CDKs by testing two additional proline-directed kinases. Neither glycogen synthase kinase 3β (GSK3β) nor p42 mitogen-activated protein kinase (MAPK) phosphorylated eEF2 (), whereas they phosphorylated known substrates (GSK3β-cyclin E and MAPK) (c-Jun and 4EBP1) ( to ).
Because site-directed mutations can impact phosphorylation of other sites, we used two-dimensional mapping of phosphopeptides generated by trypsin digestion to identify eEF2 phosphorylations. The immunoprecipitated FLAG-eEF2 proteins shown in were excised from the membrane, digested with trypsin, and subjected to phosphopeptide mapping. The predicted eEF2 tryptic map is shown in , whereas the mapping procedures used and the properties of the major peptides discussed below are represented in and . Phosphorylation of eEF2 by cyclin A-CDK2 produced two major phosphopeptides (, map 1, spots A and B). Both spots were eliminated by the S595A mutation (, map 2) but were unaffected by a triple S279A/T435A/S502A mutation (, map 3), suggesting that they both contained S595. In addition to the lack of spots A and B, the S595A map revealed three minor spots (, map 2, arrows) that were abrogated by a S279A/T435A/S502A mutation (, maps 3 and 4), indicating these were likely minor CDK2-phosphorylation sites. Although our initial proteomic screen identified eEF2 T435 phosphorylation by engineered cyclin A-CDK2 in cell lysates (22
), the in vitro
phosphorylation experiments and peptide maps revealed that S595 is the major phosphorylation site and that the contribution of additional residues, including T435, to overall eEF2 phosphorylation is minor. This discrepancy likely resulted from properties of the small S595 peptide (SPNK) that prevented its efficient identification by our proteomic methods, as well as from methodological differences between the screen and our current studies.
The presence of a single phosphorylation site in two spots often results from partial tryptic digestion. We hypothesized that the more positively charged spot B was derived from spot A by fusion with the next C-terminal peptide, since partial cleavage at the N terminus would result in the production of a neutral peptide (oxidized cysteine residues are negatively charged under pH 1.9 conditions). We thus mutated histidine 599 to proline (H599P), which prevents trypsin cleavage at lysine 598 and mimics the partial digestion product by extending the S595-containing peptide to arginine 601 ( and ). As predicted, the H599P mutant resulted in a single major S595 phosphopeptide corresponding to spot B () that was eliminated when S595 was also mutated (). A minor spot that comigrates with spot A, or migrates nearby, was seen in the H599 maps (asterisks, to ) that likely corresponds to a minor CDK site seen with eEF2 S595A (, map 2). Of note, the partial trypsin digestion of the S595 peptide resulting in spot B differs somewhat between experiments, and in some cases, nearly complete digestion leads to reduced amounts of spot B (not shown).
In order to prove that S595 is phosphorylated by cyclin A-CDK2 in vitro, we mutated S595 to threonine (S595T) in both the wild-type (WT) and H599P backgrounds, which rescued normal amounts of eEF2 phosphorylation (). We purposely overexpressed the S595A mutants relative to the WT and S595T eEF2 proteins to reveal residual phosphorylations () and to highlight the reduced eEF2 phosphorylation caused by S595A mutants even when they are relatively overexpressed (). Importantly, phosphoamino acid analysis demonstrated that spot B was converted from phosphoserine to phosphothreonine when S595 was mutated to T595, thereby demonstrating that S595 is the phosphorylated residue in this phosphopeptide ( and ). In sum, these data indicate that S595 is the major site of eEF2 phosphorylation by cyclin A-CDK2 in vitro.
eEF2 is phosphorylated on serine 595 in vivo.
We also used phosphopeptide mapping to characterize eEF2 phosphorylation in vivo by incubating transfected cells with [32P]orthophosphate. These experiments utilized physiologic amounts of FLAG-eEF2 protein that were immunoprecipitated from transfected 293T cells (), excised from membranes after electrophoresis and transfer, and subjected to phosphopeptide mapping as described above. In vivo eEF2 maps also contained spots A and B, and both were eliminated by the S595A mutation (, maps 1 and 2). These maps also revealed a third major phosphopeptide, spot C, which represents the T56 phosphopeptide, as shown by its disappearance from the map of an eEF2 T56A mutant (). Analogous to the in vitro maps, in vivo S595 phosphorylation shifted to spot B in the H599P mutant (, map 3) and was eliminated by a concordant S595A/H599P mutation (, map 4). Although it is difficult to quantitatively compare the amounts of phosphorylation of a specific peptide between independent maps, spot C appeared reduced in S595A and almost absent from H599P, suggesting a relationship between T56 phosphorylation and S595 (see below).
Fig 3 eEF2 S595 phosphorylation in vivo. (A) Phosphopeptide maps of eEF2 (map 1) and the indicated mutants (maps 2 to 4) isolated from 293T cells labeled with [32P]orthophosphate. The peptides representing S595 (spots A and B) and T56 (spot C) are indicated. (more ...)
To determine if cyclin A-CDK2 activity increases S595 phosphorylation in vivo, we labeled U2OS cells that were cotransfected with eEF2 and cyclin A-CDK2 with [-32P]orthophosphate. Ectopic cyclin A-CDK2 increased eEF2 phosphorylation in vivo (, left), and this involved spots A and B as well as three additional spots (, right, arrows) that correspond to the minor in vitro phosphorylation sites (, map 2). Cyclin A-CDK2 similarly increased eEF2 H599P phosphorylation on S595 and the three minor spots (). We speculate that the remaining phosphopeptides seen with eEF2 but not eEF2 H599P, which are not sensitive to cyclin A-CDK2, are likely derived from the T56 peptide. We conclude that eEF2 is phosphorylated on S595 in vivo and that eEF2 phosphorylation is augmented by ectopic cyclin A-CDK2 expression.
eEF2 S595 phosphorylation is increased in mitosis.
Cyclin A-CDK2 is active in the nucleus, whereas eEF2 is cytoplasmic. Thus, unlike the results seen with our initial lysate screen or in vitro
experiments, subcellular localization may limit eEF2 access to cyclin A-CDK2 in vivo
. Cyclin A-CDK2 shuttles from the cytoplasm to the nucleus and could phosphorylate eEF2 in interphase cells (28
). However, we considered that cyclin A-CDK2 might have greatest access to eEF2 in mitosis, when nuclear compartmentalization is lost, and examined eEF2 S595 phosphorylation in different stages of the cell cycle. We do not have a phospho-S595-specific antibody, and we used the extent to which eEF2 isolated from cells can be further phosphorylated by cyclin A-CDK2 in vitro
as a surrogate for preexisting phosphorylation, since in vitro
phosphorylation occurs almost exclusively on S595. and show that eEF2 immunoprecipitated from nocodazole-arrested prometaphase cells (M) was poorly phosphorylated by cyclin A-CDK2 in vitro
compared with eEF2 isolated from asynchronous (A) or S-phase-arrested (S) cells. shows data from a single representative experiment, whereas shows the combined data from four independent experiments. A representative cell cycle distribution of the various conditions is shown in .
To determine if reduced in vitro phosphorylation of mitotic eEF2 resulted from its increased phosphorylation in vivo, we treated the immunoprecipitated mitotic eEF2 with phosphatase to remove preexisting in vivo phosphorylation. Phosphatase treatment restored the amount of mitotic eEF2 phosphorylation by cyclin A-CDK2 in vitro to nearly the amount seen with S-phase-derived eEF2, indicating that reduced in vitro phosphorylation did indeed reflect increased preexisting in vivo eEF2 phosphorylation in mitotic cells ( and , M+λ). Finally, treating mitotic cells with roscovitine prior to lysis to inhibit endogenous CDKs also restored in vitro phosphorylation of mitotic eEF2, indicating that mitotic S595 phosphorylation is sensitive to roscovitine ( and , M+Ros). These data demonstrate that eEF2 S595 is hyperphosphorylated in mitotic cells and that this is likely mediated by a CDK. In vitro phosphorylation of S-phase-derived eEF2 was modestly reduced compared with that seen with asynchronous cells, but this is not sensitive to the presence of phosphatase and is presently not well understood.
We also examined the extent to which endogenous eEF2 T56 phosphorylation differed under the cell cycle conditions described above in three cell types: Hct116, 293A, and SK-N-AS cells (). Each of these cell lines exhibited large increases in T56 phosphorylation in prometaphase cells, consistent with the idea that eEF2 is inhibited in mitosis. Thus, both T56 and S595 are hyperphosphorylated eEF2 sites in mitotic cells.
S595 region mutants are poorly phosphorylated by eEF2K.
The in vivo maps suggested that eEF2 T56 phosphorylation is reduced by the S595A and H599P mutations (), and we therefore used a phospho-T56-specific eEF2 antibody to assess their inhibitory phosphorylation in vivo. The S595A and H599 mutations each greatly reduced eEF2 T56 phosphorylation in vivo, which was normal in the S595T mutant (). As expected, the pT56-specific antibody did not detect eEF2 when T56 was mutated. We also tested if glutamic acid could mimic S595 phosphorylation and restore T56 phosphorylation, but it did not (, lane 6).
Fig 5 An intact S595 region is required for efficient T56 phosphorylation. (A) eEF2 T56 phosphorylation in vivo is reduced by the S595A and H599P mutations. The indicated FLAG-eEF2 proteins were immunoprecipitated from transfected 293A cells and immunoblotted (more ...)
Because reduced in vivo T56 phosphorylation of eEF2 S595A and eEF2 H599P could reflect many cellular processes, we determined if these mutations directly inhibit T56 phosphorylation in vitro. shows FLAG-wt-eEF2 and FLAG-eEF2 S595A proteins that were eluted from immunoprecipitates, subjected to in vitro phosphorylation by eEF2K, and analyzed either by autoradiography or by blotting with the T56 phospho-specific antibody. Remarkably, the S595A mutation prevented eEF2 phosphorylation by eEF2K.
We also immunoprecipitated a series of FLAG-eEF2 mutants from transfected 293T cells and subjected the bound eEF2 proteins to phosphorylation with recombinant cyclin A-CDK2 or eEF2K in vitro
or to immunoblotting to reveal preexisting in vivo
T56 phosphorylation and eEF2 abundance. As expected, S595A was poorly phosphorylated by cyclin A-CDK2 (, top panel, lane 2). Moreover, T56 phosphorylation of the S595A, H599P, and T56A eEF2 mutants by recombinant eEF2K was greatly reduced compared with WT eEF2 (lane 1) or S595T eEF2 (lane 3) (, fourth panel; eEF2K autophosphorylation is also indicated). In each case, the amount of in vitro
T56 phosphorylation by eEF2K (fourth panel) matched the amount of in vivo
T56 phosphorylation (second panel), demonstrating that the S595A and H599P mutations directly impede T56 phosphorylation by eEF2K. The ability of recombinant eEF2K to further phosphorylate eEF2 isolated from cells is consistent with the fact that T56 phosphorylation in vivo
is substoichiometric. The residual in vitro
phosphorylation of eEF2 T56A by eEF2K (lane 5, fourth panel) likely reflects low-level phosphorylation of T58, a known minor eE2FK site (8
). Overall, these data show that mutations of the S595 region greatly inhibit eEF2 T56 phosphorylation by eEF2K in vivo
and in vitro
Reduced T56 phosphorylation of the S595A and H599P mutants could simply reflect their deleterious structural consequences. To address this possibility, we examined eEF2 S595A function by measuring its ability to catalyze protein synthesis in reticulocyte extracts. Diphtheria toxin inactivates eEF2 by catalyzing its ADP ribosylation, and this requires NAD (NAD+). We used diphtheria toxin and NAD+ to inactivate endogenous reticulocyte eEF2 and then rescued the inhibited extracts with equal amounts of either eEF2 or eEF2 S595A (). The amount of diphtheria toxin activity was titrated to inhibit endogenous eEF2 but not the exogenous eEF2 by limiting the amount of NAD+ (not shown). WT eEF2 and eEF2 S595A each restored translation of a luciferase plasmid in the inhibited extracts. eEF2 S595A is thus active as a translocase, strongly suggesting that its reduced T56 phosphorylation does not result from gross structural anomalies.
eEF2 S595 phosphorylation is required for efficient eEF2 T56 phosphorylation.
The previous experiments relied on mutants to demonstrate a role for S595 phosphorylation in regulating T56 phosphorylation. We next determined if S595 phosphorylation per se stimulates T56 phosphorylation. To accomplish this, WT eEF2 was prephosphorylated with cyclin A-CDK2 (or mock treated), the cyclin A-CDK2 was removed, and eEF2 was subjected to an eEF2K assay that included [32P]ATP and roscovitine (to inhibit any residual cyclin A-CDK2 activity). eEF2 phosphorylation by cyclin A-CDK2 greatly stimulated its phosphorylation by eEF2K, and this was not seen with eEF2 T56A, indicating that the enhanced phosphorylation occurred on T56 (). We tested if cyclin A-CDK2 catalytic activity was required to stimulate T56 phosphorylation by inclusion of roscovitine in the prephosphorylation step to inhibit CDK2. Roscovitine completely prevented the stimulation of T56 phosphorylation by cyclin A-CDK2, indicating that CDK2 activity is required to stimulate eEF2 phosphorylation by eEF2K (, compare lanes 2 and 3). Finally, we performed the sequential cyclin A-CDK2/eEF2K reaction using eEF2 S595A and found that the S595A mutation completely abrogated the impact of cyclin A-CDK2 (). In sum, these data indicate that phosphorylation of eEF2 on S595 by cyclin A-CDK2 directly stimulates eEF2 phosphorylation by eEF2K on T56.
Fig 6 Phosphorylation of S595 by cyclin A-CDK2 facilitates phosphorylation of eEF2 by eEF2K. (A) S595 phosphorylation stimulates T56 phosphorylation by eEF2K. eEF2 (lanes 1 to 3) or eEF2-T56A (lanes 4 to 6) was phosphorylated with cyclin A-CDK2 or mock treated (more ...)
In addition to the S595A mutation, T56 phosphorylation is also inhibited by the H599P mutation but does not affect S595 phosphorylation but instead likely introduces a local structural alteration caused by the proline substitution. We thus speculated that the S595 phosphorylation serves to recruit eEF2K to eEF2 and that this is inhibited when phosphorylation is prevented by the S595A mutations or by a structural change imposed by the H599P mutation. Unfortunately, our efforts to coprecipitate eEF2 with eEF2K failed for technical reasons (not shown). We therefore synthesized 30-mer peptides spanning the eEF2 S595 region, with or without phosphorylated S595, and determined if they inhibited eEF2 phosphorylation by eEF2K. When present in 30-fold molar excess over eEF2, the phosphorylated peptide was a more effective inhibitor of T56 phosphorylation than the unphosphorylated peptide ().