Protein phosphatase (EC 3.1.3.16); elongation factor-. 2 kinase (EC 2.7.1.37); trypsin (EC 3.4.21.4). lation system [3, 51 and in reticulocyte lysates [6]. Increased.
Eur. J. Biochem. 213, 689-699 (1993) 0 FEBS 1993
Regulation of elongation factor-2 by multisite phosphorylation Nicholas T. REDPATH, Nigel T.PRICE, Konstantin V. SEVERINOV and Christopher G. PROUD Department of Biochemistry, School of Medical Sciences, University of Bristol, England (Received November 6, 1992February 1, 1993) - EJB 921584/1
We have studied the phosphorylation of protein synthesis elongation factor eEF-2, the effects of phosphorylation on its activity and the dephosphorylation of phosphorylated eEF-2 by protein phosphatases-2A and -2C. Extensive analysis of phosphopeptides generated from eEF-2 phosphorylated in v i m by subsequent digestion with CNBr and trypsin indicated that Thr56 and Thr58 are the only residues significantly phosphorylated, consistent with our earlier report. They are also the only two residues to be significantly phosphorylated in reticulocyte lysates : in this system monophosphorylated eEF-2 corresponded only to phosphorylation of Thr56, no factor phosphorylated at only Thr58 being detected. Phosphorylation of Thr56 and Thr58 was found to be an ordered process, modification of Thr.56 preceding, and apparently being required for, phosphorylation of Thr58. This presumably explains why the only species of mono-phosphorylated eEF-2 detected are phosphorylated at Thr56. The eEF-2 kinase could phosphorylate a synthetic peptide based on residues 49-60 of eEF-2 (RAGETRFTDTRK), albeit only at a very low rate, and with a very high K,, compared to eEF-2 itself. The kinase phosphorylated the residues corresponding to Thr56 and Thr58, apparently in a random manner, but not Thr53. In the light of the existence of two phosphorylation sites in eEF-2, the relationship between phosphorylation and activity was investigated. Activity was measured in the poly(U)-directed synthesis of polyphenylalanine, where both the bis- and mono-phosphorylated (mono at Thr56) forms of the factor were found to be completely inactive. Indeed, the phosphorylated species appeared to be able to impair the activity of non-phosphorylated eEF-2 in this system. Experiments using reticulocyte lysates also indicated that both phosphorylated forms of eEF-2 were inactive in the translation of physiological templates, but no evidence for dominant inhibition by these species was obtained. Protein phosphatases-2A and -2C (PP-2A and PP-2C) can each efficiently dephosphorylate phosphorylated eEF-2. While bis-phosphorylated eEF-2 was a better substrate for PP-2A than monophosphorylated factor (phosphorylated at Thr56), the converse was true for PP-2C. This seemed to be due, at least in part, to the inhibition of dephosphorylation of Thr56 by PP-2C by the presence of phosphate on Thr58. Nevertheless, PP-2C exhibited a preference for dephosphorylation of Thr56 in bis-phosphorylated eEF-2, while PP-2A showed no such preference. These findings are discussed in terms of current knowledge of the specificity of these two protein phosphatases.
Elongation factor-2 (eEF-2) mediates the translocation step of peptide-chain elongation in eukaryotic cells. It is a monomeric protein of apparent M,100000 which binds guanine nucleotides and also possesses ribosome-stimulated GTPase activity [l]. eEF-2 is phosphorylated by a specific eEF-2 kinase on threonine residues [2, 31. The eEF-2 kinase is absolutely dependent on Ca2+/calmodulin for activity and is widely distributed in mammalian tissues [4]. Phosphorylation of eEF-2 inhibits its activity both in the reconstituted poly(U) transCorrespondence to C . G . Proud, Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol, BS8 1TD, England Fax: +44 0272 303497. Abbreviations. eF-2, eukaryotic elongation factor-2; eIF-2a, the a-subunit of eukaryotic initiation factor-2; PP, protein phosphatase. Enzymes. Protein phosphatase (EC 3.1.3.16); elongation factor2 kinase (EC 2.7.1.37); trypsin (EC 3.4.21.4).
lation system [3, 51 and in reticulocyte lysates [6]. Increased phosphorylation of eEF-2 occurs in several types of cells in response to stimuli which elevate cellular Ca” levels [7-91. However the significance of such changes in eEF-2 phosphorylation in the regulation of translation under such conditions remains unclear. The major protein phosphatase responsible for dephosphorylating eEF-2 in several types of cells is protein phosphatase-2A [lo- 121, although protein phosphatase-2C can also dephosphorylate it [lo, 121. Recently we have shown that phosphorylation of eEF-2 by the eEF-2 kinase occurs on two adjacent threonine residues (Thr56 and Thr58) both in vitro and in reticulocyte lysates [13]. Here we address a number of questions relating to the phosphorylation of these residues in eEF-2 and the specificity of eEF-2 kinase, the effects of phosphorylation at one or both of these sites on the properties of eEF-2 and the ability of protein phosphatases to dephosphorylate eEF-2 phosphorylated on one or both of these threonine residues.
690
MATERIALS AND METHODS Chemicals and biochemicals These items were obtained as previously described [ l l , 131. Reticulocyte lysates were prepared as described in [6]. Okadaic acid was kindly provided by Professor Philip Cohen (University of Dundee, Scotland). Protein phosphatase-2C was a generous gift from Dr. Gottfried Mieskes (Department of Clinical Biochemistry, University of Gottingen, Germany).
Protein preparations eEF-2 and eEF-2 kinase were purified from rabbit reticulocytes as described in 1131. Phosphorylation of eEF-2 The in vitro phosphorylation of purified eEF-2 and of eEF-2 in situ in reticulocyte lysates has been described earlier [ 131. The peptide-mapping procedures are also described in this reference.
no bis-phosphorylated eEF-2 had been generated, as assessed by isoelectric focusing analysis [ 131. eEF-2 phosphorylated at both sites, but labelled only at Thr56, was prepared from the mono-phosphorylated material described above. The mono-phosphorylated and radiolabelled factor was subjected to gel filtration on Sephadex G-25 and then re-incubated with eEF-2 kinase using unlabelled ATP for sufficient time to achieve maximal phosphorylation; 75 % of the resulting factor was in the bis-phosphorylated form, the remainder being equally divided between the mono- and un-phosphorylated forms, as judged by IEF analysis.
Other procedures Sodium dodecyl sulphate/polyacrylamide gel electrophoresis was performed as in [13] using 15% (masshol.) acrylamide. Slab-gel isoelectric focusing was performed as described in [13]. Protein concentrations were determined by the method of Bradford with bovine serum albumin as standard [14]. Protein synthesis in reticulocyte lysates was measured as described in [6] using [3H]leucineas the radiolabelled amino acid.
Phosphorylation of a synthetic eEF-2 peptide A 12-residue synthetic peptide corresponding to the phosphorylation site of eEF-2 (residues 49 -60, sequence RAGETRFTDTRK) was prepared by Dr. Vyacheslav Medvedev (Pushchino, Russia) using fluoren-9-ylmethoxycarbony1 (Fmoc) chemistry. Phosphorylation was performed as for eEF-2 for 30 min, using 1.25 mM peptide. Radiolabelled ATP was removed by spotting the samples onto Whatman P81 paper followed by extensive washing with 150 mM phosphoric acid. The peptide was eluted with 1 M ammonia solution, recovered in a SpeedVac centrifugal concentrator and two-dimensional peptide mapping was performed with electrophoresis at pH 3.6 [13]. Unlabelled peptide was located by ninhydrin staining. Phosphorylated peptide was detected by autoradiography, scraped from the thin-layer plates and eluted from the cellulose powder with 70% (by vol.) formic acid. Digestion with trypsin andor CNBr was performed as described [18], prior to further peptide mapping.
PolyfU) translation This was performed essentially as described by Ryazanov et al. IS]. The ionic conditions for poly(U) translation differ from those used for the reticulocyte lysates system mainly in the Mg” concentration which is substantially higher (10 mM as compared to 2 mM): the KCI concentration in both cases is 100 mM and other conditions are similar. Dephosphorylation of eEF-2 The conditions used for dephosphorylation of purified eEF-2 and the assay itself have been described previously [lo]. Reticulocyte lysate, diluted 1:400 (final value), was used as the source of PP-2A. Assays contained a synthetic peptide inhibitor of PP-1 [lo]. An appropriate amount of protein phosphatase-2C from rat brain was added to give 50% overall dephosphorylation of eEF-2 after 30 min. The preparation of eEF-2 labelled in both sites has been described previously [ 101. For the preparation of eEF-2 phosphorylated only at Thr56, the factor was incubated with eEF2 kinase for only 50 s, by which time about 30% of the factor had been converted to the mono-phosphorylated species and
RESULTS Mapping of tryptic phosphopeptides from eEF-2 As reported earlier [13], phosphorylated eEF-2 yielded three major phosphopeptide species when subjected to tryptic digestion. These were termed peptides A, B and C and corresponded to Phe-Thr(P)56-Asp-Thr(P)58-Arg, Phe-Thr(P)%Asp-Thr(P)58-Arg-Lys and Phe-Thr(P)56-Asp-Thr58-Arg, respectively. However, in some maps of phosphopeptides from purified eEF-2 phosphorylated in vitro, additional species were present. Most frequently observed was peptide D (Fig. lA), which migrated close to peptide B. It was important to establish whether or not it and the other phosphopeptides contained phosphorylated residues other than Thr56 and Thr58. More extensive tryptic digestion of peptide D resulted in its, albeit partial, conversion to A and B (Fig. 1B). Peptide D therefore seems to be a product of incomplete tryptic digestion of eEF-2 phosphorylated at Thr56 and Thr58 rather than being derived from an additional phosphorylation site. It must represent peptide B [Phe-Thr(P)56-Asp-Th(P)58-ArgLys] extended in either the C- or the N-terminal direction towards adjacent tryptic cleavage sites. Digestion of bis-phosphorylated eEF-2 with relatively large amounts of trypsin (an enzyme/substrate mass ratio of 1: 10) for 18 h yielded a species which co-migrated with peptide A (not shown). This result is in keeping with the earlier identification of peptides A and B as alternative tryptic cleavage products, the latter containing an additional Lys which is presumably removed by ‘harder’ tryptic digestion. The relative proportions of A, B and D varied between different digests under ‘standard‘ conditions (results not shown). Under ‘standard’ conditions, peptide C was the only species seen for mono-phosphorylated eEF-2. When trypsinolysis of eEF-2 preparations containing mono- and bis-phosphorylated factor was performed in the presence of 0.1 M urea (a condition used by other workers for carboxiodomethylated proteins), further phosphopeptides (E, F), named in order of their elution from the reverse-phase chromatography, were observed in varying amounts. A typical phosphopeptide map obtained under such conditions is
691
Fig. 1. Two-dimensional mapping of phosphopeptides from purified eEF-2 labelled in vitro. The figures show autoradiographs of twodimensional peptide maps of tryptic digests of phosphorylated eEF-2. The position of the origin, polarity of electrophoresis, direction of chromatography and position of migration of the uncharged dinitrophenyl-lysine marker are indicated. Peptide D was observed in some digests of bis-phosphorylated eEF-2 (A). Further tryptic digestion lead to the conversion of peptide D to a mixture containing A, B and D (B). When tryptic digests of eEF-2 (containing both mono- and bis-phosphorylated forms) were performed in the presence of 0.1 M urea, in addition to peptides C and D, two further phosphopeptides were observed, peptides E and F (C). Further tryptic digestion of E in the absence of urea lead to the formation of a mixture containing peptides A, B and D (not shown). Peptide F was completely converted to peptide C by this treatment (D).
shown in Fig. lC, which resembles that reported by Nairn and co-workers [8]. Again one can conclude that these peptides (E, F) do not represent additional phosphorylation sites but, rather, other products of partial trypsinolysis. The sequences adjacent to Thr56 and Thr58 contain potential tryptic cleavage sites on both the N- and C-terminal sides. To confirm this, E and F were scraped off the plate, eluted and redigested with trypsin. This resulted in the conversion of F to a peptide which co-migrated with the mono-phosphorylated peptide C (Fig. lD), and E was converted to a mixture of the bis-phosphorylated peptides A, B and D (not shown). These partial trypsinolysis products have been discussed in detail to emphasise the importance of their identification if one is to compare the extents of phosphorylation of eEF-2 and the identities of sites of phosphorylation in eEF-2 under different conditions. Even our standard digests contain a peptide which is not a limit tryptic peptide (peptide B). Phosphorylation of eEF-2 in reticulocyte lysates
Phosphopeptides derived from eEF-2 phosphorylated in reticulocyte lysates were analysed by two-dimensional mapping. eEF-2 from the control lysate shown yielded only a single peptide, peptide C (Fig. 2A), which is also obtained from mono-phosphorylated eEF-2 prepared in vitro [131. (The degree of bis-phosphorylated factor present under control conditions varied somewhat from one lysate to another.
In our earlier published work we have sometimes seen essentially no bis-phosphorylated factor [ l l ] in some preparations and a small but significant proportion in others [13, 151: the reason for this variability is unclear, but the level of Ca2+ ions may be an important variable factor.) Given its sequence (Phe-Thr-Asp-Thr-Arg), peptide C from eEF-2 phosphorylated in situ in reticulocyte lysates could be phosphorylated at either the first or the second threonine (Thr56 or Thr58). To distinguish between these possibilities, peptide C was subjected to three rounds of the automated Edman degradation as described previously [13], and the material on the sequencing membrane was analysed to determine how much of the radioactivity had now been released as "P, and how much was still present as phosphopeptide [I 31. All the radioactivity was found to be associated with P,, and none with the remaining peptide (as demonstrated by electrophoresis at pH 1.9, Fig. 2B) showing that in the initial peptide C , all the label was on the residue corresponding to Thr56, and none on Thr58. This is consistent with the observation that phosphorylation of eEF-2 in vitro is an ordered process, phosphorylation of Thr56 preceding that of Thr58 [13]. Relative rates of phosphorylation of Thr56 and Thr58 in vitro
The phosphorylation of eEF-2 over a time course was examined using slab-gel IEF to resolve the non-phosphory-
692
0
5
10
15
20
25
30
TIME /MINUTES
Fig. 2. Mapping of phosphopeptides from eEF-2 labelled in sifu in reticulocyte lysates. Reticulocyte lysate was incubated in the presence of ["PIATP for 10min. The lysate was fractionated by SDSFAGE and the excised eEF-2 band subjected to tryptic digestion and two-dimensional phosphopeptide mapping (A). The figure is annotated as in Fig. 1: the broken circle in each panel shows the position of the dinitrophenyl-lysine marker and the unbroken circle marks the origin. The single peptide observed (peptide C) was subjected to three rounds of the Edman degradation. The recovered peptide was analysed by electrophoresis at pH 1.9 to see if the radiolabel was still present on the peptide or had been released as 32Pi (B).
lated, mono- and bis-phosphorylated forms of eEF-2 (Fig. 3). The rapid appearance of the mono-phosphorylated species is followed by the generation of the bis-phosphorylated form. Based on results obtained after three rounds of the Edman degradation (see above), only Thr56 was phosphorylated in the mono-phosphorylated species at all times tested (not shown). When the appearance of the mono- and bis-phosphorylated forms of eEF-2 is plotted against time, it can be seen that whereas the mono-phosphorylated species is generated at a rapid initial rate, the appearance of the bis-phosphorylated species occurs only after an apparent lag period (Fig. 3). This was reproducible. After the lag period, the rate of appearance of the bis-phosphorylated species is quite rapid, and when one takes into account that the substrate for the second phosphorylation (mono-phosphorylated eEF-2) is present as only a small proportion of the total eEF-2, the actual rate of phosphorylation of the second site in mono-phosphorylated eEF-2 is higher than that of the initial phosphorylation event. Consideration of the rates of appearance of the monoand bis-phosphorylated species, together with the assumption that phosphorylation of Thr56 is indeed a prerequisite for phosphorylation of Thr58, allows us to estimate the relative rates of phosphorylation of the two Thr residues. The rate of
Fig. 3. Time course of phosphorylation of eEF-2 by purified eEF2 kinase in vitro. The phosphorylation of purified eEF-2 by eEF-2 kinase was followed over time, samples being removed at the indicated times for analysis by slab-gel IEF. Chips containing the mono,).( bis-).( or tris-(A) phosphorylated species of eEF-2 were excised and the associated radioactivity was determined by Cerenkov counting.
phosphorylation of Thr58 (in factor already phosphorylated at Thr56) actually appears to be about four times faster (calculated from the data in Fig. 3) than the initial phosphorylation of Thr.56 in unphosphorylated factor. Thus Thr58 only becomes a good phosphorylation site for eEF-2 kinase after phosphorylation of Thr56. At longer time points (15 and 30 min in Fig. 3B) a small proportion of a tris-phosphorylated form of eEF-2 could be observed. This may correspond to phosphorylation of Thr53, as has been reported by Ovchinnikov et al. [16]. We have only seen tris-phosphorylated factor when eEF-2 was phosphorylated in vitro for extended periods with relatively large amounts of kinase. It therefore appears unlikely that trisphosphorylated eEF-2 occurs physiologically, especially since the phosphorylation of eEF-2 which follows stimulation of cells with agents which elevate Ca" levels is not only rapid but also very transient [8]. In intact cells we have never observed this species and in reticulocyte lysates it is only seen in the presence of high concentrations of protein phosphatase inhibitors [ ll] . We ourselves have not therefore attempted to determine the location of the third phosphorylation site in eEF-2.
Ability of eEF-2 kinase to phosphorylate a synthetic peptide A synthetic peptide corresponding to the sequence in eEF-2 around Thr56 and Thr58 was prepared (sequence ArgAla-Gly-Glu-Thr-Arg-Phe-Thr-Asp-Thr-Arg-Lys). It was phosphorylated by eEF-2 kinase under the standard phosphorylation conditions used for eEF-2 and analysed by peptide mapping (Fig. 4A). A single radiolabelled peptide species was generated upon phosphorylation, which had a more
693 2 kinase does not phosphorylate the equivalent of Thr53 in the synthetic peptide, since the tryptic product generated in that case would not be expected to co-migrate with peptide C. Three cycles of the Edman degradation were performed on the tryptic fragment to determine whether the residue corresponding to Thr56 orThr58 was labelled. When the resulting material was analysed by electrophoresis at pH 3.6, about half the label was found to migrate as orthophosphate, while the remainder migrated as a single species, with a slight net positive charge. This compares with the small net positive charge on the starting material, and is compatible with it having the structure Thr(P)-Arg (data not shown).
Effect of phosphorylation on the activity of eEF-2 There is a considerable body of information which indicates that mono-phosphorylation of eEF-2 (presumably on Thr56) results in its inactivation. We have however shown the existence of two major phosphorylation sites in the factor and this raises the question of their individual roles in modulating its activity. Two technical problems arise in evaluating the relative activities of the factor phosphorylated at Thr56 orThr58. First, while it is relatively straightforward to compare the activities of the unphosphorylated and bis-phosFig.4. Phosphorylation of a synthetic peptide based on the se- phorylated factors, it is much more difficult to study the quence around the phosphorylation sites in eEF-2 by purified properties of the mono-phosphorylated factor. This is beeEF-2 kinase. A synthetic peptide derived from the phosphorylation site sequence for eEF-2 was incubated with eEF-2 kinase. The reac- cause, although it is possible to prepare factor which is pretion was analysed by two-dimensional peptide mapping and sub- dominantly in the mono-phosphorylated form, such prepjected to autoradiography (A). The position of the (non-phosphory- arations are always contaminated with significant amounts of lated) ninhydrin-detected peptide (N) is indicated. Dinitrophenyl-ly- both the unphosphorylated and bis-phosphorylated forms of sine (D, neutral charge) and xylene cyanol FF (X, charge -1 at the protein. We have expended a great deal of effort in trying, pH3.6) were run as markers. The resulting phosphopeptide was in vain, to resolve these species satisfactorily by chromatoscraped from the plate, digested with trypsin and again subjected to focusing. We had felt it was very important to achieve this peptide mapping (B). since the mono-phosphorylated factor might still possess partial activity, or, conversely, phosphorylated forms of eEF-2 might exert dominant inhibition (see also below). Our innegative charge than the corresponding non-phosphorylated ability to do this has led us to examine the activities of factor peptide (detected by ninhydrin staining). Ninhydrin staining preparations in differing states of phosphorylation (rather revealed that the proportion of peptide phosphorylated was than preparations which are entirely mono-phosphorylated) very small (50% unphosphorylated eEF-2, see Table l), there is no increase in the rate of translation, indicating that the availability of active eEF-2 is not restricting the overall rate of protein synthesis. Further evidence that the phosphorylated species of eEF2 do not exert a dominant inhibition in the reticulocyte lysate system is the observation that addition of phosphorylated eEF-2, which had been prepared using ATP[yS], to prevent its subsequent dephosphorylation, did not inhibit protein synthesis in this system (C. G. Proud, unpublished observation). Dephosphorylation of Thr56 and Thr58 by protein phosphatases-2A and -2C Protein phosphatase-2A (PP-2A) is the major protein phosphatase acting on phosphorylated eEF-2 in extracts of many cell types [lo, 111. PP-1 has little activity against eEF2, while PP-2C can dephosphorylate it. These experiments were performed with eEF-2 phosphorylated in both sites, and we have not previously studied the dephosphorylation of mono-phosphorylated eEF-2 or the relative rates of dephosphorylation of the two sites. We prepared samples of eEF-2 phosphorylated either for a short period to obtain a preparation in which the only phosphorylated species was the mono-phosphorylated factor or for a longer period to obtain predominantly bis-phosphorylated eEF-2. Their dephosphorylation by PP-2A (reticulocyte lysate under conditions where only PP-2A is active) or purified PP-2C was monitored under conditions designed to give similar concentrations of phosphorylated factor : since the mono-phosphorylated factor was incompletely phosphorylated, it was important to match the concentrations of phosphorylated factor (the phosphatase substrate) rather than the total eEF-2 concentrations. Preliminary experiments indicated that the rate of dephosphorylation was linearly related to the concentration of eEF-2 used as substrate (data not shown). The rate of dephosphorylation of bis-phosphorylated eEF-2 by PP-2A in the experiment depicted in Fig. 6 was more than 2.5-fold faster than the rate for the mono-phosphorylated factor. The difference in the concentrations of eEF-2-bound phosphate in the two assays (13nM for the mono- and 20 nM for the bis-phosphorylated factor) would be expected to give a 1.5-fold faster rate of dephosphorylation for the latter. However, a 2.5-fold increase in the rate of dephosphorylation was observed and it is therefore clear that there is an overall increase in the relative rate of dephosphorylation for the factor phosphorylated at both Thr residues. Thus it appears that the bis-phosphorylated factor is a better substrate for PP-2A than the mono-phosphorylated species. For PP-2C the initial rate of dephosphorylation of bisphosphorylated eEF-2 was scarcely higher (1.1-fold) than for the mono-phosphorylated factor, despite the increase in substrate concentration (Fig. 6). The bis-phosphorylated factor seems to be a worse substrate for PP-2C than the monophosphorylated form. The differences in the relative effectiveness of the different phosphorylated species of eEF-2 as substrates for PP-2A and PP-2C could be a consequence either of the effects of phosphorylation at one site on the dephosphorylation of the other or of differences in the intrinsic efficiencies of the sites
696
f, -I
W
oc 0.8 E" Q
w
- 0.6 L
6 a
0.4
v)
52
0.2
v)
9 0
10
20
30
TlMElMlNUTES
TI ME/MINUTES
Fig. 7. Dephosphorylation of phosphorylated eEF-2 labelled only Fig. 6. Dephosphorylation of phosphorylated eEF-2 labelled in at Thr56 by protein phosphatases3A and -2C. eEF-2, labelled both sites, Thr56 and Thr58, by protein phosphatases3A and with 32Pon Thr56 alone ('mono-phosphorylated' ; 0 , W) or phos-2C. eEF-2, labelled with 32Pon Thr56 alone ('mono-phosphorylat- phorylated on both Thr56 and Thr58 but labelled with 32Pon Thr56 ed', 0, B) or Thr56 and Thr58 ('bis-phosphorylated', 0, 0),was only ('bis-phosphorylated' ; 0, 0) were prepared as described. prepared as described. Dephosphorylations were carried out in a 2.2 pmol eEF-2 (2.2 pmol phosphorylated Thr56) were incubated as volume of 0.1 ml containing 3.8 pmol mono-phosphorylated eEF-2 above with PP2A (0, 0 )or PP2C (0, m). (1.3 pmol phosphate since 30% of eEF-2 was phosphorylated) or 1 pmol bis-phosphorylatedeEF-2 (2 pmol phosphate) and PP2A (0, 0 ) or PP2C (0, W). Samples were removed at the indicated times and processed for the measurement of released [3zP]phosphate.
themselves. To investigate this we prepared mono- and bisphosphorylated samples of eEF-2 radiolabelled only in Thr56. In order to prepare the former sample, phosphorylation was carried out only for a short period and this material contained only 0.3 mol phosphate/mol eEF-2. When the rates of dephosphorylation of Thr56 in monoand bis-phosphorylated eEF-2 were compared, allowing for the differences in the concentrations of this phosphorylated site in the two samples used, the rate for PP-2A was slightly lower than expected for the bis-phosphorylated species (Fig. 7). However, a much more marked inhibition of the dephosphorylation of Thr56 was observed for PP-2C, a twofold reduction being seen. Thus the phosphorylation of Thr58 substantially inhibits the dephosphorylation of Thr56 by this enzyme. Although the above experiment suggests that phosphorylation of Thr58 can inhibit the dephosphorylation of Thr56 by PP-2C it provides no information about the relative activities of PP-2C and PP-2A towards the two phosphorylated residues. In order to study this, the bis-phosphorylated factor radiolabelled on Thr56 was analysed on an IEF gel after dephosphorylation by PP-2A or PP-2C. The rationale behind this was that, if the phosphatase dephosphorylated mainly Thr56, then the mono-phosphorylated factor produced, separated by IEF, would have a relatively low specific radioactivity whereas if mainly Thr58 was dephosphorylated then the specific radioactivity of the mono-phosphorylated factor would be higher than expected if the dephosphorylation was random. The patterns observed for protein phosphatases-2A and -2C were quite different. For PP-2A, as the reaction pro-
Fig. 8. Dephosphorylation of bis-phosphorylated eEF-2 labelled only at Thr56 by protein phosphatases3A and -2C: analysis by isoelectric focusing. Bis-phosphorylatedeEF-2, 3ZP-labelledonly on Thr56, was prepared as described. This was incubated with either PP-2A (lanes 2-4) or PP-2C (lanes 5-7). Samples were removed at 10 min (lanes 2 and 5), 20 min (lanes 3 and 6) and 40 min (lanes 4 and 7) and ran on an isoelectric focusing gel. The gel was then subjected to autoradiography. Lane 1 contained bis-phosphorylated eEF-2 prior to dephosphorylation. The migrating positions of the bis- and mono-phosphorylated eEF-2 are indicated (B andM respectively).
ceeded, the relative intensities of the bands corresponding to the mono- and bis-phosphorylated species changed from a higher intensity for the bis- to a greater intensity of the mono-phosphorylated band (Fig. 8, lanes 2-4). In the case of PP-2C, on the other hand, the intensity of the bis-phosphorylated band was always the stronger even after lengthy dephosphorylation (Fig. 8, lanes 5-7). The relative selectivities of PP-2A and PP-2C for Thr56 and Thr58 therefore
697 Table 2. Alignment of sequences around residues corresponding to Thr56 and Thr58 of eEF-2 from various euakryotic species. Sections from the derived amino acid sequence of eEF-2 from the indicated species are shown. The residues corresponding to Thr56 and Thr58 from mammalian eEF-2 are shown bold, residues identical in all sequences are shown in upper case and underlined, those representing conservative replacements are shown in upper case and other residues are in lower case. The single letter code is used. Species
Sequence
Reference
Hamster Rat Human Drosophila melanogaster Chlorella kessleri Saccharomyces cerevisiae
ASARAGETRFTDTRKDEOERC I T I
[21] [22] [23]
ASARAGETRFrnTRKDEOERCITI ASARAGETRFTDT RKDEOERCITI AGAKAGETR FTDTRKDEOERC I T I
[24]
Af e a D q _ R L T D T R a D E O E R G I T I
[25]
sAAKAAEaRFTDTRKDEOSRGISp
[26]
clearly differ: it appears that PP-2C has relatively greater activity against Thr56 compared to Thr58 than PP-2A has. It is likely that PP-2A either dephosphorylates the bis-phosphorylated factor in random manner or exhibits a preference for Thr58.
DISCUSSION The data obtained from following a time course of the appearance of the mono- and bis-phosphorylated species of eEF-2 show that Thr56 is the first residue to be phosphorylated. Given that the mono-phosphorylated species is only phosphorylated on Thr56, it seems that phosphorylation of Thr56 is a prerequisite for phosphorylation of Thr58, i.e. that there is ordered phosphorylation of these two residues in eEF-2. Ordered phosphorylation of adjacent residues is also seen in other phosphoproteins, e.g. ribosomal protein S6, pyruvate dehydrogenase and glycogen synthase (reviewed by Roach [20]). In the first two cases the same kinase phosphorylates all of the sites. It seems likely that Thr58 is a poor initial substrate for the kinase. This is consistent with the fact that Thr53, which is in a sequence (Glu-Thr-Arg) closely resembling that around Thr58 (Asp-Thr-Arg), is a poor substrate for eEF-2 kinase, judging from our data. Thus phosphorylation of Thr56, which is at the N-terminal side of Thr58, somehow favous the phosphorylation at Thr58. However, phosphorylation at Thr53 is clearly not facilitated in this way. Following the phosphorylation of Thr56, phosphorylation at Thr58 is rapid. In this context, it also is interesting to note that residues corresponding to Thr56 and Thr58 are present in the sequences of eEF-2 from the alga Chlorellu kessleri and from Succhuromyces cerevisiae, but that these sequences lack a phosphorylatable residue at the position corresponding to Thr53 in mammalian eEF-2 (Table 2), which does not seem to be a significant phosphorylation site in rabbit eEF-2. In these two species, however, the sequences around the equivalents of Thr56 and Thr58 are very similar to those of eEF2 from vertebrates. The known eEF-2 sequences from mammals (three examples), birds (one) and insects (one) are almost entirely identical across the > 20 residues containing Thr56 and Thr58.
It is interesting to note, however, that phosphorylation of Thr56 does not place Thr58 in a sequence context similar to that around Thr56: Thr56 has a positively charged residue at positions -2 and +3, and a negatively charged residue at position +l.In contrast, in eEF-2 phosphorylated on T~I-56, Thr58 has negatively charged residues at -2 and -1, and positively charged ones at - 3 and + 1. Therefore, despite the observation that eEF-2 kinase can phosphorylate a peptide substrate apparently in a random manner (see below), features other than simply the sequence would seem to play an important role in the site-selectivity of the kinase. This is in contrast to another, better-known, example of ordered phosphorylation, in glycogen synthase, where phosphorylation of certain Ser residues makes the adjacent Ser at -4 a substrate for glycogen synthase kinase-3, so that a serial phosphorylation of residues termed C46, C42, C38 and C34 occurs (for review, see [20]). This appears to be simply a consequence of the sequence specificity of this protein kinase, which utilises Ser(P) at +4 as the recognition 'motif'. Such a simple situation does not seem to apply to eEF-2 kinase, or to the 70kDa S6 kinase [27]. This enzyme catalyses the ordered phosphorylation of five serine residues. When the context of consecutive phosphorylation sites is examined, the consensus sequence -(Arg)-Arg-Xaa-Xaa-Ser-Xaa- (determined for the first phosphorylation site using synthetic peptides [27]) is not adhered to for subsequent sites. Indeed, in some cases, Ser(P) replaces one of the Arg residues, as is seen for Thr(P) in the place of Arg at position -2 in eEF-2. An alternative possibility is that the phosphorylation of the two sites is catalysed by distinct active sites, either located in the same polypeptide chain (the 92-kDa S6 kinase apparently possesses two catalytic sites [28]) or in two different but co-purifying polypeptides. In the case of eEF-2 this might be similar to the properties of casein kinases-1 and -2, where, for example, a phosphorylation carried out by casein kinase-2, can provide a recognition motif, Ser(P), for casein kinase-1 [20]. Our recent data indicate that eEF-2 kinase activity is associated with a single polypeptide of about 103 kDa (N. T. Redpath, unpublished work); only the complete sequence of this polypeptide will reveal whether it possesses more than one potential catalytic site. As discussed in Results, Thr53 does not seem to be phosphorylated in the synthetic peptide, which is consistent with our data for intact eEF-2. However, in contrast to the situation with intact eEF-2, the kinase does not appear to exhibit an absolute preference for the equivalent of Thr56 over Thr58 when phosphorylating the synthetic peptide. Indeed, the data in Fig. 4B suggest random phosphorylation at the positions corresponding to Thr56 and Thr58. The selectivity of the kinase for phosphorylation at Thr56 in the unphosphorylated eEF-2 protein presumably reflects distal features of the protein primary sequence or its higher-order structure. As a consequence of the apparently ordered nature of the phosphorylation of eEF-2, it is only possible to examine the properties of one of the two possible mono-phosphorylated species of eEF-2, i.e. that phosphorylated at Thr56, since it is not possible to prepare factor phosphorylated only at Thr58. We are currently using site-directed mutagenesis to generate species of eEF-2 which lack one or other of the phosphorylatable Thr residues, and studies of these mutants should enable us to examine in greater detail the effects of phosphorylation of individual sites on the activity of the factor. Detailed analyses of the activities of eEF-2 containing different proportions of mono- and bis-phosphorylated factor
698 have not previously been reported. However, the data obtained by Nygkd and coworkers [19], who apparently observed only a single phosphorylated species of eEF-2, also suggested that the mono-phosphorylated protein was inactive. This conclusion was based on the inability of the phosphorylated factor to ‘rescue’ protein synthesis in a reticulocyte lysate pretreated with diphtheria toxin and seemed to be due to the markedly decreased affinity of the phosphorylated eEF-2 for the ribosome [19]. Ryazanov et al. [5] presented data which suggested that phosphorylated eEF-2 might exert a dominant inhibitory effect in the poly(U)-translation system and our more detailed data are consistent with this. However, the occurrence of a dominant inhibitory effect of phosphorylated eEF-2 is surprising in view of the decreased affinity of the phosphorylated factor for the ribosome, but as our data show, this dominant inhibitory effect seems to be a phenomenon of the poly(U)-translation system, and is not seen under more ‘physiological’ conditions, viz., in reticulocyte lysates (see below). Since eEF-2 is a monomeric protein, the explanation for the dominant inhibitory effect cannot lie in co-operative intersubunit interactions, through which phosphorylation of one subunit could modulate the activities of the others. One possibility, perhaps the most likely, is that phosphorylated eEF-2 can interact with the ribosome under the conditions of the poly(U)-translation system, but cannot mediate translocation, and thus blocks access of unphosphorylated eEF-2 to its functional binding site in eEF-2. If the interaction of the phosphorylated factor were tighter than that of non-phosphorylated eEF-2 it could then exert a dominant inhibitory effect by blocking the binding, and thus the action, of nonphosphorylated eEF-2. However, as mentioned above, Nyg h d and co-workers have reported that phosphorylated eEF2 (apparently mono-phosphorylated) failed to bind efficiently to ribosomes [19]. This was due to a 10-100-fold decrease in affinity for ribosomes in complexes resembling the pretranslocational complexes which arise in elongation. As pointed out above, the situation in the poly(U)-translation system would appear to differ greatly from that in reticulocyte lysates, where the data strongly indicate that no such dominant inhibitory effect occurs. Even when the bulk of the eEF-2 is phosphorylated (>97 %), translation still proceeded at about 50% of the control rate. There does not therefore seem to be an inhibitory effect of eEF-2 phosphorylation in reticulocyte lysates under standard translation conditions, or, by implication therefore, in intact cells. It is not immediately clear why there should be such a marked difference between the reticulocyte lysate and the poly(U)dependent translation systems. The most obvious difference between them is the much higher Mg2+ concentrations employed in the latter (10 mM vs 2 mM) which might influence the interaction of eEF-2 with the ribosome. Previous reports have suggested that phosphorylation of only one site in eEF-2 is required to completely inhibit its activity [3, 5, 191. The results presented here suggest that this site is likely to be Thr56, since it is the first site to be phosphorylated in vitro. This raises the question of the possible roles of the additional, second, phosphorylation site at Thr58. Phosphorylation of Thr58 might itself inactivate eEF2 but could also influence the dephosphorylation of Thr56. The rate at which phosphorylated eEF-2 is reactivated will depend on the rates at which the inhibitory site(s) are dephosphorylated. First, when both sites are phosphorylated, it seems likely that the time taken for complete dephosphorylation would be increased. This would be the case, in
particular, if the sites of phosphorylation were adjacent, as they are in eEF-2, since the binding of the phosphatase to one phosphorylation site would hinder access to the other, whether or not both sites are dephosphorylated by the same protein phosphatase. This consideration applies whether or not phosphorylation of Thr58 is inhibitory. A related point is that, if dephosphorylation of Thr56 alone is required for reactivation of eEF-2, a preferential attack by the protein phosphatase at Thr58 might be expected to impede reactivation of the factor. This situation may occur with PP-2A, although from the results presented here it is difficult to tell whether PP-2A exhibits a preference for Thr58 or dephosphorylates both sites in a random manner. Although it is unknown at this time, it is possible that phosphorylation of Thr58 is also inhibitory, therefore even if Thr56 in the bisphosphorylated factor became dephosphorylated, then the factor would still be inactive and thus complete dephosphorylation would be necessary to reactivate eEF-2 rather than just dephosphorylation of Thr56. Therefore, again, the rate of reactivation would be decreased. A second possible effect of multiple phosphorylation might be that phosphorylation of one site could inhibit the activity of the phosphatase against another site. It has been reported that negatively charged groups on the carboxyl-terminus side of the target phosphoamino acid inhibits PP-2C [29]. The results presented here are consistent with this in that they suggest that PP-2C preferentially dephosphorylates Thr56 but that phosphorylation of Thr58 appears to inhibit the phosphatase against this site. Thus, even if phosphorylation of Thr58 did not itself inhibit the activity of eEF-2, it could still retard its reactivation by hindering the dephosphorylation of the inhibitory site, Thr56. This work was supported by Grants to CGP from the Science and Engineering Research Council, and by funds from the British Diabetic Association. The visit of KVS to Bristol was made possible by awards from the Ministry of Education of the former USSR and the British Council. We are very grateful to Dr. Alexey Ryazanov, Dr. Elena Davydova and Elena Melnikova for advice on and materials for the poly(U)-translation assay.
REFERENCES 1. Riis, B., Rattan, S. I. S., Clark, B. F. C. & Memck, W. C. (1990) Trends Biochem. Sci. 15, 420-424. 2. Ryazanov, A. G. (1987) FEBS Lett. 214, 331-334. 3. Nairn, A. C. & Palfrey, H. C. (1987) J. Biol. Chem. 262, 17 299 - 17303. 4. Palfrey, H. C. (1983) FEBS Lett. 157, 183-190. 5. Ryazanov, A. G., Shestakova, E. A. & Natapov, P. G. (1988) Nature 334, 170- 173. 6. Redpath, N. T. & Proud, C. G. (1989) Biochem. J. 262,69-75. 7. Haycock, J. W., Browning, M. D. & Greengard, P. (1987) Proc. Natl Acad. Sci. USA 85,1677-1681. 8. Palfrey, H. C., Naim, A. C., Muldoon, L. L. & Villareal, M. L. (1987) J. Biol. Chem. 262, 9785-9792. 9. Mackie, K. P., Naim, A. C., Hampel, G., Lam, G. & Jaffe, E. A. (1989) J. Biol. Chem. 264, 1748-1753. 10. Redpath, N. T. & Proud, C. G. (1990) Biochem. J. 272, 175180. 11. Redpath, N. T. & Proud, C. G. (1991) Biochim. Biophys. Acta 1093, 36-41. 12. Gschwendt, M., Kittstein, W., Mieskes, G. & Marks, F. (1989) FEBS Lett. 257, 357-360. 13. Price, N. T., Redpath, N. T., Severinov, K. V., Campbell, D. G., Russell, R. J. M. & Proud, C. G. (1991) FEBS Lett. 282, 253 -258. 14. Bradford, M. M. (1976) Anal. Biochem. 77, 248-254.
699 15. Redpath, N. T. (1992) Anal. Biochem. 202, 340-343. 16. Ovchinnikov, L. P., Motuz, L. P., Natapov, P. G., Averbuch, L. J., Wettenhall, R. E. H., Szyszka, R., Kramer, G. & Hardesty, B. (1990) FEBS Lett. 275,209-212. 17. Nilsson, A., Carlberg, U. & Nygird, 0. (1991) Eul: J. Biochem. 195, 377-383. 18. Mellor, H. & Proud, C. G . (1991) Biochem. Biophys. Res. Commun. 178,430-437. 19. Carlberg, U., Nilsson, A,, & Nygird, 0. (1990) E m J. Biochem. 191, 639-645. 20. Roach, P. J. (1991) J. Biol. Chem. 266, 14139-14142. 21. Kohno, K., Uchida, T., Ohkubo, H., Nakanishi, T., Fukui, T., Ohtsuki, E., Ikehara, M. & Okada, Y. (1986) Proc. Nut1 Acud. Sci. USA 83, 4978-4982. 22. Oleinikov, A. V., Jokhadze, G. G. & Alakhov, Y. B. (1989) FEBS Lett. 248, 6384-6391.
23. Rapp, G., Klaudiny, J., Hagendorff, G., Luck, M. R. & Scheit, K. H. (1989) Biol. Chem. Hoppe-Seyler 370, 1071-1075. 24. Grinblat, Ye., Brown, N. H. & Kafatos, F. C. (1989) Nucleic Acids Res. 17, 7303-7314. 25. Schnelbogl, G. & Tanner, W. (1991) Plant Physiol. 97, 469471. 26. Perentesis, J. P., Phan, L. D., Gleason, W. B., LaPorte, D. C., Livingston, D. M. & Bodley, J. W. (1992) J. Biol. Chem. 267, 1190- 1197. 27. Flotow, H. & Thomas, G. (1992) J. Biol. Chem. 267, 30743078. 28. Jones, S . W., Erickson, E., Blenis, J., Maller, J. L. & Erikson, R. L. (1988) Proc. Natl Acud. Sci. USA 85, 3377-3381. 29. Donella Deana, A,, Macgowan, C. H., Cohen, P., Marchiori, F., Meyer, H. E. & Pinna, L. A. (1990) Biochim. Biophys. Actu 1051, 199-202.
