Our results of the present study confirm and expand earlier findings [19
] that eEF2K undergoes extensive Ca2+
/CaM-dependent autophosphorylation. Importantly, we now identify nine sites of autophosphorylation in human eEF2K, consistent with the observed stoichiometry of approximately 8 mol of phosphate/mol of enzyme and show that at least three of them affect phosphorylation of the MH-1 substrate peptide.
Interestingly, our results reveal for the first time that eEF2K can use serine as well as threonine as a phosphoacceptor, at least within the eEF2K polypeptide itself, whereas an earlier study which employed peptides as substrates found that eEF2K exhibited an overwhelming preference for phosphorylation of threonine residues in that context [38
]. In different species, the physiological substrate eEF2 always has a threonine residue at the position corresponding to Thr56
in human eEF2, never a serine residue. That study also indicated that eEF2K strongly preferred to phosphorylate residues with basic amino acids at the +1 and +3 positions. Consistent with this, six of the autophosphorylation sites that we have identified in the present study have a basic residue in close proximity to the C-terminal side [arginine residues at +3 (Thr348
) or +2 (Ser491
, a less prominent site), or lysine at +3 (Thr353
also have a lysine residue at +4. Finally, for Ser474
, the basic residue is located at +6. Autophosphorylation does not require a basic residue at +1 (none of the autophosphorylation sites we have identified has a basic residue at this position, and neither does Thr56
in eEF2 itself).
Several of the major phosphorylation sites are conserved among vertebrate eEF2K sequences (), but only one, Thr348, a critical site for eEF2K activity, is generally conserved in other phyla as well (e.g. arthropods, echinoderms, molluscs, cnidaria and nematodes; it is replaced by another phosphoacceptor, serine, in some nematodes).
Mutation of Ser78, Thr348, Ser366 or Ser491 to an alanine residue reduced the stoichiometry of autophosphorylation (A and B), but activity measured towards the MH-1 peptide was most drastically reduced by mutation of Thr348 or Ser366. However, this may be the consequence of different mechanisms as the HPLC profiles and activity measurements differ considerably for these three mutants. One must also bear in mind that mutations may alter the folding of eEF2K and exert effects unrelated to the role of given residues as autophosphorylation sites. The T348A mutation had a very marked impact on autophosphorylation and activity against both the MH-1 peptide and eEF2. Thus Thr348 may be considered as a master site whose phosphorylation is important for phosphorylation at the other sites. It should be noted that Thr348 is the autophosphorylation site that is closest to the catalytic domain and is also the most widely conserved one among known eEF2K sequences from species as divergent as nematodes and sea slugs (see further discussion of this site below).
Mutation of eEF2K Ser366
to alanine decreased the extent of overall 32
P-incorporation (A and D). This mutant also showed a substantial decrease in activity towards the MH-1 peptide similar to that observed for the eEF2K[T348A] mutant. Thus Ser366
is important for kinase activity against the MH-1 peptide, although it does not apparently affect activity against eEF2 itself [15
Although the eEF2K[T348A] (and eEF2K[S366A]) mutant proteins showed drastically reduced activities towards MH-1 (C), they still underwent autophosphorylation (A), showing an important difference between autokinase activity and the ability to phosphorylate substrates in trans
. The results for the activity of the S366A mutant against the MH-1 peptide are surprising given that earlier work found that this mutant did not show a marked change in activity against eEF2 itself [15
]; this may reflect differences in the requirements for phosphorylation of the MH-1 peptide and eEF2, which are clearly distinct [35
]. The eEF2K[S491A] variant showed a similar behaviour to that of eEF2K[S366A] as regards its impact on the level of autophosphorylation and the HPLC profile, but it is clearly different in terms of catalytic activity towards MH-1, which was only slightly altered. This again indicates that the autophosphorylation and transphosphorylation processes against different substrates, although sharing common regulatory mechanisms, also have specific requirements.
We have shown in a recent study that, although fragments lacking the C-terminal part of eEF2K cannot phosphorylate eEF2 or the MH-1 peptide, addition of the eEF2K[478–725] fragment restores their ability to do so, and we suggest that the C-terminal SEL1-containing region helps to recruit substrates for phosphorylation by the kinase domain [35
]. The sequence connecting the two domains contains a number of phosphorylation sites for kinases that regulate the activity of eEF2K (e.g. Ser359
] and Ser398
]). It is possible that phosphorylation of these sites affects eEF2K activity by altering the conformation of the ‘linker’ region and thereby the efficiency of the ‘coupling’ between the C-terminal and kinase domains of eEF2K. In this context, it is notable that mutation of Ser366
markedly alters activity against the MH-1 peptide.
Interestingly, other α-kinases also undergo autophosphorylation. TRPM6 and TRPM7 contain many sites of autophosphorylation [20
] and MHCKA also undergoes autophosphorylation [24
]. Where known, many of these autophosphorylation sites lie in regions that are not homologous with eEF2K; of those that do lie in their homologous kinase domains, only one corresponds to a potential phosphorylation site in eEF2K (Ser135
), and we did not find this as an autophosphorylation site, even though it should yield a readily detectable tryptic phosphopeptide. Crawley et al. [45
] identified several sites of autophosphorylation in MHCKA. Our finding in the present study that Thr348
is a major site of autophosphorylation in eEF2K is precisely in accord with their data and predictions [45
]. These authors showed that Thr825
is constitutively autophosphorylated in MHCKA. This residue, the equivalent of Thr348
in eEF2K, lies just C-terminal to the catalytic domain. On the basis of the crystal structure of its catalytic domain together with biochemical data, they proposed that phosphorylated Thr825
docks with a phosphate-binding pocket in the C-terminal lobe, and that this interaction stimulates the catalytic activity of MHCKA. They suggest that a similar mechanism may operate in other α-kinases [45
]. The results of the present study are entirely consistent with this; Thr348
, which we have identified as a major site of autophosphorylation, lies close to, and on the C-terminal side of, the catalytic domain, and, in common with Thr825
in MHCKA, is followed by a hydrophobic residue (isoleucine in many vertebrate eEF2Ks; replaced by other branched-chain residues in some other species). Both the phosphate-binding pocket and the proposed ‘partner’ for the adjacent hydrophobic residue are conserved among MHCKs and eEF2Ks. It thus appears likely that autophosphorylation promotes the activity of these enzymes in similar ways, through the interaction of a constitutively phosphorylated threonine residue with a conserved phosphate-binding region. Our observation that replacement of Thr348
with either a glutamate or aspartate residue generated a catalytically inactive enzyme is in accordance with the data of Crawley et al. [45
], who found that adding phosphate or phosphothreonine, but not glutamate or aspartate, could activate a truncated version of MHCKA that lacks the region containing Thr825
. It is interesting that Thr348
is basally phosphorylated in human cells; this probably ensures that eEF2K is already ‘primed’ and poised to phosphorylate eEF2.
A surprising finding from our studies is that eEF2K undergoes autophosphorylation on two sites, which inhibit its activity, Ser78
; both sites are phosphorylated in vivo
in response to, e.g., insulin in an mTORC1 (mammalian target of rapamycin complex 1)-dependent manner [15
]. An analogous situation is found for the catalytic α-/β-subunits of AMP-activated protein kinase, which autophosphorylate on Ser485
]. However, protein kinase B also phosphorylates this site, thereby decreasing the activation of AMP-activated protein kinase by LKB1 [46
]. Phosphorylation of Ser78
impairs the interaction of eEF2K with CaM [33
], whereas phosphorylation of Ser366
impairs its activation by Ca2+
]. Both of these sites are also autophosphorylated, and, in the case of Ser78
, apparently only at a low level in vitro
or in vivo
(results not shown). In contrast, the 2D peptide maps show that Ser366
is a major site of autophosphorylation. It is possible that its slow phosphorylation serves to turn off eEF2K after its activation in response to cellular stresses, by desensitizing eEF2K to activation by Ca2+
/CaM, thereby allowing translation elongation to resume.
In conclusion, the results from the present study reveal that Thr348, and presumably its autophosphorylation, are critical for the ability of eEF2K to phosphorylate substrates in trans, having similar effects on the phosphorylation of the MH-1 substrate peptide. These results suggest that autophosphorylation of eEF2K may be a prerequisite for the activity of eEF2K, in line with other α-kinases that have been studied.