The present data reveal differing roles for three important regulatory features located in the hitherto poorly studied C terminus of 4E-BP1. Firstly, we identify a novel in vivo phosphorylation site at S101 and show that this site plays a key role in the insulin-induced phosphorylation of S65, a site near the eIF4E-binding motif of 4E-BP1 that affects the affinity of 4E-BP1 for eIF4E. Secondly, we demonstrate that S112 is required for insulin-induced release of 4E-BP1 from eIF4E and that this is due to a direct effect on eIF4E-4E-BP1 binding rather than to an influence of this site on phosphorylation of other residues in 4E-BP1. Since S112 is constitutively phosphorylated, and a mutant with alanine at this position is not released efficiently in response to insulin, we infer that phosphorylation at this site is required for release, although other explanations are formally possible. Thirdly, we show for the first time that the extreme C terminus, which contains the TOR signaling (TOS) motif reported by Schalm and Blenis (39
), is critical for phosphorylation of several sites in 4E-BP1. However, this input is not strictly linked to inputs from mTOR, as discussed in more detail below. These findings point to key roles for the C-terminal region of 4E-BP1 in its regulation. Given that recombinant 4E-BP1 has little, if any, folded structure, it is surprising that sites far from the 4E-binding motif have effects on its association with eIF4E or the phosphorylation of other sites distant in its primary sequence, and this issue is discussed further below.
It is thus now clear that phosphorylation of S65 requires at least two priming events—phosphorylation at S101 as well as at T70. Certain other protein kinases such as glycogen synthase kinase 3 and casein kinase 1 also require priming phosphorylation events for their action, but in these cases priming phosphorylation occurs only a few residues C- or N-terminal (respectively) from their sites of action. In the former case, structural studies have revealed that this priming mechanism involves a phosphate-binding site that interacts with the priming phosphoserine in the +4 position relative to the target residue (5
). The kinase acting at S65 is clearly dependent on a phosphorylation event much further away than this (at S101). Although S101 in human 4E-BP1 is phosphorylated by DYRK isoforms in vitro, these kinases may not be responsible for phosphorylating this site in vivo. Indeed, although the 4E-BP1 proteins from the rat and mouse each have a serine at this position, these residues do not lie in a consensus for phosphorylation by DYRK, suggesting that other kinases act on these sites. It is important to note that the sequence around S101 and its equivalents in rodent 4E-BP1 is generally highly conserved: it is identical at 16 out of 20 residues around this serine. In fact the following 10 residues are identical in rat, mouse, and human 4E-BP1.
Phosphorylation at S65 also requires the C-terminal QFEMDI motif, but this probably reflects the fact that this feature is needed for phosphorylation at the corresponding priming site, T70. The fact that two widely separated phosphorylation sites (T70 and S101) are required for phosphorylation at S65 could imply either that the kinase acting at this site has a docking site that recognizes both phosphoseryl residues individually or that phosphorylation of the otherwise unfolded 4E-BP1 polypeptide induces structure which is recognized by the S65 kinase. Extensive work will be required to investigate these possibilities.
Our data show that phosphorylation at S112 influences the binding of 4E-BP1 to eIF4E without affecting its phosphorylation at other sites, i.e., directly, despite the fact that S112 also lies quite far from the eIF4E-binding site. This may again imply the existence either of long-range interactions within the 4E-BP1 molecule or the fact that when phosphorylated, or when associated with eIF4E, 4E-BP1 may adopt a more-structured conformation in which, e.g., S112 is adjacent to eIF4E. These new data indicating a direct role for phosphorylation of S112 in modulating the binding of 4E-BP1 are interesting in the light of recent findings that phosphorylation of S65 alone or at S65 and T70 did not suffice to effect release of 4E-BP1-based peptides from eIF4E (29
). These authors interpreted this effect as indicating a role for phosphorylation at T37 and T46 in release, and the present data indicate that phosphorylation at S112 also plays an important role in modulating eIF4E-4E-BP1 binding, thus helping to explain their data. Thus, release of 4E-BP1 from eIF4E appears to require its phosphorylation at S65 and T70, which is enhanced by insulin, and at S112, which is constitutively phosphorylated in HEK293 cells.
An important related point is that the sequences of 4E-BP2 and -3 do not have a serine at the position corresponding to S112, even though in other respects their C termini are very similar to that of 4E-BP1—in both proteins, alanine is present at this position (Fig. ). Furthermore, neither protein contains a residue equivalent to S101. These differences raise important questions about the regulation of these 4E-BPs. Interestingly, neither protein has so far been shown to dissociate from eIF4E in response to stimuli such as insulin. Recent data clearly show that insulin fails to induce release of 4E-BP3 from eIF4E under conditions where 4E-BP1 does dissociate (40
). For almost all the phosphorylation sites in 4E-BP2 and -3, phosphospecific antisera are not available, and we have not explored the phosphorylation of these proteins in any detail. Converting alanine 84 in 4E-BP3 to serine or glutamate did not allow 4E-BP3 to be released from eIF4E in response to insulin. It therefore seems that a phosphorylatable residue at this position is insufficient to allow insulin to bring about release and that additional inputs are likely required. Another major difference between 4E-BP1 and 4E-BP3 is that the latter contains only one residue equivalent to T37 and T46. This may impair the phosphorylation of other sites in 4E-BP3 (e.g., those corresponding to S65 and T70 in 4E-BP1) and could also in part underlie its failure to be released in response to insulin.
To study the phosphorylation of S112 in 4E-BP1, we developed an appropriate phosphospecific antiserum. By use of this reagent, it was clear that S112 is constitutively phosphorylated in HEK293 cells. This contrasts with an earlier report for rat adipocytes (10
) which indicated that this site was not phosphorylated. That study involved 32
P labeling of the protein in vivo, and it may be that the rate of turnover of phosphate on this constitutively phosphorylated residue is so low that no significant labeling occurred. In our studies, we found that phosphorylation of S112 was not increased by insulin, in contrast to a second report for rat adipocytes, where an increase was observed (20
). Again, this study employed in vivo labeling with [32
P]orthophosphate, and it may be that insulin increases the turnover of phosphate on S112 without a change in its net level of phosphorylation. Contrary to the suggestion made by Heesom et al. (20
), S112 does not act to “prime” phosphorylation of other sites in 4E-BP1. A further study suggested that S112 was a target for phosphorylation by ATM (47
). This link is surprising given that DNA-damaging agents cause dephosphorylation of 4E-BP1 rather than inducing an increase (40
) (although it should be noted that the conditions used by these workers do not activate ATM). The present study shows that the level of phosphorylation of S112 is not affected by wortmannin at concentrations reported to inhibit phosphorylation of p53 by ATM in vivo (37
). Indeed, phosphorylation of p53 by ATM was almost completely inhibited by wortmannin concentrations 10 times lower than that used here. Nevertheless, some reports have indicated that (in vitro) ATM is inhibited only by higher concentrations of wortmannin (4
). However, even at 1 μM (which does completely inhibit ATM in vitro), wortmannin did not reduce the level of signal seen with the anti-S112[P] antibody. These data suggest that ATM is not responsible for the basal phosphorylation of S112 in HEK293 cells, implying the existence of another S112 kinase(s). ATM is presumably able to phosphorylate S112 in vitro because the following residue is Gln, creating a consensus site for phosphorylation by ATM. Our data also show that phosphorylation of S112 is also not influenced by the mTOR pathway, since phosphorylation of S112 was not affected by rapamycin.
Our analysis of the effect of deletion of the C-terminal QFEMDI motif confirms the initial observation of Schalm and Blenis (39
) that a single point mutation within this motif eliminates the ability of insulin to induce the mobility shift in 4E-BP1 that normally accompanies its phosphorylation. These authors interpreted their data as indicating that this region was required for signaling from mTOR to 4E-BP1, and they called the FEMDI sequence a TOR signaling (TOS) motif. However, they did not establish whether, and how, it modulated the regulation of 4E-BP1. We demonstrate here that absence of the QFEMDI motif drastically affects the phosphorylation of 4E-BP1 at multiple sites (T37, T46, T70, and S65) and therefore its regulation. However, our data indicate that this is not specifically a motif required for mTOR signaling. Firstly, deletion of the motif affects the phosphorylation of two basal phosphorylation sites that are not sensitive to rapamycin in vivo, i.e., T37 and T46. Secondly, the increase in phosphorylation of T37, T46, and T70 in response to insulin observed in this Δ6 mutant is blocked by rapamycin, illustrating that mTOR can still regulate 4E-BP1 in the absence of this motif. Phosphorylation at T37 and T46 is actually more prone to inhibition by rapamycin in the Δ6 mutant (where it is eliminated by this drug) than in full-length 4E-BP1. It is possible, indeed likely, that S112 does not undergo phosphorylation in the Δ6 mutant (but we cannot easily check this because the Δ6 protein is not recognized by our anti-S112[P] antibody, due to the loss of the part of the epitope that it binds). However, lack of phosphorylation of S112 cannot be the cause of the effects of the Δ6 truncation on the phosphorylation of other sites, as they are still phosphorylated in the S112A mutant. Our data are consistent with the idea that the QFEMDI motif is needed for phosphorylation of basal sites in 4E-BP1, which are required for insulin-induced phosphorylation at other sites, rather than specifically for rapamycin-sensitive inputs from mTOR. In view of our data, further work to explore the precise role of the TOS motif in the S6 kinases (39
) will be required.
It is now clear that the hierarchical phosphorylation of 4E-BP1 is even more complex than previously thought, with constitutive phosphorylation sites in the N (T37 and T46) and C (S101/112) termini playing crucial roles in its regulation. An increasing number of proteins are now known to be modulated by complex multisite phosphorylation. Two of many possible examples are p53 (37
) and the SCFCdc4
ubiquitin ligase (8
), both of which are, like 4E-BP1, key regulators of cell function. The existence of multiple phosphorylation sites in these proteins presumably facilitates their control by multiple inputs. Our data also identify a C-terminal motif required for phosphorylation of many sites in 4E-BP1. This is reminiscent of the N-terminal “RAIP” sequence which was recently shown to be required for phosphorylation of T37, T46, T70, and S65 in 4E-BP1 (40
). The protein raptor (18
), which binds to mTOR, was recently identified as a potential scaffold that interacts with 4E-BP1and facilitates its phosphorylation. This raises the possibility that raptor may do so by binding the RAIP and/or QFEMDI motif. However, the findings that these motifs are required for phosphorylation of sites in 4E-BP1 that are not sensitive to rapamycin in vivo (40
; this report) appear inconsistent with this idea.
The data presented here show that two phosphorylation sites near the C terminus of 4E-BP1 influence the functions or phosphorylation of the central region of 4E-BP1, which is surprising given that recombinant 4E-BP1 has no folded structure (12
). It is possible that phosphorylation of 4E-BP1 induces a more-ordered structure: for example, as discussed above, phosphorylation at S101 and T70 may induce a structure that creates a docking site for the kinase that acts at S65, a residue whose phosphorylation in vivo requires prior phosphorylation at these two sites. The evidence that phosphorylation of S112 is required for release of 4E-BP1 from eIF4E even when T70 is phosphorylated suggests that this part of 4E-BP1 may contact eIF4E in the binary eIF4E-4E-BP1 complex, even though the interaction does not depend upon the C-terminal QFEMDI motif.