The role of the five (S/T)P sites in controlling PHAS-I was investigated by expressing mutant PHAS-I proteins in HEK293 cells. As with all overexpression studies, there is the issue of whether the effects observed are representative of events that occur with the endogenous proteins. The present finding that phosphorylation was confined to the same five (S/T)P sites previously identified in endogenous PHAS-I in rat adipocytes indicates that ectopic phosphorylation of the proteins expressed in HEK293 cells did not occur (8
). Furthermore, insulin stimulated the phosphorylation of PHAS-I expressed in HEK293 cells in the same four sites that were phosphorylated in response to the hormone in adipocytes (8
), and phosphorylation occurred via a rapamycin-sensitive pathway, as with the endogenous PHAS-I proteins in other cell types (3
). These findings argue that regulation of the phosphorylation of the expressed PHAS-I proteins occurs by normal signaling mechanisms. The results obtained with these proteins have important implications with respect to the mechanisms involved in the regulation of phosphorylation and the roles of the different sites in controlling the function of the PHAS-I protein.
It has become common practice to use a gel shift assay to evaluate the phosphorylation of PHAS-I. A caveat in using this assay to evaluate functional control of PHAS-I is that sites having the largest influence on electrophoretic mobility do not necessarily have the largest effects on eIF4E binding. Phosphorylation of either Thr45 (40
) or Ser64 alone did not affect mobility, although phosphorylation of either of these two sites abolished eIF4E binding in vitro. PHAS-1β is generated by phosphorylation of Thr69 or Ser82. PHAS-Iγ results from the phosphorylation of Thr69 and either Ser64 or Ser82. Phosphorylated Thr36 and Thr45 may appear in the α, β, and γ forms, since these two sites do not significantly influence electrophoretic mobility.
With multisite phosphorylation, the potential exists for interactions among phosphorylation sites. It was recently proposed that PHAS-I phosphorylation involves a two-step mechanism in which phosphorylation of Thr36 and Thr45, occurring when PHAS-I is bound to eIF4E, is required for the phosphorylation of the other sites (10
). We have confirmed that mutating Thr36 or Thr45 decreases the phosphorylation of Ser64 in HEK293 cells. However, other findings are clearly incompatible with certain aspects of the two-step model (10
). For example, mutating Thr69 also markedly decreased the phosphorylation of Ser64, indicating that Ser64 phosphorylation depends on the phosphorylation of all three TP sites. Also, PHAS-I proteins lacking Thr36 and Thr45 could be phosphorylated in both Thr69 and Ser82. Indeed, the absence of Thr36 or Thr45 actually enhanced the phosphorylation of Thr69, and the phosphorylation of Thr69 was regulated in a rapamycin-sensitive manner by amino acids and insulin in the mutant lacking the four other sites. The finding that the three PHAS-I proteins having an individual TP site with mutations in the four remaining sites were phosphorylated implies that there is not an obligate order in the phosphorylation of Thr36, Thr45, and Thr69. S64 NBPHAS-I was not phosphorylated, indicating that dissociation of the PHAS-I–eIF4E complex, which occurs in response to phosphorylation of the TP sites, is not sufficient for Ser64 phosphorylation. Moreover, since phosphorylation of Ser64 still required the TP sites when PHAS-I was rendered incapable of high-affinity binding to eIF4E, the ordered phosphorylation of Ser64 does not have to occur when PHAS-I is bound to eIF4E as proposed in the two-step model (10
Hierarchal phosphorylation, a mechanism in which phosphorylation of one site creates a consensus site for phosphorylation of a second site (32
), might explain the dependence of Ser64 on prior phosphorylation of the TP sites. Phosphorylated Ser or Thr residues ([S/T]*
) are found in the consensus motifs, [S/T]XXX[S/T]*
XX[S/T], for phosphorylation by glycogen synthase kinase 3 and casein kinase I, respectively (32
). If such a mechanism is involved in the control of Ser64 phosphorylation, the consensus motif is likely to be complex, since three sites were required for the phosphorylation of Ser64. As suggested previously (10
), phosphorylation might create a recognition motif that recruits the Ser64 kinase and/or other regulatory factors to PHAS-I. However, the ordered-phosphorylation model is based on studies in intact cells, and there are other potential explanations of the data. For example, phosphorylation could appear to be ordered if phosphorylation of the TP sites protected the Ser64 site from dephosphorylation. Interestingly, evidence for such a mechanism was recently obtained for the control of nPKCδ, another downstream protein in the mTOR signaling pathway (26
). In this case, phosphorylation of Thr505 in the activation loop of the kinase appears to markedly decrease the rate of Ser662 dephosphorylation.
In view of the complexities in the control of PHAS-I, the simple hypothesis that the five (S/T)P sites in PHAS-I are regulated by a common mechanism can be eliminated. Ser82 was not phosphorylated in response to insulin or amino acids, indicating that Ser82 is not subject to the same control as the other sites whose phosphorylation was increased by these agents. Insulin and amino acids increased the phosphorylation of T36 PHAS-I, T45 PHAS-I, and T69 PHAS-I, indicating that the stimulatory effects of these agents on the phosphorylation of Thr36, Thr45, and Thr69 occurred independently of other sites. Although the accumulation of phosphate in Ser64 is complicated by the dependence on prior phosphorylation of the TP sites, the finding that amino acids and insulin increased the phosphorylation of the same four sites is consistent with the hypothesis that these agents act via a common upstream effector. The effect of insulin on increasing the phosphorylation of PHAS-I is mediated by the protein kinase B (PKB) signaling pathway (11
); however, PKB is not activated by amino acids (14
). mTOR is a more likely common effector, since the effects of both insulin and amino acids are attenuated by rapamycin. The PHAS-I kinase activity of mTOR is increased in response to insulin (33
). Activation of mTOR occurs by a PKB-dependent pathway, which leads to an increase in phosphorylation of Ser2448 in the COOH-terminal region of mTOR (25
). Amino acids appear to have a permissive effect on the phosphorylation of mTOR by PKB (25
The marked effects of rapamycin on decreasing the phosphorylation of Thr69 suggest that this site might be directly phosphorylated in cells by mTOR, which is able to phosphorylate Thr69 in vitro (4
). Decreasing Thr69 phosphorylation by rapamycin would be expected to reduce the phosphorylation of Ser64. Nevertheless, it is paradoxical that phosphorylation of Thr36 and Thr45, the sites preferred by mTOR in vitro (6
), is less sensitive to rapamycin than is the phosphorylation of Thr69 and Ser64. A kinase that associates with mTOR and that can be released upon incubation with an mTOR antiserum was recently described (16
). This enzyme was reported to specifically phosphorylate Ser64 and to promote the dissociation of the PHAS-I–eIF4E complex, but the kinase phosphorylated PHAS-I only when it was bound to eIF4E, a finding that would seem to exclude a role in phosphorylating Ser64 in the free PHAS-I protein. It was recently concluded that MAP kinase participates in the control of PHAS-I (30
), as was proposed several years ago (20
). However, it has been argued that MAP kinase activation is neither necessary nor sufficient for the phosphorylation of PHAS-I (38
). Additional work is needed to identify the kinases that phosphorylate PHAS-I in cells.
Far Western analysis was used to investigate the effect of phosphorylating PHAS-I in vitro on eIF4E binding. An advantage of this method is that it allows a direct assessment of eIF4E binding to purified PHAS-I proteins phosphorylated in defined sites. We recently demonstrated that phosphorylation of Thr45 in T45 PHAS-I markedly decreased FLAG-eIF4E binding whereas phosphorylating Thr36 in T36 PHAS-I or Ser82 in S82 PHAS-I had less pronounced inhibitory effects on binding (40
). In the present study, phosphorylation of S64 PHAS-I by MAP kinase in vitro was found to abolish FLAG-eIF4E binding. Interestingly, introducing Asp at position 64 in 5A PHAS-I did not decrease eIF4E binding, indicating that acidic substitutions do not mimic the effect of phosphorylating the mutant protein in Ser64. The excellent correlation between the loss of eIF4E binding and the stoichiometry of S64 PHAS-I phosphorylation leaves little doubt that Ser64 phosphorylation inhibits binding. In an earlier study in which WT PHAS-I was phosphorylated by MAP kinase, the loss of binding did not appear to correlate with the extent of phosphorylation of Ser64, and it was concluded that phosphorylating Ser64 did not inhibit eIF4E binding (8
). Imprecision in measuring the stoichiometry of phosphorylation of Ser64, which was complicated by the presence of other sites in this previous study, is the likely reason for the erroneous conclusion. We have been unable to efficiently phosphorylate Thr69 in vitro; however, Thr69 in T69 PHAS-I may be almost completely phosphorylated in cells, as evidenced by the accumulation of most of the protein in the β electrophoretic form. FLAG-eIF4E binding to this phosphorylated form was attenuated (Mothe-Satney and Lawrence, unpublished), indicating that phosphorylation of Thr69 decreases the affinity of PHAS-I for eIF4E. Taken together, the results of far Western analyses of the phosphorylated forms of T36, T45, S64, T69, and S82 PHAS-I indicate the following order for the influence of phosphorylation on eIF4E binding in vitro: Ser64 > Thr45 > Thr69 > Thr36 > Ser82. Thr45 and Ser64 flank the eIF4E-binding motif (23
), which may explain their greater influence relative to the other sites.
The effects of mutating the different sites on eIF4E binding of PHAS-I proteins in cells were assessed by both far Western analyses and copurification of mutant proteins with endogenous eIF4E. Presumably, the presence of other endogenous proteins, such as eIF4G, that influence the binding of PHAS-I to eIF4E in cells could lead to differences between binding assessed by the two methods. It is reassuring that when expressed relative to the binding of 5A PHAS-I, there was reasonably good agreement between binding results obtained by far Western analysis and by copurification with eIF4E. In contrast, the relative influence of in vitro phosphorylation of the different sites on eIF4E binding did not correlate with the relative effects of mutating the sites on eIF4E binding of PHAS-I proteins expressed in cells. For example, mutating Ser64 had little, if any, effect on the amount of PHAS-I bound to eIF4E in cells. To investigate the possibility that the Ala mutation itself might have decreased binding, we expressed proteins with Asn, Cys, and Thr substitutions at position 64. Our choice of these substitutions was influenced by the recent discussion of unpublished structural studies in which Ser64 was placed in close proximity to Glu70 in eIF4E. If they are positioned appropriately, a hydrogen bond could form between these two residues. If this were the case, introducing an Ala mutation in place of Ser64 would reduce the binding affinity, since Ala cannot participate in hydrogen bonding. The three alternative substitutions have the potential to participate in hydrogen bonding but (except for Thr) cannot be phosphorylated. The result that binding of C64 PHAS-I and N64 PHAS-I to eIF4E in cells was very similar to that of A64 PHAS-I supports the conclusion that phosphorylation of Ser64 is not necessary for the dissociation of the PHAS-I–eIF4E complex. Whether Ser64 phosphorylation is sufficient to inhibit binding in cells is still not clear.
We also found that mutating Ala36 increased eIF4E binding much more than expected on the basis of the modest effect of Thr36 phosphorylation on eIF4E binding in vitro observed previously (40
). The fact that the Ala36 mutation not only ablates the Thr36 site but also decreases the phosphorylation of Ser64 in cells is a potential explanation. The result that binding of eIF4E to PHAS-Iγ, which may be generated by the phosphorylation of Ser64 in combination with Thr69, is never observed supports the concept that phosphorylation of Ser64 may act in combination with the phosphorylation of other sites to modulate the affinity of PHAS-I for eIF4E. While such interactions also complicate the interpretation of the findings with A45 PHAS-I and A69 PHAS-I, they provide an elegant mechanism through which the phosphorylation of the TP sites can control eIF4E binding by facilitating the accumulation of phosphate in Ser64.
Mutating any individual site except Ser64 increased eIF4E binding, and there was a very good correlation between the amount of PHAS-I bound to eIF4E and the inhibition of cap-dependent mRNA translation. Thus, each of the TP sites appears to be able to influence mRNA translation, either directly by modulating the binding affinity of PHAS-I and eIF4E or indirectly by affecting the phosphorylation of Ser64. The equivalents of Thr36, Thr45, Ser64, and Thr69 are found in all members of the PHAS family thus far discovered in species ranging from slime mold to humans on the evolutionary scale. Presumably, the functional importance explains why the sites are so highly conserved.