Phosphorylation is a well-known regulatory event that plays a key role in controlling the functions of cellular and viral proteins (1
). The modulation of protein function can occur by multiple mechanisms, including regulation of subcellular localization, protein-protein interactions, protein stability, and protein-nucleic acid binding. Previously, it was demonstrated that phosphorylation of HTLV-2 Rex, predominantly on serine residues, was correlated with an altered conformation, as observed by a gel mobility shift and the detection of p24Rex
protein species (16
). Rex-2 phosphorylation and the presence of the p26Rex
species were correlated with functional activity, including specific binding to its viral mRNA target sequence, cytoplasmic export, and translation of the target mRNA (31
). Moreover, we recently reported that the carboxy terminus of Rex-2 contains a stability/inhibitory domain that is positively regulated through phosphorylation (54
). However, a complete map of site-specific phosphorylation of Rex-2 is lacking, and the precise role it plays in protein function remains unclear. The goal of this study was to use affinity purification and LC-MS/MS to identify all phosphorylation sites in both p24Rex
to provide insight into a possible phosphorylation continuum and the mechanism that controls Rex-2 function and ultimately the “on/off” switch that regulates productive viral replication. Consistent with previous reports, we confirmed that Rex-2 is phosphorylated predominantly on serine residues and some threonine residues, and although the p26Rex
species is heavily phosphorylated, p24Rex
contains minimal phosphorylation. We reported the phosphorylation of p24Rex
at two sites, Ser-117 and Thr-164. We also reconfirmed phosphorylation of Ser-151 in p26Rex
) and identified five additional phosphorylation sites, Thr-19, Ser-117, Ser-125, Ser-153, and Thr-164.
Using mutational analysis replacing the identified phosphor acceptor residues in Rex-2 with alanine or aspartic acid (which mimics phosphoserine or threonine), we showed that (i) the site-specific phosphorylations at Ser-151, Ser-153, and Thr-164 all positively contributed to Rex-2 function; (ii) the phosphorylation of Ser-151, Thr-164, and, to a lesser extent, Ser-153 dictate the p24Rex
ratio and thus Rex conformational switching; and (iii) the phosphorylation of Ser-151 regulates the ability to efficiently localize to the nucleus. Both p24Rex
are phosphorylated at Ser-117 and Thr-164, but only phosphorylation at Thr-164 is functionally significant. Thus, since we do not have evidence that Ser-151 or Ser-153 is phosphorylated without prior phosphorylation of Thr-164, a logical conclusion is that phosphorylation occurs on a continuum, with phosphorylation of Thr-164 an earlier initiating event that likely facilitates subsequent phosphorylation of Ser-151 and/or Ser-153 and Rex function. In Fig. , we present a model, consistent with the published literature and our data, showing how phosphorylation regulates Rex-2 function. The initial translation product of Rex-2, p24Rex
, is located primarily in the cytoplasm. It is an inactive species in a structural conformation in which the C terminus is configured in such a way that it prevents efficient phosphorylation of Ser-151 and Ser 153 and generation of an active p26Rex
mRNA binding protein. We propose that the phosphorylation continuum starts with the phosphorylation of Thr-164, which begins the rotation of the C-terminal inhibitory domain, exposing Ser-151 and/or Ser-153 and making them available to be phosphorylated. This is also consistent with the phenotype of a previously characterized Rex-2 truncation mutant (S158Term) in which the 13 carboxy-terminal amino acids, including the Thr-164 phosphor acceptor, are deleted; Rex S158Term, although less stable, is functionally more active than the wt Rex-2 (54
). However, since proteins are dynamic and not fixed in space, as long as one or more key phosphor acceptor sites are available within the C terminus, a hierarchical phosphorylation pattern could be sidestepped at a low frequency in mutant Rex proteins. This is consistent with the functional and Western blot results of our single (S151A or T164A) and double (S151A T164A or S153A T164A) alanine mutants, which maintain some function, and the expression of the active p26Rex
. However, the triple alanine mutant (T164A S151A S153A), which contains none of the identified key phosphor acceptor residues, shows minimal functional activity and very little detection of the active p26Rex
species. We have yet to experimentally identify the cellular kinase(s) that phosphorylates Rex on these key functional residues. However, using bioinformatics analyses, we previously reported (31
) that Ser-151 falls in a predicted consensus site for phosphorylation by casein kinase 1 (Sp/Tp-X2-3
-S/T-X). Ser-153 also shows high homology to the casein kinase 1 consensus sequence, and Thr-164 is predicted to be phosphorylated by either GSK3β or casein kinase 1 (data not shown).
FIG. 7. Rex-2 phosphorylation continuum and function model. The newly Rex-2-translated product p24Rex is inactive due to the structural and spatial inhibition of the carboxy-terminal domain (black ribbon) that masks the RBD and nuclear localization signal. Initial (more ...)
Typically, stable protein conformational changes require bond rotations. Interestingly, using proteomic protein identification (mudpit) analysis, we recently identified the association of the cellular protein Pin1 in the Rex-2 pulldown complex (data not shown). Pin1 is a peptidylproline cis-trans
isomerase of Ser/Thr peptide bonds N-terminal to proline residues. Pin1 is known to induce conformational changes that can affect enzymatic activities, phosphorylation status, subcellular localization, protein stability, and protein/protein interactions (8
). One hypothesis consistent with our data is that once Thr-164 is phosphorylated, Pin1 could bind and cause a structural conformational change, rotating the inhibitory C-terminal domain and allowing access of the cellular kinase to phosphorylate Ser-151. The single mutants alone only hinder the ability of the kinase to reach its target consensus sequence. For example, S151A or S153A could still be phosphorylated at Thr-164, facilitating subsequent phosphorylation of either Ser-153 or Ser-151, respectively, resulting in the formation of p26Rex
and modest function. In the case of the mutant T164A, the C-terminal domain is not efficiently dislocated, and since a biological protein is not static, a minimal amount of phosphorylation can occur on both Ser-151 and Ser-153. Whereas in the double mutant we not only inhibit Thr-164 phosphorylation, and thus the conformational change of the C terminus, but also block any phosphorylation of Ser-151, with this mutant we detected a greater increase in the amount of p24Rex
, which correlates with the reduced function of the mutant protein, with the only possible phosphorylation being Ser-153.
The question arises as to whether this C-terminal phosphorylation domain is unique to Rex-2 or if it is also contained in the closely related Rex-1. Although the carboxy termini of the two related proteins at the amino acid level are quite divergent with no apparent Ser-151, Ser-153, and Thr-164 counterpart in Rex-1 (31
), we have previously shown that, like that of Rex-2, the carboxy terminus of Rex-1 does contain a stability domain (54
). Rex-1 is known to be phosphorylated (1
), but further studies are needed to determine whether Rex-1 site-specific phosphorylation has a significant functional role.
Taken together, the data presented in this study provide a wealth of knowledge about the cellular signaling controlling Rex-2 protein function, structure, and localization. Further studies will be aimed at identifying the cellular proteins, including the kinase(s), involved in the regulation of Rex-2 function.