TI - Discussion . AB - PHOSphorylation plays a key role in regulating the function of cellular and viral proteins [28 , 38 , 39 , 47 , 48] . Previously , it was demonstrated that Rex-1 is a phosPHOprotein and that PHOSphorylation may play a role in Rex-1 function [39,40] . It also has been shown that Rex-1 is essential for efficient viral replication and survival in vivo [45] . Given the importance of this protein in HTLV biology , we sought to understand how Rex-1 function is regulated . Multiple studies have been directed at understanding the importance of PHOSphorylation in HTLV Rex-2 function [26,33,41,42,49] . These studies reported that phosphorylaTION at the carboxy terminus of Rex-2 is critical for protein stability , shuttling , and cellular localization , all of which are positively regulated through PHOSphorylation [28,33,49] . There have been some efforts aimed at determining the role of PHOSphorylation in the regulation of HTLV Rex-1 [39,40] . The first studies used thin layer chromatography and tryptic peptide analysis . The studies reported that the native protein was PHOSphorylated mainly on serine and threonine . Subsequently , it was reported that Rex-1 was PHOSphorylated on three residues ; Ser-70 , Ser-177 and Thr-174 . This group also speculated that protein kinase C may play a role in Rex-1 PHOSphorylation , which was supported by drug studies using the more global kinase inhibitor H-7 [40] . Neither study could conclusively identify all PHOSphorylation sites within Rex-1 , nor were any of the sites further tested for their biological relevance . In the current study , we were able to not only identify phosphorylaTED Rex-1 , but also assign PHOSphorylation to site -specific residues by peptide sequencing using tandem mass spectrometry . Consistent with previous reports , we confirmed that Rex-1 is PHOSphorylated predominantly on serine and threonine residues . We report the identification of five novel phosphorylaTION sites , Thr-22 , Ser-36 , Thr-37 , Ser-97 and Ser-106 and also confirmed the PHOSphorylation on Ser-70 and Thr-174 . Furthermore , we identified specific PHOSphorylation sites that are critical for Rex-1 function in vivo . These PHOSphorylation sites specifically include Ser-97 and Thr-174 . We previously showed that PHOSphorylation of a specific residue of Rex-2 at the carboxy terminus (Ser-151) is important for proper protein nuclear localization [28,33] . Evaluation of the functionally disrupted substitution mutants S97A and T174A for subcellular localization revealed no difference when compared to wild-type . It is important to note that Ser-97 falls within the previously characterized central core activation domain/nuclear export signal [50] , and that PHOSphorylation of this residue may be pivotal for proper Rex-1 function . Previous studies of both HIV-1 Rev and HTLV-1 Rex showed that mutations within the NES interfere with the ability of these proteins to associate with CRM1 , a cellular protein that belongs to the importin beta family and functions as a nuclear export receptor for NES-containing proteins and the Rev - and Rex -dependent viral mRNAs encoded by these complex retroviruses [29,50-54] . An important direction for future studies is to evaluate whether the non-functional mutants are defective for CRM1 binding or the efficient interaction with the Rex-response element RNA target . Thr-174 , which is located in the carboxy terminus of Rex-1 , was identified as a critical PHOSphorylation site . It was shown previously that Ser-151 , located in the carboxy terminus of Rex-2 , is a key PHOSphorylation site important for Rex-2 function in vivo [26,33] . We also demonstrated that Rex-1 and Rex-2 share a similar stability domain located within their carboxy terminus [28] . We hypothesized that PHOSphorylation of Thr-174 of Rex-1 (Fig 4B) could play a similar role in regulating Rex-1 function similar to Rex-2 Ser-151 . Further C-terminal comparison analysis is on-going to elucidate further homology between these two related proteins . One previous study identified PHOSphorylation on Ser-177 of Rex-1 [39] . Throughout our studies , we were unable to confirm this finding , but we did identify multiple new sites . One explanation for why these new phosphorylaTION sites were not identified in the earlier studies could be that the high performance liquid chromatography fraction procedure used may have resulted in a loss of other PHOSphopeptides within the protein . The selective loss of PHOSphopeptides can result from the addition of a phosphate group , thus reducing hydrophobicity , which may cause failure of the protein to be retained on the reverse-phase material used in purification [55] . An additional consideration is that the previous study analyzed Rex-1 protein derived from a different cell type ( COS-7 or HTLV-1 transformed T-cell lines ) , which may produce alternative post-translational modification patterns when compared to 293 T cells . Although it is not without its own caveat and limitations , LC-MS/MS provides a more robust method for the comprehensive mapping of PHOSphorylation sites [55-58] .