TI - Substitutional Mutational Analysis of the Identified Rex-1 PHOSphorylation Sites . AB - To determine possible regulatory roles of the seven identified PHOSphorylation sites , we generated single alanine amino acid substitutions and tested these Rex-1 mutants to see if they retain their ability to function in our quantitative reporter bioassay . The Rex-1 mutants were transiently co-transfected into 293T cells with pcTat and pCgagRxRE-I , along with CMV-luciferase to account for transfection efficiency . We indentified two mutants S97A and T174A that displayed significantly reduced function ( Fig 4A ) . Further mutational analysis of these two residues by converting them to phosphomimetic aspartic acid ( S97D and T174D ) restored functional activity to wtRex-1 levels , which indicated that PHOSphorylation plays a positive functional role ( Fig 4A ) . Although we did not detect PHOSphorylation of Ser-177 in our analysis , we subjected this residue to a similar mutational and functional analysis . Our results indicated that mutant S177A or S177D maintained wild type Rex functional activity ( Fig 4A ) . Moreover , aspartic acid substitution of Thr-22 , Ser-36 , Thr-37 , Ser-70 or Ser-106 had no effect on protein function , which is consistent with the conclusion that PHOSphorylation of any of these five residues does not negatively regulate function , but is silent ( data not shown ) . The steady state expression levels of the wild-type and mutant Rex-1 proteins were determined for each mutant by Western blot analysis and detected using rabbit polyclonal alpha-Rex-1 antisera ( Fig 4B ) . All of the Rex-1 mutants were stably expressed . 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] . However , evaluation of the functionally disrupted substitution mutants S97A and T174A for subcellular localization revealed no difference when compared to wild-type Rex-1 (Fig 4C) . Together , we concluded that although PHOSphorylation of Ser-97 and Thr-174 are pivotal for Rex-1 function , the substitution for alanine did not result in a significant change in subcellular localization to the cytoplasm . Since the individual mutations ( S97A and T174A ) still maintain partial function , it remained a possibility that PHOSphorylation of both residues are required for optimal biologic activity . To test this hypothesis and determine if there is a functional relationship between Ser-97 and Thr-174 , we generated and characterized the double mutant for function and protein expression . As shown in Figure 5A , the double mutant S97A , T174A displayed significantly reduced functional activity as compared to wtRex-1 , but a similar activity as the single mutants . Lastly , the nonfunctional Rex-1 mutants were next tested for their capacity to block the biological action of wtRex-1 using the pCgagRxRE-I reporter assay described above . The single mutants ( S97A and T174A ) or the double mutant ( S97A , T174A ) displayed a recessive negative phenotype , as the action of wtRex-1 was not significantly altered in their presence ( Figure 5B and data not shown ) .