TI - Discussion . AB - In this study , we have shown that two independent mechanisms : trans-activation and mRNA stabilization are responsible for the dramatic up-regulation of p21 by Tax . Our data are in agreement with other studies showing that Tax up-regulates p21 expression [40,45-49] . We have mapped the cis-elements in the p21 promoter that mediate Tax trans-activation to two Sp1 -binding sites ( -83 to -64 ) near the p21 mRNA start site . This region has been shown previously to be critical for TGF-beta -mediated trans-activation of p21 . Tax does not alter the intracellular level of Sp1 . Rather it binds Sp1 directly . Analyses of Tax mutants further suggest that the interaction between Tax and CBP/p300 is important for p21 promoter trans-activation . Our results are consistent with a model in which Tax may serve as a tether between Sp1 and CBP/p300 , and possibly help recruit the latter to the p21 promoter to activate transcription . These results are in agreement with a previous report that Tax can interact with Sp1 to activate c-sis/platelet-derived growth factor-B promoter [50] . Importantly , the 5-fold trans-activation of the p21 promoter cannot account fully for the dramatic 40-fold up-regulation of p21 by Tax [23] . Our data further indicate that Tax also induces post-transcriptional stabilization of p21 mRNA . The level of p21 , like that of p27 , is cell cycle-regulated . It increases transiently after cellular exit from mitosis and entry into G1 , and declines during G1/S transition . The increase in p21 and p27 after mitotic exit serves to maintain cells in G1 until such time when a new round of DNA replication is warranted . P21 also functions as a scaffold/chaperone for Cdk4-Cdk6/cyclin D assembly , thus helps set the stage for a new round of cell cycle . Since APC/C is intimately involved in regulating mitotic progression and mitotic exit , it is not surprising that the transient increase of p21 and p27 during early G1 is linked to the APC/C activity . P27 is a SUBstrate of the E3 ubiquitin ligase , SCFSkp2 [28-30] , which targets p27 for degradation . Recent evidence has indicated that Skp2 and another component of the SCF complex , Cks1 , are both SUBstrates of APC/C . During mitotic exit , APCCdh1 targets the degradation of Skp2 and Cks1 , thereby rendering SCFSkp2 inactive [51,52] . This in turn leads to the transient stabilization of p27 during G1 . We have found that APC/C becomes activated by Tax in an unscheduled manner during S phase [23] , thus causing premature degradation of a number of mitotic and cell cycle regulators including cyclin B1 and Skp2 . The loss of Skp2 ( and possibly Cks1 ) in turn causes Tax-expressing cells to accumulate p27 throughout S , G2 and M , leading to rapid senescence . Indeed , the half-life of p27 protein becomes greatly increased by Tax ( Fig 5A and ref [23] ) . Based on earlier reports that p21 may be a SUBstrate of SCFSkp2 [44] , we originally thought that the dramatic increase in p21 induced by Tax is also mediated by the same mechanism as that for p27 , i.e.through the inactivation of SCFSkp2 . Contrary to earlier reports , our present data suggest that p21 is not a SUBstrate of SCFSkp2 , and the protein stability of p21 , unlike that of p27 , is not affected by Tax (Fig 5B) . Rather , in addition to the aforementioned p21 promoter trans-activation , Tax also causes p21 mRNA half-life to increase significantly . Our data further suggest that APC/C activation by Tax is responsible for the dramatic stabilization of p21 mRNA . We have previously isolated several Tax mutants whose expression did not cause S.cerevisiae to undergo cell cycle arrest . Most of these mutants , including L235F and A108T , are attenuated in up-regulating p21 and p27 [27] . Importantly , they were found to be impaired in activating the APC/C [27] . As shown in Fig. 4 , the inability of APC/C-deficient L235F and A108T mutants to fully up-regulate p21 is not due to a deficit in p21 promoter trans-activation . An in-depth analysis of L235F indicates that it is attenuated in stabilizing p21 mRNA ( Fig 6 ) . Based on these results , we suggest that the stabilization of p21 mRNA during normal cell cycle progression or by Tax may be causally linked to the activity of the APC/C E3 ligase . Alternatively , since IKK-NF-kappaB activation and APC/C activation by Tax appear correlated [27] , the stabilization of p21 may be a result of IKK or NF-kappaB activation . How APC/C activity or IKK/NF-kappaB activation leads to p21 mRNA stabilization mechanistically remains to be determined . It has been reported previously that the 3' untranslated region ( 3'-UTR ) of the p21 transcript contains AU-rich elements ( AREs ) that destabilizes p21 mRNA [53] . RNA -binding proteins of the HuR family including HuR and its homologues HuB , HuC and HuD , on the other hand , bind AREs and prevent ARE-containing mRNAs such as that of p21 from degradation [54,55] . HuR proteins reside in the nucleus and their interaction with mRNA is facilitated by increased cytoplasmic localization of HuR or through post-translational modifications of HuR that increases its RNA binding affinity [56-58] . Whether Tax-APC/C -mediated and HuR-related mechanisms of p21 mRNA stabilization are connected is currently under investigation .