Modulation of glucocorticoid receptor-interacting protein 1 (GRIP1) transactivation and co-activation activities through its C-terminal repression and self-association domains

Glucocorticoid receptor-interacting protein 1 (GRIP1), a p160 family nuclear receptor co-activator, possesses at least two autonomous activation domains (AD1 and AD2) in the C-terminal region. AD1 activity appears to be mediated by CBP/p300, whereas AD2 activity is apparently mediated through co-activator-associated arginine methyltransferase 1 (CARM1). The mechanisms responsible for regulating the activities of AD1 and AD2 are not well understood. We provide evidence that the GRIP1 C-terminal region may be involved in regulating its own transactivation and nuclear receptor co-activation activities through primary self-association and a repression domain. We also compared the effects of the GRIP1 C terminus with those of other factors that functionally interact with the GRIP1 C terminus, such as CARM1. Based on our results, we propose a regulatory mechanism involving conformational changes to GRIP1 mediated through its intramolecular and intermolecular interactions, and through modulation of the effects of co-repressors on its repression domains. These are the first results to indicate that the structural components of GRIP1, especially those of the C terminus, might functionally modulate its putative transactivation activities and nuclear receptor co-activator functions.     

Glucocorticoid receptor-interacting protein 1 mediates ligand-independent nuclear translocation and activation of constitutive androstane receptor in vivo

Phenobarbital (PB) induction of CYP2B genes is mediated by translocation of the constitutively active androstane receptor (CAR) to the nucleus. Interaction of CAR with p160 coactivators and enhancement of CAR transactivation by the coactivators have been shown in cultured cells. In the present studies, the interaction of CAR with the p160 coactivator glucocorticoid receptor-interacting protein 1 (GRIP1) was examined in vitro and in vivo. Binding of GRIP1 to CAR was shown by glutathione S-transferase (GST) pull-down and affinity DNA binding. N- or C-terminal fragments of GRIP1 that contained the central receptor-interacting domain bound to GST-CAR, but the presence of ligand increased the binding to GST-CAR of only the fragments containing the C-terminal region. In gel shift analysis, binding to CAR was observed only with GRIP1 fragments containing the C-terminal region, and the binding was increased by a CAR agonist and decreased by a CAR antagonist. Expression of GRIP1 enhanced CAR-mediated transactivation in cultured hepatic-derived cells 2-3-fold. In hepatocytes transfected in vivo, expression of exogenous GRIP1 alone induced transactivation of the CYP2B1 PB-dependent enhancer 15-fold, whereas CAR expression alone resulted in only a 3-fold enhancement in untreated mice. Remarkably, CAR and GRIP1 together synergistically transactivated the enhancer about 150-fold, which is approximately equal to activation by PB treatment. In PB-treated mice, expression of exogenous CAR alone had little effect, expression of GRIP1 increased transactivation about 2-fold, and with CAR and GRIP, a 4-fold activation was observed. In untreated mice, expression of GRIP resulted in nuclear translocation of green fluorescent protein-CAR. These results strongly suggest that a p160 coactivator functions in CAR-mediated transactivation in vivo in response to PB treatment and that the synergistic activation of CAR by GRIP in untreated animals results from both nuclear translocation and activation of CAR.     

Modification of Daxx by small ubiquitin-related modifier-1

Small ubiquitin-related modifier-1 (SUMO-1) is a protein that is covalently modified to various cellular proteins and protects cells against both anti-Fas and TNF-induced cell death. Previously, we reported that the C-terminus of Daxx interacted with Ubc9, an E2 type SUMO-1 conjugating enzyme, as well as with SUMO-1. In BOSC23 cells expressing FLAG-Daxx together with HA-SUMO-1, 110 and 130kDa Daxx appeared and the 130kDa band bound to both anti-HA and anti-FLAG antibodies. This means that Daxx can be covalently modified by SUMO-1. Substitution of K630 and K631 abrogated the modification of Daxx by SUMO-1, implying that K630 and K631 were essential for sumoylation. Daxx (K630, 631A) and Daxx (K634, 636, 637A) in which the putative C-terminal nuclear localization signals (NLSs) were disrupted appeared in the nucleus, suggesting that the C-terminal NLS was not functional. Daxx (K630, 631A), the sumoylation defective mutant, was able to interact with PML and co-localized with PML in the PML oncogenic domains (PODs). Thus, our data show that sumoylation status of Daxx does not affect its presence in PODs.     

Characterization of a family of nucleolar SUMO-specific proteases with preference for SUMO-2 or SUMO-3

SUMOylation is a reversible process regulated by a family of sentrin/SUMO-specific proteases (SENPs). Of the six SENP family members, except for SENP1 and SENP2, the substrate specificities of the rest of SENPs are not well defined. Here, we have described SENP5, which has restricted substrate specificity. SENP5 showed SUMO-3 C-terminal hydrolase activity but could not process pro-SUMO-1 in vitro. Furthermore, SENP5 showed more limited isopeptidase activity in vitro. In vivo, SENP5 showed isopeptidase activity against SUMO-2 and SUMO-3 conjugates but not against SUMO-1 conjugates. Native SENP5 localized mainly to the nucleolus but was also found in the nucleus. The N terminus of SENP5 contains a stretch of amino acids responsible for the nucleolar localization of SENP5. N-terminal-truncated SENP5 co-localized with PML, a known SUMO substrate. Using PML SUMOylation mutants as model substrates, we showed that SENP5 can remove poly-SUMO-2 or poly-SUMO-3 from the Lys160 or Lys490 positions of PML. However, SENP5 could not remove SUMO-1 from the Lys160 or Lys490 positions of PML. Nonetheless, SENP5 could remove SUMO-1, -2, and -3 from the Lys65 position of PML. Thus, SENP5 also possesses limited SUMO-1 isopeptidase activity. We were also able to show that SENP3 has substrate specificity similar to that of SENP5. Thus, SENP3 and SENP5 constitute a subfamily of SENPs that regulate the formation of SUMO-2 or SUMO-3 conjugates and, to a less extent, SUMO-1 modification.     

Protein arginine methylation of SERBP1 by protein arginine methyltransferase 1 affects cytoplasmic/nuclear distribution

Protein arginine methylation regulates a broad array of cellular processes. SERBP1 implicated in tumor progression through its putative involvement in the plaminogen activator protease cascade, is an RNA-binding protein containing an RG-rich domain and an RGG box domain that might be methylated by protein arginine N-methyltransferases (PRMTs). Asymmetric dimethylarginine (aDMA) was detected in SERBP1 and an indirect methyltransferase inhibitor adenosine dialdehyde (AdOx) significantly reduced the methylation signals. Arginines in the middle RG and C-terminal RGG region of SERBP1 are methylated based on the analyses of different deletion constructs. The predominant type I protein arginine methyltransferase PRMT1 co-immunoprecipitated with SERBP1 and the level of bound PRMT1 decreased upon the addition of AdOx. Recombinant PRMT1 methylated SERBP1 and knockdown of PRMT1 significantly reduced the aDMA level of SERBP1, indicating that SERBP1 is specifically methylated by PRMT1. Immunofluorescent analyses of endogenous SERBP1 showed predominant cytoplasmic localization of SERBP1. Treatment of AdOx or PRMT1 siRNA increased the nuclear localization of SERBP1. Analyses of different deletions indicated that the middle RG region is important for the nuclear localization while both N- and C- terminus are required for nuclear export. Low methylation of the C-terminal RGG region also favors nuclear localization. In conclusion, the RG-rich and RGG box of SERBP1 is asymmetrically dimethylated by PRMT1 and the modification affects protein interaction and intracellular localization of the protein. These findings provide the basis for dissecting the roles of SERBP1.