MDM2 promotes SUMO-2/3 modification of p53 to modulate transcriptional activity

The tumor suppressor p53 is extensively regulated by post-translational modification, including modification by the small ubiquitin-related modifier SUMO. We show here that MDM2, previously shown to promote ubiquitin, Nedd8 and SUMO-1 modification of p53, can also enhance conjugation of endogenous SUMO-2/3 to p53. Sumoylation activity requires p53-MDM2 binding but does not depend on an intact RING finger. Both ARF and L11 can promote SUMO-2/3 conjugation of p53. However, unlike the previously described SUMO-1 conjugation of p53 by an MDM2-ARF complex, this activity does not depend on the ability of MDM2 to relocalize to the nucleolus. Interestingly, the SUMO consensus is not conserved in mouse p53, which is therefore not modified by SUMO-2/3. Finally, we show that conjugation of SUMO-2/3 to p53 correlates with a reduction of both activation and repression of a subset of p53-target genes.     

Activation of p53 by conjugation to the ubiquitin-like protein SUMO-1

The growth-suppressive properties of p53 are controlled by posttranslational modifications and by regulation of its turnover rate. Here we show that p53 can be modified in vitro and in vivo by conjugation to the small ubiquitin-like protein SUMO-1. A lysine residue at amino acid position 386 of p53 is required for this previously undescribed modification, strongly suggesting that this lysine residue serves as the major attachment site for SUMO-1. Unlike ubiquitin, attachment of SUMO-1 does not appear to target proteins for rapid degradation but rather, has been proposed to change the ability of the modified protein to interact with other cellular proteins. Accordingly, we provide evidence that conjugation of SUMO-1 to wild-type p53 results in an increased transactivation ability of p53. We suggest that posttranslational modification of p53 by SUMO-1 conjugation provides a novel mechanism to regulate p53 activity.     

MDM2 promotes SUMO-2/3 modification of p53 to modulate transcriptional activity

The tumor suppressor p53 is extensively regulated by post-translational modification, including modification by the small ubiquitin-related modifier SUMO. We show here that MDM2, previously shown to promote ubiquitin, Nedd8 and SUMO-1 modification of p53, can also enhance conjugation of endogenous SUMO-2/3 to p53. Sumoylation activity requires p53-MDM2 binding but does not depend on an intact RING finger. Both ARF and L11 can promote SUMO-2/3 conjugation of p53. However, unlike the previously described SUMO-1 conjugation of p53 by an MDM2-ARF complex, this activity does not depend on the ability of MDM2 to relocalize to the nucleolus. Interestingly, the SUMO consensus is not conserved in mouse p53, which is therefore not modified by SUMO-2/3. Finally, we show that conjugation of SUMO-2/3 to p53 correlates with a reduction of both activation and repression of a subset of p53-target genes.Key words: p53, SUMO-2/3, sumoylation, MDM2, ARF, L11     

SUMO-1 modification of Mdm2 prevents its self-ubiquitination and increases Mdm2 ability to ubiquitinate p53

Mdm2 is an E3 ubiquitin ligase for the p53 tumor suppressor protein. We demonstrate that Mdm2 is conjugated with SUMO-1 (sumoylated) at Lys-446, which is located within the RING finger domain and plays a critical role in Mdm2 self-ubiquitination. Whereas mutant Mdm2(K446R) is stabilized, it elicits increased degradation of p53 and concomitant inhibition of p53-mediated apoptosis. In vitro sumoylation of Mdm2 abrogates its self-ubiquitination and increases its ubiquitin ligase activity toward p53. Radiation caused a dose- and time-dependent decrease in the degree of Mdm2 SUMO-1 modification, which is inversely correlated with the levels of p53. Our results suggest that the maintenance of the intrinsic activity of a RING finger E3 ubiquitin ligase is sumoylation dependent and that reduced Mdm2 sumoylation in response to DNA damage contributes to p53 stability.     

Identification of a substrate recognition site on Ubc9

Human Ubc9 is homologous to ubiquitin-conjugating enzymes. However, instead of conjugating ubiquitin, it conjugates a ubiquitin homologue, small ubiquitin-like modifier 1 (SUMO-1), also known as UBL1, GMP1, SMTP3, PIC1, and sentrin. The SUMO-1 conjugation pathway is very similar to that of ubiquitin with regard to the primary sequences of the ubiquitin-activating enzymes (E1), the three-dimensional structures of the ubiquitin-conjugating enzymes (E2), and the chemistry of the overall conjugation pathway. The interaction of substrates with Ubc9 has been studied using NMR spectroscopy. Peptides with sequences that correspond to those of the SUMO-1 conjugation sites from p53 and c-Jun both bind to a surface adjacent to the active site Cys93 of human Ubc9, which has been previously shown to include residues that demonstrate the most significant dynamics on the microsecond to millisecond time scale. Mutations in this region, Q126A, Q130A, A131D, E132A, Y134A, and T135A, were constructed to evaluate the role of these residues in SUMO-1 conjugation. These alterations have significant effects on the conjugation of SUMO-1 with the target proteins p53, E1B, and promyelocytic leukemia protein and define a substrate binding site on Ubc9. Furthermore, the SUMO-1 conjugation site of p53 does not form any defined secondary structure when either free or bound to Ubc9. This suggests that a defined secondary structure at SUMO-1 conjugation sites in target proteins is not necessary for recognition and conjugation by the SUMO-1 pathway.     

PRIMA-1 induces apoptosis by inhibiting JNK signaling but promoting the activation of Bax

The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 40% of breast cancers and are indicative of tumor resistance to chemotherapeutic agents. Recently, there has been a high degree of interest in pharmacological approaches for restoring the normal function to mutant p53. The low molecular weight compound p53 reactivation and induction of massive apoptosis (PRIMA-1) was shown to induce cytotoxic effects and apoptosis in human tumor cells with mutant p53. Here, we studied the molecular mechanisms of PRIMA-1-induced apoptosis in human breast cancer cells with p53 mutations such as MDA-231 and GI-101A as compared to MCF-7 cells. We show that PRIMA-1 selectively induces apoptosis in human breast cancer cells MDA-231 and GI-101A compared to the MCF-7. This effect was paralleled by an increase in total p53 level in the nucleus and the induction of its phosphorylation at Ser-15 site. Using the chromatin immunoprecipitation (ChIP) assays, we show that PRIMA-1 restored p53 DNA binding activity to the promoters of the proapoptotic genes such as Bax and PUMA, but inhibited the binding activity to the promoters of the MAP4K4 gene. Knockdown of p53 protein in breast cancer cells using siRNA followed by PRIMA-1 treatment resulted in decline of Bax and PUMA proteins expression. Cell incubation with either PRIMA-1 or SP600125 (c-Jun NH2-terminal kinase inhibitor) resulted in the abrogation of adriamycin-induced c-Jun NH2-terminal kinase (JNK) activation, whereas Bax activation was not inhibited. We conclude that both Bax and PUMA but not JNK signaling are involved in PRIMA-1-induced apoptosis in breast cancer cells with p53 mutation.     

Immunosuppressant FK506 activates NF-kappaB through the proteasome-mediated degradation of IkappaBalpha. Requirement for Ikappabalpha n-terminal phosphorylation but not ubiquitination sites

The immunosuppressant FK506 activates NF-kappaB through IkappaBalpha degradation in nonlymphoid cells. In the present study, we analyzed mechanisms by which FK506 induces IkappaBalpha degradation. We found that FK506 induces the degradation of both IkappaBalpha and IkappaBbeta and that the time courses of the FK506-induced degradation are quite different from degradation induced by interleukin 1 (IL-1). Despite this difference, FK506-induced IkappaBalpha degradation was dependent on the N-terminal Ser-32 and Ser-36 phosphorylation sites and was mediated by proteasomes, as is the case for IL-1-induced IkappaBalpha degradation. We further showed that FK506 induces weak and slow phosphorylation of IkappaBalpha at Ser-32. However, unlike IL-1-induced degradation, IKK-1 and IKK-2 were not activated significantly nor was FK506-induced IkappaBalpha degradation dependent on the N-terminal ubiquitination sites (Lys-21 and Lys-22). These results therefore indicate that FK506 and IL-1 utilize similar but distinct mechanisms to induce the phosphorylation and degradation of IkappaBalpha.     

The NEDD8 pathway is essential for SCF(beta -TrCP)-mediated ubiquitination and processing of the NF-kappa B precursor p105

The p50 subunit of NF-kappaB is generated by limited processing of the precursor p105. IkappaB kinase-mediated phosphorylation of the C-terminal domain of p105 recruits the SCF(beta-TrCP) ubiquitin ligase, resulting in rapid ubiquitination and subsequent processing/degradation of p105. NEDD8 is known to activate SCF ligases following modification of their cullin component. Here we show that NEDDylation is required for conjugation and processing of p105 by SCF(beta-TrCP) following phosphorylation of the molecule. In a crude extract, a dominant negative E2 enzyme, UBC12, inhibits both conjugation and processing of p105, and inhibition is alleviated by an excess of WT- UBC12. In a reconstituted cell-free system, ubiquitination of p105 was stimulated only in the presence of all three components of the NEDD8 pathway, E1, E2, and NEDD8. A Cul-1 mutant that cannot be NEDDylated could not stimulate ubiquitination and processing of p105. Similar findings were observed also in cells. It should be noted that NEDDylation is required only for the stimulated but not for basal processing of p105. Although the mechanisms that underlie processing of p105 are largely obscure, it is clear that NEDDylation and the coordinated activity of SCF(beta-TrCP) on both p105 and IkappaBalpha serve as an important regulatory mechanism controlling NF-kappaB activity.     

Identification of the enzyme required for activation of the small ubiquitin-like protein SUMO-1

The ubiquitin-like protein SUMO-1 is conjugated to a variety of proteins including Ran GTPase-activating protein 1 (RanGAP1), IkappaBalpha, and PML. SUMO-1-modified proteins display altered subcellular targeting and/or stability. We have purified the SUMO-1-activating enzyme from human cells and shown that it contains two subunits of 38 and 72 kDa. Isolation of cDNAs for each subunit indicates that they are homologous to ubiquitin-activating enzymes and to the Saccharomyces cerevisiae enzymes responsible for conjugation of Smt3p and Rub-1p. In vitro, recombinant SAE1/SAE2 (SUMO-1-activating enzyme) was capable of catalyzing the ATP-dependent formation of a thioester linkage between SUMO-1 and SAE2. The addition of the SUMO-1-conjugating enzyme Ubch9 resulted in efficient transfer of the thioester-linked SUMO-1 from SAE2 to Ubch9. In the presence of SAE1/SAE2, Ubch9, and ATP, SUMO-1 was efficiently conjugated to the protein substrate IkappaBalpha. As SAE1/SAE2, Ubch9, SUMO-1, and IkappaBalpha are all homogeneous, recombinant proteins, it appears that SUMO-1 conjugation of IkappaBalpha in vitro does not require the equivalent of an E3 ubiquitin protein ligase activity.     

SUMO-1 conjugation in vivo requires both a consensus modification motif and nuclear targeting

SUMO-1 is a small ubiquitin-related modifier that is covalently linked to many cellular protein targets. Proteins modified by SUMO-1 and the SUMO-1-activating and -conjugating enzymes are located predominantly in the nucleus. Here we define a transferable sequence containing the PsiKXE motif, where Psi represents a large hydrophobic amino acid, that confers the ability to be SUMO-1-modified on proteins to which it is linked. Whereas addition of short sequences from p53 and IkappaBalpha, containing the PsiKXE motif, to a carrier protein is sufficient for modification in vitro, modification in vivo requires the additional presence of a nuclear localization signal. Thus, protein substrates must be targeted to the nucleus to undergo SUMO-1 conjugation.     

Common pathway for the ubiquitination of IkappaBalpha, IkappaBbeta, and IkappaBepsilon mediated by the F-box protein FWD1.

FWD1 (the mouse homolog of Drosophila Slimb and Xenopus betaTrCP, a member of the F-box- and WD40 repeat-containing family of proteins, and a component of the SCF ubiquitin ligase complex) was recently shown to interact with IkappaBalpha and thereby to promote its ubiquitination and degradation. This protein has now been shown also to bind to IkappaBbeta and IkappaBepsilon as well as to induce their ubiquitination and proteolysis. FWD1 was shown to recognize the conserved DSGPsiXS motif (where Psi represents the hydrophobic residue) present in the NH(2)-terminal regions of these three IkappaB proteins only when the component serine residues are phosphorylated. However, in contrast to IkappaBalpha and IkappaBbeta, the recognition site in IkappaBepsilon for FWD1 is not restricted to the DSGPsiXS motif; FWD1 also interacts with other sites in the NH(2)-terminal region of IkappaBepsilon. Substitution of the critical serine residues in the NH(2)-terminal regions of IkappaBalpha, IkappaBbeta, and IkappaBepsilon with alanines also markedly reduced the extent of FWD1-mediated ubiquitination of these proteins and increased their stability. These data indicate that the three IkappaB proteins, despite their substantial structural and functional differences, all undergo ubiquitination mediated by the SCF(FWD1) complex. FWD1 may thus play an important role in NF-kappaB signal transduction through regulation of the stability of multiple IkappaB proteins.     

Conjugation of Nedd8 to CUL1 enhances the ability of the ROC1-CUL1 complex to promote ubiquitin polymerization

The SCF-ROC1 ubiquitin-protein isopeptide ligase (E3) ubiquitin ligase complex targets the ubiquitination and subsequent degradation of protein substrates required for the regulation of cell cycle progression and signal transduction pathways. We have previously shown that ROC1-CUL1 is a core subassembly within the SCF-ROC1 complex, capable of supporting the polymerization of ubiquitin. This report describes that the CUL1 subunit of the bacterially expressed, unmodified ROC1-CUL1 complex is conjugated with Nedd8 at Lys-720 by HeLa cell extracts or by a purified Nedd8 conjugation system (consisting of APP-BP1/Uba3, Ubc12, and Nedd8). This covalent linkage of Nedd8 to CUL1 is both necessary and sufficient to markedly enhance the ability of the ROC1-CUL1 complex to promote ubiquitin polymerization. A mutation of Lys-720 to arginine in CUL1 eliminates the Nedd8 modification, abolishes the activation of the ROC1-CUL1 ubiquitin ligase complex, and significantly reduces the ability of SCF(HOS/beta)(-TRCP)-ROC1 to support the ubiquitination of phosphorylated IkappaBalpha. Thus, although regulation of the SCF-ROC1 action has been previously shown to preside at the level of recognition of a phosphorylated substrate, we demonstrate that Nedd8 is a novel regulator of the efficiency of polyubiquitin chain synthesis and, hence, promotes rapid turnover of protein substrates.