Members of the PIAS family act as SUMO ligases for c-Jun and p53 and repress p53 activity

Abstract

The activity of the p53 tumor suppressor protein and the c-Jun protooncogene is regulated by posttranslational modifications, such as phosphorylation or ubiquitination. In addition, covalent attachment of the ubiquitin-like modifier SUMO appears to modulate their transcriptional activity. Sumoylation proceeds via an enzymatic pathway that is mechanistically analogous to ubiquitination, but requires a different E1-activating enzyme and Ubc9, a SUMO-specific E2-conjugating enzyme. Here, we show that two members of the PIAS family, PIAS1 and PIASx␤, act as specific E3-like ligases that promote sumoylation of p53 and c-Jun in vitro and in vivo. The PIAS proteins physically interact with both p53 and c-Jun. In addition, they bind to Ubc9, suggesting that they recruit the E2 enzyme to their respective substrate. The SUMO ligase activity requires the conserved zinc-finger domain, which is distantly related to the essential RING-finger motif, found in a subset of ubiquitin ligases. Furthermore, similar to RING-type ubiquitin ligases, PIASx␤ can catalyze its own modification. Hence, these data further extend the analogy between the ubiquitin and SUMO pathway. Strikingly, PIAS proteins strongly repress the transcriptional activity of p53, suggesting that the PIAS-SUMO pathway plays a crucial role in the regulation of p53 and presumably other transcription factors. Many cellular key functions are regulated by the covalent modification of proteins with the ubiquitin-like SUMO-1 modifier (1-3). Two important examples are the transcriptional activity of the c-Jun protooncogene and the p53 tumor suppressor, which seem to be regulated by sumoylation (4-6). Mammalian SUMO-1 and its close relatives, SUMO-2 and SUMO-3, are about 18% identical to ubiquitin and are conjugated via an enzymatic cascade that requires a SUMO-specific, heterodimeric E1-activating enzyme (Aos1͞Uba2) and a single E2-type conjugating enzyme, Ubc9. In the ubiquitin pathway, at least one additional factor, called E3 or ubiquitin ligase, is required for substrate recognition and formation of an isopeptide bond between ubiquitin and the target protein. Because the SUMO-specific E2 enzyme Ubc9 has been shown to physically interact with almost all known SUMO substrates in yeast twohybrid assays, Ubc9 was considered to be sufficient for substrate recognition.Very recently, however, in the yeast Saccharomyces cerevisiae, the Siz1 and Siz2 proteins were identified as E3-like factors that promote SUMO conjugation to yeast proteins, in particular to proteins of the septin family (7-9). Siz proteins are homologous to members of the mammalian PIAS (protein inhibitor of activated Stat) family of proteins. In humans, this family consists of at least five members: PIAS1, PIAS3, the ␣ and ␤ splice variants of PIASx, and PIASy (10). PIAS proteins were initially identified as specific inhibitors of Stat transcription factors. PIAS1 and PIAS3 block the DNA binding activity of activated Stat1 and Stat3, respectively, and inhibit Stat-mediated transcription (11, 12). Subsequentl...

SUMO modification regulates inactivation of the voltage-gated potassium channel Kv1.5

“…In this regard, the K ϩ channelassociated protein KChAP (35) has been shown to be a member of the PIAS family of proteins originally identified as negative regulators of several transcriptional responses (33). PIAS proteins possess intrinsic SUMO-specific E3 activity (48), and most of their transcriptional effects depend on this function. Although the regulatory consequences of KChAP association with Kv1.5 remain to be determined, the effects on surface expression observed for other Kv channels occur in a transcriptionindependent manner (33).…”

Body language: the function of PML nuclear bodies in apoptosis regulation

“…135 Several groups reported that p53 is a target protein for SUMO-1. 42,43,143,144 SUMO-1 is covalently attached to specific lysine residues of its target proteins by an enzymatic machinery, which is similar to that of ubiquitin modification. 45 p53 is SUMOlated at Lys386 within its regulatory domain through a complex containing the SUMO-1 conjugating enzyme Ubc9 and the SUMO-1 ligase protein inhibitor of activated STAT (PIAS)1.…”

“…45 p53 is SUMOlated at Lys386 within its regulatory domain through a complex containing the SUMO-1 conjugating enzyme Ubc9 and the SUMO-1 ligase protein inhibitor of activated STAT (PIAS)1. 51,144 However, addressing the function of p53 SUMOlation on p53 activity led to contradictory results, reaching from increased activity, 42,43 no effect 143 to decreased transcriptional activity of p53. 144 The reason for these discrepancies should be clarified in the future, although it is currently not clear whether SUMOlation of p53 might take place in PML-NBs.…”

“…51,144 However, addressing the function of p53 SUMOlation on p53 activity led to contradictory results, reaching from increased activity, 42,43 no effect 143 to decreased transcriptional activity of p53. 144 The reason for these discrepancies should be clarified in the future, although it is currently not clear whether SUMOlation of p53 might take place in PML-NBs. Interestingly, similar to SUMO-1, PIAS proteins also appear to localise to PML-NBs.…”

Identification of a molecular recognition feature in the E1A oncoprotein that binds the SUMO conjugase UBC9 and likely interferes with polySUMOylation

“…Conjugation requires an E1-activating enzyme and the E2 conjugase Ubc9. Ubc9 has a role in substrate recognition by binding a CKxE/D sequence often present in target proteins (Sampson et al, 2001), and SUMO conjugation may involve E3 ligases (Schmidt and Muller, 2002). SUMOylation has an important role in regulating transcription, protein trafficking, innate immunity and cell proliferation (Kerscher et al, 2006).…”