PIAS proteins can bind to distinct classes of nuclear proteins and enhance or repress transcriptional activities of structurally unrelated factors, such as steroid receptors and STATs (4
), and their biological functions are clearly not restricted to the inhibition of STAT signaling. Even though PIAS1 and Miz1 possess modest intrinsic transcription activation functions and ARIP3 and PIAS3 lack this activity, the pleiotropic effects of PIAS proteins on transcriptional regulation can hardly derive from these functions (25
). The mechanism of PIAS protein action has remained elusive, and one study has suggested that it involves formation of complexes with other coregulatory proteins (25
Using PIASxα/ARIP3 as the main representative of PIAS proteins, we demonstrate in this work that ARIP3 interacts via separate conserved structural domains with both SUMO-1 and its E2 conjugase, Ubc9. In addition, ARIP3, Miz1, and PIAS1 are heavily sumoylated even though their sequences do not harbor recognizable consensus SUMO-1 attachment motifs. Many distinct proteins have recently been shown to be modified by SUMO-1 (31
), and the ability of a given protein to interact with Ubc9 and SUMO-1 generally reflects its capability of becoming sumoylated. The fact that ARIP3, Miz1, and PIAS1 interact with other sumoylated proteins and possess a peculiar sumoylation feature, i.e., they lack consensus SUMO-1 acceptor sites, suggests that these proteins participate in the sumoylation process itself.
It has been suggested that protein motifs that contain Ser-rich and Glu- and Asp-rich stretches preceded by hydrophobic residues mediate interactions with free SUMO-1 (32
). In agreement with this notion, deletion of the region comprising amino acids 467 to 487, fulfilling the characteristics of a potential SUMO-1 binding motif, abolished the interaction of ARIP3 with unconjugated SUMO-1 in vitro. This deletion did not, however, reduce sumoylation of ARIP3 in vivo or in vitro, but it influenced the recruitment of other sumoylated proteins. By contrast, ARIP3Δ347-418, which is devoid of the zinc finger-like region, was both very weakly sumoylated and incapable of tethering other sumoylated proteins. Colocalization of SUMO-1 and PIAS proteins in the same subnuclear sites is likely to reflect both the covalent attachment of SUMO-1 to PIAS proteins and their ability to bind other sumoylated proteins at those subnuclear domains. These results are in line with the findings that deletion of the SUMO-1-binding motif weakened the formation of nuclear cogranules by ARIP3 and SUMO-1 and that removal of the zinc finger-like structure abolished the nuclear granules.
In addition to binding proteins in a zinc finger-like domain-dependent fashion, ARIP3, Miz1, and PIAS1 clearly promoted sumoylation of other proteins in a manner that was also dependent on this domain. Both ARIP3 and PIAS1 were capable of stimulating SUMO-1 attachment to AR in intact cells, whereas PIAS1, but not ARIP3, enhanced sumoylation of GRIP1. Furthermore, ARIP3 enhanced sumoylation of AR and c-Jun in a RING-like domain-dependent manner in a cell-free system utilizing purified recombinant proteins. The activity of many E3-type ubiquitin protein ligases relies on their RING finger structures. It is, therefore, intriguing that the central regions of mammalian PIAS proteins, except for PIAS3, harbor seven cysteine residues and a histidine which are mandatory for a C3HC4-type RING finger motif. Indeed, threading analysis of this PIAS region suggests that its three-dimensional structure is similar to a C3HC4 RING finger fold. However, the spacing between potential zinc-coordinating residues and the amino acid composition of the mammalian PIAS RING-like structure differ substantially from those of the RING motifs of E3 ubiquitin ligases. Interestingly, PIAS3, which is poorly sumoylated in cell-free conditions and in intact cells, does not have a cysteine residue at the fourth position of the putative C3HC4 motif.
The crystal structure of c-Cbl E3 ubiquitin ligase bound to a cognate E2 and a substrate peptide suggests that the RING finger domains not only recruit the substrate and the E2 enzyme but also serve as scaffolds that position and orient them optimally for ubiquitin transfer (64
). A number of RING finger proteins which function as ubiquitin E3 ligases or which have been implicated in protein degradation possess a conserved Trp residue between cysteines at the fifth and sixth positions of their RING (17
). This Trp in c-Cbl forms part of the RING interface for the interaction with E2 ligase, and Trp mutations eliminate the E3 activity. All PIAS proteins contain a Trp residue in their central Cys-rich regions. It is conserved in more distantly related proteins possessing a similar Cys-rich structure. Conversion of the Trp to Ala in PIASxα/ARIP3 (W383A) and PIAS1 (W372A) abolished their ability to promote protein sumoylation, indicating that this amino acid residue plays an important role in the sumoylation process. Thus, PIAS proteins harboring a Miz zinc finger structure seem to function as E3 SUMO-1 ligases, facilitating Ubc9-dependent sumoylation in a fashion that resembles the action of RING-type E3 ligases in ubiquitination.
Ubc9 interacts physically with almost all known SUMO acceptors, which may indicate that it is sufficient for substrate recognition in vivo. It has been suggested that sumoylation sites per se act as major determinants of Ubc9 binding and SUMO-1 modification, which argues against the need for E3 ligase activity in sumoylation (47
). However, even though purified or recombinant E1 and E2 enzymes are sufficient to sumoylate target proteins in vitro, these reactions are slow and inefficient compared to reactions in intact cells (1
). The notion that Ubc9 alone is not likely to be able to discriminate between different SUMO proteins and, therefore, is not capable of conferring specificity also strongly points to the existence of additional regulatory factors in sumoylation (58
). The high degree of homology between Ubc9 and ubiquitin E2 ligases suggests that the transfer of SUMO-1 to target proteins is very similar to that in ubiquitination and that substrate specificity in sumoylation requires additional E3-type ligases. Our results showing that PIAS proteins differ in their abilities to promote SUMO-1 attachment to GRIP1 indeed support the notion that different PIAS proteins act as E3 SUMO ligases with dissimilar substrate preferences for target proteins.
Enzymes of sumoylation are predominantly nuclear, and proteins involved in various nuclear functions (DNA replication and repair, mitosis, and transcriptional regulation) are substrates for SUMO attachment (31
). There is compelling evidence for a critical role of SUMO modifications in the regulation of protein-protein interactions and subcellular and subnuclear localization (31
). Conjugation of SUMO-1 to RanGAP1 targets it to the nuclear pore complex (30
), and modification of PML by SUMO-1 directs it to subnuclear domains termed PML nuclear bodies (38
). The latter modification is also needed for PML-mediated recruitment of other proteins (15
), which may also be the case with PIAS proteins. Sumoylation may also prevent degradation of the target protein, as exemplified by IκBα (5
). In view of the role of SUMO-1 in protein complex formation, one can envision that sumoylation modulates transcription factor-coregulator interactions (12
, 41, 42
). Our present data on the PIAS1-promoted sumoylation of coactivator GRIP1 support this possibility. In addition to interacting with various sequence-specific transcription factors and influencing their sumoylation state, PIAS proteins may act as platforms to recruit other sumoylated proteins or proteins to be sumoylated, such as transcriptional coactivators and corepressors. Notably, the effects of ARIP3 and PIAS1 on AR-dependent transcription are dependent on both the SUMO-binding motif and the ligase domain. However, all effects of PIAS proteins on AR-dependent transcription cannot be explained through events based solely on the sumoylation of AR. It is important to note that PIAS proteins have multiple interaction partners in transcriptional regulation, including steroid receptor coactivator GRIP1, and their effects (positive or negative) on a given promoter are most likely combinatorial and reflect the overall effects of the interaction partners. In agreement with this notion, we have recently shown that PIAS proteins and GRIP1 exhibit synergistic effects on steroid receptor-dependent signaling and that the RING-like domain of PIAS proteins plays a crucial role also in this process (26
The function of PIAS or PIAS-like proteins in the regulation of chromosome structure and function may also be linked to protein sumoylation. The Drosophila
PIAS protein homologue Su(var)2-10 (Zimp) is required for proper chromosome structure and inheritance, and one study suggested that it controls multiple aspects of chromosome function by maintaining chromosome organization in interphase nuclei (13
). The finding that defects in chromosome condensation in S. cerevisiae
caused by the loss of the SMT4
gene are bypassed when the SIZ1
gene is overexpressed (52
) connects a PIAS-like protein to sumoylation processes. Specifically, SMT
encodes an evolutionarily conserved protease exhibiting SUMO-cleaving isopeptidase activity and Siz1 shows extensive similarity to animal PIAS proteins. Indeed, while this work was being finalized for publication, Siz1 and related protein Siz2 (Nfi1) were reported to promote sumoylation in yeast (20
) and Siz1 was shown to enhance sumoylation of septins in vitro (20
). Moreover, contemporaneous studies suggest that PIAS1 and PIASy function as SUMO ligases for p53 and LEF1, respectively (22
In summary, our results along with very recent data from other laboratories convincingly argue that proteins with the conserved Miz zinc finger function as E3 SUMO-1 ligases. Mammalian PIAS proteins themselves undergo sumoylation in a fashion that resembles automodification of RING finger E3 ubiquitin ligases (29
), which may play a self-regulatory role. Furthermore, the domains responsible for SUMO-1 binding and SUMO-1 conjugation are segregated, and both of these structures are critical for nuclear targeting of and transcriptional regulation by the PIAS proteins. Importantly, mammalian PIAS proteins display target protein selectivity in intact cells. In view of these considerations, we suggest that many of the effects of the PIAS protein on various transcription factors are exerted through their SUMO-tethering and SUMO ligase activities and that these properties are linked to their ability to interact with and modulate the activities of distinct transcription factors.