Regulation by PIAS Proteins

The activity of STAT proteins can be regulated by the protein inhibitor of activated STAT (PIAS) family (Shuai and Liu, 2003). The involvement of PIAS proteins in the regulation of STATs was first revealed through yeast two-hybrid assays. Using STATip as the bait, PIAS1 was isolated as a STAT 1-interacting protein (Chung et al., 1997; Liu et al., 1998). Subsequent cDNA library screening and sequence analysis have identified additional members of the PIAS family. The mammalian PIAS family consists of four members: PIAS1, PIAS3, PIASx, and PIASy (Shuai and Liu, 2003). Except for PIAS1, each member of the PIAS protein family has two splice isoforms. Recent studies suggest that PIAS proteins possess SUMO E3 ligase activity (Jackson, 2001; Schmidt and Muller, 2003).

The PIAS protein family contains several conserved domains (Shuai and Liu, 2003). A domain named as SAP (SAF-A/B, Acinus and PIAS) is present in the NH2-terminal region of all PIAS proteins (Aravind and Koonin, 2000). The SAP domain binds to non-specific AT-rich DNA sequences in scaffold/matrix attachment regions (S/MARs) of the chromatin (Kipp et al., 2000). PIAS proteins contain a C3HC4-type RING-finger-like zinc-binding domain (RLD) that is required for the SUMO E3 ligase activity of PIAS proteins. An LXXLL signature motif, which is known to mediate interactions between nuclear receptors and their co-regulators (Glass and Rosenfeld, 2000), is located within the SAP domain (Liu et al., 2001). The PINIT motif, located within a highly conserved region of PIAS proteins, may be involved in the nuclear retention of PIAS proteins

(Duval et al., 2003). The carboxyl-terminal regions of PIAS proteins are the most diversified, which contain a highly acidic region (AD), a serine/threonine rich (S/T) region, and a putative SUMOl interaction motif (SIM). Interestingly, the S/T region and SIM motif are absent in PIASy. The functional roles of AD, S/T, or SIM of PIAS proteins remain to be defined.

PIAS proteins, normally expressing in the nucleus, do not interact with STATs in unstimulated cells. Upon cytokine stimulation, tyrosine phosphorylated STATs translocate into the nucleus where they interact with PIAS proteins. In vivo co-immunoprecipitation studies using specific antibodies against PIAS proteins suggest that there is specificity as well as redundancy in PIAS-STAT interactions (Shuai and Liu, 2003). PIAS1, PIAS3, and PIASx interact with STAT1, STAT3, and STAT4 respectively, in response to cytokine stimulation (Arora et al., 2003; Chung et al., 1997; Liu et al., 1998). In addition, PIASy also interacts with tyrosine phosphorylated STAT1 (Liu et al., 2001). PIAS1 binds to the dimeric, but not the monomeric form of STAT 1, which may explain why PIAS-STAT interaction is cytokine-dependent (Liao et al., 2000). Members of the PIAS family have been shown to inhibit STAT-mediated gene activation through several distinct mechanisms. First, PIAS proteins can block the DNA binding activity of STAT. For example, PIAS1 and PIAS3 can inhibit the DNA binding activity of STAT1 and STAT3 in vitro respectively (Chung et al., 1997; Liu et al., 1998). Second, PIAS proteins may inhibit STAT-dependent transcription by recruiting other transcriptional co-repressors such as histone deacetyl transferases (HDACs). For example, PIASy and PIASx inhibit STAT1- and STAT4-dependent transcription without affecting their DNA binding activities (Arora et al., 2003; Liu et al., 2001). PIASx and PIASy act as transcriptional co-repressors of STATs, possibly by recruiting HDACs (Arora et al., 2003; Liu et al., 2001). Finally, PIAS proteins may inhibit the transcriptional activity of STATs by promoting SUMO modification of STATs. However, contradictory results have been reported on the role sumoylation in the regulation of STAT activity (Rogers et al., 2003; Ungureanu et al., 2005; Ungureanu et al., 2003). Further studies are needed to clarify the physiological role of PIAS SUMO ligase activity in STAT signaling.

Recently, gene-targeting analysis has been performed to understand the physiological role of PIAS proteins in cytokine signaling. Piasl null mice were runted and showed perinatal lethality (Liu et al., 2004). In PIAS1-deficient cells, the induction of a subgroup of IFN-responsive genes was enhanced, suggesting that PIAS1

has specificity in regulating IFN signaling. Consistently, Piasl null mice displayed increased protection against viral and bacterial infection. In addition, Piasl null mice were hypersensitive to LPS-induced endotoxic shock (Liu et al., 2004). These studies reveal an important role of PIAS1 in the regulation of innate immune responses and demonstrate that PIAS1 is a physiological negative regulator of STAT1.

C: Regulation by Protein Tyrosine Phosphatases (PTPs)

The activity of STATs is regulated by PTPs in both the cytoplasm and nucleus (Shuai and Liu, 2003). Since tyrosine phosphorylation is required to form the active STAT dimmer structure, the dephosphorylation of STATs in the nucleus is believed to be important in terminating the transcriptional activity of STATs. The inactivation of STAT1 in the nucleus has been extensively investigated. Through biochemical purification, TC45, the nuclear isoform of T-cell protein tyrosine phosphatase (TC-PTP), has been identified as a PTP responsible for the nuclear dephosphorylation of STAT1 (ten Hoeve et al., 2002). In vitro, TC45 can directly dephosphorylate STAT1. In Tc-ptp null mouse embryonic fibroblasts (MEFs) and primary lymphocytes, the dephosphorylation of STAT1 in the nucleus is defective. Interestingly, TC45 is also found to be involved in the nuclear dephosphorylation of STAT3, but not STAT5 or STAT6 (ten Hoeve et al.,

2002). These results suggest that there exists specificity in the nuclear dephosphorylation of STATs. Future studies are needed to identify the PTPs involved in the dephosphorylation of other STATs in the nucleus. In addition to TC45, SHP-2, an SH2-containing PTP, is also involved in the nuclear dephosphorylation of STAT1 (Wu et al., 2002).

In the cytoplasm, SHP-2 has been suggested in the dephosphorylation of STAT5. SHP-2 interacts with STAT5 and can directly dephosphorylate STAT5 (Chen et al.,

2003). In addition, the dephosphorylation of STAT5 in the cytoplasm is inhibited in Shp-2 null cells (Chen et al., 2003).

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