PIAS proteins promote SUMO-1 conjugation to STAT1

Abstract

IntroductionRegulation of signal transducer and activator of transcription 1 (STAT1) involves posttranslational modifications such as phosphorylation of Tyr701 and Ser727 as well as arginine methylation. 1,2 STAT1 signaling is negatively regulated by protein tyrosine phosphatases (PTPs), suppressors of cytokine signaling (SOCS), and protein inhibitor of activated STAT1 (PIAS) proteins. 1-3 The family of PIAS proteins consists of 5 members that have been implicated in the regulation of several nuclear proteins. PIAS1 and PIAS3 were identified as interaction partners for STAT1 and STAT3, respectively, and they were found to inhibit the DNA-binding activity of activated STATs. [3][4][5] The other members, PIASx␣/ARIP3 (androgen receptor-interacting protein 3), PIASx␤/ Miz1, and PIASy, function as transcriptional coregulators for steroid hormone receptors. [6][7][8] Recently, several PIAS proteins have been shown to function as E3-type small ubiquitin-like modifier (SUMO) ligases. 9-12 SUMO-1, -2, and -3 are small modifier proteins that are covalently conjugated to specific lysine residues of target proteins. 13 A number of nuclear proteins, such as transcription factors AR, p53, and c-Jun, become modified by SUMO-1 conjugation, and this reversible posttranslational modification has been implicated in regulation of proteinprotein interactions, protein stability, localization, or activity. 9,13,14 However, it is currently unknown whether STAT factors are modified through sumoylation. Study design ReagentsAntibodies used include anti-SUMO-1 (mouse anti-GMP-1) (Zymed, San Francisco, CA); anti-HA (clone 16B12) (Berkeley-Antibody, Richmond, CA); anti-Flag (anti-Flag M2) (Sigma Aldrich, St Louis, MO); anti-STAT1 (N terminus) (Transduction Laboratories, BD Biosciences); anti-STAT3 (Santa Cruz Biotechnology, Santa Cruz, CA) biotinylated antimouse (Dako, Glustrup, Denmark) and streptavidin-biotin horseradish peroxidase conjugate (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom). Human interferon-␥ (huIFN-␥) was purchased from Immugenex, Los Angeles, CA. Plasmid constructsSUMO-1-Flag and SUMO-1-Flag-Flag were a kind gift from Dr H. Yasuda. 15 The SUMO-1, Flag-PIAS1, Flag-PIAS1mut (PIAS1⌬310-407), Flag-PIAS3, Flag-ARIP3 Flag-ARIP3mut (ARIP3⌬347-418) plasmids 4,9 as well as STAT1-WT-HA and GAS-luc reporter construct have been previously described. 16 The STAT1 Lys703Arg mutation was created from STAT1-WT-HA using direct polymerase chain reaction (PCR) mutagenesis with the following primers: 5ЈGGAACTGGATATATCAGGACTGAGTTGATTTCTGTGTCTG-3Ј and 5Ј-CAGACACAGAAATCAACTCAGTCCTGATATATCCAGTTCC-3Ј. Transfections, luciferase assay, and immunodetectionCOS-7 cells were electroporated using a Bio-Rad (Hercules, CA) gene pulser at 260 V and 960 microfarads (F) and lysed in Triton X lysis buffer supplemented with 5 mM N-ethylmaleimide (NEM) (Sigma Aldrich). HeLa cells were transfected using a calcium phosphate method. For luciferase assay, U3A cells were transfected using a calcium phosphate method as described. 16 Immunodetection wa...

PIASy-Deficient Mice Display Modest Defects in IFN and Wnt Signaling

“…Notably, the sumoylation of proteins by PIAS proteins does not necessarily correlate with changes in their activity. For example, PIAS1 and PIASx␣ both modulate the activity of STAT1 in transfection assays (36,40). However, the sumoylation of STAT1 by PIASx␣ does not correlate with changes in transcriptional activation as a mutation of the SUMO acceptor site at lysine 703 of STAT1 has no effect on STAT1 activity (36).…”

“…Second, PIAS proteins contain a cysteine-rich RING domain, which is a hallmark for ubiquitin ligases and is functionally important for the sumoylation activity of PIAS proteins (3,22,(33)(34)(35). Finally, PIAS proteins display some specificity of protein substrates, although the specificity is not as pronounced as for ubiquitin ligases (5,22,40).…”

Regulation of cytokine signaling pathways by PIAS proteins

“…It has been shown that STAT1 can be sumoylated on Lys703, which can be strongly enhanced by PIAS proteins [34,35]. In addition, IFN-γ treatment appears to induce STAT1 sumoylation [35].…”

“…In addition, IFN-γ treatment appears to induce STAT1 sumoylation [35]. However, whether the SUMO E3 ligase activity of PIAS1 is involved in regulating STAT1 signaling is highly controversial.…”

“…However, whether the SUMO E3 ligase activity of PIAS1 is involved in regulating STAT1 signaling is highly controversial. While Rogers et al, showed that the mutation of Lys703 to Arg did not affect the activation of STAT1 or the ability of PIAS1 to repress STAT1-mediated gene activation [34], Ungureanu et al, showed that the same mutation resulted in increased STAT1-dependent gene activation in response to IFN-γ [35]. It is possible that the effect of sumoylation on the activity of STAT may be dependent on the promoter context of individual genes.…”