SUMO modification of STAT1 and its role in PIAS-mediated inhibition of gene activation

The PIAS (protein inhibitors of activated STAT) family of proteins were first discovered as inhibitors of activated signal transducers and activators of transcription (STATs). More recently these proteins have been shown to function as E3 ligases that promote the SUMO modification of a number of transcription regulators. We have investigated the relationship between the effects of PIAS proteins on STAT1 transcriptional activity and the ability of the PIAS proteins to function as SUMO E3 ligases. We demonstrate that STAT1 is a substrate for SUMO modification and that PIASx-alpha, but not PIAS1, functions as an E3 ligase to promote STAT1 modification. In addition, we have mapped the major site for SUMO modification on STAT1 to lysine 703. This lysine residue is in close proximity to the regulatory tyrosine residue at position 701, whose phosphorylation mediates STAT1 activation in response to cytokine signaling. Mutation of lysine 703 to arginine abolishes SUMO modification of STAT1 both in vitro and in vivo. However, this mutation does not affect the activation of STAT1 or the ability of either PIAS1 or PIASx-alpha to function as an inhibitor of STAT1-mediated transcription activation. Our findings demonstrate that inhibition of STAT1 by PIAS proteins does not require SUMO modification of STAT1 itself. SUMO modification of STAT1 may nonetheless be functionally important given the close proximity between the SUMO modification site and tyrosine 701.