Also to their function as damaging regulators of STAT signalling, PIAS proteins also can act as SUMO E3 ligases, improving sumoylation of target proteins. This action is dependent on a RING finger domain present in PIAS proteins. Given that sumoylation of transcriptional regulators regularly leads to inhibition of their activity, PIAS proteins have already been described as damaging regulators of transcription. On the other hand, PIAS proteins can also be regarded to act as co activators. It has also been reported that PIAS pro teins can bind to and alter the subcellular localization of various proteins. Interestingly, these actions of PIAS proteins as modulators of transcription may possibly come about each in SUMO dependent or independent man ners. In an energy to considerably better realize FLASH action, we searched for new interaction partners for FLASH. Within a yeast two hybrid screening making use of FLASH as bait, we identified PIAS1 like a binding partner.
Here we present that PIAS1 enhances FLASH inhibitor LY294002 activity and its potential to co activate c Myb. PIAS1 together with FLASH is in a position to even further improve the transcriptional action of c Myb. Constant together with the up regulation of exercise of both FLASH and c Myb, all 3 proteins, FLASH, c Myb and PIAS1, are co localized in active RNA polymerase II foci. These success propose that FLASH and PIAS1 coop erate in enhancing c Myb transcriptional action in foci that resemble transcription factories. Effects FLASH interacts with PIAS1 To advance our understanding of FLASH perform, we performed a yeast two hybrid screening with FLASH as bait, employing a human bone marrow cDNA library. Because of the substantial level of autoactiva tion of complete length FLASH, even when using the centro meric reduced copy vector pDBT, we selected as bait a C terminal fragment of FLASH encoding amino acid residues 1508 1982.
This portion within the protein incorporates the DED recruiting domain and in addition incorporates the region that interacts with c Myb. The N terminal border of this fragment was positioned within a ser ineproline rich location, predicted to become found concerning globular domains de. Between the describes it beneficial clones obtained within the Y2H screening we identi fied the SUMO E3 ligase PIAS1. The interaction amongst FLASH D and PIAS1 was veri fied by retransformation in yeast and testing for activa tion in the HIS3 and LacZ reporter genes. Further Y2H mating assays applying FLASH D as bait indi cated that PIAS1 didn’t interact with other regions of FLASH. Then again, total length PIAS1 was able to interact not simply with FLASH D, but additionally together with the N terminal fragment of FLASH, FLASH A. This indicates that the C terminal aspect of PIAS1 probably represents a second interaction surface, associating together with the N terminal component of FLASH. The interaction concerning FLASH and PIAS1 was con firmed by GST pulldown assays.