Since ruxolitinib structure we were unable to detect expression of isoform 1d in the tissues examined, it probably does not represent a true isoform. However, the remaining AP 2a isoforms are expressed at significant levels in breast cell lines and tis sue with the AP 2a 1c protein being found at levels at least comparable to those of the initially identi fied isoform 1a, suggesting that Inhibitors,Modulators,Libraries its activity in the breast should be investigated. Our analysis focused on testing whether differences in amino terminal sequence lead to distinct biological characteristics for the AP 2a isoforms. The N terminus of AP 2a encompasses the transactivation domain, and is not involved either in DNA binding or in protein dimerisation. Therefore, functional differences are more likely to be found in transactivation activity, although the limited difference in sequence might sug gest subtle variations.
With a synthetic reporter, iso forms 1a, 1b and 1c showed similar transactivation activity indicating a similar ability to interact function ally with CITED2 or CITED4 and p300 or CBP. This agrees with the finding that the domain essential for interaction with CITED2 lies in the central region of AP 2a. However, Inhibitors,Modulators,Libraries the picture changed when the transactivation activity of the isoforms was compared using natural promoters. The cyclin D3 pro moter was differentially regulated by the different iso forms with AP 2a 1b and 1c having a minimal effect, whereas isoform 1a exerted a significant inhibitory activ ity, similar to the effect exerted by AP 2g. Lysine 10, which lies within a putative sumoylation motif, was essential for this inhibi tory activity.
An interaction between AP 2a and UBC9 has been demonstrated Inhibitors,Modulators,Libraries previously and our subse quent Inhibitors,Modulators,Libraries experiments showed that isoform 1a alone could be sumoylated in HepG2 cells. However, sumoylation of endogenous AP 2a in breast lines could not be con firmed. When a similar experiment was performed for AP 2g using MCF7 cells, which express it at high levels, only a small fraction of the protein was found to be sumoylated, in accord with similar studies on other Inhibitors,Modulators,Libraries sumoylated proteins. By extrapolation, since in breast lines levels of isoform 1a represent only a propor tion of the total AP 2a protein, it is likely that the fraction of sumoylated sellectchem isoform 1a falls below the level detectable using Western blotting or immuno precipitation. Co transfection of SUMO 1 or SUMO 2 with isoform 1a resulted in reduced transactivation activity, similar to observations made for AP 2g. The finding that for AP 2a only isoform 1a can act as a repressor strengthens the hypothesis that sumoylation is necessary for AP 2 transcriptional repression.