AP 2a has previously been demonstrated to interact with the SUMO E2 conjugating enzyme, UBC9, in a yeast two hybrid screen, but to confirm that AP 2a can be sumoylated in vivo, expression constructs for UBC9 and SUMO 1 or SUMO 2 were cotransfected in HepG2 cells together with the different AP 2a isoforms. This resulted in the appearance, for isoform 1a only, of a slower considering migrating band at approximately 75 kDa, compatible with a mono sumoylated form of AP 2a. Further more, mutation of lysine 10 led to a very pronounced reduction in intensity of this sumoylated band, thus confirming that lysine 10 is the predominant sumoyla tion site Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries in isoform 1a, and explaining why the other isoforms were not significantly modified.

To confirm that the inhibitory activity of isoform 1a is dependent on sumoylation, HepG2 cells Inhibitors,Modulators,Libraries were cotrans fected with the 3xAP2 Bluc reporter and increasing doses of SUMO 1 or SUMO 2. The transactivation activ ity was reduced with SUMO co transfection, particularly by SUMO 2, while the transactivation activity of the K10R mutant or AP 2a 1c was not altered significantly, suggesting that the most important sumoylation site is indeed lysine 10 of isoform 1a. To confirm that the inhibitory effect exerted Inhibitors,Modulators,Libraries by isoform 1a on the cyclin D3 reporter is due to sumoylation, we co trans fected with either wild type UBC9 or its sumoylation defective mutant, C93S. While transfection of wt UBC9 did not result in further inhibition of cyclin D3 reporter activity, cotransfection of UBC9 C93S partially reverted the inhibitory activity of isoform 1a.

In addition, a clear reduction in the level of sumoylated Inhibitors,Modulators,Libraries http://www.selleckchem.com/products/brefeldin-a.html AP 2a was observed by western blot when the 1a isoform and UBC9 C93S were co expressed in the same propor tion in HepG2 cells. TFAP2A isoform 1a is a weaker transactivator of the ERBB2 promoter The transactivation activity of the different AP 2a iso forms was further tested on the ERBB2 promoter, which is known to possess two functional AP 2 binding sites, mapping at 210 and 500 upstream of the transcription start site, which are required for positive regulation of ERBB2 by AP 2a in breast tumour lines. A repor ter carrying ERBB2 promoter sequences was co transfected at different ratios with the AP 2a isoform expression constructs together with Cited2 p300. Isoform 1a significantly induced reporter activity at a 1,2 and 1,4 reporter,AP 2a ratio. In contrast, the transactivation activity of isoforms 1b and 1c was already significant at a ratio of 1,0. 5, and further increased at higher ratios, reaching a plateau at 1,4. Iso form 1d was the most potent transactivator and its activ ity increased at higher ratios in an almost linear manner.