A fourth SUMOylation site (K955) was identified inside of the considerably less frequent TKEE sequence, the place a hydrophilic residue, namely a threonine, precedes the focus on lysine. The TKXE consensus, although unusual, has also been described for TIF1b, p45-NF-E2 and TEL/ETV6 [37,38,39]. The 4 SUMOylation internet sites recognized in murine FOG-2 are conserved across the species examined (Fig. 4B) suggesting functional conservation. Most substrates include only just one or 22368-21-4two SUMO acceptor residues [forty]. There are some components, nonetheless, with multiple SUMOylation web-sites these include PML, GRIP1 and ELK-1 with three SUMOylation sites each and every [39,41,forty two] and TIF1b which is modified by SUMO at six positions [37]. Furthermore, there appears to be preferential modification of specific residues, for instance BKLF is modified at one significant and a single small website [19] while TIF1b is made up of three key and a few small SUMOylation web-sites [37]. In FOG-2, K471 and K955 are modified strongly by SUMO-one while K324 and K915 are SUMOylated to a lesser extent (Fig. two and 3 and information not shown). SUMOylation of endogenous FOG-2 in C2C12 cells uncovered only just one primary SUMOylated species.
SUMO E3 ligases these kinds of as PIAS1 and PIAS2 are expressed in the coronary heart [34] and GATA-four SUMOylation is controlled by PIAS1 [35,36]. Nevertheless, co-expression of FOG-2 with SUMO-one and the E3 ligases PIAS1, PIAS2 (Miz1), PIAS3 (ARIP-three) and PIAS4 (PIASy) did not enrich FOG-2 SUMOylation (Fig. S1A). In addition, co-expression of the SUMO E2 ligase Ubc9, did not increase FOG-two SUMOylation, suggesting that this enzyme is not a restricting aspect in COS-seven cells (Fig. S1A, lanes 2 and seven). Even so, we observed that co-expression of FOG-two and GATA4 led to more robust FOG-2 SUMO modification. As observed in Fig. 8, co-expression of increasing amounts of GATA-four resulted in a corresponding increase in FOG-2 SUMOylation (Fig. 8A, lanes 2 to 4 and Fig. 8B). This is reminiscent of the boost in FOG-1 SUMOylation viewed in the presence of GATA-1 or GATA-two ( [22] and our unpublished observations). Therefore, the presence of GATA-four favours FOG-2 SUMO modification and could depict a system by which GATA elements could modulate FOG-29s activity.
FOG-2 SUMOylation and de-SUMOylation have antagonistic outcomes on its repression activity. (A) HeLa cells were being cotransfected with the BNP-Luciferase reporter and wt FOG-two or FOG-2-4KR collectively with growing amounts of SUMO-one. Growing expression of SUMO-one resulted in lowered repression by FOG-two. Expression of SUMO-one did not affect the repression ability of the non-SUMOylatable 4KR mutant. (B) HeLa cells were co-transfected with the BNP-Luciferase reporter and wt FOG-two alongside one another with SUMO-1 and FLAG-SENP-1 as indicated in the Determine. As shown in A, SUMOylation of FOG-two by GFP-SUMO-1 lowered its repression action. Conversely, de-SUMOylation by SENP-one or SENP-eight improved FOG-29s repression potential. (C) Western blot showing FOG-2 de-SUMOylation by SENP-1 and SENP-eight from an experiment operate in parallel (take note that SUMOylation is not noticed in the24135074 extracts applied for luciferase assays simply because the inhibitor NEM is not incorporated in the luciferase lysis buffer). Knowledge signify the suggest 6 SD from two unbiased experiments. Asterisks suggest non-distinct bands detected by the FOG-2 antibody. IB, immunoblot nr, no reporter. GATA-4 improves FOG-2 SUMOylation. (A) COS-seven cells ended up transfected with constructs containing FOG-two, GFP-SUMO-one and GATA-4 as indicated in the determine. Cells ended up boiled specifically in Laemmli buffer, run for Western blotting and probed with the indicated antibodies. (B) The improve in FOG-two SUMOylation was quantitated by densitometry using ImageQuant TL 1D, edition seven. (GE Healthcare). The graph reveals the ratio of complete SUMOylated FOG-two to total FOG-two (share). Asterisks point out non-specific bands detected by the FOG-two antibody. IB, immunoblot.
