However, unlike the result with the ZASP PDZ domain, the interaction between ZM1 and Ursolic-acid Ankrd2 did not occur if p53 was over-expressed in the cells suggesting a competition between these proteins for the same binding site. Whether p53 competes by blocking the binding site on Ankrd2 for ZM1 or vice versa is not known. Although the triple complex can be formed in vitro more experimental evidence is needed to confirm its existence in vivo. It should be noted from the literature that under certain circumstances, such as stress conditions, p53 can be found in the cytoplasm. It is possible that interaction with ZASP could sequester p53 in the cytoplasm, however this is purely speculative and we have no proof that this occurs in vivo. From our results we can deduce that the ZASP6 PDZ domain binds to Ankrd2 via its ankyrin repeat region whereas the ZASP6 ZM1 could have the same binding site as p53 on Ankrd2. We previously showed that, in addition to ankyrin repeats, N-terminal region of Ankrd2 is required for interaction with p53 as well as transcription factors YB-1 and PML. We can speculate that the ankyrin repeats are able to accommodate more than one binding partner and that binding specificity is achieved by concurrent interaction with the N terminal region that is proximal to ankyrin repeats. An intriguing finding is that p53 is modified by SUMO when present in the triple complex. This endogenous Ganoderic-acid-G polySUMOylation of p53 was detectable only in the presence of fulllength ZASP6. If the ZASP6 PDZ domain or ZM1 region were co-expressed with Ankrd2 and p53, SUMO modified p53 was never detected in the complex. However which SUMO family member is involved in the modification of p53 in the trimeric complex is not known since the mixture of antibodies specific for SUMO1 and SUMO2/3 was used. p53 is SUMOylated at a single site K386 by SUMO-1 and SUMO-2/3. Modification of p53 via SUMO-2/3 or SUMO-1 can have different consequences. SUMO-1 modification of p53 results in nuclear export of p53, however that does not appear to happen when p53 is modified by SUMO-2/3. The SUMOylation of p53 seen in the presence of the triple complex could possibly result in a modification of the action of p53 probably by facilitating its cytoplasmic export whereas a block of the SUMOylation could probably result in p53 retention in the nucleus. The ZASP6 ZM-motif mutants unable to bind Ankrd2 could disrupt the formation of the triple complex and therefore SUMOylation of p53 by interfering with the binding of p53 and Ankrd2. Another possibility is that SUMOylation of p53 is needed as an additional platform to enable interaction with full length proteins, while unmodified p53 is able to accommodate Ankrd2 and ZASP deletants. A achment of SUMO facilitates protein�Cprotein interactions, either by creating new interaction surfaces or by indirectly affecting the conformation of the modified target, thereby facilitating the assembly of multi-protein complexes, the recruitment of regulatory factors to the site of modification or the sequestration of the target into different subcellular compartments. Promoter assays were performed to determine if the interaction of ZASP6 with Ankrd2 and p53 could have any functional consequence on the ability of p53 to transactivate responsive promot