n in its N-terminus. Importantly, the interaction between Glis3 with endogenous Itch seemed to be almost completely ablated by the PY461 mutation indicating that the interaction between Glis3 and Itch largely depends on a short region containing the PPPY461 motif. WW-domains have been reported to make contacts with proline 16 / 22 Regulation of Glis3 Activity by the HECT E3 Ubiquitin Ligases residues flanking the core PPxY motif of substrates. The PY461 motif is flanked on either side by a number of conserved proline residues interspersed with non-polar or basic residues. It is possible that Itch may make weak contacts with these flanking regions thereby enhancing its affinity for the PY461 motif. The weak interactions with PY461 flanking regions may play a more prominent role at high Itch concentrations as under conditions of exogenous Itch overexpression. Disruption of the core PY461 motif of Glis3 by either mutation or removal of the motif by truncation was sufficient to stabilize Glis3 in the presence of the ubiquitin ligase. Interestingly, disruption of the tetrahedral configuration of Glis3 ZF3 and ZF4 protected Glis3 against Itchdirected degradation without significantly affecting the nuclear localization of Glis3, its interaction with Itch, or its Itch-mediated ubiquitination. These observations suggest that, in addition to requiring the PY461 motif, Itch-mediated degradation also required the presence of the Glis3 ZFD. Although mutation of ZF3 and ZF4 disrupted DNA binding and transactivation by Glis3, so too did mutation of ZF1-2 and 5 without affecting protein degradation by Itch. These data indicate that targeted destruction of Glis3 by Itch does not appear to require DNA binding or transactivation by Glis3 but may require the presence of ancillary proteins that interact with the ZFD of Glis3. Indeed, previous reports have identified substrate-transferring proteins that may bind ubiquitinated substrates and PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19740905 facilitate the transfer 
of targeted proteins to the proteasome. This hypothesis is supported by the fact that ZF3-4 mutants of Glis3 have higher levels of basal ubiquitination in the absence of Itch, suggesting that ubiquitinated Glis3 may not be turning over. Alternatively, transfer of ubiquitin to a specific Lys residue within the zinc finger domains of Glis3 may be required for proper recognition by the proteasome. Although Itch does appear capable of transferring ubiquitin to the N-terminus of Glis3 lacking the ZFD, previous reports have indicated that ubiquitin can be promiscuously transferred to even non-native Lys residues by an E3 ubiquitin ligase. Maximally efficient degradation of Glis3 by Itch may therefore require interaction with a complex of proteins associating with the Glis3 ZFD in addition to the N-terminal PY461 motif. It is well established that substrates are directed to the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19743978 26S proteasome through polyubiquitination, which occurs when chains of ubiquitin attached to the substrate by connecting subsequent ubiquitin molecules to one of seven lysine residues within the KU55933 preceding ubiquitin molecule. Most often proteins are targeted to the 26S proteasome for degradation when modified with K48-linked polyubiquitin chains or less commonly, K29-linked chains. Surprisingly, despite evidence indicating that Itch targets Glis3 for proteasomal degradation, we found that mutation of ubiquitin K63, but not K48 significantly reduced the amount of polyubiquitinated Glis3 in the presence of Itch. Previous reports have