Complex Interaction of the MoeA-N and MoeA-C Domains of Gephyrin Is Required for the Immortalization Activity of MLL-Gephyrin.

More than 40 proteins have been identified as partners of MLL in acute leukemia. The partner proteins are categorized based on their subcellular localization into either the nucleus or cytoplasm. Recent studies have proposed that the mechanism of transformation of MLL-cytoplasmic protein fusion products is mediated by the oligomerization ability of partner proteins. Gephyrin, which is involved in synaptic anchoring of glycine receptor and certain GABAA receptor subtypes, is a rare partner of MLL in patients with AML. Using an in vivo cross linking assay, we confirmed the dimerization activity of a domain within the MoeA-N domain of Gephyrin, which is located at amino acids 456–476. However, we found that this domain was dispensable for immortalization in the methylcellulose colony-forming assay, indicating that the presence of a dimerization domain was not sufficient for transformation. We also observed that the Tubulin binding domain, which is located at amino acids 322–376, was also dispensable. To evaluate which domain of Gephyrin is critical for the immortalization activity of MLL-Gephyrin, we performed a colony-forming assay in methylcellulose and found that both the MoeA-N (amino acids 354–528) and the MoeA-C (amino acids 691–769) domains of Gephyrin are essential for immortalization. In addition, we observed that a MLL- Gephyrin (444–476) deletion mutant formed dimers using the in vivo cross linking assay. To explore the possibility that both MoeA-N and MoeA-C domains are associated with dimer formation of MLL- Gephyrin, we performed co-immunoprecipitation experiments with three fragments of Gephyrin, MoeA-N, MoCF (amino acids 532–690) and MoeA-C. Our data showed that both the MoeA-N and MoeA-C domains interacted with the MoCF domain of Gephyrin. Taken together, these findings show that Gephyrin has multiple dimerization domains, and that the interaction that links the MoeA-N, MoeA-C, and MoCF domains is critical for the immortalization activity of the MLL- Gephyrin fusion protein. Our data indicate that complex patterns of dimerization exist among MLL partners and suggest that specific types of dimerization domains might be critical for MLL-associated transforming activity.