The interaction between mixed-lineage kinase 3 and the tumor suppressor protein merlin

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Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder of the nervous system which affects 1 in 30,000 individuals.The NF2 gene encodes a 595 amino acid tumor suppressor protein called Merlin that is a member of the ezrin, radixin, moesin (ERM) family of proteins.Patients with Neurofibromatosis Type 2 exhibit loss of function mutations in the NF2 gene and develop schwannomas and meningiomas.Mixed Lineage Kinase 3 (MLK3) is a mitogen activated protein kinase kinase kinase (MAP3K) that activates multiple MAPK signaling pathways.Stable overexpression of wildtype MLK3 in NIH3T3 cells is transforming and we previously observed that depletion of MLK3 inhibits proliferation of different types of normal and tumor cells.We also found that overexpression of Merlin inhibited MLK3 kinase activity and blocked the interaction between MLK3 and B-Raf.The goal of this research is to identify the region of Merlin that is required for the interaction with MLK3 and the inhibition of MLK3 kinase activity.We purified GST fusion proteins of Merlin expressed in bacteria consisting of the full length (aa 1-590), the N-terminus (aa 1-332) and two additional deletion mutants lacking the N-terminus (aa 308-590 and aa-340-590).Full-length histidine-tagged MLK3 (His-MLK3) was also expressed and purified from bacteria and in vitro binding assays were performed with His-MLK3 and the different deletion mutants of GST-Merlin.We observed that His-MLK3 protein bound to full length Merlin and the Merlin deletion mutants lacking the N-terminus (aa 308-590 and aa 340-590).However, the Merlin deletion mutant containing the alpha helical region and the C-terminus (aa 1-332) did not interact with His-MLK3.We conclude that the region of Merlin that is required for the interaction with MLK3 lies within aa 340-590. List of

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