Study on cellular signal transduction of Neurofibromatosis Type 2 tumor suppressor gene product (Merlin)

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The neurofibromatosis type 2 (NF2) is an autosomal dominantly inherited disorder, and strongly associated with development of benign intracranial tumors including bilateral vestibular schwannomas and meningiomas. NF2 gene was recently cloned, and the protein it encodes (termed as merlin) was found to be striking similarity to the moesin-ezrin-radixin (MER) family of cytoskeleton-associated proteins. To elucidate the biological function of NF2 protein (merlin), we analyzed the cellular localization and signal transduction of merlin. 1) Cellular expression systems of mutants or wild NF2 were established, and their cellular localization was analyzed by con focal lazer scanning microscopy (CLSM). The wild merlin showed cytoplasmic and submembranous localization that was found to be directed by its nuclear export signal (NES) dependent transport mechanism. The N-terminal mutation resulted in nuclear accumulation of merlin. 2) Cellular binding proteins of merlin were purified from bovine bra … More in extracts and identified as poly ADP-ribose polymerase, DNA-PK subunits Ku-antigen 85 and 70 by the analysis of their internal ammo-acid sequences. The N-terminal (19-339) region of merlin is essential for their interaction. The immuno-precipitation, western blotting, and CLMS study confirmed their cellular co-localization. NF2 mutations impair the merlin-related complex formation and their cellular signal transport. 3) Subcellular co-localizations of merlin and PARP, overexpressed in VA13 cells and mouse embryonic fibroblast (MEF), were observed mainly in their nuclear region when cells were treated with leptomycin B (LMB), an inhibitor of NES receptor Crm1. This nuclear accumulation of merlin increased with cellular DNA damages induced by bleomycin. 4) In marked contrast to wild type cells, PARP gene deficient MEF could not accumulate merlin in their nuclear region, even after the treatments of LMB and bleomycin, whereas the overexpression of PARP in those cells worked complementary to the cellular localization of merlin. These results suggest that merlin is a cytoplasmic-nuclear shuttling protein directed by its NES-dependent transport pathway being associated with PARP. The tumor suppressive function of merlin may be linked to cellular DNA-repair and related signals via the interaction of cellular merlin binding proteins such as PARP and DNA-PKs. Less