Binding of the merlin-I product of the neurofibromatosis type 2 tumour suppressor gene to a novel site in beta-fodrin is regulated by association between merlin domains.
The mechanism underlying the tumour-suppressor activity of the neurofibromatosis type 2 (NF2) gene product, merlin, is largely undefined but there is evidence that the biological function of the protein might be mediated partly through interactions with the cytoskeleton. Merlin is expressed predominantly as two isoforms that differ at their C-termini owing to alternative splicing of exon 16. By expressing merlin isoform I as bait in a yeast two-hybrid screen, we isolated a clone encoding a region of the cytoskeletal protein beta-fodrin. Confirmation of the merlin-fodrin interaction was provided by using the mammalian two-hybrid system and binding assays in vitro. In addition, these assays and co-immunoprecipitation from mammalian cells revealed that the binding site for fodrin is located in the C-terminal half of merlin at a site that is masked in the native protein. Co-expression of the N-terminus of merlin decreased the interaction of its C-terminus with fodrin, implicating homophilic interactions of merlin isoform I in masking the fodrin-binding site. The effect of three disease-associated mutations on the merlin-fodrin interaction and merlin dimerization was also investigated. The mutation L535P, but not L360P or K413E, significantly decreased the merlin-fodrin interaction but not dimerization, indicating that the tumour suppressor ability of merlin might reside partly in its ability to interact with the cytoskeleton via fodrin.
Neill
G W
GW
Centre for Cutaneous Research, St Bartholomew's and the Royal London, Queen Mary and Westfield College, 2 Newark Street, London E1 2AT, UK.
Crompton
M R
MR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D016515:Genes, Neurofibromatosis 2; D006801:Humans; D008322:Mammals; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008840:Microfilament Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D025581:Neurofibromin 2; D014176:Protein Biosynthesis; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013049:Spectrin / Q000737:chemistry* / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D002352:Carrier Proteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D025581:Neurofibromin 2; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013049:Spectrin