Structural Analysis of Drosophila Merlin Reveals Functional Domains Important for Growth Control and Subcellular Localization

Structural Analysis of Drosophila Merlin Reveals Functional Domains Important for Growth Control... Merlin, the product of the Neurofibromatosis type 2 ( NF2 ) tumor-suppressor gene, is a member of the protein 4.1 superfamily that is most closely related to ezrin, radixin, and moesin (ERM). NF2 is a dominantly inherited disease characterized by the formation of bilateral acoustic schwannomas and other benign tumors associated with the central nervous system. To understand its cellular functions, we are studying a Merlin homologue in Drosophila . As is the case for NF2 tumors, Drosophila cells lacking Merlin function overproliferate relative to their neighbors. Using in vitro mutagenesis, we define functional domains within Merlin required for proper subcellular localization and for genetic rescue of lethal Merlin alleles. Remarkably, the results of these experiments demonstrate that all essential genetic functions reside in the plasma membrane– associated NH 2 -terminal 350 amino acids of Merlin. Removal of a seven–amino acid conserved sequence within this domain results in a dominant-negative form of Merlin that is stably associated with the plasma membrane and causes overproliferation when expressed ectopically in the wing. In addition, we provide evidence that the COOH-terminal region of Merlin has a negative regulatory role, as has been shown for ERM proteins. These results provide insights into the functions and functional organization of a novel tumor suppressor gene. Footnotes Address all correspondence to Richard G. Fehon, B361 LSRC, Research Drive, Duke University, Durham, NC 27708-1000. Tel.: (919) 613-8192. Fax: (919) 613-8177. E-mail: rfehon@acpub.duke.edu Abbreviations used in this paper: AEL after egg laying AHS after heat shock BB Blue Box region BBA Merlin with the seven Blue Box residues changed to alanine CNS central nervous system CNTR conserved NH 2 -terminal region ΔBB Merlin with Blue Box region removed ERM ezrin-radixin-moesin FLP yeast 2 micron Flipase enzyme FRT Flipase Recognition Target site GFP green fluorescent protein Mer Merlin NF2 Neurofibromatosis type 2 S2 cells Schneider line 2 cells UAS upstream activation sequences of yeast Gal4 transcription factor Submitted: 3 March 1998 Revision received 14 May 1998 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Cell Biology Rockefeller University Press

Structural Analysis of Drosophila Merlin Reveals Functional Domains Important for Growth Control and Subcellular Localization

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Publisher
Rockefeller University Press
Copyright
© 1998 Rockefeller University Press
ISSN
0021-9525
eISSN
1540-8140
D.O.I.
10.1083/jcb.141.7.1589
Publisher site
See Article on Publisher Site

Abstract

Merlin, the product of the Neurofibromatosis type 2 ( NF2 ) tumor-suppressor gene, is a member of the protein 4.1 superfamily that is most closely related to ezrin, radixin, and moesin (ERM). NF2 is a dominantly inherited disease characterized by the formation of bilateral acoustic schwannomas and other benign tumors associated with the central nervous system. To understand its cellular functions, we are studying a Merlin homologue in Drosophila . As is the case for NF2 tumors, Drosophila cells lacking Merlin function overproliferate relative to their neighbors. Using in vitro mutagenesis, we define functional domains within Merlin required for proper subcellular localization and for genetic rescue of lethal Merlin alleles. Remarkably, the results of these experiments demonstrate that all essential genetic functions reside in the plasma membrane– associated NH 2 -terminal 350 amino acids of Merlin. Removal of a seven–amino acid conserved sequence within this domain results in a dominant-negative form of Merlin that is stably associated with the plasma membrane and causes overproliferation when expressed ectopically in the wing. In addition, we provide evidence that the COOH-terminal region of Merlin has a negative regulatory role, as has been shown for ERM proteins. These results provide insights into the functions and functional organization of a novel tumor suppressor gene. Footnotes Address all correspondence to Richard G. Fehon, B361 LSRC, Research Drive, Duke University, Durham, NC 27708-1000. Tel.: (919) 613-8192. Fax: (919) 613-8177. E-mail: rfehon@acpub.duke.edu Abbreviations used in this paper: AEL after egg laying AHS after heat shock BB Blue Box region BBA Merlin with the seven Blue Box residues changed to alanine CNS central nervous system CNTR conserved NH 2 -terminal region ΔBB Merlin with Blue Box region removed ERM ezrin-radixin-moesin FLP yeast 2 micron Flipase enzyme FRT Flipase Recognition Target site GFP green fluorescent protein Mer Merlin NF2 Neurofibromatosis type 2 S2 cells Schneider line 2 cells UAS upstream activation sequences of yeast Gal4 transcription factor Submitted: 3 March 1998 Revision received 14 May 1998

Journal

The Journal of Cell BiologyRockefeller University Press

Published: Jun 29, 1998

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