Impaired interaction of naturally occurring mutant NF2 protein with actin-based cytoskeleton and membrane

Abstract

Although schwannomin, the product of the neurofibromatosis type 2 gene, shares homology with three cytoskeleton-to-membrane protein linkers defining the ERM family, the mechanism by which it exerts a tumor suppressive activity remains elusive. Based on the knowledge of naturally occurring mutations, a functional study of schwannomin was initiated. Constructs encoding the two wild-type isoforms and nine mutant forms were transfected into HeLa cells. Transiently expressed wild-type isoforms were both observed underneath the plasma membrane. At this location they were detergent insoluble and redistributed by a cytochalasin D treatment, suggesting interaction with actin-based cytoskeletal structures. Proteins with single amino acid substitutions at positions 219 and 220 demonstrated identical properties. Three different truncated schwannomins, that are prototypic for most naturally occurring NF2 mutations, were affected neither in their location nor in their cytochalasin D sensitivity. However, they were revealed to be detergent soluble, indicating a relaxed interaction with the actin-based structures. An increased solubility was also observed for a mutant with a single amino acid substitution at position 360 in the C-terminal half of the protein. Mutant proteins with either a single amino acid deletion at position 118 or an 83 amino acid deletion within the N-terminal domain had lost the submembraneous localization and tended to accumulate in perinuclear patches that were unaffected by cytochalasin D treatment. A similar behavior was observed when the N-terminal domain was entirely deleted. Taken together these observations suggest that the N-terminal domain is the main determinant that localizes the protein at the membrane where it interacts weakly with actin-based cytoskeletal structures. The C-terminal domain potentiates this interaction. With rare exceptions, most naturally occurring mutant schwannomins that have lost their tumor suppressive activity are impaired in an interaction involving actin-based structures and are no longer firmly maintained at the membrane.

Inhibition of the hyaluronan-CD44 interaction by merlin contributes to the tumor-suppressor activity of merlin

“…The first 50 amino acids of merlin are required for the binding of merlin to CD44 and for inhibition of the CD44-HA interaction Studies have shown that merlin mutants lacking different portions of the NH 2 -terminal ERM-homology domain lose their ability to interact with the plasma membrane (Deguen et al, 1998;Koga et al, 1998), a finding that is consistent with the possibility that merlin engages CD44 using the segment(s) located in its NH 2 -terminal half. To define the domain of merlin that is required for its inhibitory effect on the CD44-HA interaction, we established five mutants each of which bore a 50-amino acid deletion within the first 250 residues of the NH 2 -terminal portion of merlin.…”

“…These mutants are often associated with a more severe clinical outcome (Merel et al, 1995;Parry et al, 1996;Ruttledge et al, 1996;Deguen et al, 1998;Evans et al, 1998;Koga et al, 1998). It is widely held that tumor-suppressor mutants often act in a dominant-negative fashion to promote tumorigenesis (Nigro et al, 1989).…”

“…Expression of merlinDel-1 alters the interaction between CD44 and ezrin, and between CD44 and the actin cytoskeleton Increased expression of merlin impairs cell adhesion, spreading and motility, possibly because of its interaction with the actin cytoskeleton (Huynh and Pulst, 1996;Deguen et al, 1998;Pelton et al, 1998;Gutmann et al, 1999b). As a receptor for a major ECM component, CD44 plays an important role in regulating cell adhesion and migration (Lesley et al, 1993;Sherman et al, 1994;Stamenkovic 2000).…”

“…The NH 2 -terminus of merlin shares high sequence homology with the NH 2 -terminal halves of ERM proteins that mediate the binding of these proteins to a variety of molecules in the plasma membrane (Tsukita et al, 1994;Hirao et al, 1996;Yonemura et al, 1998). Accordingly, merlin mutants that lack part of the NH 2 -terminal ERM-homology domains lose their ability to associate with the plasma membrane (Deguen et al, 1998;Koga et al, 1998). Genetic analysis of NF2 patients has shown that deletions in the NH 2 -terminal FERM domain occur frequently and are associated with early tumor onset and poor prognosis (Ruttledge et al, 1996).…”

Schwann cell hyperplasia and tumors in transgenic mice expressing a naturally occurring mutant NF2 protein

“…In contrast, mutants with an intact carboxy-terminal domain but with a deleted or altered amino-terminal domain are delocalized mainly in the perinuclear cytoplasmic region. Such delocalization was observed for a mutant protein modeled from naturally occurring mutations in which exons 2-3 are spliced out without frameshift, Sch-⌬(39-121) (Deguen et al 1998).…”