Mutant Products of the NF2 Tumor Suppressor Gene Are Degraded by the Ubiquitin-Proteasome Pathway

Abstract

Neurofibromatosis type 2 (NF2), a syndrome associated with multiple tumors of the nervous system, mostly schwannomas, is caused by mutations in the NF2 tumor suppressor gene that encodes schwannomin (Sch). Here we examined NF2 pathogenetic mutations that result in misfolding of the FERM domain. We found that these mutant forms of Sch were efficiently degraded by the ubiquitin-proteasome pathway. In transfected cells, Sch⌬F118 was 3-fold more efficiently degraded than the related molecule ezrin bearing the equivalent mutation. In heterozygous Nf2 knock-out mouse fibroblasts, endogenous mutant Sch⌬81-121, but not wild type Sch, was also degraded by proteasomes. We further show that this degradation pathway is functional in primary Schwann cells. We analyzed Sch⌬39 -121 expressed in a transgenic mouse model of NF2 and found that Sch⌬39 -121, but not the endogenous wild type Sch, was unstable due to proteasome-mediated degradation. Altogether these results suggest that degradation of mutant Sch mediated by the ubiquitin-proteasome pathway is a physiopathological pathway contributing to the loss of Sch function in NF2 patients. Neurofibromatosis type 2 (NF2)1 is a dominantly inherited disorder characterized by the predisposition to develop multiple nervous system tumors, in particular schwannomas. Tumor development in NF2 patients is in accordance with Knudson's two-hit hypothesis for tumor suppressor genes. As NF2 patients inherit one mutant NF2 allele, a second hit in the remaining wild type allele is sufficient to induce tumorigenesis. The two NF2 alleles are also inactivated in the majority of sporadic schwannomas (1).The NF2 tumor suppressor gene product, Schwannomin (Sch), also known as merlin, displays 45% identity with ERM proteins, which are cytoskeletal linkers between cortical actin filaments and the plasma membrane (2, 3). Like ERM proteins, Sch has an amino-terminal FERM domain and a carboxylterminal domain that associate in an intramolecular manner to form closed monomers and in an intermolecular manner to form oligomers (4 -6). The FERM domain of Sch binds to the plasma membrane and to filamentous actin (7,8). Sch regulates cell adhesion and motility (9, 10) and mediates contact inhibition (11).In human tumors, levels of mutant Sch are consistently below limits of detection although NF2 mutant alleles were detected at the mRNA level (12-15). Therefore, we reasoned that mutant schwannomin might be degraded quickly. To examine this hypothesis, we studied the stability of mutant schwannomin in transfected cells or in primary cultures derived from mouse models developed to examine the pathogenesis of NF2 (16, 17). We focused on pathogenetic mutations in the conserved FERM domain of Sch. The deletion of the phenylalanine 118 codon in exon 3 has been described in two unrelated families affected by NF2 as well as in a sporadic meningioma (Ref. 12 and references therein). Mutations that lead to the skipping of exon 3 (Sch⌬81-121) or exons 2-3 (Sch⌬39 -121) have been observed in the germ line or in the tumors o...

L166P Mutant DJ-1, Causative for Recessive Parkinson's Disease, Is Degraded through the Ubiquitin-Proteasome System

“…2 These data indicate that L166P would produce insufficient protein as a result of proteasomal degradation. This situation is similar to that seen with mutant forms of other recessive gene products such as the cystic fibrosis transmembrane receptor (28) or the neurofibromatosis type 2 gene product, schwannomin (29). The ubiquitin-proteasome system also degrades some dominant negative mutations such as caveolin-3 mutations associated with limb-girdle muscular dystrophy type 1C (30).…”

Proteasome inhibition rescues clinically significant unstable variants of the mismatch repair protein Msh2

“…The pathology of ubiquitin-mediated turnover of missense variants of Msh2 is in keeping with mounting evidence that other human diseases, such as cystic fibrosis and Huntington, Alzheimer's, and Parkinson diseases are linked to proteinfolding abnormalities resulting in instability and ubiquitin-positive aggregates (18,19). Interestingly, diseases including neurofibromatosis (20), multiple endocrine neoplasia Type 1 (21), α-1 antitrypsin deficiency (22), classic late-infantile neuronal ceroid lipofuscinosis (23), and phenylketonuria (24) all have incidences in which missense variants are targeted by the ubiquitin-proteasome pathway. Thus, small-molecule stabilization of missense variants or the targeted inhibition of the ubiquitin pathway represent exciting avenues for therapeutic exploration for many diseases, not just Lynch syndrome.…”

Correlation of mutant menin stability with clinical expression of multiple endocrine neoplasia type 1 and its incomplete forms

“…(38) These mutants, lacking the properly folded sequences required for binding to the plasma membrane and filamentous actin, were shown to be degraded rapidly, suggesting an important role of the ubiquitin-proteasome pathway in eliminating potentially dominant-negative mutant proteins. (39) These examples and rapid degradation of MEN1-associated menin mutants imply that misfolding and ⁄ or loss of protein binding might lead to ubiquitination and rapid degradation of mutants of certain tumor suppressor proteins including menin. Because menin is a multi-facet scaffold protein with widely dispersed multiple binding domains, this view is consistent with the fact that disease-causing missense mutations are distributed, although not evenly, throughout the whole menin molecule.…”