Molecular insights into NF2/Merlin tumor suppressor function

Abstract

The FERM domain protein Merlin, encoded by the NF2 tumor suppressor gene, regulates cell proliferation in response to adhesive signaling. The growth inhibitory function of Merlin is induced by intercellular adhesion and inactivated by joint integrin/receptor tyrosine kinase signaling. Merlin contributes to the formation of cell junctions in polarized tissues, activates anti-mitogenic signaling at tight-junctions, and inhibits oncogenic gene expression. Thus, inactivation of Merlin causes uncontrolled mitogenic signaling and tumorigenesis. Merlin's predominant tumor suppressive functions are attributable to its control of oncogenic gene expression through regulation of Hippo signaling. Notably, Merlin translocates to the nucleus where it directly inhibits the CRL4(DCAF1) E3 ubiquitin ligase, thereby suppressing inhibition of the Lats kinases. A dichotomy in NF2 function has emerged whereby Merlin acts at the cell cortex to organize cell junctions and propagate anti-mitogenic signaling, whereas it inhibits oncogenic gene expression through the inhibition of CRL4(DCAF1) and activation of Hippo signaling. The biochemical events underlying Merlin's normal function and tumor suppressive activity will be discussed in this Review, with emphasis on recent discoveries that have greatly influenced our understanding of Merlin biology.

Keywords: CRL4 E3 ubiquitin ligase; Contact inhibition; DDB1 and Cul4-Associated Factor 1; Hippo signaling pathway; Merlin; Neurofibromatosis Type 2.

Figure 1

(A) Merlin structure and overview of pathogenic mutations. Merlin is a 595-residue protein divided into three structurally distinct regions – an amino-terminal FERM domain, an α-helical coiled-coil domain, and a carboxy-terminal hydrophilic tail. Merlin's FERM domain is further subdivided into three globular subdomains. Nf2 encodes 16 exons, terminating with exon 17 in the canonical Merlin isoform. The positions of patient-derived missense mutations or single residue deletions are indicated below the exon schematic, with mutation frequency indicated by a heat map. Either these mutations underlie NF2 tracked within a family, were found in two or more unrelated patients, or experimental evidence was obtained to confirm their pathogenicity. Mutational data were obtained from Ahronowitz et al, 2007 [48] and Li et al, 2010 [29]. (B) Adhesion-mediated regulation of the Merlin activation cycle. (Left) In contact-inhibited cells, dephosphorylated Merlin accumulates as a result of intercellular adhesions which lead to PAK inhibition. Merlin may also be activated via MYPT1-mediated dephosphorylation. (Right) Conversely, in proliferating cells, integrin-mediated anchorage to the cell matrix and stimulation of receptor tyrosine kinases (RTKs) activate Rac, in turn activating PAK and leading to phosphorylation of Merlin at Serine 518. In response to high cyclic AMP levels, PKA also phosphorylates Merlin at serine 518. Serine 518 phosphorylation increases the interdomain binding between Merlin's carboxy-terminus and FERM domain, maintaining Merlin in a more closed, inactive form.

Figure 2

(A) Merlin activates the Hippo pathway to suppress YAP/TAZ through regulation of core kinase components at the plasma membrane, through inhibition of the CRL4^DCAF1 E3 ubiquitin ligase in the nucleus, and by regulating cell junction-associated proteins that modulate Hippo signaling. (1) At adherens junctions, E-cadheren promotes Hippo signaling while α-catenin sequesters 14-3-3 protein-bound YAP. Merlin interacts with α-catenin at maturing adherens junctions in skin epithelium, plausibly influencing Hippo signaling at this subcellular compartment. (2) At the plasma membrane, Mn recruits the Lats kinases and coordinates their activation by Mst1, driving phosphorylation and inhibition of YAP/TAZ. (3) The Crumbs homolog (CRB) complex recruits Angiomotin (AMOT), which directly binds Hippo pathway components. AMOT serves as a scaffold for Mst1/2 and Lats1/2 activation and also directly binds and inhibits YAP. Conversely, the p180 isoform of AMOT promotes YAP function in the nucleus. Merlin interacts with AMOT at tight junctions to suppress Rac activity, however, it remains to be determined how Merlin influences Angiomotin's inhibition of YAP at tight junctions or YAP-activating functions in the nucleus. (4) Dephosphorylated Merlin translocates to the nucleus and inhibits CRL4^DCAF1, preventing this E3 ligase from ubiquitinating and inhibiting Lats1/2. These core Hippo pathway components, activated by an upstream kinase, phosphorylate and inactivate YAP/TAZ. (B) Merlin's regulation of CRL4^DCAF1 impinges on multiple downstream epigenetic mechanisms and blocks oncogenic gene expression. In contact inhibited cells, active Merlin translocates to the nucleus where it binds to and inhibits the CRL4^DCAF1 E3 Ubiquitin ligase. Active or dysregulated CRL4^DCAF1 induces an oncogenic gene expression program by promoting downstream Hippo pathway targets via inhibition of the Lats1/2 kinases and through modulation of epigenetic modifiers and transcription factors.

Figure 3

(A) Active Merlin translocates to the nucleus and binds to the carboxy-terminus of DCAF1, acting as a pseudo-substrate to block CRL4^DCAF1 from recruiting and ubiquitinating target proteins. Merlin's inhibition of CRL4^DCAF1 suppresses oncogenic gene expression. (B) CRL4^DCAF1 is an E3 ubiquitin ligase consisting of the substrate-recruiting DCAF1 protein which is linked to the amino-terminal domain of a cullin (Cul4A/B) backbone through DDB1, which is itself a large multi-domain protein. Bound to the cullin carboxy-terminal domain, Rbx1 uses its RING domain to recruit a ubiquitin-charged E2, which directs conjugation of ubiquitin to a DCAF1-bound target protein.