Localization to the cortical cytoskeleton is necessary for Nf2/merlin-dependent epidermal growth factor receptor silencing

Abstract

Merlin, the product of the NF2 tumor suppressor gene, is closely related to the ERM (ezrin, radixin, moesin) proteins, which provide anchorage between membrane proteins and the underlying cortical cytoskeleton; all four proteins are members of the band 4.1 superfamily. Despite their similarity, the subcellular distributions and functional properties of merlin and the ERM proteins are largely distinct. Upon cell-cell contact merlin prevents internalization of and signaling from the epidermal growth factor receptor (EGFR) by sequestering it into an insoluble membrane compartment. Here we show that the extreme amino (N) terminus directs merlin biochemically to an insoluble membrane compartment and physically to the cortical actin network, with a marked concentration along cell-cell boundaries. This insoluble-membrane distribution is required for the growth-suppressing function of merlin and for the functional association of merlin with EGFR and other membrane receptors. Our data support a model whereby locally activated merlin sequesters membrane receptors such as EGFR at the cortical network, contributing to the long-held observation that the cortical actin cytoskeleton can control the lateral mobility of and signaling from certain membrane receptors.

FIG. 1.

The extreme N-terminal residues of merlin confer insoluble-membrane localization. (A) Merlin orthologues (shown are mouse and Drosophila orthologues) contain an N-terminal sequence (black) that precedes the FERM domain (red) and is not present in the ERM proteins. The Drosophila N-terminal extension is smaller than that in mammals but contains both potential phosphorylation sites and charged residues (see Fig. 6A; see also Fig. S1A in the supplemental material). The ERM proteins contain a carboxy (C)-terminal F-actin binding domain (green). (B) Subcellular fractionation of confluent Nf2^−/− mouse LDCs and Nf2^−/− mouse immortalized fibroblasts expressing the indicated versions of Nf2 revealed that little or no Nf2^18-595 is present in the Triton X-100-insoluble fractions while Nf2^L64P is enriched in the cytosol. TCL, total cell lysate; Cyt, cytosol; TX Sol, Triton X-100 soluble; TX Insol, Triton X-100 insoluble; pellet, remaining insoluble pellet. Samples were probed with an antiactin antibody to control for loading. (C) Immunostaining of confluent Nf2^−/− LDCs infected with pBMN-Nf2^wt, -Nf2^18-595, or -Nf2^L64P revealed that, in contrast to Nf2^wt, both Nf2^18-595 and Nf2^L64P fail to localize to cell-cell boundaries. Bar = 10 μm; n ≥ 3.

FIG. 2.

Nf2^18-595 cannot restore contact-dependent inhibition of proliferation or prevent EGFR internalization in Nf2^−/− cells. (A) Confluent mosaic populations of Nf2-expressing and Nf2-deficient LDCs were scored for BrdU incorporation (20 μM for 12 h) as a measure of contact-dependent inhibition of proliferation (Nf2, green; BrdU, red; DAPI, blue). White lines demarcate the interface between Nf2-expressing and Nf2-deficient cells. Bars = 10 μm. (B) Quantitation reveals that both Nf2^L64P and Nf2^18-595 are defective in restoring contact-dependent inhibition of proliferation. Approximately 300 cells were scored in each of five experiments. (C) Confluent mosaic populations of LDCs were serum starved and treated with Tr-EGF (2 μg/ml, 1 h, 37°C), and ligand internalization was monitored as a measure of EGFR internalization (Nf2, green; Tr-EGF, red; DAPI, blue). (D) Quantitation reveals that, in contrast to Nf2^wt, neither Nf2^L64P nor Nf2^18-595 prevented EGFR internalization in confluent cells. Approximately 300 cells were scored in each of five experiments. Data represent means ± standard deviations. (E) In contrast to Nf2^wt, Nf2^18-595 failed to restore contact-dependent inhibition of proliferation to primary Nf2^−/− Schwann cells. (F) Persistent activation of EGFR and its downstream effectors is apparent in the membranes of LDCs expressing Ad-Nf2^18-595 but not Ad-Nf2^wt. Phosphorylated proteins are indicated by the letter “p” preceding the name. Membrane pellets including nuclei were extracted in 0.5% sodium dodecyl sulfate in radioimmunoprecipitation assay buffer.

FIG. 3.

The N terminus of merlin is essential for merlin interaction with membrane-associated proteins. (A) IP of EGFR from total membrane extracts revealed that Nf2^wt, but not Nf2^18-595 or Nf2^L64P, associates with the EGFR. Both Nf2^wt and Nf2^18-595 are present in the total membrane extracts, but Nf2^L64P, which is largely cytosolic, is not. (B) IP of NHE-RF1 from total cell extracts (tce) revealed that Nf2^wt, but not Nf2^18-595, associates with NHE-RF1. Total immunoglobulin G (IgG), representing the NHE-RF1 IPs, is shown as NHE-RF1 runs at the same 50-kDa molecular mass as the IgG heavy chain. (C) IP of E-cadherin from total membrane extracts revealed that Nf2^wt, but not Nf2^18-595 or Nf2^L64P, associates with E-cadherin. (D) Surface proteins in confluent Nf2^−/− LDCs or in LDCs expressing pBMN-Nf2^wt or -Nf2^18-595 were labeled with biotin and collected at 0 or 30 min after release to 37°C. Biotin pull-downs from total membrane extracts revealed that, in contrast to Nf2^wt, Nf2^18-595 fails to associate with any surface proteins at either time point. (E) IP of ezrin from total cell extracts revealed that Nf2^18-595 retains the ability to interact with ezrin. n ≥ 3.

FIG. 4.

The N terminus directs merlin to an insoluble apical network. Simultaneous fixation and permeabilization (fix 2) revealed that Nf2^wt (A) and ezrin (B) decorate the same cortical network in LDCs. Merge of the images is shown in panel C; lower magnification of images in panels A and B are represented in panels E and F, respectively. (D) Nf2^18-595 exhibits a fragmented localization to this network (see also Fig. S2D in the supplemental material). (E) The network localization of ezrin is not altered in the presence or absence of Nf2^wt (E and F). (G) LDCs expressing Nf2^wt were treated with jasplakinolide (2 μM, 1 h, 37°C). Jasplakinolide treatment yielded markedly enlarged compartments within the Nf2^wt-decorated cortical actin network. Upon detergent extraction prior to fixation (fix 3), Nf2^wt (H), but not ezrin (I) or Nf2^18-595 (J), retained network staining. (K) Immunofluorescence localization of NHE-RF1 in LDCs fixed and then permeabilized (fix 1) revealed that NHE-RF1 localizes to a similar cortical network. (L) Confocal imaging of confluent LDCs expressing various versions of Nf2 (fix 1) revealed that Nf2^wt colocalizes with apically distributed concanavalin A (ConA; a lectin that binds polysaccharides on the cell surface) and ezrin (an established apical marker); Nf2^wt is also enriched at cell-cell boundaries (arrowheads). However, careful examination revealed that Nf2^18-595 is not retained at the apical compartment and instead extends basally; Nf2^18-595 also fails to be enriched at cell-cell junctions (arrowheads). Little overlap is seen between apically distributed Nf2^wt and the basal marker, paxillin; in contrast, Nf2^18-595 does overlap with paxillin. Nuclei in panel L are stained with TOTO 3 (blue). Bars = 5 μm. n ≥ 3.

FIG. 5.

Residues 1 to 18 confer insoluble-membrane localization to ezrin. (A) Subcellular fractionation of immortalized Nf2^−/− fibroblasts with and without exogenous ezrin (Ez) or Ez^Nf21-18 revealed that neither endogenous (long exposure) nor exogenous ezrin is enriched in the insoluble fractions; however, Ez^Nf21-18 is specifically enriched in the insoluble fractions. The same extracts were probed with an anti-Nf2 antibody (sc-331) that recognizes residues 1 to 18 of merlin (NT Nf2); only the Ez^Nf21-18 protein is detected. TCL, total cell lysate; Cyt, cytosol, TX Sol, Triton X-100 soluble; TX Insol, Triton X-100 insoluble; pellet, remaining insoluble pellet. Samples were probed with an antiactin antibody to control for loading. (B) Immunostaining of confluent Nf2^−/− LDCs expressing pBMN-Ez or Ez^Nf21-18 revealed that Ez^Nf21-18 is now enriched along cell-cell boundaries. Bar = 10 μm. (C) Ez^Nf21-18 cannot restore contact-dependent inhibition of proliferation, as measured with BrdU (20 μM for 12 h), or prevent EGF internalization (Tr-EGF; 2 μg/ml, 1 h, 37°C) in Nf2^−/− cells. Approximately 300 cells were scored in each experiment. Data represent means ± standard deviations. n ≥ 3.

FIG. 6.

Membrane targeting via the N-terminal residues of merlin. (A) Schematic of residues 1 to 18 of merlin depicting putative sites of phosphorylation and basic charged residues. (B) Subcellular fractionation of confluent Nf2^−/− LDCs expressing pBMN-Nf2^wt, -Nf2^S7-13D, -Nf2^S7-13A, or -Nf2^15-17A revealed that both Nf2^S7-13D and Nf2^15-17A are underrepresented in the insoluble fractions. TCL, total cell lysate; Cyt, cytosol; TX Sol, Triton X-100 soluble; TX Insol, Triton X-100 insoluble; pellet, remaining insoluble pellet. Immunoblotting with an antiactin antibody served as a loading control. (C) Immunostaining of confluent Nf2^−/− LDCs expressing pBMN-Nf2^wt, -Nf2^S7-13D, or -Nf2^S7-13A revealed that Nf2^S7-13D failed to concentrate at cell-cell boundaries. Bar = 10 μm. n ≥ 3.