Distinct overlapping sequences at the carboxy-terminus of merlin regulate its tumour suppressor and morphogenic activity

Abstract

The Neurofibromatosis 2 (NF2) gene product merlin is a tumour suppressor, which in addition to inhibiting cell proliferation regulates cell morphology. The morphogenic properties of merlin may play a role in tumour suppression, as patient-derived tumour cells demonstrate cytoskeletal abnormalities. However, it is still unclear how these functions are linked. The N-terminal FERM-domain of merlin is highly homologous to the oncogenic protein ezrin, while the C-termini are less conserved, suggesting that the opposite effect of the proteins on proliferation could be mediated by their distinct C-terminal regions. In this study we characterize the role of the most C-terminal residues of merlin in the regulation of proliferation, cytoskeletal organization, phosphorylation and intramolecular associations. In addition to the two full-length merlin isoforms and truncating mutations found in patients, we focused on the evolutionally conserved C-terminal residues 545-547, also harbouring disease-causing mutations. We demonstrate that merlin induces cell extensions, which result from impaired retraction of protrusions rather than from increased formation of filopodia. The residues 538-568 were found particularly important for this morphogenic activity. The results further show that both merlin isoforms are able to equally inhibit proliferation, whereas C-terminal mutants affecting residues 545-547 are less effective in growth suppression. This study demonstrates that the C-terminus contains distinct but overlapping functional domains important for regulation of the morphogenic activity, intramolecular associations and cell proliferation.

Fig 1

Characterization of the merlin C-terminal constructs. (A) Schematic overview of merlin's exon composition and structural domains (upper figure) and the constructs in this study (lower figure). Merlin wild-type (WT) isoform 1, isoform 2, several C-terminally truncated molecules of isoform 1, constructs with C-terminal point mutations (arrowheads) and deletion of residues 50-70 in exon 2 (Δ2) were used. The unique C-terminus of isoform 2 is indicated in grey, and numbers represent amino acids. (B) Lysates from COS-7 cells transfected with merlin constructs were separated into insoluble (I) and soluble (S) fractions and detected with the N-terminal A-19 merlin antibody. Merlin WT is mainly soluble whereas isoform 2 and the truncated constructs are distributed to both the insoluble and soluble fraction. All merlin constructs migrate at expected molecular size. (C) Schematic picture of merlin C-terminus based on moesin structure and merlin predictions (combined from [39, 41]). Alpha-helices are indicated with helical structures, and the predicted conserved α-helical region is shown by arrows. Arrowheads point out the termination of the truncations and the missense mutations are marked with X. (D) Expression of a truncated merlin protein corresponding to residues 1-548 in a patient-derived tumour with a mutation in the NF2 gene. The tumour material was run in SDS-PAGE together with COS-7 cell lysates expressing WT and 1-547 merlin as controls, and detected with various merlin antibodies. The N-terminal antibodies A-19 and HB7 and the phospho-serine 518 antibody (p-S518 Ab) detect merlin in the tumour (arrowheads), whereas C-terminal merlin antibodies KF10 and C-18 do not. Tubulin is used as a loading control.

Fig 2

The morphogenic activity of different merlin constructs. COS-7 cells (A), mouse embryonic fibroblasts lacking merlin (Nf2−/− MEFs) (B), and Schwann cells (C) were transiently transfected with merlin constructs and stained for merlin. Untransfected cells were stained for ezrin (COS-7 cells and Nf2−/− MEFs) or phalloidin (Schwann cells). The total extension length per cell was quantified from Nf2−/− MEFs, and the values indicate the average length of extensions per cell (B, right panel). Merlin induces the formation of cell extensions and a significant increase in the total extension length is observed in isoform 2, 1-587 and 1-547 expressing cells compared to WT. Extensions over 50 μm in length in Nf2−/− MEFs are marked with arrows. Scale bars 20 μm. *P < 0.05, **P < 0.01.

Fig 3

Cytoskeletal components and time-lapse analysis of merlin-induced extensions. (A) Nf2−/− MEFs expressing merlin isoform 2 and 1-547 were stained for merlin and either phalloidin or α-tubulin. Both actin and tubulin are present in longer merlin-induced extensions (arrows). (B) Still pictures from live cell imaging of Nf2−/− MEFs transfected with merlin isoform 2. Arrowheads show the direction of movement and arrows the forming extension. The long extensions are formed when protrusions cannot detach as the cell moves in the opposite direction. (C) Migrating cells used in live cell imaging expressing isoform 2 (left picture) and WT expressing cells (right picture) were stained for merlin. GFP-α-actinin was used to identify isoform 2 expressing cells used in live cell imaging. A gradient of merlin isoform 2 is observed in moving cells in the trailing edge, in addition merlin is present at the leading edge (inset). (D) Nf2−/− MEFs expressing merlin isoform 2 were stained for merlin and endogenous ezrin (left panel). Ezrin does not form a gradient but is present in membrane structures which are devoid of merlin (arrowhead). Mouse embryonic fibroblasts lacking ezrin (ezrin−/− MEFs) expressing merlin isoform 2 were stained for merlin and phalloidin (right picture). A gradient of merlin is detected also in ezrin−/− MEFs.

Fig 4

The E545 + E547 motif and its impact on morphogenic properties. (A) Sequence alignment of residues 541-551 of merlin proteins from different species and the ERM proteins of human and Drosophila. (B) Nf2−/− MEFs (upper panel) and COS-7 cells (lower panel) transfected with merlin E547K and E545K + E547K were stained for merlin. Mutation of E547K or E545K + E547K leads to the formation of cell extensions in both cell types (arrows). (C) Nf2−/− MEFs expressing WT or E545K + E547K with or without a deletion in exon 2, containing a cytoplasmic retention factor, were stained for merlin (green) and the nuclei (red). Mutation of E545K + E547K does not disrupt the nuclear export sequence, as the protein shows a cytoplasmic distribution. (D) Enlargement of Nf2−/− MEFs expressing merlin WT Δ2 and E545K + E547K Δ2. WT Δ2 cells display a punctate distribution of merlin which is absent from E545K + E547K Δ2 cells.

Fig 5

The role of merlin-ezrin association in extension formation. (A) Nf2−/− MEFs expressing endogenous ezrin were transfected with merlin constructs and stained for merlin (green) and ezrin (red). Merlin and ezrin co-localize only partially at the cell membrane. Ezrin is present at the tip of extensions and membrane regions which lack merlin (arrows). (B) Ezrin−/− MEFs transfected with merlin constructs were stained for merlin and untransfected cells with phalloidin. The total extension length per cell was quantified, and values indicate the average length of extensions (right panel). Merlin induces cell-extension formation in ezrin−/− MEFs, but the extensions are shorter than in Nf2−/− MEFs. An increase in the total extension length is observed in isoform 2 expressing cells, whereas the truncated constructs did not induce a significant increase in extensions compared to WT merlin. Scale bar 20 μm. *P < 0.05. (C) Pull-down analysis of COS-7 cell lysates expressing merlin constructs (upper panel) incubated with GST-ezrin and merlin fusion proteins. Bound proteins were separated on SDS–PAGE and immunoblotted with merlin A-19 Ab or GST to ensure equal loading of the GST-fusion proteins. All C-terminally deleted merlin constructs bind full-length ezrin (left panels), but not the GST-control (right lower panels). Merlin isoform 2, 1-547, and E545K + E547K bind to merlin 1-314 FERM-domain whereas only weak binding was observed with WT merlin (right upper panel).

Fig 6

Electrophoretic mobility and phosphorylation status of merlin constructs. (A) 293 lysates transfected with merlin constructs were separated into insoluble (I) and soluble fractions (S), run in SDS–PAGE and detected with merlin A-19 Ab. The migration pattern is shown for the constructs in the long exposure (upper panel), and in the short exposure (lower panel) for WT and isoform 2. Merlin isoform 2 migrates as a single band whereas the longest truncated construct 1-587 migrates as a triplet like WT. (B) S518 phosphorylation of merlin constructs. WT and S518A merlin expressed in 293 cells were detected with A-19 and phospho-serine 518 Ab (p-S518 Ab, upper panel). S518 phosphorylated merlin is detected both in the hyperphosphorylated (upper) and phosphorylated (middle) band (arrows), but not in the hypophosphorylated (lower) band. All merlin truncations and missense mutants expressed in COS-7 cells are S518 phosphorylated (lower panel). (C) The C-terminal sequence of merlin isoforms 1 and 2. Isoform 2 and merlin 1-587 differ only in their last eight residues (580-587, underlined). Potential phosphorylation sites in this region are bolded. (D) Immunoblot analysis of lysates from 293 cells expressing WT merlin or missense mutants T581A, S584A and S587A detected with A-19 Ab. The hyperphosphorylated band (arrow) is detected in all constructs and the alanine mutations of the indicated residues do not change the electrophoretic mobility of merlin.

Fig 7

The effect of C-terminal mutations on growth inhibition and proliferation. (A) Soft agar colony formation of Nf2−/− MEFs. Cells transfected with c-Ha-Ras C61L and merlin were grown in soft agar (left panel). Merlin expression levels of transfectants were verified (upper right panel). The number of colonies was quantified from triplicate plates of each construct from three independent experiments (lower right panel). The chart shows the number of colonies in comparison to control (cells transfected with c-Ha-Ras C61L and empty vector), and average number of colonies in control (206) is regarded as 100%. Merlin WT and isoform 2 suppress Ras-induced growth, whereas 1-547 and E545K + E547K have reduced growth inhibitory effect. (B) Nf2−/− MEFs transfected with GFP or merlin were serum starved and then serum induced for ∼12 hrs. Cells were stained for merlin and the proliferation marker Ki-67, and Ki-67 positivity was quantified. Both merlin WT and isoform 2 decrease proliferation compared to GFP whereas merlin 1-547 and E545K + E547K do not suppress proliferation. ***P < 0.001; **P < 0.01.