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Phosphorylation and activity of the tumor suppressor Merlin and the ERM protein Moesin are coordinately regulated by the Slik kinase
Correspondence to Richard G. Fehon: rfehon{at}uchicago.edu
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Merlin and Moesin are closely related members of the 4.1 Ezrin/Radixin/Moesin domain superfamily implicated in regulating proliferation and epithelial integrity, respectively. The activity of both proteins is regulated by head to tail folding that is controlled, in part, by phosphorylation. Few upstream regulators of these phosphorylation events are known. In this study, we demonstrate that in Drosophila melanogaster, Slik, a Ste20 kinase, controls subcellular localization and phosphorylation of Merlin, resulting in the coordinate but opposite regulation of Merlin and Moesin. These results suggest the existence of a novel mechanism for coordinate regulation of cell proliferation and epithelial integrity in developing tissues.
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Introduction |
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The neurofibromatosis 2 tumor suppressor protein Merlin and its close relatives Ezrin/Radixin/Moesin (ERM; Trofatter et al., 1993b; Bretscher et al., 2002) function as membrane-cytoskeletal linkers and regulators of multiple signaling pathways (Shaw et al., 2001; Bretscher et al., 2002; Speck et al., 2003). Merlin and ERMs share 
45% sequence identity and a similar domain organization with an N-terminal 4.1 ERM domain, a putative coiled-coil spacer, and a C-terminal domain that in ERMs binds to filamentous actin (Bretscher et al., 2002). Merlin has a clear role in regulating proliferation (Rouleau et al., 1993; Trofatter et al., 1993a), whereas Moesin and its paralogues Ezrin and Radixin are thought to maintain epithelial integrity by organizing the apical cytoskeleton (Speck et al., 2003).
A central question in the study of these proteins has been how their interaction with binding partners is regulated. For both Merlin and ERMs, there is abundant evidence for an intramolecular interaction between the 4.1 ERM domain and the C-terminal domain (Gary and Bretscher, 1995; Sherman et al., 1997; Gonzalez-Agosti et al., 1999; Gronholm et al., 1999; Meng et al., 2000; Nguyen et al., 2001). In ERM proteins, this interaction produces a closed, inactive form of the protein that does not interact with either transmembrane binding partners or filamentous actin (Matsui et al., 1998; Nakamura et al., 1999). For Merlin, studies in mammalian cells suggest that the closed form is active in inhibiting proliferation (Sherman et al., 1997; Shaw et al., 1998; Gutmann et al., 1999; Morrison et al., 2001), whereas studies in Drosophila suggest that, as with ERMs, the open form of Merlin retains all essential genetic functions (LaJeunesse et al., 1998). Whether this apparent distinction between flies and mammals represents a true functional difference or reflects methodological differences remains to be resolved.
Phosphorylation of a conserved threonine (Thr) in the actin-binding domain of ERM proteins has been demonstrated to be important for their activation by relieving the head to tail interaction (Nakamura et al., 1995; Matsui et al., 1998; Oshiro et al., 1998; Hayashi et al., 1999; Tran Quang et al., 2000). The precise kinase responsible for this event is unclear, although its activity seems to be positively regulated by Rho activation in mammalian cells. In Drosophila, the Ste20 family kinase Slik is necessary for the phosphorylation of Moesin, although, again, it is not clear whether Slik phosphorylates Moesin directly or via intermediate kinases (Hipfner et al., 2004). In mammalian cells, Merlin activity is regulated by a phosphorylation event at serine 518 that blocks head to tail interactions (Shaw et al., 2001). However, unlike ERMs, it appears that the phosphorylated form of Merlin is inactive in that it does not suppress growth (Shaw et al., 2001). In contrast, hypophosphorylated Merlin is enriched under conditions of serum starvation or confluency, suggesting that this form is growth suppressive (Sherman et al., 1997; Shaw et al., 1998; Gutmann et al., 1999). Serine 518 is thought to be phosphorylated by the p21-activated kinase (PAK) downstream of Rac activity (Kissil et al., 2002; Xiao et al., 2002), although the possibility of other mechanisms regulating Merlin phosphorylation cannot be excluded. In addition, evidence to date has failed to demonstrate phosphorylation of the equivalent Thr residue to the one phosphorylated in ERMs, although this residue is conserved in both mammalian and fly Merlin.
Many questions remain about the regulation of Merlin activity, particularly in the context of developing tissues undergoing normal proliferation. To better understand how Merlin is regulated, we have investigated the mechanism by which Merlin phosphorylation and, thus, its activity are controlled in Drosophila. In particular, we have examined the possibility that Merlin and Moesin are regulated by the same molecular mechanism. In this study, we show that Slik kinase, which positively regulates Moesin function, also regulates Merlin but in the opposite direction. In addition, our observations suggest a competitive interaction between Moesin and Merlin for Slik activity. These results provide in vivo evidence of a kinase-based regulation of Drosophila Merlin and suggest that Merlin and Moesin are coordinately regulated in developing tissues.
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Results |
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To examine the effect of the loss of Slik function on Merlin subcellular localization, we used FLP/FRT (Flip recombinase/Flip recombination target)-mediated mitotic recombination to generate clones of slik–/– cells in heterozygous slik–/+ (wild type) imaginal epithelia. Induction of a homozygous slik mutant clone by mitotic recombination simultaneously produces a homozygous wild-type (slik+/+) sister clone, thus allowing side by side comparisons between cells containing two, one, or no functional copies of the slik gene. Wild-type cells within the epithelium are positively marked by the expression of either one copy (slik–/+) or two copies (slik+/+) of a GFP transgene, whereas slik–/– cells lack this marker. Optical sections taken below the apical surface of the epithelium (Fig. 1, A–A'') show a clear inverse correlation between slik gene dosage and Merlin staining. Merlin staining was increased in homozygous slik– clones and decreased in homozygous wild-type sister clones relative to the surrounding heterozygous slik–/+ cells.
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Slik affects Merlin localization and trafficking in cultured cells
To further examine the effects of Slik activity on Merlin subcellular localization, we performed coexpression experiments in cultured Drosophila S2 cells. Previous studies (McCartney and Fehon, 1996; LaJeunesse et al., 1998) have shown that upon induction, Merlin initially localizes to the membrane of S2 cells and then, within 3 h, traffics into punctate cytoplasmic structures that are associated with endocytic vesicles (McCartney and Fehon, 1996). Perturbation of the C-terminal domain of Merlin alters its localization and trafficking pattern (LaJeunesse et al., 1998). To determine whether Slik affects the subcellular localization and movement of Merlin, a pulse-chase assay was performed in S2 cells using a heat shock–inducible GFP-tagged Merlin expression construct (LaJeunesse et al., 1998). Control experiments in which cells were induced to express a pulse of Mer+ exhibited a similar pattern of Merlin localization to that reported previously (Fig. 2, B–E; LaJeunesse et al., 1998).
In contrast, the coexpression of Slik with Mer+ results in a shift in the temporal pattern of Merlin localization. In this case, a substantial proportion of cells displayed Merlin that associated with the plasma membrane even 6 h after induction (Fig. 2, A and F). Thus, Slik activity prevents the normal trafficking of Merlin off the plasma membrane and into cytoplasmic punctate structures. Coexpression of a kinase-inactive version of Slik has no effect on Merlin localization or trafficking (Fig. 2, compare G with E). Together with the loss of function clonal analysis, these results indicate that Slik kinase activity controls the localization and trafficking of Merlin.
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phosphatase converted the slower migrating bands to the most rapidly migrating form (Fig. 3 B), indicating that the slower migrating forms represent differentially phosphorylated forms of the protein.
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To better characterize Slik effects on Merlin phosphorylation, we next examined these proteins when expressed in Drosophila cultured S2 cells. A similar pattern of Merlin isoforms is observed on immunoblots when Merlin is expressed in S2 cells, as was seen in wing imaginal discs (unpublished data). Increased phosphorylation of Merlin in the presence of Slik kinase is also observed in S2 cells, albeit with a more subtle effect.
As the Thr residue near the C terminus of Moesin (Thr559) is also conserved in Merlin (Thr616; McCartney and Fehon, 1996), we wondered whether Slik activity might control the phosphorylation of this site in Merlin. To address this question, we used site-directed mutagenesis to construct phosphomimetic (MerT616D) and nonphosphorylatable (MerT616A) versions of the Merlin protein and examined their effect on Merlin phosphorylation in the presence of Slik kinase in S2 cells. Expressed MerT616D displays a prominent hyperphosphorylated band, whereas this band is much less prominent in expressed MerT616A (Fig. 3 C). These results indicate that sites in addition to Thr616 are phosphorylated in Merlin and suggest that the phosphorylation state of Thr616 may regulate the phosphorylation of these sites by other kinases. The addition of Slik kinase does not appear to alter the phosphorylation pattern of either mutant (Fig. 3 C), which is consistent with the notion that Slik acts on Merlin via phosphorylation of the Thr616 residue.
If Slik's effects on Merlin localization are mediated by phosphorylation, phosphomimetic Merlin mutations should affect subcellular localization in a similar manner to the cotransfection of wild-type Merlin with Slik. To examine this, MerT616D and MerT616A were tested in the aforementioned S2 cell trafficking assay. As we observed for wild-type Merlin in the presence of Slik kinase (Fig. 2 F), MerT616D alone trafficked very slowly off the plasma membrane (Fig. 2 H). However, MerT616A internalized from the plasma membrane to the cytoplasm with even faster kinetics than Mer+ or Mer+ coexpressed with kinase-inactive Slik (Fig. 2, compare I with E and G). These results indicate that one effect of phosphorylation is to regulate Merlin trafficking and subcellular localization. They also suggest that phosphorylated Merlin remains closely associated with the plasma membrane, whereas hypophosphorylated Merlin rapidly traffics off of the membrane, possibly in association with transmembrane proteins.
To ask whether Slik interacts directly with Moesin and Merlin, we used an in vitro GST pull-down assay (Fig. 3 D). The results indicate that bacterially expressed Merlin and Moesin both bind to Slik in vitro. In addition, we attempted to determine whether purified Slik can phosphorylate either Moesin or Merlin in vitro. However, as previously shown for Moesin (Hipfner et al., 2004), we were unable to detect direct phosphorylation of Merlin or Moesin by Slik kinase (unpublished data). Whether this indicates that Slik acts in vivo via intermediary kinases or requires unidentified cofactors not present in our experiments is unknown, but the observation that Slik interacts directly with both Moesin and Merlin is consistent with the idea that they serve as substrates for Slik's kinase activity.
Slik interacts genetically with Merlin
As a further test of functional interaction between Merlin and Slik, we examined genetic interactions between Merlin and slik mutations. Specifically, we asked whether reducing slik function genetically modifies the phenotype of an activated Merlin transgene (Mer1-600; LaJeunesse et al., 1998) that confers growth suppression. The expression of Mer1-600 in wild- type wings causes a reduction in size by a mean of 15% from wild type (P = 0.01; Fig. 4, A vs. B).
Using this sensitized genetic background, we asked whether manipulating slik gene dose affects the activity of endogenously expressed wild-type Merlin. The reduction of slik dose (slik is completely recessive) by one half in this genetic background reduced wing size by a mean of 18% (P = 0.002; Fig. 4, A vs. C). Thus, reduction in Slik function enhances the phenotype from expressing an activated form of Merlin, suggesting that Slik antagonizes Merlin function. This phenotypic interaction is most likely mediated through Slik's effects on endogenously expressed wild-type Merlin acting synergistically with the coexpressed Mer1-600, which lacks the Thr616 residue.
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We also addressed the relationship between Merlin and Moesin using the aforementioned S2 cell trafficking assay. Coexpression of Merlin and Moesin does not alter the subcellular trafficking of Merlin (Fig. 2, J vs. E). However, the coexpression of Moesin blocks the effect of Slik on Merlin trafficking (Fig. 2, compare E with K), which is consistent with the hypothesis that Moesin and Merlin act as competitive substrates.
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Discussion |
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This study also provides the first genetic evidence that Moesin and Merlin functionally interact through competition for Slik kinase activity, although previous studies have shown physical interactions between these proteins (Gonzalez-Agosti et al., 1999; Gronholm et al., 1999; Meng et al., 2000). It is interesting to note that in mammalian Schwann RT4 cell lines, expressing constitutively phosphorylated Merlin not only impairs the ability of Merlin to suppress proliferation and motility but also induces a novel ERM-like phenotype (Surace et al., 2004). Surace et al. (2004) attribute this phenotype to the conversion of Merlin to an ERM-like molecule. However, if Merlin and Moesin are also coordinately regulated in mammalian cells, an alternative possibility is that overexpression of a phosphomimetic Merlin could affect the phosphorylation state of endogenous ERM proteins, thereby increasing their level of activity.
We found that the loss of slik function results in a dramatic shift in Merlin localization from the apical plasma membrane to punctate cytoplasmic structures. We have previously shown that Merlin traffics from the plasma membrane with endocytic vesicles in cultured cells (McCartney and Fehon, 1996), raising the possibility that in the absence of Slik, activated Merlin is more stably associated with endocytic compartments than in normal cells. If this is so, inactive Merlin may reside at the plasma membrane and, in response to activation, traffics internally, presumably in association with transmembrane proteins. If this model is correct, it suggests that Merlin may function in tumor suppression by facilitating removal from the plasma membrane of receptors that promote cell proliferation. This model fits well with our recent observation that several receptors, including Notch and the EGF receptor, accumulate to abnormal levels on the surface of cells that are mutant for Merlin and the functionally redundant related tumor suppressor expanded (Maitra et al., 2006).
Several important questions remain regarding the regulation of Moesin and Merlin that we have described in this study. It remains unclear whether Slik itself can directly phosphorylate either protein or whether there are one or more kinases operating downstream of Slik. Additionally, the dual functions described here may provide novel insights into the role of the mammalian orthologues of Slik, such as PAK, in the malignant transformation of epithelial cells. Equally important will be to elucidate how Slik activity is itself controlled. Given its ability to simultaneously regulate epithelial integrity and proliferation in developing epithelial tissues, Slik may function as a central integrator of the multitude of signals that converge to regulate growth and morphology during development.
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Materials and methods |
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Pulse-chase of Merlin protein in Drosophila Schneider cells
S2 cells were cotransfected with either UAS-slik+ or UAS-slikkd and pCasper ubiquitin GAL4 along with pCasper-hs Mer+ to allow the simultaneous expression of wild-type Slik ubiquitously and a heat shock–driven pulse (30 min at 37°C) of expression of wild-type hsGFP-tagged Merlin (hsMer+). hsMer+ retains wild-type function (LaJeunesse et al., 1998). pCasperHS Myc MerT616A and pCasperHS Myc MerT616D were made by mutating Thr 616 to alanine or aspartic acid using complementary oligonucleotides and the QuikChange method (Stratagene; constructed by R. Kulikauskas, Duke University, Durham, NC). Mutations were confirmed by sequencing. Cells were collected, fixed in 2% PFA for 20 min at room temperature, and Merlin GFP patterns were analyzed at 1, 3, and 6 h after heat shock. At least three independent replicates were scored for each experiment. For each combination and time point analyzed, a minimum of 150 transfected cells were counted. Myc-tagged constructs were detected using monoclonal anti-Myc at 1:4,000 (9B10; Cell Signaling). Slik was detected using a polyclonal antibody (Hipfner and Cohen, 2003). Myc and Slik were then visualized using cyanine dye CY3, FITC secondary antibodies (Jackson ImmunoResearch Laboratories), and cells mounted in ProLong (Invitrogen). Cells were analyzed using a confocal microscope (LSM510; Carl Zeiss MicroImaging, Inc.) and a plan-Apo 63x NA 1.4 lens.
Immunoblotting and immunolocalization
To characterize the phosphorylation patterns of Merlin protein, late third instar wing imaginal discs were dissected in Drosophila serum-free media (Invitrogen) and homogenized in lysis buffer (20 mM Hepes, pH 7.0, 50 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM DTT, 1.0% Triton X-100, Complete Protease Inhibitor [Roche], 50 mM NaF, 30 mM Na pyrophosphate, and 100 µm Na orthovanadate). Merlin protein complexes were subsequently immunoprecipitated using guinea pig anti-Merlin linked to Sepharose protein A beads (McCartney and Fehon, 1996) and separated on 8% (118:1) polyacrylamide gels (Scheid et al., 1999). For phosphatase treatment after immunoprecipitation, the protein A beads were precipitated, and one half was then treated with 400 U phosphatase (New England Biolabs, Inc.) at 30°C for 45 min followed by Western blot analysis.
Wandering third instar larvae were dissected in serum-free Drosophila media and fixed in either 4% PFA or ice-cold 10% TCA (Hayashi et al., 1999) for 20 min. For Western analysis (W) and immunolocalization (I), antibodies used were as follows: guinea pig anti-Slik at 1:40,000 (W) and 1:10,000 (I; provided by S. Cohen and D. Hipfner, European Molecular Biology Laboratory, Heidelberg, Germany), rabbit anti-Moesin D44 at 1:40,000 (W) and 1:20,000 (I; provided by D. Kiehart, Duke University, Durham, NC), rabbit antiphospho-Moesin at 1:10,000 (I; obtained from D. Ready, Purdue University, West Lafayette, IN), guinea pig anti-Merlin at 1:10,000, rhodamine phalloidin at 1:1,000 (Invitrogen), mouse anti-coracle at 1:500, and mouse anti–ß-tubulin at 1:5,000 (W; E7; developed by M. Klymkowsky and obtained from the Developmental Studies Hybridoma Bank, The University of Iowa, Iowa City, IO). Appropriate secondary fluorescent antibodies (FITC and cyanine dyes CY3 and CY5) were obtained from Jackson ImmunoResearch Laboratories and were used at 1:1,000. Western blots were visualized and quantified using an infrared imaging system (Odyssey; LI-COR). Immunostained tissues were mounted in ProLong (Invitrogen) and analyzed using either an LSM410 or LSM510 confocal microscope (Carl Zeiss MicroImaging, Inc.) with a plan-Apo 63x NA 1.4 lens. Figures were compiled in Photoshop 7.0.1 (Adobe).
In vitro GST pull-down assay
GST, GST-Merlin, and GST-Moesin fusion proteins were grown in BL21 cells overnight at 37°C. Cultures were diluted 1:100, grown to an OD260 of 1, and GST constructs were induced by adding 1 mM IPTG and grown at 18°C for 3 h. Lysates were sonicated and batch incubated with glutathione–Sepharose 4B for 3 h at 4°C and washed in columns with an excess of 10 bed volumes of 1x PBS. [S35]methionine-labeled probe protein (Slik) was prepared using the T7 TNT Quick Coupled Transcription/Translation System (Promega) according to the manufacturer's instructions. Proteins were incubated at 4°C for 4 h and boiled in SDS sample buffer, and proteins were separated on a 10% SDS-PAGE gel, transferred to nitrocellulose, and exposed to film.
Generation of mosaic and MARCM clones
Larvae of the genotype w; 42DFRT Ubi-GFPnls/42DFRT slik1; hsFLP or UAS-CD8-GFP hsFlp; FRT42 Gal80; TubG4/FRT 42D slik1; UAS MYC-MoeT559D were heat shocked at 36 ± 12 h after egg laying for 1 h at 37°C, 1 h at 25°C, and 1 h at 37°C. Wing imaginal discs were dissected from wandering third instar larval stages and fixed in 4% PFA. GFP was visualized directly. Moesin was detected with rabbit anti-Moesin D44 at 1:20,000 (provided by D. Kiehart), and Merlin was detected with guinea pig anti-Merlin at 1:10,000. Moesin and Merlin were then visualized using cyanine dye CY3 and FITC secondary antibodies, respectively (Jackson ImmunoResearch Laboratories), and cells were mounted in ProLong (Invitrogen). Cells were analyzed using a confocal microscope (LSM510; Carl Zeiss MicroImaging, Inc.) with a plan-Apo 63x NA 1.4 lens.
Wing measurements
Crosses with flies of the appropriate genotypes were raised at 25°C, and wings were analyzed as described previously (LaJeunesse et al., 2001). Images were collected on a camera (AxioCam HRm; Carl Zeiss MicroImaging, Inc.) mounted on a microscope (Axiovert 200M; Carl Zeiss MicroImaging, Inc.) using a Fluar 5x NA 0.25 lens. Area measurements of each wing were obtained from images using the free draw tool in ImageJ software (National Institutes of Health). Statistics were calculated using Excel (Microsoft), and figures were compiled in Photoshop 7.0.1 (Adobe).
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Acknowledgments |
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S.C. Hughes was the recipient of a Young Investigator Award from the Children's Tumor Foundation (formerly known as the National Neurofibromatosis Foundation). This work was funded by grant NS034783 from the National Institutes of Health.
Submitted: 2 August 2006
Accepted: 19 September 2006
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