Department of Biological Sciences, Stanford University, 385 Serra Mall, Stanford, CA 94305-5020, USA
* Present address: Department of Applied Genomics, Bristol-Myers Squibb, P.O. Box 5400, Princeton, NJ 08543-5400, USA
Present address: Inflammatory Diseases Unit, Roche Bioscience, 3401 Hillview Ave, Palo Alto, CA 94304-1397, USA
Author for correspondence (e-mail: msimon{at}stanford.edu)
Accepted 10 October 2001
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SUMMARY |
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Key words: Receptor tyrosine kinase, Corkscrew, Ankyrin repeat, KH domain, Photoreceptor development, Drosophila
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INTRODUCTION |
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One system that has proved useful in the study of RTK signaling is the specification of photoreceptors in the developing Drosophila eye [reviewed by Raabe (Raabe, 2000)]. Proper Drosophila eye development requires signaling through the RTKs EGFR and Sevenless (SEV) (Hafen et al., 1987; Freeman, 1996). The adult eye is composed of approximately 800 clusters of cells called ommatidia arrayed in a uniform repeating pattern. Each ommatidium contains six outer photoreceptors (R1-R6), two inner photoreceptors (R7 and R8), and a fixed number of accessory cells. Cell fate specification begins with the selection of the R8 photoreceptor, followed by a series of cell-cell communication events resulting in the recruitment of R1-R7 [reviewed by Tomlinson and Ready (Tomlinson, 1988; Ready, 1989)]. The differentiation of R1-R7 requires signaling through EGFR, with the R7 also requiring additional signals through Sevenless (Freeman, 1996). In addition to its role in cell fate specification, EGFR activity is also necessary for promoting proliferation and preventing apoptosis among the undifferentiated cells after patterning has begun (Baker and Yu, 2001).
Through the study of signal-dependent processes such as photoreceptor differentiation, the roles of many components of the RTK signaling pathway have been well characterized [reviewed by Schlessinger (Schlessinger, 2000)]. Upon ligand binding, the receptor dimerizes and auto-phosphorylates, initiating signaling through the canonical Ras/MAPK pathway. In addition to recruiting adapter molecules that stimulate Ras signaling, tyrosine phosphorylation also creates docking sites on scaffolding molecules such as Daughter of Sevenless (DOS/Gab1) [reviewed by Huyer and Alexander (Huyer and Alexander, 1999)]. These scaffolding proteins provide additional binding sites for signaling components, including the protein tyrosine phosphatase CSW/Shp2 [reviewed by Pawson and Scott, and Huyer and Alexander (Pawson and Scott, 1997; Huyer and Alexander, 1999)].
The mechanism by which CSW/Shp2 contributes to the transduction of the RTK signal is not completely understood. While CSW/Shp2 is required for RTK signaling and MAPK activation, genetic and biochemical experiments have not placed CSW/Shp2 activity at any one specific point within the linear Ras/MAPK pathway [reviewed by Huyer and Alexander (Huyer and Alexander, 1999)]. One proposed mechanism for CSW/Shp2 signal transduction is that it serves as an adapter between the receptor and DRK/Grb2, leading to Ras activation (Li et al., 1994; Bennett et al., 1994; Cleghon et al., 1998, Ronnstrand et al., 1999). However, several lines of evidence demonstrate that CSW/Shp2 has additional signaling roles. First, the putative DRK/Grb2 binding site of CSW/Shp2 may be removed without affecting signaling (Allard et al., 1998; OReilly and Neel, 1998). Second, the effects of dominantly activated Raf can be blocked with a dominant negative CSW, suggesting a role for CSW downstream of or in parallel to Ras/Raf signaling (Allard et al., 1996). Third, the phosphatase activity of CSW/Shp2 is essential for signaling (Tang et al., 1995; Allard et al., 1998; Deb et al., 1998; Frearson and Alexander, 1998). Therefore, CSW/Shp2 must have a role in addition to that of an adapter and must have substrates whose dephosphorylation is critical for signal transduction.
In order to elucidate the role of CSW during RTK signaling and identify potential targets, we conducted a sensitized genetic screen using the Drosophila eye. This screen identified mutations that dominantly modify the phenotype caused by the expression of a dominant negative form of CSW. Many known components of the RTK signaling pathway were identified, including DOS (Herbst et al., 1996). Since DOS plays a key role in activating CSW in response to RTK signaling, other genes isolated through this screen may also encode novel components of this pathway, perhaps including those involved in transducing the signal through CSW. Here we describe a second protein identified through this screen. MASK (Multiple Ankyrin repeats Single KH domain) is a novel, predominantly cytoplasmic protein that genetically interacts with components of RTK signaling pathways. MASK is crucial for photoreceptor differentiation, cell survival and cell proliferation in a manner similar to other signaling molecules that transduce the EGFR and SEV signals. However, unlike other pathway components, MASK is not absolutely required for the activation of MAPK, and potentially acts downstream of MAPK or defines a new branch of the RTK signaling pathway.
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MATERIALS AND METHODS |
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Clonal analysis
Homozygous mutant clones in the eye were generated as previously described using FRT sites and the hs-flp recombinase (Xu and Rubin, 1993) or the ey-flp recombinase (Newsome et al., 2000). Homozygous wild-type tissue was eliminated using the l(3)cl-R31 allele (Newsome et al., 2000). Non-clonal tissue in the eye imaginal disc contained GFP. Non-clonal tissue in the adult eye contained pigment granules due to the presence of a functional w+ transgene. The comparison between the homozygous mutant clone and homozygous wild-type twin spot was quantified using the Histogram function of Adobe Photoshop. Follicle cell clones were generated as previously described (Duffy et al., 1998). Eggs were collected on molasses/agar plates and prepared as previously described (Wasserman and Freeman, 1998).
Histology
Scanning electron microscopy was performed as previously described (Kimmel et al., 1990). Tangential sections (2 µm) of adult eyes were prepared as described previously (Tomlinson and Ready, 1987).
Immunohistochemistry
Third instar larval eye discs were dissected and stained essentially as described previously (Gaul et al., 1992) and mounted in Vectashield (Vector Laboratories, Inc.). Constructs and SL2 cells expressing SEVS11 and SEVS11 kinase dead were described previously (Basler et al., 1991; Simon et al., 1993). Cells were maintained and heat shock expression of SEVS11 was induced as described previously (Herbst et al., 1999). Cells were fixed in PLP on hydrophobic slides with a cell adhesion surface (Denville Scientific) and stained essentially as described for eye discs.
Primary antibodies used were: rat monoclonal anti-Elav (provided by G. M. Rubin), mAb BP104 (Hortsch et al., 1990), mouse or rabbit anti-GFP (Molecular Probes), anti-phosphohistone H3 (Upstate Biotechnology), anti-diphosphorylated-ERK1 and 2 (Sigma), and anti-MASK. The MASK antibody was generated by injecting rabbits with a glutathione S-transferase/MASK fusion protein that contained amino acids 1047 to 1171 of the full-length MASK protein (Covance Research Products, Inc.). Secondary antibodies were obtained from Jackson Immunoresearch.
All immunostained tissues were observed with a Biorad MRC 1024 and Nikon Eclipse E600 confocal microscope.
TUNEL staining
TUNEL staining kits used were the In Situ Cell Death Detection Kit, Fluorescein (Roche Diagnostics GmbH) and the In Situ Cell Death Detection Kit, TMR red (Boehringer Mannheim GmbH). Eye discs were stained as described above with modifications: the secondary antibody was diluted in the TUNEL reaction solution as prepared from the kits, and the discs were incubated for 3 hours at 37 degrees. Third instar larvae were cut open, and stained with TUNEL as described for eye discs.
Cloning
The mask alleles were mapped by recombination between P[w+] elements located at chromosomal positions 95E1 and 95F14. A cosmid genomic library was used to isolate clones from this region. Probes from the genomic cosmids were used to identify restriction fragment length polymorphisms (RFLPs). Recombination events between the RFLPs and mask10.22 were used to reduce the region known to contain mask to about 35 kb. Overlapping fragments of mask cDNAs were isolated from the LD cDNA library (Berkeley Drosophila Genome Project) using probes from this small genomic region. Nucleotide changes in mutant alleles were identified by sequencing PCR amplified sections of the coding region from heterozygous, genomic DNA (Schlag and Wassarman, 1999). mask approximately corresponds to the predicted transcripts CG18671, CG6268, and CG6313 in the annotated Drosophila genome.
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RESULTS |
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mask genetically interacts with members of the receptor tyrosine kinase signaling pathway
Dominant negative and activated forms of known components of the receptor tyrosine kinase signaling pathway can affect photoreceptor differentiation when expressed in the developing eye (Basler et al., 1991; Fortini et al., 1992; Allard et al., 1996). If MASK is a component of the RTK signaling pathway, removal of one copy of the gene might be expected to modify these photoreceptor phenotypes.
As described above, mask was identified owing to its ability to significantly enhance the photoreceptor phenotype of cswCS. In addition, the alleles mask10.22 and mask5.8 suppressed the rough eye and ectopic R7 phenotypes caused by increased signaling through an activated SEV (sevS11) and an activated CSW (cswsrc90) (data not shown, Table 1). Furthermore, removal of one copy of mask weakly suppressed the rough eye phenotype caused by the expression of an activated Ras (rasV12) and weakly enhanced the rough eye phenotype caused by the expression of a dominant negative Ras (rasN17) (Fig. 1I,J,K,L). These results suggest that MASK plays a positive role in transducing the signal downstream of receptor tyrosine kinases.
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In order to establish a requirement for CSW and MASK in the prevention of apoptosis, third instar eye imaginal discs were stained by TUNEL to mark apoptotic nuclei. Discs from males hemizygous for the loss-of-function allele cswC114 (Perkins et al., 1992) showed a significant increase in TUNEL staining (Fig. 3D,H). Discs expressing cswCS under the control of the sevenless promoter also showed a significant increase in TUNEL staining posterior to the morphogenetic furrow (Fig. 3I). In addition, a zone of TUNEL staining was seen anterior to the morphogenetic furrow, where apoptosis is not observed in wild-type discs but is seen in EGFRts loss-of-function discs (Baker and Yu, 2001) (Fig. 3D,H,I). Removal of one copy of the mask gene in the cswCS background enhanced the number of cells exhibiting TUNEL staining, both posterior and anterior to the morphogenetic furrow (Fig. 3J).
The ability of mask alleles to enhance the loss of photoreceptor differentiation in eyes expressing cswCS may be due to a reduction in viability of those photoreceptors. However, an extensive search for TUNEL staining of differentiating photoreceptors in cswCS and cswCS/mask discs revealed no neurons undergoing apoptosis (data not shown). These results indicate that MASK and CSW act in a crucial signaling pathway responsible for maintaining cell viability in undifferentiated cells.
Loss of MASK reduces cell proliferation
The reduction in mask tissue size could result from reduced cell proliferation as well as increased cell death. Ras and EGFR mutant clones exhibit reduced proliferation due to a phenomenon called cell competition, in which neighboring wild-type cells grow faster and therefore divide preferentially over mutant cells (Prober and Edgar, 2000; Baker and Yu, 2001). To examine proliferation directly in homozygous mask clones, eye imaginal discs were stained with -phosphohistone H3 antibodies, which stain the nuclei of cells undergoing mitosis (Hendzel et al., 1997). Two waves of proliferation occur in wild-type third instar eye imaginal discs, one preceding the morphogenetic furrow, and a second wave following the onset of photoreceptor differentiation [reviewed by Wolff and Ready (Wolff and Ready, 1993)]. Small mask clones induced with the hs-flp very rarely contained cells in M phase (Fig. 3K). However, in larger mutant patches induced with the more efficient ey-flp the pattern of cell division within the mitotic zones continued through the tissue unaltered (Fig. 3L). These results suggest that the ability of a cell lacking MASK to undergo mitosis may depend on the genotype of its neighboring cells. In larger clones mask cells are next to other mask cells, and are able to proliferate. In smaller clones mask cells must compete with neighboring wild-type cells, and are only infrequently able to divide.
Loss of MASK inhibits photoreceptor differentiation
In addition to affecting tissue size, removal of MASK function within the developing eye also resulted in a loss of photoreceptor specification. Eye imaginal discs from transheterozygous mask6.3/mask7.29 larvae contained differentiating photoreceptors (Fig. 3B). However, when these discs were stained with -Elav, a marker for photoreceptor nuclei, many ommatidia did not contain the normal complement of eight photoreceptors (Fig. 4A,B). In order to examine photoreceptor differentiation using a stronger mask allele, homozygous mutant clones of mask10.22 were analyzed. While some cells lacking MASK function began photoreceptor differentiation, the ommatidia within the clones did not contain all eight photoreceptors (Fig. 4C,D,E). This partial block in photoreceptor differentiation is consistent with a role for MASK in EGFR and SEV signaling.
In order to assay the requirement for MASK function in individual photoreceptors, mask6.3, mask7.29 and mask10.22 clones were induced in flies with the genotype w,hs-flp; FRT82B, mask/FRT82B, P[w+]. The adult eyes were tangentially sectioned, revealing a loss of outer and inner photoreceptors in many clonal ommatidia (Fig. 4F). Ommatidia along the borders of the mutant clones contained both wild-type and mutant photoreceptors, which could be distinguished by the presence or absence of pigment granules, respectively. The photoreceptors in these normally constructed, mosaic ommatidia were assayed for their ability to differentiate without MASK function. If a particular photoreceptor does not require MASK function for differentiation, one would expect that photoreceptor to be wild-type in 50% of the ommatidia scored and mutant in 50% of the ommatidia scored. However, in mask10.22 clones, fewer than 50% of photoreceptors scored in each class (R1-R8) were mutant, indicating a partial requirement for MASK function for their differentiation (Table 2). Strikingly, no R7 cells in mosaic ommatidia in clones made with mask10.22 were mutant. Only a small number of R7 photoreceptors in mosaic ommatidia in clones generated using the hypomorphic alleles mask6.3 and mask7.29 were mutant (Table 2). In addition, most R7 cells were missing from ommatidia within the clones. The numbers of mutant R1 and R6 photoreceptors were also significantly reduced in mosaic ommatidia from mask10.22 clones. From these data, the most stringent requirement for MASK is in the differentiation of the R7 photoreceptor, followed closely by R1 and R6.
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Loss of MASK affects RTK-dependent processes other than eye development
Signaling through EGFR in the ovarian follicle cells is required for proper construction of the egg [reviewed by Ray and Schupbach (Ray and Schupbach, 1996)]. Follicle cells that are mutant for many components of the RTK pathway results in dorsal appendages that are spaced too closely, are completely fused, or are absent altogether (Wasserman and Freeman, 1998). In order to establish a requirement for mask in egg shell patterning, homozygous mask10.22 clones were generated in the ovarian follicle cells. Eggs from females containing mask10.22 clones had dorsal appendages that were close together or fused (Fig. 5). No fused dorsal appendages were seen in the absence of a mask allele (data not shown). A role for MASK in this EGFR-signaling-dependent process further supports MASK as a common component of this signaling pathway.
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In order to determine whether MASK is required for MAPK activation and translocation, homozygous mask10.22 clones were generated in third instar eye discs and stained with an antibody that specifically recognizes diphosphorylated, activated MAPK (Yung et al., 1997). In differentiating photoreceptors, activated MAPK is in the cytoplasm of cells in the morphogenetic furrow, and moves into the nucleus after the furrow has advanced several rows (Kumar et al., 1998) (Fig. 6A). Diphosphorylated MAPK was seen both in the cytoplasm and in the nuclei of cells within mask clones (Fig. 6B,C). While MAPK activation and localization appeared unaltered, the possibility remains that a few individual cells failed to activate MAPK. This result indicated that MASK function is not absolutely required for the activation or translocation of MAPK.
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MASK is a large protein containing ankyrin repeats and a KH domain
The EMS-induced alleles of mask were mapped by recombination between P-elements and restriction fragment length polymorphisms (RFLPs). The mutations were localized to a small region within chromosomal bands 95E-F. Probes from this region were then used to isolate cDNAs from a library, which were cloned and sequenced. Several overlapping cDNAs were isolated and a long open reading frame of approximately 13 kb was identified. By sequencing mutant alleles, single nucleotide changes resulting in premature stop codons were found in mask10.22, mask5.8 and mask8.7 (Fig. 7A). These three mask alleles with stop codons in the same gene confirmed the identity of this transcript as mask.
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MASK predominantly localizes to the cellular cytoplasm
An antibody was raised against a portion of MASK that lies between the two blocks of ankyrin repeats. The specificity of the antibody was tested by staining third instar eye imaginal discs containing mask10.22 clones. This allele contains a stop codon before the sequence used as the antigen, and therefore clones should contain no protein recognized by the antibody. This antibody clearly stained wild-type tissue, but did not stain within the mask10.22 clones (Fig. 8A,B). Upon closer examination of eye imaginal discs stained with the -MASK antibody, MASK appeared to be ubiquitously expressed, and was localized primarily in the cytoplasm (Fig. 8C). Expression appeared to be somewhat higher in presumptive photoreceptors (Fig. 8D).
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DISCUSSION |
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Loss of MASK compromises cell differentiation, cell survival and cell proliferation
MASK activity is required for the differentiation of all developing photoreceptors, although the penetrance of this phenotype varies depending on the photoreceptor type examined. The strongest requirement for MASK appears to be in R1, R6 and R7. The R7 photoreceptor is often the most sensitive to decreases in RTK signaling, since its specification requires the activation of both EGFR and SEV (Freeman, 1996). R1, R6 and R7 are all recruited from cells that have undergone mitosis during the second mitotic wave, and these cells are often related (Hofbauer and Campos-Ortega, 1976). This relatedness may account for the apparent increased sensitivity of R1 and R6 to a reduction in signaling.
EGFR and Ras are important in actively promoting cell survival in Drosophila and in mammalian systems [reviewed by McNeill and Downward (McNeill and Downward, 1999)]. EGFR is required in developing Drosophila eyes to maintain the viability of undifferentiated cells surrounding the ommatidial preclusters (Baker and Yu, 2001). Removal of Ras in clones in the wing imaginal disc and the eye imaginal disc leads to an increase in apoptosis (Prober and Edgar, 2000; Halfar et al., 2001). Developmental apoptosis is promoted through the activity of three Drosophila genes: hid, grim and reaper [reviewed by Bangs and White (Bangs and White, 2000)]. Ras activity leads to the downregulation of Hid, both through transcriptional regulation and phosphorylation by MAPK (Bergmann et al., 1998; Kurada and White, 1998).
The role of CSW in preventing apoptosis is not as clearly defined. However, recent studies in mammalian systems have implicated the CSW homolog Shp2 in signal-mediated cell survival (Chauhan et al., 2000; Wu et al., 2000). As demonstrated here, CSW and MASK also play an important role in maintaining cell viability. Whether this anti-apoptotic signal is mediated by Ras and Hid or defines a new regulatory mechanism remains to be seen.
Recent studies have demonstrated that signaling through EGFR and Ras is important for proliferation in developing Drosophila imaginal discs (Prober and Edgar, 2000; Baker and Yu, 2001). Cells in clones lacking Ras in the wing imaginal disc do not grow or undergo division at the same rate as their wild-type neighbors (Prober and Edgar, 2000). An inability to transduce the Ras signal leads to a disadvantage among these competing cells. Similarly, studies of EGFR signaling in the developing eye imaginal disc have demonstrated that undifferentiated cells posterior to the morphogenetic furrow undergo cell division only if they receive a signal from neighboring developing photoreceptors (Baker and Yu, 2001). Such developmental regulation of cell division is essential for the proper patterning of the adult eye. Likewise, cells that lack MASK function seem to undergo mitosis at a lower rate than their neighbors, suggesting that the mask cells may be at a similar competitive disadvantage. This requirement for MASK activity in specifying cell fate, maintaining cell viability, and promoting cell proliferation is consistent with the known roles of RTK signaling during Drosophila eye development.
How does MASK transduce the RTK signal?
The phenotypic characterization of MASK presented here has not determined the mechanism by which MASK mediates signaling downstream of RTKs. MASK was isolated through a genetic screen designed to identify proteins that interact with CSW. While DOS, the first novel signaling component isolated through this screen, does interact directly with CSW, no evidence presented here suggests that MASK can as well (Herbst et al., 1996). mask alleles genetically interact with both dominant negative and activated alleles of ras and csw. However, unlike both Ras and CSW, MASK function does not seem to be required for normal levels of MAPK activation.
Several lines of evidence suggest that Shp2, the vertebrate homolog of CSW, can transduce signals through mechanisms that do not affect MAPK activation. First, dissociation of Shp2 from the scaffolding protein Gab2 (a vertebrate homolog of DOS) prevents signal-induced transcription without affecting MAPK activation (Gu et al., 1998). Second, Shp2 is required for Insulin-like Growth Factor stimulated FAK dephosphorylation and cellular chemotaxis, processes that are unperturbed when MAPK activation is eliminated (Manes et al., 1999). Third, expression of activated Shp2 in Xenopus animal caps induces elongation without significantly increasing MAPK activity (OReilly et al., 2000). Also, Shp2 negatively regulates gene expression induced by Leukemia Inhibitory Factor signaling in a MAPK-independent manner (Bartoe and Nathanson, 2000). In addition, Shp2 has been shown to act upstream of the small GTPase RhoA during filamentous actin remodeling in response to the activity of growth factor receptors (Schoenwaelder et al., 2000). Finally, mutations in Shp2 eliminate NF-B signaling and IL-6 induction in response to IL-1
and TNF-
signaling, without affecting MAPK activation (You et al., 2001). MASK may mediate a part of the CSW/Shp2 signal that is downstream of or parallel to MAPK. Alternatively, MASK may respond to RTK signaling through an entirely new mechanism.
MASK does not show overall homology to any protein of known function, although it does contain several well-characterized domains. The presence of the ankyrin repeats and KH domain, the predominantly cytoplasmic localization of the protein, and the largely unaffected MAPK activation allow for some speculation as to the function of this protein.
Signaling through RTKs such as EGFR and SEV affect the Ets-domain transcription factors Yan (also known as AOP), a transcriptional inhibitor, and PNT, a transcriptional activator (Brunner et al., 1994a; ONeill et al., 1994; Rebay and Rubin, 1995). Since MASK is not absolutely required for MAPK activity it may act downstream, perhaps as a transcriptional cofactor of PNT or a repressor of Yan. Such a role in transcription is also suggested by the fact that MASK contains a KH domain, many long stretches of glutamines, and a highly basic region that could be a bi-partite nuclear localization sequence. One example of such a transcriptional cofactor is found in mammalian cells. The GA Binding Protein (GABP
) is an Ets-domain transcription factor that is only active when bound to its partner GABPß, an ankyrin repeat containing transcriptional cofactor (LaMarco et al., 1991; Thompson et al., 1991; Batchelor et al., 1998). The antibody staining presented here shows that MASK is localized predominantly in the cytoplasm, and therefore a nuclear function for this protein is unlikely. However, MASK levels in the nucleus may be extremely low or transient and therefore difficult to detect. Alternatively, the protein may be cleaved, with a portion not recognized by the antibody entering the nucleus. MASK could also act downstream of MAPK by binding to Yan while localized in the cytoplasm, sequestering the transcriptional repressor.
Signaling may be transduced not only through transcriptional activation, but also through regulating the translation and mRNA stability of downstream effectors. The nuclear signaling molecule Split ends (SPEN) contains RNA binding motifs and may act to downregulate Yan translation (Chen and Rebay, 2000; Kuang et al., 2000; Rebay et al., 2000). Since MASK contains a potential RNA-binding KH domain, it may also act post-transcriptionally, mediating the effects of RTK signaling on mRNA in the cytoplasm.
Alternatively, the presence of two blocks of ankyrin repeats suggests a role for MASK as a scaffolding protein, bringing together many signaling molecules, perhaps at particular DNA sequences or on particular mRNAs. The exact role of MASK in transducing the RTK signal will be determined by further study. The presence of close homologs in other systems indicates that MASK is a conserved, essential signaling molecule. Understanding the role of this novel protein in Drosophila will add to our understanding of RTK signaling in general.
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ACKNOWLEDGMENTS |
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