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Molecular and Cellular Biology, December 2004, p. 10397-10405, Vol. 24, No. 23
0270-7306/04/$08.00+0 DOI: 10.1128/MCB.24.23.10397-10405.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover,1 Norris Cotton Cancer Center, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire,3 Department of Molecular Genetics, Graduate Program, University of Cincinnati Medical Center, Cincinnati, Ohio2
Received 21 July 2004/ Returned for modification 3 September 2004/ Accepted 14 September 2004
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Fu) and its carboxyl-terminal domain (Fu-tail). We show that Fu and Fu-tail can interact in trans, with or without the kinesin-related protein Costal 2 (Cos2). However, since the majority of Fu is found associated with Cos2 in vivo, we hypothesized that Fu-tail, which binds Cos2 directly, would be able to tether Fu to Cos2. We demonstrate that Fu colocalizes with Cos2 in the presence of Fu-tail and that this colocalization occurs on a subset of membrane vesicles previously characterized to be important for Hh signal transduction. Additionally, expression of Fu-tail in fu mutant flies that normally express only the kinase domain rescues the fu wing phenotype. Therefore, reestablishing the association between these two domains of Fu in trans is sufficient to restore Hh signal transduction in vivo. In such a manner we validate our hypothesis, demonstrating that Fu self-associates and is functional in an Hh-dependent manner. Our results here enhance our understanding of one of the least characterized, yet critical, components of Hh signal transduction. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Fu is a critical component of the HSC, since mutations of fu result in deregulation of all forms of Ci processing, ultimately leading to a loss of Hh signaling (1, 16, 30, 45). fu flies, which reach adulthood through a maternal rescue (26, 34), exhibit a number of phenotypes, including a fusion of longitudinal veins 3 and 4 (LV3 and LV4) in the wing blade and a posterior expansion of the double-row wing margin bristles (7). The development of these two wing structures depends on at least two different Hh target genes. The LV3-LV4 intervein region requires decapentaplegic (dpp) expression, whereas the margin bristles in this same region are specified by late third-instar anterior engrailed (en) expression (4, 23, 38). These downstream target genes require two distinct transcriptional activator forms of Ci, Ciact and Ci*, respectively. Loss of Fu kinase activity results in the failure to convert Ci to either Ciact or Ci* (1, 16, 20, 30, 45).
Two major classes of fu alleles, class I and class II, were identified based on genetic interactions with Su(fu) (35, 43). In the presence of Su(fu), the phenotype of class I fu mutants is indistinguishable from that of class II mutants. However, while loss of Su(fu) rescues the fu phenotype of both classes of alleles, the resulting phenotype of the double-mutant animal differs depending on the class of the rescued fu allele. In a Su(fu) mutant background, class I fu flies appear to be phenotypically wild type, while class II fu mutant flies exhibit a neomorphic cos2-like phenotype. The two classes of mutations either affect the amino-terminal protein kinase domain, class I, or truncate the large carboxyl-terminal domain, class II. Based on these studies, it was proposed that the kinase domain of Fu functions, primarily, in direct opposition to Su(fu) function. Additionally, the combined loss of Su(fu) and the carboxyl-terminal domain of Fu (Fu-tail) appeared to overcome the ability of Cos2 to regulate Ci. The carboxyl-terminal domain of Fu has now been shown to associate directly with Cos2 (2, 22). This interaction is absolutely required for HSC function, since its disruption leads to a loss of Hh signaling (2). The carboxyl-terminal domain is the only domain of Fu necessary for processing Ci to Ci75, since loss of Fu kinase activity does not appear to affect production of Ci75 (1, 2, 16, 20). Hence, the carboxyl-terminal domain of Fu is required to tether the protein to the HSC and facilitates the production of Ci75 in a kinase-independent manner. The kinase domain of Fu is less well characterized. Although Cos2 and Su(fu) are phosphorylated in an Hh- and Fu-dependent manner, it has not been established that this phosphorylation is direct (18, 27, 44). Taken together, it is clear that a central role of Fu is to regulate Cos2 and Su(fu) function. Despite this, the mechanism that leads to Fu activation is not understood. Part of the difficulty in identifying the substrates of Fu stems from the inability to develop an in vitro kinase assay for it (8, 24, 27, 44) (unpublished data). Thus, to characterize the role that Fu plays in Hh signaling, we decided to explore possible regulatory mechanisms that might control Fu kinase activity.
A number of protein kinases are regulated through self-association, in either an inter- or an intramolecular fashion (40). We hypothesized that Fu may be regulated through such a mechanism. Here, we report that Fu can associate with itself through an intramolecular association between its kinase and carboxyl-terminal domains. We demonstrate that the two domains of Fu, when expressed separately, stably interact. Additionally, we show that they colocalize with Cos2 in Drosophila cells in a manner consistent with vesicular localization. To substantiate the functional significance of Fu self-association, we rescue the wing phenotype of fu mutant flies lacking the carboxyl-terminal domain by transgenically providing it. Thus, by expressing the exogenous Fu carboxyl-terminal domain in these flies, we restore patterning to regions of the wing which requires Hh-dependent Fu kinase activity.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Fu (aa 1 to 305) (see Fig. 1) baculovirus was described previously (2). Fu cDNA (aa 1 to 305) (Fig. 2 and 3) was also subcloned into the pIZT insect expression vector, where it is in frame with a 3' V5--six-His epitope tag. Generation of the six-His-tagged Fu-tail (aa 421 to 805 of Fu) (see Fig. 1 and 2) construct and baculovirus was described previously (2). Fu-tail was also subcloned into the pIZT expression vector containing a 5' Flag epitope tag and used in Fig. 3. A larger Fu-tail construct (aa 270 to 805) was used to generate the Fu-tail transgenic fly strain (Fig. 4) (2). Full-length Flag-tagged Cos2 (aa 1 to 1201) baculovirus (Fig. 2) (2) and pAct eGFP-Cos2N (Fig. 3) (41) have been described previously. Enhanced green fluorescent protein (eGFP)-Cos2N is a fusion construct containing amino acids 500 to 1201 of Cos2 fused to the cDNA of eGFP. Generation of glutathione S-transferase (GST) baculovirus was described previously (2).
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Cell culture and transfection.
Sf21 cells were cultured in Grace's insect medium (Invitrogen, Inc.). S2 cells were cultured in Schneider's Drosophila medium (Invitrogen, Inc.). Both cell lines were also supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Sf21 and S2 cells were transfected by using Cellfectin (Invitrogen, Inc.) according to the manufacturer's instructions. pIZT-Fu V5-HIS, pIZT-Flag-Fu-tail, and pAct-eGFP-Cos2N were used in various amounts depending on the experiment. Further details are available upon request. Where appropriate, empty pIZT or pAc 5.1A vectors (Invitrogen) were used to normalize the total amount of DNA.
Cellular lysates. Sf21 or S2 cells were lysed in a Nonidet P-40 (NP-40) buffer as previously described (2). The lysates were centrifuged at 6,000 x g for 20 min at 4°C. The resulting supernatants were either subjected to immunoprecipitation analysis or resolved directly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted for the appropriate proteins.
Batch purification of Fu. Twenty million Sf21 cells were infected with Flag-Fu baculovirus at a MOI of 10. The cells were lysed 44 h postinfection, using an NP-40 lysis buffer as previously described (2). The postnuclear supernatant was diluted 10-fold, using the NP-40 lysis buffer, and 0.5 ml of a 50:50 slurry of anti-Flag M2 resin was added. The M2 resin was rocked in the diluted supernatant for 16 h at 4°C. The Flag resin was then collected by low-speed centrifugation and washed three times using 15 ml of lysis buffer. The Flag resin was then incubated in 2 ml of elution buffer (NP-40 lysis buffer containing 0.65 M NaCl and 200 mM Flag peptide [Sigma]) overnight with rocking at 4°C. Purified Fu was dialyzed in NP-40 lysis buffer containing 10% glycerol.
Isolation of the trimolecular complex by Flag purification.
Fifty million Sf21 cells were infected with wt, Flag-Cos2, and His-Fu-tail baculovirus in various combinations at a MOI of 5 each for a total of 10 (see Results and Discussion). Cells were lysed 40 to 44 h postinfection in an NP-40 lysis buffer as before. Anti-Flag M2 resin (0.25 ml) was then applied to each lysate and incubated overnight as described above. The beads were then washed with a total of 25 ml of lysis buffer. Meanwhile, Sf21 cells transfected with pIZT-Fu V5-HIS were lysed in the same lysis buffer, aliquoted evenly to the three pools of washed beads, and then allowed to incubate overnight as before. The beads were then washed with 2.5 ml of lysis buffer and then 5 ml of lysis buffer supplemented with NaCl for a final concentration of 0.5 M. Flag complexes were then eluted in the 0.5 M NaCl lysis buffer with 0.2 mg of 3x Flag peptide (Sigma)/ml, collecting 10 0.25-ml fractions. The first three eluted fractions are shown in Fig 2; the first eluted fraction contained none of the three proteins of interest, whereas fractions following the third did not have Fu.
Antibodies. The following antibodies were used to immunoblot the appropriate proteins: affinity-purified polyclonal rabbit anti-Fu kinase domain (anti-FuKD) (0.1 µg/ml) (44), affinity-purified polyclonal rabbit anti-Fu hinge domain (anti-FuH) (0.044 µg/ml) (36), mouse monoclonal antibody (MAb) 5D6 directed against Cos2 (0.073 µg/ml) (2), mouse MAb M2 directed against the Flag epitope (anti-Flag) (0.2 µg/ml) (Sigma), mouse MAb Penta-His directed against the six-His epitope (0.2 µg/ml) (QIAGEN), mouse MAb directed against the V5 epitope (0.2 µg/ml) (Invitrogen), and mouse MAb directed against the GST epitope (anti-GST) (0.1 µg/ml) (Santa Cruz Biotechnology). Anti-FuH antibodies were used to immunoblot Fu proteins unless otherwise noted.
Immunoprecipitation of cytosolic lysates. Immunoprecipitations of baculovirus-infected Sf21 lysates and S2 cellular lysates were carried out essentially as described previously (2). The appropriate antibody was used for each immunoprecipitation reaction at 2.0 µg. Mouse immunoglobulin G1 (IgG1) (Jackson ImmunoResearch) was used as the isotype-matched control for the various MAbs used in this work. Rabbit serum IgG was used as an immunoprecipitation control when using either of the polyclonal rabbit anti-Fu antibodies.
Size exclusion chromatography. Gel filtration analyses were carried out by using a Superose 12 gel filtration column installed on an AKTA fast-performance liquid chromatography system (Amersham-Pharmacia). Affinity-purified Flag-Fu was fractionated over the column equilibrated in isotonic running buffer (150 mM NaCl, 40 mM HEPES [pH 7.6], 1 mM EDTA, 10% glycerol, 0.001% NP-40, 1 mM dithiothreitol, 1:1,000 protease inhibitor cocktail [2]). The following molecular mass standards (Amersham-Pharmacia) were used to calibrate the column: ferritin, 440 kDa; catalase, 232 kDa; aldolase, 158 kDa; albumin, 67 kDa; ovalbumin, 48 kDa; chymotrypsinogen A, 25 kDa; and RNase A, 13.7 kDa. A standard curve was constructed as described in the calibration kit manual.
Drosophila strains and generation of transgenic flies. Drosophila strains were maintained as previously described (9). Upstream activation sequence (UAS)-6xHis fu-tail transgenic flies were generated as previously described (2). The fuG3 (class II fu) (43) flies were kindly provided by P. Therond (Nice, France). The fuG3 mutant allele is missing aa 407 to 805 of Fu but adds 3 aa due to a frameshift mutation. ptc-GAL4 (12) was kindly provided by the Kornberg laboratory (University of California, San Francisco). ap-GAL4 (11) was a kind gift from X. Lin (Cincinnati, Ohio). Crosses were conducted at 18, 25, and 29°C; the crosses performed at 25°C provided the best results. The wings which corresponded to the genotype fuG3/Y; GAL4/UAS-fu-tail were examined for their vein and wing margin development. Adult wings were mounted using Euparal (Asco Laboratories) for observation. A more detailed description of the mating scheme is available upon request.
Immunofluorescence of Drosophila S2 cells. Indirect immunofluorescence analysis of S2 cells was performed essentially as described before (29). Polyclonal rabbit anti-FuH (2.2 µg/ml) and monoclonal mouse anti-V5 (Invitrogen) (2 µg/ml) primary antibodies were used. Goat anti-mouse Alexa-647 (4 µg/ml) and goat anti-rabbit Alexa-594 (4 µg/ml) (Molecular Probes) secondary antibodies were used. Green fluorescent protein signal was detected by the appropriate excitation and filter parameters. Confocal images were collected by using a Leica Confocal laser scanning microscope. Images were processed with Adobe Photoshop 7.0. Colocalization analyses were performed essentially as described previously except with use of Zeiss LSM colocalization analysis software (29).
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RESULTS AND DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Fu or Fu-tail. Fu contains only the kinase domain of Fu, whereas Fu-tail contains the carboxyl-terminal domain. We found that both Fu and Fu-tail coimmunoprecipitate with full-length Fu (Fig. 1B). These interactions were specific, since neither Fu nor Fu-tail is found in isotype matched IgG control immunoprecipitates. Additionally, none of the Fu proteins were coimmunoprecipitated, using anti-GST antibodies, from lysates where they were coexpressed with GST (Fig. 1B). Since both domains of Fu bind full-length Fu, we tested whether the two distinct domains of Fu could associate with each other. We prepared lysates from Sf21 cells expressing Fu or Fu-tail on their own, with each other, or with a control baculovirus expressing GST. Immunoprecipitations from these lysates were performed, using antibodies that specifically recognize Fu, Fu-tail, or GST (as a negative control). We found that Fu and Fu-tail associated with each other but not with GST (Fig. 1C). Our results clearly demonstrate that Fu can associate with itself. However, while our size exclusion analysis of purified Fu is consistent with Fu behaving like a dimer, our results do not rule out that Fu might be monomeric in vivo. We hypothesized two models of how Fu might exist in vivo, as a dimer or a monomer (Fig. 2A). In this first case, a dimerization interface would exist between the carboxyl-terminal and kinase domains of adjacent Fu proteins (head-to-tail dimer). The second case predicts that Fu is normally monomeric, with the kinase domain interacting with its own carboxyl-terminal domain (head-to-tail monomer). In this scenario, purifying overexpressed Fu or coexpressing it with truncated domains of Fu would force the normally monomeric protein to interact with a neighboring Fu molecule. To distinguish between these models, we decided to identify the form of Fu that associates with Cos2. In Drosophila cell lysates, Fu and Cos2 are predominantly found associated with each other (36). The Fu-Cos2 association is direct and absolutely necessary for proper Hh signaling. Therefore, the form of Fu bound to Cos2 would represent the physiologically relevant form of the protein kinase. We expressed full-length Fu and six-His-tagged Fu-tail, with increasing levels of Cos2, in Sf21 cells (Fig. 2B, left panel). In this experiment Fu-tail represents a monomeric form of Fu, since it cannot form a dimer with itself (data not shown), which is capable of binding directly to Cos2. Since Fu and Fu-tail associate with each other quantitatively (Fig. 1B), we reasoned that we could utilize their interaction to determine what form of Fu binds Cos2. If a dimer of Fu interacts with Cos2, then immunoprecipitating Fu-tail should coimmunoprecipitate full-length Fu equally well in the absence or presence of increasing amounts of Cos2. That is, the Fu-tail-Fu association should not be affected by Cos2. If a Fu monomer, in this case Fu-tail, normally associates with Cos2, then Cos2 would bind to Fu-tail at the expense of the Fu-tail-Fu association. We find that the association between Fu-tail and Fu is significantly reduced in the presence of increasing levels of Cos2 (Fig. 2B, right panel), with a corresponding increase in the Fu-tail-Cos2 association. Therefore, our results indicate that a Fu monomer preferentially binds to Cos2.
We have previously shown that truncated Fu proteins which lack the carboxyl-terminal domain do not significantly interact with Cos2 (2, 36). Since Fu-tail can bind to both Fu and Cos2, we tested whether the two truncated domains of Fu could associate with Cos2 as a trimolecular complex. However, when we coexpressed all three proteins in Sf21 or Drosophila S2 cells, we did not observe a significant interaction by coimmunoprecipitation assays. This may be due to a change in the affinity between Fu-tail and Fu once Fu-tail binds Cos2, destabilizing the trimolecular complex under the conditions used. To overcome this difficulty, we subjected lysates containing either Flag-Cos2 or Flag-Cos2 and Fu-tail to affinity purification with anti-Flag M2 chromatography resin. We also took lysate from Sf21 cells infected with wt baculovirus and mock purified it as a negative control. In this manner we immobilized Flag-Cos2, with or without associated Fu-tail, in separate affinity columns to act as bait. We then made a lysate from Sf21 cells expressing Fu and distributed it equally between the Flag-Cos2, Flag-Cos2-Fu-tail, and mock affinity columns. After successive washes, we selectively eluted the Flag complexes using Flag peptide and immunoblotted the resulting fractions (Fig. 2C). We found that Fu enriched specifically on the Flag-Cos2-Fu-tail column, compared to results with the Flag-Cos2 or control affinity columns. Taken together, our results are consistent with a model where the kinase and carboxyl-terminal domain of Fu bind each other in an intramolecular head-to-tail manner in the presence of Cos2.
Since Fu and Fu-tail associate in a heterologous expression system, we next wanted to determine if these proteins could interact in a Drosophila cell line and do so in subcellular locations important for Hh signaling. An important aspect of the Hh pathway is the subcellular localization of the various signaling components and how these locations affect Hh activation (14, 17, 18, 28, 29, 36, 37, 41, 42). We found that when Fu was expressed alone in S2 cells, it localized within the cytoplasm of cells in a diffuse manner (Fig. 3A). When Fu was coexpressed with Fu-tail, both proteins colocalized with each other and have a punctate appearance, consistent with localizing to various membrane vesicles (Fig. 3E to G). Previously, we found that Cos2 colocalized with the vesicle marker, Rab11, giving a similar punctate staining pattern (41). Vesicular localization of Cos2 and Fu was found to be biologically relevant, since this pool of the HSC is responsive to Hh activation. We hypothesized that Fu-tail-dependent redistribution of Fu might be representative of the kinase domain regaining the ability to interact with Cos2 and hence the HSC. To test this hypothesis, we expressed eGFP-Cos2N (41), Fu-tail, and Fu singly or in combination and prepared S2 cells for immunofluorescence analysis. eGFP-Cos2N localizes in cells in a punctate manner similar to that of full-length Cos2 (41), and Fu binds Cos2 within this region (22). When all three proteins are coexpressed, they colocalize in regions of the cytoplasm consistent with vesicular association (Fig. 3H to K, compare to panels B to D). Our results here indicate that we can recapitulate a trimolecular complex between the two domains of Fu and Cos2 in a biologically relevant cell line. More importantly, we show that this trimolecular complex localizes in a manner similar to that of the endogenous HSC.
To address if Fu self-association is important to its function in the Hh pathway, we transgenically expressed Fu-tail in a class II fu mutant fly. fuG3 is a particularly strong class II fu allele that is missing the last 398 amino acids of Fu but still encodes an intact kinase domain (43). fuG3 mutants exhibit a severe fusion of LV3 and LV4 across the entire wing blade and an abnormal extension of double-row bristles from LV3 to LV4 (Fig. 4B; compare to panel A). We expressed Fu-tail by using the GAL4/UAS system (5), under the control of either the ptc-GAL4 driver (Fig. 4C) or the ap-GAL4 driver (data not shown). We observed a rescue of the fu wing vein fusion phenotype in 15% of the flies expressing Fu-tail in the fuG3 background, under the control of the ptc-GAL4 driver. The other 85% of the flies of the same genotype exhibited a phenotype indistinguishable from that of the fuG3 mutant flies. Using the ap-GAL4 promoter, only 1% of the fu wing phenotype was rescued. The wings of the rescued transgenic flies, under the control of either GAL4 driver, show only minor fusions near the cross vein between LV3 and LV4 or small ectopic veins in the distal intervein region. However, the transgenic flies still developed a double-row bristle extension (Fig. 4C'; compare to panels A' and B'). Thus, by providing Fu-tail in trans, we were able to restore the majority of Fu function to a fu mutant fly expressing an endogenous Fu-like molecule.
Our in vivo results indicate that expression of Fu-tail in the fuG3 mutant background allows the proper development of the LV3-LV4 intervein space, thus rescuing one of the regions disrupted by the loss of fu function. Although the fuG3 allele encodes an intact kinase domain, it is not sufficient for Fu function. Since the addition of Fu-tail rescues the LV3-LV4 intervein region, we propose that recapitulating the interaction between Fu-tail and the kinase domain in vivo allows the conversion of Ci to Ciact. We therefore predict that this interaction must restore some functional kinase activity, since kinase-inactive Fu mutants cannot convert Ci to Ciact. Although the association between the two domains of Fu is able to produce Ciact, it does not appear to do so in a constitutive manner. When Fu-tail is expressed using the ap-GAL4 driver, it is present throughout the ventral-anterior compartment of the wing disk (2; also data not shown). This would afford FuG3 and Fu-tail the opportunity to interact in a large field of cells that receive the full spectrum of Hh activation. If their interaction is the only event necessary to promote Ci to Ciact, then Ciact would be ectopically produced in far-anterior sections of the wing disk. This would lead to constitutive activation of the Hh pathway, a prediction inconsistent with the rescued phenotype of the transgenic flies. While we only observed a small number of flies rescued using our ap-GAL4, none of the nonrescued flies exhibited any anterior ectopic wing growth. Thus, when Fu-tail and FuG3 interact, they appear capable of correctly regulating Ciact production and do so in a manner that depends on Hh signaling. However, the rescue of functional Fu kinase activity does not appear sufficient to produce Ci*, since the wing margin bristles still develop abnormally. Our inability to rescue margin bristle development may be due to incomplete recapitulation of all Fu kinase activities. Alternately, high levels of overexpressed Fu-tail may prevent Ci* formation by competing with the recapitulated Fu monomer for binding of Cos2. Overexpression of Fu-tail in wt flies causes marked fusion of LV3 and LV4, indicating that it has the capacity to shut down the Hh pathway (2). Thus, Fu-tail appears capable of disrupting or promoting Hh signaling under different genetic contexts. Therefore, a fine balance might exist between providing enough Fu-tail to allow the kinase domain reentry into the HSC and too much Fu-tail, which would bind Cos2 and the kinase domain independently of each other.
We propose a model in which Fu, associated with Cos2, exists in equilibrium between kinase-active and kinase-inactive states, with the activity states being dependent on the interaction between its catalytic and carboxyl-terminal domains. We suggest that in the inactive state, the carboxyl-terminal domain associates stably with the kinase domain, thereby preventing substrate access (Fig. 5). In the absence of Hh, Fu bound to Cos2 is held in an inactive, but poised, state. Hh signaling would promote dissociation of the Fu carboxyl-terminal domain from its kinase domain, thereby activating the protein kinase. In a wild-type setting, dissociation of the kinase domain from the carboxyl-terminal domain would not completely remove it from the HSC, allowing the kinase domain access to all its various substrates. While we present a model where Fu is a monomeric self-associating molecule, we cannot rule out the possibility that Fu might also exist as a dimer in vivo. Given that Fu associates with Cos2 preferentially as a monomer, a Fu dimer would likely represent a minor inactive fraction. Purified Fu migrates at a size consistent with it being a dimer, at least by size exclusion chromatography. In agreement with this possibility, size fractionation of endogenous HSC complexes from Drosophila extracts reveal a minor peak (peak C), absent of all HSC components except Fu, that migrates with a molecular size consistent with it being dimeric (36).
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ACKNOWLEDGMENTS |
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This work was supported by National Institutes of Health grant CA82628 (to D.J.R.), NCI training grant 5T32 ES07250 (to M.A.), and the Albert J. Ryan Foundation (to M.A.).
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FOOTNOTES |
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