Characterization and Functional Analysis of the Murine Frat2 Geneстатья из журнала
Аннотация: The Frat1 proto-oncogene was first identified as a gene contributing to tumor progression in T-cell lymphomas induced by retroviral insertional mutagenesis with the Moloney murine leukemia virus. The biological function of Frat remained elusive until its Xenopus homologue GBP was isolated as a glycogen synthase kinase 3 (GSK3)-binding protein and was shown to be an essential component of the maternal Wnt-signaling pathway. To date two Frat homologues have been described in the mouse, Frat1 and Frat3. The proteins encoded by these two genes are 84% identical. Here we describe the cloning and characterization of a third murine Frat homologue, Frat2, which is the mouse ortholog of human FRAT2. Frat1 and Frat2 are juxtaposed on chromosome 19 in a chromosomal organization conserved between man and mouse. We show that Frat1 and Frat2 are phosphorylated, which is the first evidence that these proteins are subject to posttranslational modification. Like Frat1, Frat2 is able to bind to GSK3β. However, a side-by-side comparison of the murine Frat proteins for their capacity to induce signaling through β-catenin/T-cell factor reveals that Frat2 is a less potent activator of the canonical Wnt pathway. Frat2 protein accumulates to higher levels upon transfection into 293T cells than either Frat1 or Frat3. Thus, whereas Frat1 may be a core component of canonical Wnt-signaling, Frat2 might very well be part of a divergent intracellular GSK3β pathway. The Frat1 proto-oncogene was first identified as a gene contributing to tumor progression in T-cell lymphomas induced by retroviral insertional mutagenesis with the Moloney murine leukemia virus. The biological function of Frat remained elusive until its Xenopus homologue GBP was isolated as a glycogen synthase kinase 3 (GSK3)-binding protein and was shown to be an essential component of the maternal Wnt-signaling pathway. To date two Frat homologues have been described in the mouse, Frat1 and Frat3. The proteins encoded by these two genes are 84% identical. Here we describe the cloning and characterization of a third murine Frat homologue, Frat2, which is the mouse ortholog of human FRAT2. Frat1 and Frat2 are juxtaposed on chromosome 19 in a chromosomal organization conserved between man and mouse. We show that Frat1 and Frat2 are phosphorylated, which is the first evidence that these proteins are subject to posttranslational modification. Like Frat1, Frat2 is able to bind to GSK3β. However, a side-by-side comparison of the murine Frat proteins for their capacity to induce signaling through β-catenin/T-cell factor reveals that Frat2 is a less potent activator of the canonical Wnt pathway. Frat2 protein accumulates to higher levels upon transfection into 293T cells than either Frat1 or Frat3. Thus, whereas Frat1 may be a core component of canonical Wnt-signaling, Frat2 might very well be part of a divergent intracellular GSK3β pathway. The Frat1 proto-oncogene was originally isolated in a retroviral insertional mutagenesis screen aimed at the identification of genes that were involved in later stages of T-cell lymphomagenesis (1Jonkers J. Korswagen H.C. Acton D. Breuer M. Berns A. EMBO J. 1997; 16: 441-450Crossref PubMed Scopus (115) Google Scholar). After infection of newborn Eμ-Pim1 or H2K-Myc transgenic mice with the Moloney murine leukemia virus, the resulting primary tumors were grafted to syngeneic hosts. When the proviral integration patterns of primary and transplanted tumors were compared up to 30% of the transplanted tumors showed outgrowth of a population of cells with a similar common integration site. Cloning of the gene affected by integration of the provirus into this genomic locus resulted in the identification of Frat1. Although Frat1 was shown to convey a strong selective advantage to lymphoma cells in vivo (1Jonkers J. Korswagen H.C. Acton D. Breuer M. Berns A. EMBO J. 1997; 16: 441-450Crossref PubMed Scopus (115) Google Scholar), its biological function remained unknown until its Xenopus homologue GBP was identified as a GSK3 1The abbreviations used are: GSK3β, glycogen synthase kinase 3β; GBP, GSK3-binding protein; CHX, cycloheximide; Frat, frequently rearranged in advanced T-cell lymphomas; GST, glutathione S-transferase; RT, reverse transcriptase; UTR, untranslated region; TCF, T-cell factor; contig, group of overlapping clones.1The abbreviations used are: GSK3β, glycogen synthase kinase 3β; GBP, GSK3-binding protein; CHX, cycloheximide; Frat, frequently rearranged in advanced T-cell lymphomas; GST, glutathione S-transferase; RT, reverse transcriptase; UTR, untranslated region; TCF, T-cell factor; contig, group of overlapping clones.-binding protein (2Yost C. Farr III, G.H. Pierce S.B. Ferkey D.M. Chen M.M. Kimelman D. Cell. 1998; 93: 1031-1041Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar).GBP was shown to be part of the maternal Wnt pathway in Xenopus, since depletion of the endogenous pool of GBP in the oocyte prevented the formation of a normal body axis in developing embryos. Like GBP, Frat is able to induce secondary axis formation upon ectopic expression in developing Xenopus embryos by stabilizing β-catenin levels (2Yost C. Farr III, G.H. Pierce S.B. Ferkey D.M. Chen M.M. Kimelman D. Cell. 1998; 93: 1031-1041Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 3Jonkers J. van Amerongen R. van der Valk M. Robanus-Maandag E. Molenaar M. Destree O. Berns A. Mech. Dev. 1999; 88: 183-194Crossref PubMed Scopus (34) Google Scholar). Frat/GBP competes with axin for the same binding site on GSK3β (4Thomas G.M. Frame S. Goedert M. Nathke I. Polakis P. Cohen P. FEBS Lett. 1999; 458: 247-251Crossref PubMed Scopus (202) Google Scholar, 5Bax B. Carter P.S. Lewis C. Guy A.R. Bridges A. Tanner R. Pettman G. Mannix C. Culbert A.A. Brown M.J. Smith D.G. Reith A.D. Structure (Camb). 2001; 9: 1143-1152Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 6Fraser E. Young N. Dajani R. Franca-Koh J. Ryves J. Williams R.S. Yeo M. Webster M.T. Richardson C. Smalley M.J. Pearl L.H. Harwood A. Dale T.C. J. Biol. Chem. 2002; 277: 2176-2185Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 7Dajani R. Fraser E. Roe S.M. Yeo M. Good V.M. Thompson V. Dale T.C. Pearl L.H. EMBO J. 2003; 22: 494-501Crossref PubMed Scopus (254) Google Scholar). It is generally presumed that Frat functions to titrate GSK3β away from the scaffolding complex containing APC and axin, thus preventing the phosphorylation and subsequent degradation of β-catenin (8Hino S. Michiue T. Asashima M. Kikuchi A. J. Biol. Chem. 2003; 278: 14066-14073Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). As a result, Frat/GBP is a potent activator of the canonical Wnt pathway.To learn more about the function of Frat in mammalian development, we have generated Frat1 knockout mice in which most of the Frat1-coding sequence has been replaced by a lacZ marker gene (3Jonkers J. van Amerongen R. van der Valk M. Robanus-Maandag E. Molenaar M. Destree O. Berns A. Mech. Dev. 1999; 88: 183-194Crossref PubMed Scopus (34) Google Scholar). Despite the fact that Frat1 is expressed in a broad range of neural and epithelial tissues during embryonic development as well as in adult animals, Frat1-deficient animals showed no gross abnormalities. At that time we described the existence of Frat3. Because it shares 84% amino acid identity with Frat1, it was hypothesized to exhibit compensatory activity in its absence. Frat3, however, appears to be present as an imprinted gene only in mice and rats due to a relatively recent transposition event (9Chai J.H. Locke D.P. Ohta T. Greally J.M. Nicholls R.D. Mamm. Genome. 2001; 12: 813-821Crossref PubMed Scopus (52) Google Scholar, 10Kobayashi S. Kohda T. Ichikawa H. Ogura A. Ohki M. Kaneko-Ishino T. Ishino F. Biochem. Biophys. Res. Commun. 2002; 290: 403-408Crossref PubMed Scopus (26) Google Scholar). The human genome instead harbors FRAT2, which is less conserved to FRAT1 (2Yost C. Farr III, G.H. Pierce S.B. Ferkey D.M. Chen M.M. Kimelman D. Cell. 1998; 93: 1031-1041Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 11Saitoh T. Moriwaki J. Koike J. Takagi A. Miwa T. Shiokawa K. Katoh M. Biochem. Biophys. Res. Commun. 2001; 281: 815-820Crossref PubMed Scopus (46) Google Scholar, 12Freemantle S.J. Portland H.B. Ewings K. Dmitrovsky F. DiPetrillo K. Spinella M.J. Dmitrovsky E. Gene (Amst.). 2002; 291: 17-27Crossref PubMed Scopus (26) Google Scholar).Here we report the cloning and characterization of the murine Frat2 gene. It bears close resemblance to its human counterpart and is less homologous to Frat1 than Frat3. We have performed an extensive side-by-side analysis of all three murine Frat genes with regard to their ability to induce canonical Wnt signaling in mammalian cells and show that Frat2 is less able to do so in comparison to Frat1 despite the fact that Frat2 protein accumulates to much higher levels. In addition, we show that Frat1 and Frat2 are phosphorylated, which is the first evidence for posttranslational modification of this family of proteins.MATERIALS AND METHODSCloning of Frat2—A Frat2 cDNA clone was identified in the NCBI EST data base (IMAGE clone 318660) and used to design primers. Using Frat2-ForA (5′-CGGTAGATCCCAGGTCCTC-3′) and Frat2-RevA (5′-AGAGACCGGGAACCTTGC-3′) Frat2 was then PCR-amplified from murine kidney cDNA. The Frat2 sequence has been deposited in the NCBI data base under GenBank™ accession number AY518895.RT-PCR—Total RNA from mouse organs or whole embryos was isolated with Trizol (Invitrogen) according to the manufacturer's instructions. After treatment with RQ1 RNase-free DNase (Promega), 1 μg of RNA was used for cDNA synthesis using avian myeloblastosis virus reverse transcriptase (Roche Applied Science). First-strand cDNA was used as input for PCR using gene-specific primers Frat1-For (5′-GTGGTGGACTCGAGCTTATTTG-3′) and Frat1-Rev (5′-AGGAGAGTCTGCGTGGAATTGC-3′), Frat2-For (5′-GAATCGGGAGGGCTTCTAAC-3′) and Frat2-Rev (5′-CAAGTTGGCAAGGTCGAATC-3′), Frat3-For (5′-GAGGCAGCAGTGAGCAGTGCTG-3′) and Frat3-Rev (5′-GCAGCTGTTCCCCGCAGAGCCG-3′), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-For (5′-ACCGGATTTGGCCGTATT-3′) and GAPDH-Rev (5′-TCTGGGATGGAAATTGTGAG-3′) in a PCR reaction of 30 cycles (30 min at 94 °C, 30 min at 65 °Cfor Frat or 60 °C for GAPDH, 50 min at 72 °C). As a negative control, an aliquot from a cDNA synthesis reaction without reverse transcriptase enzyme was subjected to the same PCR protocol for each sample (no RT control) to exclude possible DNA contamination.Generation of Expression Constructs—The complete Frat1-, Frat2-, and Frat3-coding sequences were cloned in-frame with an N-terminal Myc tag into pGlomyc3.1, which contains 5′ and 3′ globin UTRs and the pCDNA1.1 polylinker in the backbone of pCDNA3.1. All cloning procedures were carried out according to standard techniques. Hybrid fusions between Frat1 and Frat2 were cloned by swopping a BssHII/-XbaI fragment, an EcoNI/XbaI fragment, or an Eco47III/XbaI fragment. To introduce an Eco47III site by silent mutation into Frat1, a HindII-I/EcoRI fragment was replaced with an HindIII/EcoRI fragment containing this mutation, which had been generated using the forward 5′-GCAAAGCTTCCCGCACACCCGTTCCTCGGGCCTCTGAGCGCTCCAG-3′ and reverse 5′-GGTGTTCTTGAGGCTGG-3′ primers. A Frat1 C-terminal deletion mutant was cloned by deleting coding sequences downstream of the NruI site. The integrity of all constructs was verified by restriction enzyme digestion and sequence analysis on an ABI sequencer.Cell Culture and Transfection—COS7 or 293T cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen) under 5% CO2 at 37 °C in humidifying conditions. On the day before transfection cells were plated in 12-well tissue culture plates. Cells were transfected with a total amount of 500 ng of DNA per well using FuGENE (Roche Applied Science).Protein Assays—Cells were harvested 48 h after transfection by lysis in radioimmune precipitation assay buffer supplemented with protease inhibitors (Roche Applied Science). Protein concentration was determined using a colorimetric assay (Bio-Rad), and equal amounts of protein were run on a 12% SDS-PAGE gel and analyzed on Western blot using ECL (Pierce). Antibodies were used recognizing the Myc tag (9E10, 1:5000, Invitrogen), GSK3β (1:2500, Transduction Laboratories), or β-galactosidase (1:2500, Chapel). Secondary antibodies were goat-anti-mouse-horseradish peroxidase (HRP) (1:5000, BIOSOURCE) or swine-anti-rabbit-HRP (1:2000, Dako). For immunoprecipitation, COS7 lysates were incubated at 4 °C with a polyclonal antibody directed against GSK3β (Santa Cruz). Immunocomplexes were pulled down by incubation with protein G-Sepharose, after which samples were washed in radioimmune precipitation assay buffer to remove unbound protein, resuspended in radioimmune precipitation assay buffer, boiled, and analyzed on gel. For phosphatase treatment, 293T lysates were incubated with the indicated amounts of λ protein phosphatase (New England Biolabs) at 30 °C for 90 min, boiled, and analyzed on gel. For cycloheximide (CHX) experiments, 293T cells were treated for the indicated amounts of time with 100 μg/ml cycloheximide (ready made, Sigma) before lysis in radioimmune precipitation assay buffer. Protein levels were evaluated by densitometry following Western blotting.Luciferase Assay—293T cells were transfected as described above with TOPFLASH, pGlomyc-Frat1, Frat2 or Frat3, human TCF4, and β-galactosidase (Clontech). Cells were harvested 48 h after transfection in reporter lysis buffer (Promega) and analyzed with luciferase assay reagent (Promega) according to the manufacturer's instructions in a TopCounter (Packard Instrument Co.). To control for transfection efficiency β-galactosidase activity was determined using ortho-nitrophenyl-β-d-galactopyranoside (Sigma). The same lysates were analyzed on Western blot after the addition of sample buffer and electrophoresis on a 12% SDS-PAGE gel.Immunofluorescence—Cells were plated on glass chamber slides and fixed in 4% paraformaldehyde at 48 h after transfection. Cells were permeabilized with 0.2% Triton X-100 and incubated with a primary antibody recognizing the Myc tag (1:200, Invitrogen) and a fluorescein isothiocyanate-conjugated secondary antibody (1:500, Molecular Probes).RESULTSCloning and Characterization of the Frat2 Gene—In search of a putative mouse homologue of the human FRAT2 gene, we identified an EST clone in the NCBI data base with a high degree of homology to FRAT2. Using this clone to design primers, we were able to PCR-amplify Frat2 from murine kidney. Comparison of the PCR products from cDNA or genomic clones revealed no difference in size, indicating that the Frat2 gene does not contains introns, similar to murine Frat1 and Frat3 and human FRAT1 and FRAT2. The Frat2 gene (GenBank™ accession number AY518895) contains an open reading frame of 696 nucleotides encoding a polypeptide of 231 amino acids (Fig. 1A). The transcript encoded by Frat2 contains a long 3′-UTR with four destabilizing motifs ATTT(A) upstream of the polyadenylation signal (AATAAA). Sequence alignment of the three murine Frat proteins reveals that Frat2 is the most distant family member of the three with 68% amino acid identity to Frat1. By comparison, Frat3 shares 84% identity with Frat1 at the protein level (Fig. 1B). The human and mouse Frat2 homologues are 76% identical at the amino acid level (Fig. 1C), which is comparable with the degree of conservation between mouse Frat1 and human FRAT1. Despite its reduced similarity to the other Frat homologues, Frat2 does contain the conserved domains that are also present in Xenopus and zebra fish GBP. The C-terminal domain contains the conserved IKEA box, which forms the binding site for GSK3β in the Frat1 protein and Xenopus GBP (2Yost C. Farr III, G.H. Pierce S.B. Ferkey D.M. Chen M.M. Kimelman D. Cell. 1998; 93: 1031-1041Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar).A detailed Ensembl data base search revealed two Frat-related sequences that mapped to the same sequence contig. Analysis of the genomic organization at this locus revealed that Frat1 and Frat2 lie only 18 kilobases apart on mouse chromosome 19 in opposite transcriptional orientation (Fig. 1D). This genomic organization has recently been confirmed for human FRAT1 and FRAT2, which lie 16 kilobases apart in the syntenic region of human chromosome 10 (13Saitoh T. Katoh M. Int. J. Oncol. 2001; 19: 311-315PubMed Google Scholar).To monitor the expression of Frat1, Frat2, and Frat3, we set up a gene-specific RT-PCR (Fig. 1E). Primers for each of the three homologues were designed in the 3′-UTR of the genes. No RT control reactions were included for each sample to exclude DNA contamination (results not shown). All three genes are expressed in embryonic stage E12.5-E16.5. Frat1 and Frat2 also show an overlapping expression pattern in tissues from adult FVB mice. In contrast, Frat3 is expressed at much lower levels as well as in a reduced number of tissues, as has been reported previously (10Kobayashi S. Kohda T. Ichikawa H. Ogura A. Ohki M. Kaneko-Ishino T. Ishino F. Biochem. Biophys. Res. Commun. 2002; 290: 403-408Crossref PubMed Scopus (26) Google Scholar).Frat2 Binds to GSK3β—It has previously been shown that GSK3β can be co-immunoprecipitated with Frat1 using a polyclonal antibody raised against mouse Frat1 (15Li L. Yuan H. Weaver C.D. Mao J. Farr III, G.H. Sussman D.J. Jonkers J. Kimelman D. Wu D. EMBO J. 1999; 18: 4233-4240Crossref PubMed Scopus (356) Google Scholar). Because the IKEA box, which is required for binding to GSK3β, is conserved between all Frat family members across species, we expected Frat2 to bind GSK3β as well. To test this, we cloned all three murine Frat genes into a mammalian expression vector inframe with an upstream Myc tag. After transient transfection of these constructs into COS7 cells, all three proteins can be readily detected by Western blot analysis. Myc-tagged Frat2 has an apparent molecular mass of around 35 kDa on an SDS-PAGE gel compared with 40 kDa for Myc-tagged Frat1 and Frat3 (Fig. 2A). To test for binding of Frat2 to GSK3β, we sought to co-immunoprecipitate Frat2 with GSK3β. As shown in Fig. 2A, Frat2 can indeed be co-precipitated with GSK3β from the same COS7 lysates.Fig. 2Characterization of Frat2 protein.A, Frat2 binds to GSK3β. Top, detection of transiently overexpressed Myc-Frat1, Myc-Frat2, and Myc-Frat3 in COS7 cells. Bottom, Myc-tagged Frat proteins can be co-immunoprecipitated (IP) with endogenous GSK3β from the same COS7 lysates. WB, Western blot. B, Frat1 and Frat2 are phosphorylated. Myc-Frat2 is observed as a doublet on denaturing gels. After treatment with λ protein phosphatase, migration of both Frat1 and Frat2 is affected. C, phosphorylation by GSK3β is not responsible for the observed mobility shift. After treatment with LiCl, GSK3β becomes phosphorylated on Ser-9, which renders it inactive. However, Frat2 was still observed as a doublet on SDS/PAGE gels.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Frat2 Is Phosphorylated—Upon closer analysis Frat2 and Frat3 were found to migrate as a doublet on SDS-PAGE gels. Doublets often represent fractions of the same protein with a differential phosphorylation status. When protein lysates from 293T cells transfected with Frat2 were treated with λ protein phosphatase, the upper band disappeared, indicating that this band represents phosphorylated Frat2 (Fig. 2B). Because the lower band appeared to be affected by the phosphatase treatment as well, the originally observed doublet may have consisted of hypo- and hyperphosphorylated Frat2. Although Frat1 was not seen as a doublet on SDS-PAGE gels, we did observe a change in mobility after treatment with λ protein phosphatase (Fig. 2B). Thus, both Frat1 and Frat2 are phosphoproteins. Because Frat1 and Frat2 contain three and five sites, respectively, that match the reported consensus motif for GSK3 (X(S/T)XXXS, in which the C-terminal serine must be prephosphorylated (16Rylatt D.B. Aitken A. Bilham T. Condon G.D. Embi N. Cohen P. Eur. J. Biochem. 1980; 107: 529-537Crossref PubMed Scopus (175) Google Scholar, 17Doble B.W. Woodgett J.R. J. Cell Sci. 2003; 116: 1175-1186Crossref PubMed Scopus (1741) Google Scholar, 18Dajani R. Fraser E. Roe S.M. Young N. Good V. Dale T.C. Pearl L.H. Cell. 2001; 105: 721-732Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar, 19Frame S. Cohen P. Biondi R.M. Mol. Cell. 2001; 7: 1321-1327Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar, 20ter Haar E. Coll J.T. Austen D.A. Hsiao H.M. Swenson L. Jain J. Nat. Struct. Biol. 2001; 8: 593-596Crossref PubMed Scopus (319) Google Scholar)), we tested whether this was the kinase responsible for the observed phosphorylation of Frat2. After transfection of 293T cells with Myc-Frat2, the cultures were treated with LiCl (21Klein P.S. Melton D.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 455-459Google Scholar), a known inhibitor of GSK3 (Fig. 2C). The addition of LiCl resulted in phosphorylation of GSK3 on Ser-9, which is indicative of inhibition of its kinase activity (22Stambolic V. Woodgett J.R. Biochem. J. 1994; 303: 701-704Crossref PubMed Scopus (502) Google Scholar), but we did not observe a change in the migration of Myc-Frat2 in cultures treated with LiCl compared with controls that were treated with NaCl. This indicates that, although GSK3 might still phosphorylate Frat, it does not result in the hyperphosphorylated form observed on denaturing gels.Frat2 Activates the Canonical Wnt Pathway—Given the high degree of homology between the three murine Frat proteins, we compared the ability of all three to induce Wnt signaling through β-catenin/TCF in a TOPFLASH reporter assay, which uses luciferase activity as a readout for activation of consensus TCF binding sites cloned upstream of the reporter gene (23Molenaar M. van de Wetering M. Oosterwegel M. Peterson-Maduro J. Godsave S. Korinek V. Roose J. Destree O. Clevers H. Cell. 1996; 86: 391-399Abstract Full Text Full Text PDF PubMed Scopus (1599) Google Scholar). Frat1 has previously been shown to function as an activator of canonical Wnt signaling using this assay (15Li L. Yuan H. Weaver C.D. Mao J. Farr III, G.H. Sussman D.J. Jonkers J. Kimelman D. Wu D. EMBO J. 1999; 18: 4233-4240Crossref PubMed Scopus (356) Google Scholar). As shown in Fig. 3A, we found that Frat2 and Frat3 are also able to activate the TOPFLASH reporter in 293T cells, albeit to a lesser extent than Frat1 (p < 0.005 for Frat2). However, when we compared the Frat protein levels present in these 293T lysates, we found that Myc-Frat1 and Myc-Frat3 were present at much lower levels compared with Myc-Frat2 (Fig. 3A) even though β-galactosidase, which was co-transfected to control for transfection efficiency, was expressed at similar levels (not shown). By transfecting increasing amounts of Frat we studied the concentration-dependent response of the TOPFLASH reporter. Comparable amounts of Frat1 and Frat2 protein induced different levels of reporter activity (Fig. 3B). Thus, Frat2 is a less potent activator of Wnt signaling through β-catenin/TCF compared with Frat1 and Frat3 even if present at much higher protein levels. Because Frat3 is only present in mice and rats and since its behavior in the TOPFLASH assay was intermediate to that of Frat1 and Frat2, we decided to focus on the latter two homologues.Fig. 3Frat2 is less efficient in inducing canonical Wnt-signaling than Frat1.A, Frat2 is able to activate a TOP-FLASH reporter in 293T cells. All luciferase reporter assays were performed in triplicate and performed at least three times. A representative experiment is shown. Frat2 induces reporter activity less well than Frat1 (*, p < 0.005 as determined by Student's t test). When the same lysates were analyzed on Western blot (WB) for expression of the proteins Frat2 was found to accumulate to higher levels than Frat1 or Frat3 even though it was less active in inducing Wnt signaling. B, the transfection of increasing concentrations of Frat (10, 25, 50, 75, and 100 ng of DNA for each homologue) shows the concentration-dependent response of the TOPFLASH reporter. Again, the absolute protein levels of Frat2 are higher than those of Frat1 and Frat3. Similar levels of Frat protein are obtained upon the transfection of 100 ng of Frat1 and 25 ng of Frat2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Frat2 Is More Stable than Frat1—The observed difference in protein levels could be accounted for by a difference in protein half-life. To test this, we treated 293T cells expressing Myc-Frat1 or Myc-Frat2 with CHX to block protein synthesis and monitored the decay patterns by Western blot analysis. We consistently observed that substantial amounts of Frat2 remained present after prolonged CHX treatment. In contrast, Frat1 could hardly be detected at the same time point (Fig. 4A). When the change in protein levels was followed over time, both Frat1 and Frat2 levels gradually decreased in the first 2 h after CHX treatment (Fig. 4B). After 6 h of CHX treatment the decrease in Frat1 levels was more pronounced than the decrease in Frat2 levels. The observed pattern of protein degradation was independent of the starting levels of protein. Also when the initial levels of Frat1 were higher than those of Frat2 (Fig. 4B, bottom) the levels of Frat2 diminished much less than those of Frat1. There was no obvious difference in the stability of hyper- and hypophosphorylated Frat2 (Fig. 4, A and B). To determine the half-life of Frat1 and Frat2 more accurately, we quantified the amounts of protein by Western blot analysis and densitometry. Fig. 4C depicts the average of 3 or 4 experiments for each time point. After a 6- or 7-h treatment with CHX, Frat1 levels had decreased by more than 90%. In contrast, we observed that the levels of Frat2 protein had only dropped by 50%. Therefore, we conclude that Frat2 is considerably more stable than Frat1 due to a longer protein half-life.Fig. 4Frat2 is more stable than Frat1.A, transiently transfected 293T cells were treated with CHX for 7 h, resulting in a substantial decrease in Frat1 but not Frat2 protein levels. To start out with similar levels, 4–5 times more Frat1 than Frat2 DNA was transfected. WB, Western blot. B, Western blot analysis of protein stability reveals that the difference in protein levels at later time points is irrespective of the initial net levels. Two representative experiments are shown. C, quantitation of protein levels after CHX treatment of transiently transfected 293T cells by Western blot densitometry does not show a significant difference in protein half-life at early time points. After 6–7 h of CHX treatment, Frat1 levels dropped to less than 10%, whereas Frat2 levels remain at 50%. The graph shows the average of 3 or 4 experiments for each time point. D, immunofluorescence of transfected 293T cells reveals a predominantly cytoplasmic localization for Frat1 and Frat2. An overall decrease in fluorescence signal is observed after 6 h of CHX treatment, albeit more pronounced for Frat1 than for Frat2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Given its interaction with GSK3β and the increasing evidence that different pools of this kinase exist within the cell (24Franca-Koh J. Yeo M. Fraser E. Young N. Dale T.C. J. Biol. Chem. 2002; 277: 43844-43848Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) we speculated that Frat2 might be more stable as a result of its presence in an intracellular pool, where it could be shielded from destruction. After transfection of 293T cells we studied the intracellular localization of Myc-Frat1 and Myc-Frat2 by immunofluorescence (Fig. 4D). Both Frat1 and Frat2 were predominantly located in the cytoplasm when cells were left untreated. After CHX treatment for 6 h before fixation, the overall fluorescence intensity decreased. In fact, the results confirmed the earlier Western blot analysis, with a more substantial decline in Frat1 signal compared with Frat2. However, we did not observe a pool of Frat2 with a specific subcellular localization that was more resistant to destruction.The C Terminus of Frat1 Conveys Protein Instability—Given the apparent discrepancy between Frat2 protein stability on the one hand and its poor ability to induce canonical Wnt signaling on the other hand, we searched for specific regions in Frat1 and Frat2 that might determine either of these characteristics. The amino acid sequence of Frat2 is most divergent from Frat1 in the central part of the protein, where Frat2 lacks a stretch of amino acids present in Frat1 and in the C-terminal part downstream of the IKEA box (Fig. 5A). We generated fusion proteins (shown schematically in Fig. 5A) by cloning Frat1 and Frat2 sequences in-frame at a BssHII restriction site (A), at the EcoNI site in the IKEA box (B), or at an Eco47III site, which was first introduced into Frat1 by site-directed silent mutagenesis (C). All constructs were tested in the TOP-FLASH assay to ascertain that they still represented functional proteins. Although all fusion proteins retained activity, none of them mimicked the behavio
Год издания: 2004
Издательство: Elsevier BV
Источник: Journal of Biological Chemistry
Ключевые слова: Wnt/β-catenin signaling in development and cancer, Cancer-related gene regulation, Kruppel-like factors research
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Том: 279
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Страницы: 26967–26974