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Identification of a Conserved Protein That Interacts with Specific LIM Homeodomain Transcription Factors

2000; Elsevier BV; Volume: 275; Issue: 18 Linguagem: Inglês

10.1074/jbc.275.18.13336

1083-351X

Paul W. Howard, Richard A. Maurer,

Cancer-related gene regulation

Lhx3, a member of the LIM homeodomain family of transcription factors, is required for development of the pituitary and is implicated in the transcription of pituitary-specific hormone genes. In this report we describe a novel gene product, SLB, that selectively interacts with Lhx3 and the closely related LIM factor, Lhx4. The SLB cDNA encodes a 1749-residue protein that contains seven WD40 repeats near the amino terminus and a putative nuclear localization signal and does not contain other recognizable motifs. SLB is expressed in a tissue-specific manner with the highest concentrations of SLB mRNA in the testis and pituitary cells. We demonstrate that SLB specifically binds to Lhx3 and Lhx4 with high affinity both in vitro and in vivo. SLB has much lower affinity or no detectable affinity for other LIM domains. An expression vector for a fragment of SLB containing the LIM-interaction domain was shown to reduce expression of Lhx3-responsive reporter genes. The ability of the LIM-interacting domain of SLB to alter reporter gene activity as well as the tissue-specific expression and the specificity of SLB binding to LIM factors suggest a possible role in modulating the transcriptional activity of specific LIM factors. Lhx3, a member of the LIM homeodomain family of transcription factors, is required for development of the pituitary and is implicated in the transcription of pituitary-specific hormone genes. In this report we describe a novel gene product, SLB, that selectively interacts with Lhx3 and the closely related LIM factor, Lhx4. The SLB cDNA encodes a 1749-residue protein that contains seven WD40 repeats near the amino terminus and a putative nuclear localization signal and does not contain other recognizable motifs. SLB is expressed in a tissue-specific manner with the highest concentrations of SLB mRNA in the testis and pituitary cells. We demonstrate that SLB specifically binds to Lhx3 and Lhx4 with high affinity both in vitro and in vivo. SLB has much lower affinity or no detectable affinity for other LIM domains. An expression vector for a fragment of SLB containing the LIM-interaction domain was shown to reduce expression of Lhx3-responsive reporter genes. The ability of the LIM-interacting domain of SLB to alter reporter gene activity as well as the tissue-specific expression and the specificity of SLB binding to LIM factors suggest a possible role in modulating the transcriptional activity of specific LIM factors. LIM homeodomain transcription factor selective LIM domain-binding protein glutathione S-transferase Dulbecco's modified Eagle's medium LIM homeodomain proteins comprise a family of transcription factors that are important regulators of development (1.Jurata L.W. Gill G.N. Curr. Top. Microbiol. Immunol. 1998; 228: 75-113PubMed Google Scholar, 2.Dawid I.B. Breen J.J. Toyama R. Trends Genet. 1998; 14: 156-162Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, 3.Dawid I.B. Trends Genet. 1998; 14: 480-482Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Lhx1 transcription factors contain a homeodomain DNA-binding motif and two LIM domains each consisting of two cysteine/histidine zinc fingers. It has recently become clear that specific Lhx proteins are important regulators of pituitary development and gene expression (4.Taira M. Hayes W.P. Otani H. Dawid I.B. Dev. Biol. 1993; 159: 245-256Crossref PubMed Scopus (73) Google Scholar, 5.Bach I. Rhodes S.J. Pearse R.V., II Heinzel T. Gloss B. Scully K.M. Sawchenko P.E. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2720-2724Crossref PubMed Scopus (289) Google Scholar, 6.Sheng H.Z. Zhadnov A.B. Mosinger Jr., B. Fujii T. Bertuzzi S. Grinberg A. Lee E.J. Huang S.-P. Mahon K.A. Westphal H. Science. 1996; 272: 1004-1007Crossref PubMed Scopus (395) Google Scholar, 7.Sheng H.Z. Moriyama K. Yamashita T. Li H. Potter S.S. Mahon K.A. Westphal H. Science. 1997; 278: 1809-1812Crossref PubMed Scopus (319) Google Scholar, 8.Sharma K. Sheng H.Z. Lettieri K. Li H. Karavanov A. Potter S. Westphal H. Pfaff S.L. Cell. 1998; 95: 817-828Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar, 9.Thor S. Andersson S.G.E. Tomlinson A. Thomas J.B. Nature. 1999; 397: 76-80Crossref PubMed Scopus (251) Google Scholar). Disruption of thelhx3 gene in mice and Drosophila melanogaster has demonstrated a role for Lhx3 in specification of motor neuron subtype identity and pathway selection (8.Sharma K. Sheng H.Z. Lettieri K. Li H. Karavanov A. Potter S. Westphal H. Pfaff S.L. Cell. 1998; 95: 817-828Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar, 9.Thor S. Andersson S.G.E. Tomlinson A. Thomas J.B. Nature. 1999; 397: 76-80Crossref PubMed Scopus (251) Google Scholar). Studies of mutant mice with disruptions of the lhx3 and lhx4 genes have further demonstrated the role of these LIM factors in organogenesis of the pituitary gland and in differentiation and proliferation of pituitary cell lineages (6.Sheng H.Z. Zhadnov A.B. Mosinger Jr., B. Fujii T. Bertuzzi S. Grinberg A. Lee E.J. Huang S.-P. Mahon K.A. Westphal H. Science. 1996; 272: 1004-1007Crossref PubMed Scopus (395) Google Scholar, 7.Sheng H.Z. Moriyama K. Yamashita T. Li H. Potter S.S. Mahon K.A. Westphal H. Science. 1997; 278: 1809-1812Crossref PubMed Scopus (319) Google Scholar). In addition to their roles in pituitary development, Lhx2 and Lhx3 also play a role in stimulating the expression of several pituitary-specific genes (5.Bach I. Rhodes S.J. Pearse R.V., II Heinzel T. Gloss B. Scully K.M. Sawchenko P.E. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2720-2724Crossref PubMed Scopus (289) Google Scholar, 10.Roberson M.S. Schoderbek W.E. Tremml G. Maurer R.A. Mol. Cell. Biol. 1994; 14: 2985-2993Crossref PubMed Google Scholar, 11.Girardin S.E. Benjannet S. Barale J.-C. Chrétien M. Seidah N.G. FEBS Lett. 1998; 431: 333-338Crossref PubMed Scopus (16) Google Scholar). For example, basal transcription and hormonally regulated expression of the glycoprotein hormone α subunit gene involves a binding site for Lhx2 and/or Lhx3 (5.Bach I. Rhodes S.J. Pearse R.V., II Heinzel T. Gloss B. Scully K.M. Sawchenko P.E. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2720-2724Crossref PubMed Scopus (289) Google Scholar, 10.Roberson M.S. Schoderbek W.E. Tremml G. Maurer R.A. Mol. Cell. Biol. 1994; 14: 2985-2993Crossref PubMed Google Scholar, 12.Schoderbek W.E. Roberson M.S. Maurer R.A. J. Biol. Chem. 1993; 268: 3903-3910Abstract Full Text PDF PubMed Google Scholar). The homeodomain of Lhx3 can also bind to DNA elements within the thyroid-stimulating hormone β subunit gene and the prolactin gene (5.Bach I. Rhodes S.J. Pearse R.V., II Heinzel T. Gloss B. Scully K.M. Sawchenko P.E. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2720-2724Crossref PubMed Scopus (289) Google Scholar). Lhx3 can also act synergistically with Pit-1 to activate reporter genes containing promoter sequences from these same genes (5.Bach I. Rhodes S.J. Pearse R.V., II Heinzel T. Gloss B. Scully K.M. Sawchenko P.E. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2720-2724Crossref PubMed Scopus (289) Google Scholar, 13.Meier B.C. Price J.R. Parker G.E. Bridwell J.L. Rhodes S.J. Mol. Cell. Endocrinol. 1999; 147: 65-74Crossref PubMed Scopus (32) Google Scholar). Transfection of an expression vector for Lhx3 into the AtT20 pituitary cell line can induce expression of the silent, endogenous prolactin gene in the absence of Pit-1 expression (11.Girardin S.E. Benjannet S. Barale J.-C. Chrétien M. Seidah N.G. FEBS Lett. 1998; 431: 333-338Crossref PubMed Scopus (16) Google Scholar). The LIM domain likely functions as a modular protein-protein interaction surface (1.Jurata L.W. Gill G.N. Curr. Top. Microbiol. Immunol. 1998; 228: 75-113PubMed Google Scholar, 2.Dawid I.B. Breen J.J. Toyama R. Trends Genet. 1998; 14: 156-162Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, 3.Dawid I.B. Trends Genet. 1998; 14: 480-482Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). For LIM homeodomain factors, the LIM domain may modulate the DNA binding affinity of the homeodomain (1.Jurata L.W. Gill G.N. Curr. Top. Microbiol. Immunol. 1998; 228: 75-113PubMed Google Scholar, 2.Dawid I.B. Breen J.J. Toyama R. Trends Genet. 1998; 14: 156-162Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, 3.Dawid I.B. Trends Genet. 1998; 14: 480-482Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). The LIM domain has also been shown to bind to a widely expressed nuclear adapter protein designated NLI, LBD, or CLIM (14.Agulnick A.D. Taira M. Breen J.J. Tanaka T. Dawid I.B. Westphal H. Nature. 1996; 384: 270-272Crossref PubMed Scopus (292) Google Scholar, 15.Jurata L.W. Kenny D.A. Gill G.N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11693-11698Crossref PubMed Scopus (211) Google Scholar, 16.Bach I. Carrière C. Ostendorff H.P. Andersen B. Rosenfeld M.G. Genes Dev. 1997; 11: 1370-1380Crossref PubMed Scopus (264) Google Scholar, 17.Jurata L. Gill G.N. Mol. Cell. Biol. 1997; 17: 5688-5698Crossref PubMed Scopus (159) Google Scholar). Genetic experiments have provided evidence that CHIP, the Drosophilahomolog of NLI, functionally cooperates with LIM factors to modulate transcription (18.Morcillo P. Rosen C. Baylies M.K. Dorsett D. Genes Dev. 1997; 11: 2720-2740Crossref Scopus (171) Google Scholar, 19.van Meyel D.J. O'Keefe D.D. Jurata L.W. Thor S. Gill G.N. Thomas J.B. Mol. Cell. 1999; 4: 259-266Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). We have cloned a novel LIM-interacting protein that contains a WD40 repeat. The remainder of the protein other than the WD40 domain has no substantial similarity to other known proteins. This 190-kDa protein is expressed in a tissue-specific manner with the highest expression in testis and pituitary. Unlike NLI, which binds to all nuclear LIM domain factors, this protein binds selectively to Lhx3 and Lhx4. GH3 cells were maintained in DMEM supplemented with 15% equine serum and 2.5% fetal bovine serum. Rat-1 cells were maintained in DMEM containing 10% calf serum. All other cells were maintained in DMEM supplemented with 10% fetal bovine serum. Reporter genes containing 0.6 kilobase pairs of 5′-flanking region of the rat prolactin gene fused to the firefly luciferase coding sequence (20.Iverson R.A. Day K.H. D'Emden M. Day R.N. Maurer R.A. Mol. Endocrinol. 1990; 4: 1564-1571Crossref PubMed Scopus (103) Google Scholar) and 5 copies of a GAL4-binding site upstream of the E1b TATA box linked to luciferase (21.Sun P. Enslen H. Myung P.S. Maurer R.A. Genes Dev. 1994; 8: 2527-2539Crossref PubMed Scopus (635) Google Scholar) have been described previously. Mammalian expression vectors for GAL4 and VP16 fusions have been described previously (22.Sun P. Maurer R.A. J. Biol. Chem. 1995; 270: 7041-7044Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The coding sequences for various LIM domains and NLI were amplified by the polymerase chain reaction using standard protocols. The products were all confirmed by automated DNA sequencing. Cells were transfected with a total of 1 μg of DNA and 5 μl of LipofectAMINE (Life Technologies, Inc.) in 35-mm well plates using a protocol provided by the supplier. The two-hybrid screen described by Hollenberg et al. (23.Hollenberg S.M. Sternglanz R. Cheng P.F. Weintraub H. Mol. Cell. Biol. 1995; 15: 3813-3822Crossref PubMed Scopus (580) Google Scholar) was used to identify cDNAs for factors that can interact with Lhx3. Briefly, the polymerase chain reaction was used to prepare an Lhx3 cDNA fragment coding for amino acids 25–136 which was subcloned into the vector pBTM116. The ade2 gene was also subcloned into pBTM116 to allow the host strain L40 to be cured of the bait vector. A library of GH3 cDNA fused to the VP16 transcriptional activation domain was constructed using the pVP16 vector as described (23.Hollenberg S.M. Sternglanz R. Cheng P.F. Weintraub H. Mol. Cell. Biol. 1995; 15: 3813-3822Crossref PubMed Scopus (580) Google Scholar). Yeast transformations, curing, and mating to the strain AMR70 were carried out as described (23.Hollenberg S.M. Sternglanz R. Cheng P.F. Weintraub H. Mol. Cell. Biol. 1995; 15: 3813-3822Crossref PubMed Scopus (580) Google Scholar) with the exception of the inclusion of 3-amino-1,2,4-triazole to increase stringency of selection. The SLB cDNA fragment isolated from the two-hybrid screen was used to screen a λ Zap II rat testis cDNA library (Stratagene) using standard protocols. The library contains cDNA prepared from the testis of 6-week-old Sprague-Dawley rats. A polymerase chain reaction approach was used to isolate cDNAs representing the 5′ and 3′ termini of SLB using commercial reagents and protocols provided by the supplier (Marathon cDNA Amplification Kit, CLONTECH). Poly(A)-containing cellular RNA was isolated by solubilizing GH3 or HeLa cells in guanidine HCl and sedimentation through cesium chloride as described (24.Glisin V. Crkvenjakov R. Byus C. Biochemistry. 1974; 13: 2633-2637Crossref PubMed Scopus (1543) Google Scholar) followed by chromatography of oligo(dT)-cellulose (25.Aviv H. Leder P. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 1408-1412Crossref PubMed Scopus (5173) Google Scholar). The poly(A)-containing RNA (2 μg) was electrophoresed through an agarose gel containing formaldehyde (26.Thomas P.S. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 5201-5205Crossref PubMed Scopus (5845) Google Scholar) and transferred by blotting to a nylon filter. A membrane containing size-fractionated poly(A) RNA from several rat tissues was purchased from CLONTECH. The blots were hybridized with a 32P-labeled SLBcDNA fragment of about 1000 base pairs using hybridization buffers purchased from CLONTECH and following hybridization and wash conditions provided by the supplier. Cell monolayers were washed once with ice-cold 0.15 m NaCl, 10 mm Hepes, pH 7.4, and then scraped from the dish in 5 ml of 10 mm Hepes, pH 7.4, 1 mm EDTA, 5 mm dithiothreitol, 1 mm benzamidine, and 0.2 mm phenylmethylsulfonyl fluoride (homogenization buffer). Cells were homogenized with 10 strokes of the tight pestle of a Dounce homogenizer, and the homogenate was centrifuged through a cushion of 0.5 m sucrose in homogenization buffer at 1200 ×g for 10 min at 4 °C. Nuclear pellets were resuspended in homogenization buffer containing 0.4 m NaCl. After 10–20 min on ice the mixture was centrifuged at 10,000 × gfor 10 min at 4 °C and the supernatant saved as the nuclear extract. For preparation of whole cell extracts, cells were scraped from the culture dishes in 100 mm sodium phosphate, pH 7.8. The cells were pelleted in a microcentrifuge and resuspended in the same buffer but with 1% Triton X-100 or, for co-immunoprecipitation experiments, 0.1% Nonidet P-40, and then the cells were disrupted by 4 cycles through dry ice/ethanol and 37 °C water baths. After centrifugation at 10,000 × g for 5 min at 4 °C, the supernatant was saved as the whole cell extract. The antiserum to SLB was produced by immunizing rabbits with a fusion protein containing glutathioneS-transferase linked to residues 1213–1265 of SLB. The GST-SLB-(1213–1265) fusion protein was produced in Escherichia coli and purified by affinity chromatography as described (27.Smith D.B. Johnson K.S. Gene (Amst.). 1988; 67: 31-40Crossref PubMed Scopus (5028) Google Scholar). For immunoprecipitation, cell extracts were adjusted to contain 0.1% Tween 20 or 0.1% Nonidet P-40. Aliquots containing equal amounts of total protein were combined with 15 μl of a 50% slurry of protein A-agarose (Santa Cruz Biotechnology) or, in some cases, anti-FLAG agarose (Eastman Kodak Co.). The immunoprecipitation mixtures were rotated for 2 h at 4 °C, and the protein A-agarose was collected by centrifugation. The protein A or anti-FLAG-agarose was then washed 3 times with 1 ml each of 10 mm Tris, pH 7.4, 150 mm NaCl, 0.1% Tween 20 or 0.5% Nonidet P-40. Proteins bound to the protein A or anti-FLAG agarose were then analyzed by electrophoresis on a denaturing, polyacrylamide gel. For Western blotting, proteins were transferred to polyvinylidene difluoride membranes (Millipore). For blocking reactions, incubation with a 1:5,000 dilution of antiserum to SLB, incubation with a 1:10,000 dilution of horseradish peroxidase-conjugated goat anti-rabbit antibody (Santa Cruz Biotechnology), and incubation with chemiluminescent reagent (Amersham Pharmacia Biotech) were all performed as suggested by the suppliers. For immunohistochemistry experiments, cells were cultured in 8-well glass slides. Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline and then incubated in phosphate-buffered saline containing 10% fetal bovine serum and 1 mg/ml bovine serum albumin (blocking solution). Primary antibodies were diluted to 1:50 in blocking solution. Secondary Cy3-conjugated antibody (Jackson ImmunoResearch) was diluted 1:200. Hoechst 33258 nuclear stain (Molecular Probes) was also included in the secondary antibody incubation. A GST-SLB-(1213–1265) or a GST-SLB-(1213–1749) fusion protein was used for protein binding assays. Radiolabeled proteins to be tested in this assay were prepared by coupled transcription and translation reactions in the presence of [35S]methionine using protocols provided by the supplier (TNT, Promega). Typical binding reactions contained 7 μl of in vitro translated protein, 15 μl of GST or GST-SLB-agarose, and Tris-buffered saline (10 mm Tris, pH 7.4, 150 mm NaCl) with 0.1% Tween 20 in a final volume of 100 μl. Reactions were rotated at 4 °C for 2 h and then washed 3 × 1 ml each with Tris-buffered saline with 0.1% Tween 20. The radiolabeled proteins bound to the GST- or GST-SLB-agarose were then analyzed by denaturing polyacrylamide gel electrophoresis. The gels were dried and exposed to x-ray film. Previous studies have shown that the LIM homeodomain transcription factor, Lhx3, can enhance prolactin promoter activity in heterologous cells (5.Bach I. Rhodes S.J. Pearse R.V., II Heinzel T. Gloss B. Scully K.M. Sawchenko P.E. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2720-2724Crossref PubMed Scopus (289) Google Scholar). To begin to explore further the ability of Lhx3 to support transcription of the prolactin gene, we transfected several constructs into GH3pituitary tumor cells that express the endogenous prolactin gene (Fig.1). For these studies we examined both basal and Ras-induced prolactin reporter gene activity (28.Conrad K.E. Oberwetter J.M. Vaillancourt R. Johnson G.L. Gutierrez-Hartmann A. Mol. Cell. Biol. 1994; 14: 1553-1565Crossref PubMed Scopus (71) Google Scholar, 29.Howard P.W. Maurer R.A. J. Biol. Chem. 1994; 268: 28662-28669Abstract Full Text PDF Google Scholar). Transfection of an Lhx3 expression vector modestly, but reproducibly, enhanced both basal and Ras-activated expression of the prolactin reporter gene. As Lhx3 expression has been shown to have much greater effects on the prolactin promoter in heterologous cells (5.Bach I. Rhodes S.J. Pearse R.V., II Heinzel T. Gloss B. Scully K.M. Sawchenko P.E. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2720-2724Crossref PubMed Scopus (289) Google Scholar), it seems likely that the modest effects of forced Lhx3 expression in GH3 cells indicate that endogenous levels of Lhx3 (5.Bach I. Rhodes S.J. Pearse R.V., II Heinzel T. Gloss B. Scully K.M. Sawchenko P.E. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2720-2724Crossref PubMed Scopus (289) Google Scholar) are near-optimal. A more informative expression vector contained the LIM domains of Lhx3 fused to the GAL4 DNA binding domain. The Lhx3 homeodomain and a large carboxyl-terminal domain have been deleted from this construct. As the prolactin promoter does not contain binding sites for GAL4, we anticipated that any effects of GAL4-Lhx3-LIM would be indirect presumably involving the sequestration of specific proteins by the LIM domain. The finding that the GAL4-Lhx3-LIM construct substantially reduced Ras-induced expression of the prolactin reporter gene offers evidence that the LIM domain may bind to specific factors necessary for Ras responsiveness of the prolactin promoter. Based on these observations we performed a yeast two-hybrid screen to search for proteins that interact with the LIM domains of Lhx3. The LIM domain of Lhx3 was subcloned into the pLex-A vector (23.Hollenberg S.M. Sternglanz R. Cheng P.F. Weintraub H. Mol. Cell. Biol. 1995; 15: 3813-3822Crossref PubMed Scopus (580) Google Scholar) to create an expression vector for a LexA-Lhx3-LIM fusion protein. This bait was used in a yeast two-hybrid screen to search for LIM-interacting factors. Approximately 10 million yeast transformants were screened from a library made from GH3 cDNA fused to the VP16 coding sequence. About 100 colonies survived and were tested for trans-activation of the β-galactosidase gene under control of a LexA operator. False positives were identified by curing the yeast of the LexA/Lhx3 bait and mating to a yeast strain carrying a LexA/Lamin bait. Clones that interacted with lamin were excluded. A sampling of the remaining clones were then tested in a mammalian two-hybrid assay (22.Sun P. Maurer R.A. J. Biol. Chem. 1995; 270: 7041-7044Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) by subcloning the VP16/cDNA fusions from the yeast library plasmid into a eucaryotic expression vector. These were transfected into GH3 cells with an expression vector coding for a GAL4 DNA binding domain fusion to the LIM domains of Lhx3 and a GAL4-responsive luciferase reporter. Only one clone demonstrated a strong interaction (data not shown). This clone was subsequently shown to bind to a limited number of LIM homeodomain transcription factors (see below). Therefore we designated this clone as SLB for selective LIM domain-binding protein. Initial analysis of total RNA from rat tissues suggested that the highest levels of SLB transcripts were found in the testis. A near full-length cDNA was obtained by screening a rat testis cDNA library with the DNA probe isolated from the two-hybrid screen. The 5′ and 3′ ends of the SLB coding sequence were obtained by polymerase chain reaction amplification of cDNA termini from rat testis cDNA. The rat SLB cDNA encodes a 1749-amino acid protein. Comparison of the predicted protein sequence of rat SLB to the GenBankTM data base reveals that theCaenorhabditis elegans genome contains a similar open reading frame which predicts a 1758-amino gene product. The predictedC. elegans protein is 37% identical and 57% similar to the rat protein over the entire length. The coding sequences are co-linear over the entire sequence with only a few gaps. The first 250 amino acids of SLB contain a seven WD40 repeats similar to those found in a number of proteins including G protein β subunits (30.Neer E.J. Schmidt C.J. Nambudripad R. Smith T.F. Nature. 1994; 371: 297-300Crossref PubMed Scopus (1280) Google Scholar). A putative bipartite nuclear localization signal is located near the amino terminus in the first WD40 repeat (31.Robbins J. Dilworth S.M. Laskey R.A. Dingwall C. Cell. 1991; 64: 615-623Abstract Full Text PDF PubMed Scopus (1238) Google Scholar). This putative bipartite nuclear localization signal consists of the sequenceRRDKFSTDPADMKYGRK (where boldface indicates consensus residues) that fits the consensus proposed by Robbins et al. (31.Robbins J. Dilworth S.M. Laskey R.A. Dingwall C. Cell. 1991; 64: 615-623Abstract Full Text PDF PubMed Scopus (1238) Google Scholar) of two basic residues, a spacer of 10 amino acids and a second basic cluster with 3 out of 5 amino acids being basic. The remaining 1500 amino acids of SLB have no significant similarity to any known gene product in the current GenBankTM data base. Hybridization analysis of poly(A)-containing RNA (Fig.2) demonstrates the greatest expression of SLB transcripts in testis with significant expression also detectable in pituitary and the GH3 pituitary, lactotroph, cell line. Although SLB mRNA is expressed at lower levels in the pituitary and GH3 cells than in testis (less testis RNA was loaded for the right panel of Fig. 2), the significant expression of SLB in these pituitary cells is consistent with a possible function in this tissue. The apparent size difference between testis and pituitary SLB transcripts was not observed in other experiments. A GST fusion to the SLB LIM-interaction domain was used to immunize rabbits for production of antiserum. The specificity of the resulting antiserum was tested by transfecting COS-7 cells with expression vectors for carboxyl-terminal fragments of SLB, either SLB-(1213–1540) or SLB-(1213–1749). Cell extracts from the transfected cells were then resolved by denaturing gel electrophoresis, and the SLB antiserum was used to detect immunologically related proteins (Fig.3 A). The antiserum strongly recognized bands of the appropriate size in cell extracts expressing the fragments of SLB. The antiserum was then used for immunoblot analysis of nuclear extracts from GH3 pituitary cells and from Rat-1 fibroblasts (Fig. 3 B). A band of approximately 190 kDa was observed only in GH3 nuclear extract. An additional two-hybrid screen of the VP16/GH3 cDNA library was performed with the SLB-(1213–1265) LIM-interacting fragment as bait. Approximately 1 million yeast transformants were screened, and six colonies survived. Five of the colonies contained identical cDNA fragments coding for the second LIM domain of Lhx3. We were unable to isolate the pVP16 plasmid from the sixth colony. This confirms the original yeast two-hybrid interaction and demonstrates that the second LIM domain of Lhx3 is sufficient to bind SLB. To determine if SLB can directly interact with Lhx3, the GST-SLB-(1213–1749) was used for binding studies. SLB-(1213–1749) contains the region that is sufficient for interacting with Lhx3 in the yeast two-hybrid assay plus additional carboxyl-terminal residues. Radiolabeled mouse Lhx2 and Lhx3 were incubated with immobilized GST or GST-SLB fusion proteins, and the bound proteins were analyzed by denaturing gel electrophoresis (Fig.4). Neither Lhx2 nor Lhx3 bound to GST, and only Lhx3 bound to the GST-SLB fusion protein. It appears that Lhx3 has a rather high affinity for this fragment of SLB as more than 50% of the input Lhx3 was bound to SLB as determined by PhosphorImager analysis. In control experiments, neither Pit-1 nor the estrogen receptor bound to the GST-SLB fusion protein (data not shown). The failure of SLB to interact with Lhx2 suggests that SLB has considerable selectivity for interacting with specific LIM homeodomain factors. This is particularly interesting as the LIM domain of Lhx2 is 47% identical to the LIM domain Lhx3, and both factors are expressed in the pituitary. This finding contrasts with the ability of the putative LIM coactivator, NLI, to bind to a wide variety of LIM factors (14.Agulnick A.D. Taira M. Breen J.J. Tanaka T. Dawid I.B. Westphal H. Nature. 1996; 384: 270-272Crossref PubMed Scopus (292) Google Scholar, 15.Jurata L.W. Kenny D.A. Gill G.N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11693-11698Crossref PubMed Scopus (211) Google Scholar, 16.Bach I. Carrière C. Ostendorff H.P. Andersen B. Rosenfeld M.G. Genes Dev. 1997; 11: 1370-1380Crossref PubMed Scopus (264) Google Scholar). Thus the in vitro binding data confirm the two-hybrid data and demonstrate a direct and selective interaction of SLB with Lhx3. We used the SLB antiserum to examine the interaction of Lhx3 and SLBin vivo. To date, we have not been able to detect the interaction of endogenous SLB and Lhx3 by co-immunoprecipitation assays. In part, this is probably due to relatively low expression of SLB in GH3 cells. It has also been somewhat difficult to test the interaction of SLB and Lhx3 in intact cells by forced expression in heterologous cells. Experiments using expression vectors for SLB tagged with various reporters have suggested that overexpression of SLB appears to be toxic to most cells. Fortunately, 293 cells appear to be somewhat resistant to the toxic effects of SLB expression. Also, the carboxyl-terminal fragment of SLB which contains the LIM-interacting domain (SLB-(1213–1749)) is not toxic in 293 or other cells. By using 293 cells it has been possible to demonstrate co-immunoprecipitation of FLAG-tagged Lhx3 with either full-length SLB or SLB-(1213–1749) (Fig. 5). No SLB was co-immunoprecipitated in cell extracts expressing SLB alone or with untagged Lhx3. These co-immunoprecipitation experiments provide evidence that SLB can bind to Lhx3 in vivo. To examine further in vivo interaction of SLB with Lhx3, the subcellular localization of these proteins was examined after transfecting COS-7 cells (Fig. 6). COS-7 cells do not contain detectable SLB mRNA, and no immunoreactive SLB was detected in untransfected cells. To assist in identifying the nuclear compartment, DNA was visualized with Hoechst stain (Fig. 6,right panels). Transfection of an expression vector for SLB-(1213–1749) resulted in the distribution of SLB immunoreactivity throughout the cell including both the cytoplasmic and nuclear compartments. A putative nuclear localization signal that is located near the amino terminus is deleted from SLB-(1213–1749). Thus the approximately 60-kDa SLB-(1213–1749) fragment probably passively distributes throughout the cytosol and the nucleus. When SLB-(1213–1749) was co-transfected with an Lhx3 expression vector, immunoreactivity was located predominantly in the nucleus, consistent with an interaction between the two proteins in vivo. An expression vector for Lhx2, which does not bind SLB, was used as a control. Lhx2 did not result in nuclear concentration of SLB-(1213–1749). These findings offer further evidence that SLB can interact with Lhx3 in cells. Indeed, the finding that expression of Lhx3 results in localization of the majority of SLB to the nucleus is consistent with a rather high affinity interaction. To explore the specificity of SLB binding to LIM homeodomain proteins a binding assay using immobilized GST-SLB-(1213–1749) was used (Fig.7). Expression vectors for the LIM-domain GAL4 fusion proteins were transfected into 293 cells. Extracts from these cells were then incubated with agarose-bound GST-SLB-(1213–1749) fusion protein, and the bound proteins were analyzed by denaturing gel electrophoresis. The various GAL4 fusion proteins were visualized by Western blotting with a monoclonal antibody to the GAL4 DNA binding domain. The results demonstrate that substantial amounts of Lhx3 and Lhx4 bound the immobilized SLB fragment. Lhx2 and Lmx-1 bound much less efficiently, and Isl-1 and Lin-11 did not demonstrate binding. The observation of weak binding of Lhx2 to SLB in this experiment appears to contrast to the absence of binding observed in Fig. 4. This is likely due to the presence of substantially greater concentrations of the LIM proteins in extracts from transfected 293 cells as compared with the amount of protein synthesized in the in vitrotranscription/translation reactions. The higher concentration of the LIM factors in 293 extracts would facilitate detection of the weaker binding of SLB to Lhx2. These data provide additional evidence that SLB interacts selectively with specific LIM domains. The finding that SLB can bind to Lhx3 and Lhx4 suggests a possible role in modulating transcription. The ability of the LIM-interacting domain of SLB (residues 1213–1749) to function as a possible dominant negative was tested in transfection experiments. The co-immunoprecipitation and nuclear co-localization experiments provided evidence that SLB-(1213–1749) can associate with Lhx3 in the nucleus. The ability of SLB-(1213–1749) to interfere with Ras-induced activation of a prolactin reporter gene in GH3 cells was tested (Fig.8). The SLB-(1213–1749) expression vector was found to partially block Ras-induced prolactin reporter gene activity. Although the effect was somewhat modest, it has been reproducible in several experiments. Importantly, the SLB-(1213–1749) vector did not appreciably alter the ability of a GAL4-Elk1 fusion protein to activate a GAL4-dependent reporter gene in a Ras-responsive manner (Fig. 8 B). Thus, the effects of SLB-(1213–1749) are specific to the Lhx3-responsive prolactin promoter, and SLB-(1213–1749) did not inhibit a presumably Lhx3-independent, Ras-responsive transcription unit. We also tested the ability of SLB-(1213–1749) to block the function of Lhx3 (Fig.9) in a heterologous cell line. As reported previously (13.Meier B.C. Price J.R. Parker G.E. Bridwell J.L. Rhodes S.J. Mol. Cell. Endocrinol. 1999; 147: 65-74Crossref PubMed Scopus (32) Google Scholar) Pit-1 and Lhx3 strongly synergize to activate the prolactin reporter gene in 293 cells. In this experiment the effects of SLB-(1213–1749) were compared with effects of NLI. Although NLI probably functions as a positive regulator of transcription (16.Bach I. Carrière C. Ostendorff H.P. Andersen B. Rosenfeld M.G. Genes Dev. 1997; 11: 1370-1380Crossref PubMed Scopus (264) Google Scholar,19.van Meyel D.J. O'Keefe D.D. Jurata L.W. Thor S. Gill G.N. Thomas J.B. Mol. Cell. 1999; 4: 259-266Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 32.Milan M. Cohen S.M. Mol. Cell. 1999; 4: 267-273Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), it has been somewhat difficult to demonstrate positive effects on transcription in transient transfection studies. For instance, it has been reported that NLI disrupts the synergy between the LIM homeodomain protein Lmx1 and the basic helix loop helix protein E47 (17.Jurata L. Gill G.N. Mol. Cell. Biol. 1997; 17: 5688-5698Crossref PubMed Scopus (159) Google Scholar). We found that both SLB-(1213–1749) and NLI substantially reduced the ability of Pit-1 + Lhx3 to stimulate prolactin reporter gene, and both factors also reduced reporter gene activity in the presence of Lhx3 alone. When examined with Pit-1 alone, neither SLB-(1213–1749) nor NLI reduced reporter activity. Thus in this heterologous system, both NLI and SLB-(1213–1749) appear to inhibit prolactin reporter activity in an Lhx3-dependent manner.Figure 9Expression of SLB -(1213–1749) in 293 cells strongly inhibits synergistic activation of a prolactin reporter gene by Pit-1 and Lhx3. Cultured 293 cells were transfected with a reporter gene containing the proximal region and promoter of the rat prolactin gene linked to luciferase and expression vectors for Pit-1, Lhx3, NLI, or SLB-(1213–1749) (SLB COOH) as indicated. The cells also received an expression vector for β-galactosidase driven by a cytomegalovirus promoter as an internal standard. The amount of expression vector was kept constant for all transfections by the inclusion of empty expression vector. Values were corrected for β-galactosidase activity and are the average ± S.E. of three independent transfections.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We have identified SLB as a novel gene product that interacts with the LIM domains of Lhx3 and Lhx4. A partial SLB cDNA was isolated in a two-hybrid screen for factors that interact with the LIM domain of Lhx3. The coding sequence of rat SLB is similar over its entire length to a C. elegans open reading frame of unknown function. The considerable similarity from C. elegans to mammals suggests the possibility of a conserved function. The only clearly identifiable domain in SLB is the presence of seven WD40 repeats in the first 250 amino acids (30.Neer E.J. Schmidt C.J. Nambudripad R. Smith T.F. Nature. 1994; 371: 297-300Crossref PubMed Scopus (1280) Google Scholar). The WD40 repeating unit is usually about 40 amino acids long and often ends with a tryptophan followed by an aspartate. This motif occurs in a wide variety of eucaryotic proteins but is not indicative of a specific function. It has been noted that most WD40 repeat domain-containing proteins are involved in some form of regulation and are not enzymes (30.Neer E.J. Schmidt C.J. Nambudripad R. Smith T.F. Nature. 1994; 371: 297-300Crossref PubMed Scopus (1280) Google Scholar). We have used several different assays to examine the interaction of SLB with LIM factors. SLB binds to Lhx3 and Lhx4 both in vitroand in vivo. Interestingly, SLB selectively interacts with Lhx3 and Lhx4 and either does not bind or binds with much lower affinity to Lhx2, Lmx1, Isl1, or Lin11. The LIM domains of Lhx3 are most similar to the LIM domains of Lhx4, being 79% identical. Lhx3 and Lhx4 have overlapping expression patterns and functional roles in the pituitary and specific neuronal populations (6.Sheng H.Z. Zhadnov A.B. Mosinger Jr., B. Fujii T. Bertuzzi S. Grinberg A. Lee E.J. Huang S.-P. Mahon K.A. Westphal H. Science. 1996; 272: 1004-1007Crossref PubMed Scopus (395) Google Scholar, 7.Sheng H.Z. Moriyama K. Yamashita T. Li H. Potter S.S. Mahon K.A. Westphal H. Science. 1997; 278: 1809-1812Crossref PubMed Scopus (319) Google Scholar, 8.Sharma K. Sheng H.Z. Lettieri K. Li H. Karavanov A. Potter S. Westphal H. Pfaff S.L. Cell. 1998; 95: 817-828Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar, 33.Li H. Witte D.P. Branford W.W. Aronow B.J. Weinstein M. Kaur S. Wert S. Singh G. Schreiner C.M. Whitsett J.A. Scott Jr., W.J. Potter S.S. EMBO J. 1994; 13: 2876-2885Crossref PubMed Scopus (107) Google Scholar). It is possible that selective binding to SLB plays a role in mediating some shared activity of these two LIM factors. The selectivity of SLB binding to specific LIM factors contrasts to the lack of selectivity in binding by the LIM cofactor, NLI (14.Agulnick A.D. Taira M. Breen J.J. Tanaka T. Dawid I.B. Westphal H. Nature. 1996; 384: 270-272Crossref PubMed Scopus (292) Google Scholar, 15.Jurata L.W. Kenny D.A. Gill G.N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11693-11698Crossref PubMed Scopus (211) Google Scholar, 16.Bach I. Carrière C. Ostendorff H.P. Andersen B. Rosenfeld M.G. Genes Dev. 1997; 11: 1370-1380Crossref PubMed Scopus (264) Google Scholar). Importantly, co-immunoprecipitation experiments offer evidence that SLB can interact with Lhx3 in cells. Analysis of subcellular localization provided additional evidence that SLB can associate with Lhx3 in cells. Indeed, the finding that co-transfection of Lhx3 could lead to the redistribution of an SLB fragment to the nuclear compartment suggests that under these conditions the majority of the SLB fragment is associated with Lhx3. Of course, forced expression in transient transfection experiments may not reflect physiological conditions. None the less, several different experimental approaches clearly provide evidence for relatively high affinity, selective interaction of SLB with Lhx3. Transfection experiments using expression vectors encoding the LIM-interacting domain of SLB have provided evidence that SLB may play a role in modulating the transcriptional activity of Lhx3. In heterologous 293 cells, there is a strong synergism between Lhx3 and the pituitary-specific transcription factor, Pit-1, for activation of the prolactin promoter. Expression of the LIM-interaction domain of SLB is a potent suppressor of synergistic activation by Lhx3 and Pit-1. In view of these transfection studies and the binding data, it seems clear that SLB can interact with Lhx3 in intact cells and affect transcriptional activation. At the present, we have been unable to assess the effects of full-length SLB on transcriptional activity due to toxic effects of overexpressing this factor. It seems likely that approaches other than transient transfections will be required to address this issue. Perhaps the most informative studies of the function of NLI, a structurally different LIM-interacting factor, have involved genetic experiments in Drosophila (18.Morcillo P. Rosen C. Baylies M.K. Dorsett D. Genes Dev. 1997; 11: 2720-2740Crossref Scopus (171) Google Scholar, 19.van Meyel D.J. O'Keefe D.D. Jurata L.W. Thor S. Gill G.N. Thomas J.B. Mol. Cell. 1999; 4: 259-266Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 32.Milan M. Cohen S.M. Mol. Cell. 1999; 4: 267-273Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Studies have been initiated to utilize genetic model systems to explore further the functional role of SLB. We thank Dr. Stan Hollenberg for reagents and advice concerning the two-hybrid screen. We also thank Dr. Tiffani Howard for assistance with immunocytochemistry, Shall Jue for technical assistance, and B. Maurer for aid in preparing the manuscript.