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The LIM domains of hic-5 protein recognize specific DNA fragments in a zinc-dependent manner in vitro
Introduction
Materials And Methods
Construction of recombinant proteins
DNA cellulose binding of the hic-5 protein
Isolation of DNA sequences bound to hic-5 protein
Protein blot assay to detect DNA binding
Results
Affinity of hic-5 protein for DNA
Isolation of hic-5 protein binding sequences from mouse genomic DNA
Specificity of the interaction between hic-5 protein and the binding fragments
Determination of the DNA binding domain in hic-5 protein
Discussion
Acknowledgements
References
The LIM domains of hic-5 protein recognize specific DNA fragments in a zinc-dependent manner in vitro
DDBJ/EMBL/GenBank accession nos AF056072-AF056078
ABSTRACT
INTRODUCTION
The hic-5 gene was originally isolated from mouse osteoblastic cells as one of the TGF[beta]1-inducible genes, encoding a polypeptide with a molecular weight of ~55 kDa (1) whose prominent feature is the presence of four LIM domains in its C-terminal half. The LIM domain is a unique cysteine-rich motif that defines a double zinc finger structure with a consensus sequence CXXCX16-23HXXCXXCXXCX16-21CXX(D/H/C) and which is found in a variety of proteins with diverse functions and subcellular distributions, including transcription factors, components of adhesion plaques and actin-based cytoskeletal components (2). The members of the LIM proteins can be classified into several groups; a LIM homeodomain family, LIM only protein, LIM kinase, a GTPase activating protein (GAP) family and the zyxin family, which includes enigma and paxillin. Spectroscopic analysis demonstrated that the LIM domain defines a specific zinc binding structure and that zinc coordination is required for proper folding of the LIM domain (3). In spite of this structural information, it is controversial as to whether the LIM domain is involved in protein-protein or protein-nucleic acid interactions. Accumulating evidence has demonstrated that the LIM domains serve as an interface for protein-protein interactions, although the interacting molecules identified so far are so diverse that it is impossible to deduce the determinant for specificity or selectivity of the interactions. For example, the LIM domain of cysteine-rich protein (CRP) interacts with that of zyxin (4), but the LIM domain of LIM homeodomain protein (Lhx/Xlim-1) interacts with a LIM domain binding factor (Ldb1) that contains no LIM motif (5). The protein enigma interacts with the insulin receptor and Ret/ptc2 (6) and its homolog, named ENH, binds to certain members of the protein kinase C family (7).
On the other hand, a certain similarity in structure has been pointed out between the LIM consensus and DNA binding-type zinc fingers, such as the GATA transcription factor family and steroid hormone receptor superfamily (8). From this similarity, together with the above mentioned diversity of protein recognition by the LIM domain, it is likely that the LIM domain also functions as a protein-nucleic acid interface.
hic-5 protein belongs to the zyxin family and has striking similarity with paxillin in its LIM domains (9). Paxillin is a phosphoprotein which interacts with tyrosine kinases of the src family as well as with focal adhesion kinase and vinculin at focal adhesions (10). Brown et al. showed that LIM 3 of paxillin is essential for localization in focal adhesions (11), but the function of the LIM domains in hic-5 protein has not yet been determined. Interestingly, zyxin, which is another member of the family and is a low abundance phosphoprotein that accumulates with integrins at focal adhesions, has recently been shown to have a functional nuclear export signal (NES) and shuttles between the nucleus and cytoplasmic focal adhesions (12). The existence of almost the same NES amino acid sequence as zyxin in hic-5 protein and the observation that treatment of cells with leptomycin B, an inhibitor of nuclear export, induced nuclear accumulation of hic-5 protein (in preparation) tempted us to examine the DNA binding ability of the LIM domains in hic-5 protein. In the present communication, we also attempted to isolate DNA sequences that specifically bound to hic-5 protein. Our results suggest that the LIM domains of this protein bind to DNA in a zinc- and sequence-dependent manner.
MATERIALS AND METHODS
The nucleotide sequences reported in this paper have been submitted to the GenBank with accession nos: AF056072 for clone 98; AF056073, clone 10; AF056074, clone 101; AF056075, clone 19; AF056076, clone 29; AF056077, clone 78; AF056078, clone 97.
Construction of recombinant proteins
Three types of prokaryotic expression vectors for hic-5 were constructed using the pET-16b vector (Novagen, Madison, WI). As a nearly full-length hic-5 expression vector containing nt 288-1596 of the hic-5 cDNA, the previously described pET-L5 plasmid was used (1). For construction of expression vectors of C-terminal truncated (pET-N) or N-terminal truncated (pET-C) hic-5 protein, a NspI-HinfI fragment (nt 190-779) of hic-5 cDNA or a fragment of nt 756-1553 flanked by an EcoRI adaptor were obtained. The fragments were blunted and ligated with BamHI linker for in-frame insertion into the expression vector. After BamHI digestion, BamHI linker linked cDNA fragments were subcloned into the BamHI site of the pET-16b vector. For construction of deletion mutants, desired hic-5 fragments were prepared using restriction enzymes or PCR. These fragments were cloned into the BamHI site of the pGEX-5X-1 vector. These mutant fragments contain the following nucleotide sequences of hic-5 cDNA; LIM 1-4 (nt 827-1598), LIM 1-3 (nt 827-1333), LIM 1-2 (nt 827-1160), LIM 1-2 X [nt 827-1164 followed by 124 bp, which were derived from the cloning vector, pVZ-1 (13), and translated into 42 amino acids, X, unrelated to hic-5 protein], LIM 1 (nt 822-992), LIM 2-4 (nt 971-1725), LIM 3-4 (nt 1160-1725), LIM 4 (nt 1335-1523).
To engineer the expression vector of the LIM region of human paxillin, a cDNA fragment encoding the LIM domains (nt 1042-1748) was obtained by PCR and subcloned into the EcoRI site of the pGEX-5X-1 vector.
The BL21 strain of Escherichia coli (14) was transformed with expression vectors for the respective proteins. Logarithmically growing cultures were induced to produce the protein by addition of 1 mM isopropyl-[beta]-d-thiogalactopyranoside (IPTG) for 5 h.
DNA cellulose binding of the hic-5 protein
Escherichia coli cells harboring the prokaryotic expression vector of hic-5 and that had been induced to produce hic-5 protein in the presence of IPTG were suspended in lysis buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM DTT) containing 0.1 mg/ml lysozyme and 0.1% Triton X-100 and then lysed with three bursts of sonication (140 W, 10 s). The insoluble fraction was collected by centrifugation and washed with lysis buffer containing 2 M urea to partially purify hic-5 protein. This fraction contained most of the hic-5 protein expressed with a small amount of contamination (confirmed by silver staining after SDS-PAGE). After solubilizing in lysis buffer containing 4 M urea, the proteins were renatured by successive dialysis against lysis buffer containing 2, 1 and 0 M urea with 1 mM ZnCl2 or 1 mM EDTA for several hours at 4°C and then used in the binding assay as described (15). In brief, 0.4 mg protein were incubated in binding buffer (50 mM Tris-HCl, pH 7.5, 12 mM [alpha]-thioglycerol, 10% glycerol, 0.1 M NaCl) with 0.1 g double-stranded (native) or single-stranded (denatured) DNA-cellulose (Pharmacia LKB Biotechnology) for 24 h at 4°C. The DNA-cellulose had been preincubated with 3% BSA in binding buffer. After washing with binding buffer containing 0.003% NP-40 and 2% Triton X-100, the material bound to DNA-cellulose was eluted with 1.25% SDS and resolved by SDS-PAGE. The hic-5 protein was then detected by western blotting using an antibody ([alpha]C86) as described previously (1).
Isolation of DNA sequences bound to hic-5 protein
Genomic DNA from mouse osteoblastic cells (MC3T3) was digested with MboI or HaeIII. The digested DNA fragments (average size 200-300 bp) were mixed and ligated to the UNI-Amp Adapter (Clontech, Palo Alto, CA). In 125 µl binding buffer (50 mM Tris-HCl, pH 7.5, 12 mM [alpha]-thioglycerol, 10% glycerol, 0.1 M NaCl, 0.1 mM ZnCl2) containing 5 µg poly(dI-dC), 1 µg adapter-linked DNA fragments were incubated with 6 µg bacterially produced and partially purified hic-5 protein at room temperature for 1 h. DNA fragments bound to the hic-5 protein were mixed with 1.25 ml immunoprecipitation buffer (0.1 M HEPES, pH 7.5, 0.3 M KCl, 10 mM MgCl2, 20 mM ZnCl2, 2% Triton X-100, 0.1% SDS) containing anti hic-5 antibody (1011) and then incubated on ice for 1 h. The DNA-hic-5-antibody complex was precipitated by adding 25 µg protein A-Sepharose in binding buffer and incubating for 1 h at 4°C. Bound DNA fragments were separated from free DNA by centrifugation. After removal of the supernatant, the immunoprecipitate was washed four times with immunoprecipitation buffer. The DNA fragments bound to hic-5 protein were incubated in dissociation buffer (0.5 M Tris-HCl, pH 9.0, 20 mM EDTA, 10 mM NaCl, 0.2% SDS) at 50°C for 1 h, extracted with phenol-chloroform and precipitated with ethanol. Recovered DNA fragments were then amplified by PCR using UNI-Amp primers (Clontech). The bound and amplified DNA fragments were used as the substrate for additional rounds of binding to hic-5 protein. After four rounds of binding/elution, selected DNA fragments were cloned into the pCR II vector (Invitrogen, San Diego, CA). Individual clones were screened by immunoprecipitation-PCR (IP-PCR) as described above, except that the cloned plasmids were used for substrate DNA. Amplified fragments were analyzed by electrophoresis on 1% agarose gels.
Protein blot assay to detect DNA binding
This assay was performed essentially as described elsewhere (16). In brief, pellets of E.coli BL21 harboring recombinant hic-5 or paxillin cDNA as described above were lysed in lysis buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM DTT) containing 0.1% Triton X-100 and 0.1 mg/ml lysozyme. Proteins were separated by SDS-PAGE and blotted to nitrocellulose filters. Radiolabeled DNA probe was added to the preincubated filter (105 c.p.m./filter) and the filters were incubated for 5 h at 4°C. After the binding reaction, the filters were washed with reaction buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 10% glycerol, 0.1 mM ZnCl2) containing sonicated E.coli genomic DNA or yeast tRNA for 3 h five times, each for 15 min, air dried and autoradiographed.
For DNA probes, the hic-5 binding fragments were cloned into pCR II vector as described above. Cloned fragments were digested with HindIII and XhoI, resolved electrophoretically and purified from a 1% agarose gel. These fragments were labeled with Klenow fragment and [[alpha]-32P]dCTP. The labeled probe was purified by phenol-chloroform extraction and ethanol precipitation in the presence of ammonium acetate.
RESULTS
Affinity of hic-5 protein for DNA
We first investigated whether hic-5 protein has the ability to bind nucleic acids in vitro using bacterially produced and partially purified protein. hic-5 protein was incubated with double-stranded or single-stranded DNA-cellulose and the fraction of hic-5 protein bound or unbound to the DNA-cellulose was analyzed by western blotting using an anti-hic-5 antibody. Figure
Figure 1. DNA binding activity of hic-5 protein. The bacterially produced and partially purified hic-5 protein was incubated with double-stranded (ds, lanes 2, 3, 7 and 8) or single-stranded (ss, lanes 4, 5, 9 and 10) DNA-cellulose in the presence of ZnCl2 (lanes 1-5) or EDTA (lanes 6-10). The bound (lanes 3, 5, 8 and 10) and unbound fractions (lanes 2, 4, 7 and 9) were collected by centrifugation and, following SDS-PAGE electrophoresis, they were western blotted using an antibody against hic-5 protein. Lanes 1 and 6 were input proteins prepared in the presence of ZnCl2 or EDTA respectively. M, O*, S and P indicate molecular weight markers, original input, supernatant and pellet fraction, respectively. The arrow indicates the position of hic-5 protein.
Isolation of hic-5 protein binding sequences from mouse genomic DNA
The DNA binding ability of hic-5 protein tempted us to isolate the potential hic-5 protein binding sequences from mouse genomic DNA. The strategy we used was previously described and has already been used successfully for a similar purpose in several studies (17-20). First, as outlined in Figure
Figure 2. Screening procedure for hic-5 protein binding fragments by immunoprecipitation and PCR. (A) A schematic presentation of the immunoprecipitation-PCR (IP-PCR) method used for isolating mouse genomic DNA fragments that bind to hic-5 protein. (B) Isolated clones were tested for their binding activity with hic-5 protein using the IP-PCR method. For each clone, H indicates the result of the binding reaction with hic-5 protein and E indicates the control experiment using an E.coli protein extract that contain no hic-5 protein. In this experiment, seven binding clones were identified. Asterisks show the hic-5 binding clones. Some portions of clones 10, 19 and 78 were precipitated by E.coli proteins, but this may be caused by their property to bind to some proteins in a non-specific manner. To identify the enriched sequences, the DNA fragments were cloned into a plasmid vector, randomly selected and hybridized to the PCR products obtained by four round selection. We selected the clones which gave the strongest signals and tested them further for hic-5 protein binding in a similar manner to the enrichment procedure described above. In this process, we found that seven plasmids containing cloned fragments were selectively immunoprecipitated in the presence of hic-5 protein (Fig. We sequenced the fragments of these seven clones. The most outstanding feature of the sequences was the presence of an extraordinarily long poly(A)-like tract in the middle of five out of the seven fragments (Fig. Figure 3. Nucleotide sequences of the hic-5 binding clones. Underlined parts indicate A-rich tracts. We next examined the specificity of the interacton between hic-5 protein and the binding fragments using the DNA binding protein blot assay as previously described. In this assay, the protein extract from E.coli expressing hic-5 protein was electrophoretically separated by SDS-PAGE, transferred to a membrane and incubated with end-labeled 101 fragment in the presence or absence of competitor E.coli DNA. Figure
The presence of a long A/T tract in five out of the seven hic-5 protein binding sequences implies that hic-5 protein bound to a poly(A) tract such as those present in the 3[prime]-end of mRNAs. This possibility was examined by protein blot assay using riboprobes transcribed from the fragments, but hic-5 protein did not show any specific binding to riboprobes containing poly(A) tracts (data not shown). To determine the domains responsible for the DNA binding activity of hic-5 protein, we constructed prokaryotic expression vectors of N- and C terminal-truncated forms of the protein and performed a DNA binding protein blot assay together with a nearly full-length (dF) hic-5 protein as described above (Fig. To define the DNA binding domains in the C-terminal half of hic-5 protein in further detail, we constructed a series of deleted forms of the hic-5 LIM domains as illustrated in Figure 
Specificity of the interaction between hic-5 protein and the binding fragments
Figure 4. Binding specificity between hic-5 protein and an isolated clone. (A) The protein extract from E.coli harboring the hic-5 protein was resolved by 12% SDS-PAGE and blotted onto nitrocellulose filters. The DNA binding protein blot assay were performed with the end-labeled clone 101 fragment in the absence or presence of unlabeled E.coli DNA as a non-specific competitor (lanes 1-5, 0, 2, 5, 10 and 30 µg/ml, respectively). In the left end panel, the protein extract was separated by SDS-PAGE and stained with Coomassie brilliant blue. The arrows a-c indicate the position of randomly selected proteins a-c on 12% SDS-PAGE. (B) Competition for binding by E.coli DNA. The radioactivity of labeled 101 DNA fragments bound to each protein was determined with a BAS2000 image analyzer and the radioactivity relative to that in the absence of the E.coli DNA was determined. Closed squares, percentage of radioactivity bound to hic-5 protein; open circles, to protein a; open squares, to protein b; open triangles, to protein c. (C) Binding of hic-5 protein to a specific sequence. A DNA-binding protein blotting assay was performed with the end-labeled 101 fragment probe in the presence of 10 µg/ml unlabeled E.coli genomic DNA without further competitor DNA (lane -) or with a 100-fold (lanes ×100) or 200-fold (lanes ×200) molar excess each of the 101 and 5-3 fragments. In the left end panel, the protein extract was separated by SDS-PAGE and stained with Coomassie brilliant blue. The arrows indicate the band of hic-5 protein. (D) Comparison of DNA binding activity between hic-5 protein and paxillin. The protein extract from E.coli harboring the LIM domain of hic-5 protein (GST-LIM 1-4) (lanes 2, 4 and 6) or paxillin (GST-paxillin LIM) (lanes 1, 3 and 5) was subjected to the DNA binding protein blot assay using the end-labeled 101 fragment as probe in the presence of 5-20 µg/ml E.coli genomic DNA as indicated. In the left end panel, the protein extract containg GST-paxillin LIM (lane P) or that of GST-LIM 1-4 of hic-5 (lane H) was separated by SDS-PAGE and stained with Coomassie brilliant blue. The arrows labeled P indicate the position of GST-paxillin LIM and those labeled H indicate GST-LIM 1-4 of hic-5. Strong signals in the fast migrating fractions came from sequence-non-specific binding of the probe to small molecular weight proteins in E.coli.
A
B
C
D
Determination of the DNA binding domain in hic-5 protein
DISCUSSION
Although the function of the LIM domains is still obscure, several lines of evidence have emerged showing that it functions as a protein-protein interface. In this study, we found that hic-5 protein could bind to DNA in vitro in a zinc-dependent manner and that the LIM domains were responsible for the activity. The requirement for zinc ions for binding, which suggests a strict dependency of binding on the secondary structure of the LIM domains retained by zinc ions, implies that the DNA binding ability was not artificial but inherent in the LIM domains of hic-5 protein. A comparison of DNA binding of hic-5 protein with that of paxillin, whose LIM domains are highly homologous to those of hic-5 protein, further supports the assumption that the LIM domains of hic-5 protein have a unique DNA binding property (Fig.
With regard to the binding sequence of hic-5 protein, we could enrich several DNA fragments from the mouse genome as putative hic-5 protein binding sequences. The sequence specificity of hic-5 protein binding to these fragments is demonstrated in Figure
Figure 5. Zinc-dependent DNA binding activity of full-length and C-terminal LIM domains. (A) A schematic representation of the truncated forms of hic-5 protein. Dotted and striped areas show proline-rich regions and an acidic region, respectively. Checked boxes show the four LIM domains. Thick lines indicate the region expressed from each construct: dF, a nearly full-length hic-5 protein; N, a C-terminal truncated protein; C, an N-terminal truncated protein. (B) Protein extracts from E.coli harboring the dF, N and C constructs were electrophoretically separated and blotted onto a nitrocellulose filter. The filter was probed with the end-labeled 101 fragment in the presence of 10 µg/ml unlabeled E.coli genomic DNA and either 1 mM ZnCl2 (middle panel) or 50 mM EDTA, 10 mM DTT (right panel). The left panel shows the Coomassie blue staining pattern after SDS-PAGE. The arrows indicate the positions of each protein. Sequencing the hic-5 protein binding fragments isolated thus far revealed the unique properties of a high G+A content and the presence of a long A/T tract, supporting the above-mentioned idea that hic-5 protein recognizes a unique secondary DNA structure. It is well known that the Alu sequence is accompanied by a poly(A)-like tract at the 3[prime]-end, but it is usually <20 bp long (23). In this respect, the sequences of clones 19, 29, 97 and 98 are thought to be part of a unique B1 family with a long poly(A)-like tract, while the other clones, 10, 78 and 101, may be relatives. Although the significance of the B1-like sequences in hic-5 protein binding remain to be resolved, the DNA of a certain member of the mouse B1 family is reported to adopt a unique Z form secondary structure (24). One of these aspects or their combination in the sequences may contribute to recognition of the fragments by hic-5 protein. Figure 6. Deletion analysis of DNA binding by the LIM domains of hic-5 protein. (A) A schematic representation of the GST-hic-5 fusion protein derived from the LIM domains of hic-5 protein. X in lane 4 indicates 42 amino acids derived from the cloning vector. (B) Coomassie brilliant staining of protein extracts from E.coli harboring each of the deleted forms, electro-phoretically separated (lane 1, GST; lane 2, GST-LIM 1-4; lane 3, GST-LIM 1-3; lane 4, GST-LIM 1-2 X; lane 5,s GST-LIM 1-2; lane 6, GST-LIM 1; lane 7; GST-LIM 2-4; lane 8, GST-LIM 3-4; lane 9, GST-LIM 4) and blotted onto a nitrocellulose filter. (C) The filter was probed with end-labeled 101 fragment in the presence of 5 µg/ml unlabeled E.coli DNA. The significance of the DNA binding ability of hic-5 protein is unclear at this stage. Since forced expression of hic-5 increases expression of several genes, as reported previously (2), hic-5 might affect some nuclear function, including transcriptional regulation, through its DNA binding. Alternatively, the DNA binding ability of hic-5 protein may somehow be deleterious to cells and thus hic-5 protein has to be dispersed in the cytoplasm by the potential NES. In any case, further analysis of hic-5 protein binding to DNA and its effect on cellular functions at the molecular level might shed some light on the mechanism of cellular senescence and immortalization.
ACKNOWLEDGEMENTS
This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, a Grant-in-Aid from the Uehara Memorial Foundation and a Grant-in-Aid from Princess Takamatsu Cancer Research Foundation.
REFERENCES
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