Single LIM Domain Research Articles

The concerted action of SEPT9 and EPLIN modulates the adhesion and migration of human fibroblasts.

Septins are cytoskeletal proteins that participate in cell adhesion, migration, and polarity establishment. The septin subunit SEPT9 directly interacts with the single LIM domain of epithelial protein lost in neoplasm (EPLIN), an actin-bundling protein. Using a human SEPT9 KO fibroblast cell line, we show that cell adhesion and migration are regulated by the interplay between both proteins. The low motility of SEPT9-depleted cells could be partly rescued by increased levels of EPLIN. The normal organization of actin-related filopodia and stress fibers was directly dependent on the expression level of SEPT9 and EPLIN. Increased levels of SEPT9 and EPLIN enhanced the size of focal adhesions in cell protrusions, correlating with stabilization of actin bundles. Conversely, decreased levels had the opposite effect. Our work thus establishes the interaction between SEPT9 and EPLIN as an important link between the septin and the actin cytoskeleton, influencing cell adhesion, motility, and migration.

Genome-wide identification and expression analysis of the plant specific LIM genes in Gossypium arboreum under phytohormone, salt and pathogen stress

Asiatic cotton (Gossypium arboreum) cultivated as ‘desi cotton’ in India, is renowned for its climate resilience and robustness against biotic and abiotic stresses. The genome of G. arboreum is therefore, considered as a valued reserve of information for discovering novel genes or gene functions for trait improvements in the present context of cotton cultivation world-wide. In the present study, we carried out genome-wide analysis of LIM gene family in desi cotton and identified twenty LIM domain proteins (GaLIMs) which include sixteen animals CRP-like GaLIMs and four plant specific GaLIMs with presence (GaDA1) or absence (GaDAR) of UIM (Ubiquitin Interacting Motifs). Among the sixteen CRP-like GaLIMs, eleven had two conventional LIM domains while, five had single LIM domain which was not reported in LIM gene family of the plant species studied, except in Brassica rapa. Phylogenetic analysis of these twenty GaLIM proteins in comparison with LIMs of Arabidopsis, chickpea and poplar categorized them into distinct αLIM1, βLIM1, γLIM2, δLIM2 groups in CRP-like LIMs, and GaDA1 and GaDAR in plant specific LIMs group. Domain analysis had revealed consensus [(C-X2-C-X17-H-X2-C)-X2-(C-X2-C-X17-C-X2-H)] and [(C-X2-C-X17-H-X2-C)-X2-(C-X4-C-X15-C-X2-H)] being conserved as first and/or second LIM domains of animal CRP-like GaLIMs, respectively. Interestingly, single LIM domain containing GaLIM15 was found to contain unique consensus with longer inter-zinc-motif spacer but shorter second zinc finger motif. All twenty GaLIMs showed variable spatio-temporal expression patterns and accordingly further categorized into distinct groups of αLIM1, βLIM1, γLIM2 δLIM2 and plant specific LIM (DA1/DAR). For the first time, response of GaDA1/DAR under the influence of biotic and abiotic stresses were studied in cotton, involving treatments with phytohormones (Jasmonic acid and Abscisic acid), salt (NaCl) and wilt causing pathogen (Fusarium oxysporum). Expressions patterns of GaDA1/DAR showed variable response and identified GaDA2 as a probable candidate gene for stress tolerance in G. arboreum.

Abstract 992: LIMD2 is a small LIM-only protein overexpressed in metastatic lesions which regulates cell motility and tumor progression by directly binding to and activating the integrin-linked-kinase

Abstract Proteins that communicate signals from the cytoskeleton to the nucleus are prime targets for effectors of metastasis as they often transduce signals regulating adhesion, motility and invasiveness. LIM domain proteins shuttle between the cytoplasm and the nucleus, and bind to partners in both compartments, often coupling changes in gene expression to extracellular cues and hence are a prime target for deregulation during tumor progression and metastasis. The LIM domain is a modular Zn finger structure, often found tandemly repeated in proteins. These LIM arrays often serve as scaffolds for assembling signal transduction apparatus. In this work, we characterize LIMD2 which is unique in that it encodes a single LIM domain. LIMD2 was originally identified as a transcript overexpressed in metastatic lesions but absent in the matched primary tumor from the same patient suggesting that it may be either a marker or effector of metastatic spread. We have shown that LIMD2 levels in fresh and archival tumors positively correlate with cell motility, metastatic potential and tumor grade in many different tumor types including bladder, melanoma, breast and thyroid tumors. LIMD2 directly contributes to these cellular phenotypes as shown by overexpression, knockdown and reconstitution experiments in cell culture models. Tumor cells with poor metastatic capability are converted to highly motile, invasive cells by expression of LIMD2 suggesting a dominant gain of function action. To understand the molecular mechanisms of its biological effects we determined its solution structure using NMR. The structure studies of LIMD2 revealed a classic LIM-domain structure containing a rigid hydrophobic core which bound 2 molecules of Zn. The 3D structure of LIMD2 was most highly related to the LIM1 domain of PINCH1, a core component of the Integrin Linked Kinase-Parvin-Pinch (IPP) complex. The IPP complex plays a key role in cell-cell and cell matrix interaction by transducing signals from membrane bound integrins to the nucleus. Structural and biochemical analyses revealed that LIMD2 bound directly to the kinase domain of ILK near the active site and strongly activated ILK kinase activity in vitro. Immunolocalization studies showed that LIMD2 and components of the IPP complex co-existed in focal adhesion plaques. Cells which were null for ILK failed to respond to the induction of motility and invasion by ectopic expression of LIMD2. This strongly suggests that LIMD2 potentiates its biological effects through direct interactions with ILK, a signal transduction pathway firmly linked to cell motility and invasion. In summary, we have defined LIMD2 as a new component of the signal transduction cascade that links integrin-mediated signaling to cell motility/metastatic behavior and may be a promising target for controlling tumor spread. Citation Format: Hongzhuang Peng, Mehdi Taleb Zadeh Farrooji, Michael J. Osborne, Jeremy W. Prokop, Paul C. McDonald, Jayashree Karar, Zhaoyuan Hou, Mei He, Electron Kebebew, Torben Orntoft, Meenhard Herlyn, Andrew J. Caton, William Fredericks, Bruce Malkowicz, Christopher S. Paterno, Alexandra S. Carolin, David W. Speicher, Emmanuel Skordalakes, Qihong Huang, Shoukat S. Dedhar, Katherine L. B. Borden, Frank J. Rauscher. LIMD2 is a small LIM-only protein overexpressed in metastatic lesions which regulates cell motility and tumor progression by directly binding to and activating the integrin-linked-kinase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 992. doi:10.1158/1538-7445.AM2014-992

FHL family members suppress vascular endothelial growth factor expression through blockade of dimerization of HIF1α and HIF1β

Four and a half LIM domain (FHL) proteins belong to a family of LIM-only proteins that have been implicated in the development and progression of various types of cancers. However, the role of FHL proteins in tumor angiogenesis remains to be elucidated. Herein, we demonstrate that FHL1-3 decrease the promoter activity and expression of vascular endothelial growth factor (VEGF), the key regulator of angiogenesis in cancer growth and progression as well as an important target gene of the transcription factor hypoxia-inducible factor 1 (HIF1α/HIF1β). FHL1-3 interacted with HIF1α both in vitro and in vivo. A single LIM domain of FHL1 was sufficient for its interaction with HIF1α. FHL1 interacted with the HIF1α region containing basic helix-loop-helix (bHLH) motif and PER-ARNT-SIM domain, both of which aid in dimerization with HIF1β and DNA binding. FHL1-3 inhibited HIF1 transcriptional activity and HIF1-mediated VEGF expression in a hypoxia-independent manner. Moreover, FHL1 blocked HIF1α-HIF1β heterodimerization and HIF1α recruitment to the VEGF promoter. These data suggest that FHL proteins are involved in negative regulation of VEGF possibly by interfering with the dimerization and DNA binding of HIF1 subunits and may play an important role in tumor angiogenesis.

The LIM Domain Protein FHL2 Interacts with the NR5A Family of Nuclear Receptors and CREB to Activate the Inhibin-α Subunit Gene in Ovarian Granulosa Cells

Nuclear receptor transcriptional activity is enhanced by interaction with coactivators. The highly related nuclear receptor 5A (NR5A) subfamily members liver receptor homolog 1 and steroidogenic factor 1 bind to and activate several of the same genes, many of which are important for reproductive function. To better understand transcriptional activation by these nuclear receptors, we sought to identify interacting proteins that might function as coactivators. The LIM domain protein four and a half LIM domain 2 (FHL2) was identified as interacting with the NR5A receptors in a yeast two-hybrid screen of a human ovary cDNA library. FHL2, and the closely related FHL1, are both expressed in the rodent ovary and in granulosa cells. Small interfering RNA-mediated knockdown of FHL1 and FHL2 in primary mouse granulosa cells reduced expression of the NR5A target genes encoding inhibin-α and P450scc. In vitro assays confirmed the interaction between the FHL and NR5A proteins and revealed that a single LIM domain of FHL2 is sufficient for this interaction, whereas determinants in both the ligand binding domain and DNA binding domain of NR5A proteins are important. FHL2 enhances the ability of both liver receptor homolog 1 and steroidogenic factor 1 to activate the inhibin-α subunit gene promoter in granulosa cells and thus functions as a transcriptional coactivator. FHL2 also interacts with cAMP response element-binding protein and substantially augments activation of inhibin gene expression by the combination of NR5A receptors and forskolin, suggesting that FHL2 may facilitate integration of these two signals. Collectively these results identify FHL2 as a novel coactivator of NR5A nuclear receptors in ovarian granulosa cells and suggest its involvement in regulating target genes important for mammalian reproduction.

Tandem zyxin LIM sequences do not enhance force sensitive accumulation

The ability to sense mechanical forces is vital to cell physiology. Yet, the molecular basis of mechano-signaling remains unclear. Previous studies have shown that zyxin, a focal adhesion protein, is recruited at force-bearing sites on the actin cytoskeleton and, therefore, identifying zyxin as a mechano-sensing protein candidate. Furthermore, zyxin accumulation at force-bearing sites requires the LIM domain located at the C-terminus of zyxin. The zyxin LIM domain consists of three LIM motifs, each containing two zinc-binding sites. Since individual LIM motifs do not accumulate at focal adhesions or force-bearing sites, we hypothesize that multiple zyxin LIM domains increase force sensitivity. Using a miniature force sensor and GFP-tagged LIM variants, we quantified the relationship between single, tandem dimer and trimer LIM protein localization and traction forces. While the presence of extra LIM domains affected VASP recruitment to focal adhesions, force sensitivity was not enhanced over the single LIM domain. Therefore, zyxin force sensitivity is optimal with a single LIM domain, while additional LIM domains fail to enhance force sensitivity.

Symptomatic Heterozygotes and Prenatal Diagnoses in a Nonconsanguineous Family with Syndromic Combined Pituitary Hormone Deficiency Resulting from Two NovelLHX3Mutations

Only 11 mutations have been reported in the transcription factor LHX3, known to be important for the development of the pituitary and motor neurons. All patients were homozygous, with various syndromic forms of combined pituitary hormone deficiency (CPHD), hampering to allocate, in these consanguineous patients, the respective contribution of LHX3 and additional genes to each symptom. The aim of the study was to report the family history and the molecular basis of a nonconsanguineous patient with syndromic CPHD. The patient, who presented at birth with respiratory distress, had a syndromic CPHD, including severe scoliosis, and normal intelligence. His father and paternal grandmother displayed limited head rotation. Two new LHX3 defects were identified. The paternally inherited c.252-3C>G mutation, which disrupts an acceptor splice site, would lead to severely truncated proteins containing a single LIM domain, resembling LIM-only proteins. Coexpression studies revealed the dominant-negative effect of this LIM-only protein over the wild-type LHX3. The maternally inherited p.Cys118Tyr mutation results in partial loss of transcriptional activity and synergy with POU1F1. Given the severity of the patient's phenotype, two prenatal diagnoses were performed: the first led to pregnancy interruption, the second to the birth of a healthy boy. This study of the first nonconsanguineous patient with LHX3 mutations demonstrates the pleiotropic roles of LHX3 during development and its full involvement in the complex disease phenotype. Isolated limitation of head rotation may exist in heterozygous carriers and would result from a dominant-negative effect. These data allowed the first prenatal diagnoses of this severe condition to be performed.

Synergistic repression of estrogen receptor transcriptional activity by FHL2 and Smad4 in breast cancer cells

Four and a half LIM domain protein 2 (FHL2) has been implicated in development and progression of various types of cancers. However, little is known about the biological function of FHL2 in breast cancer. Here, we report that FHL2 physically and functionally interacts with estrogen receptors (ERα and ERβ), important regulators of breast cancer development and progression. The N-terminal half LIM domain or a single LIM domain of FHL2 was sufficient for its interaction with ERα and ERβ. Overexpression of FHL2 reduced ER transcriptional activity in breast cancer cells, whereas reduction of endogenous FHL2 with FHL2 small interfering RNA enhanced ER transactivation. Moreover, FHL2 cooperates with Smad4, a previously known corepressor for ERα, to inhibit ERα transcriptional activity as well as expression of the estrogen-responsive gene cathepsin D. The synergistic inhibition of ERα transcriptional activity by FHL2 and Smad4 may be due to enhanced interaction of Smad4 with ERα by FHL2, because FHL2(1-156), the FHL2 deletion mutant, which showed no synergistic effect, failed to increase such interaction. These data suggested the cooperative regulation of estrogen signaling by FHL2 and Smad4 in breast cancer cells, and might provide a new regulation mechanism underlying breast cancer development and progression.

EhLimA, a Novel LIM Protein, Localizes to the Plasma Membrane inEntamoeba histolytica

The parasitic protozoan Entamoeba histolytica relies on a very dynamic cytoskeleton in order to invade and survive in host tissues. Identification of cytoskeletal elements is key to understanding these processes. Here we present the characterization of EhLimA, the first LIM protein of E. histolytica. EhLimA consists of a single LIM domain at its N terminus and exhibits the highest degree of homology with DdLimE from Dictyostelium discoideum. Immunofluorescence localization of EhLimA using anti-EhLimA antibodies revealed that EhLimA is highly concentrated at the plasma membrane of cells. Silencing or overexpression of the EhLimA gene did not have a significant effect on the growth or morphology of the parasite. EhLimA associates with the cytoskeleton as demonstrated by the enrichment of the protein in cytoskeleton fractions as well as in pull-down assays that revealed that cytoskeleton association involves interaction with actin. EhLimA binding to actin was shown to be dependent on the N-terminal LIM domain of EhLimA, as removal of even half of the LIM domain resulted in almost complete inhibition of the binding to actin. We also found that a portion of EhLimA floats to the lower-density regions of a sucrose gradient together with portions of the Gal-lectin light subunit and actin. Treatment of cells with the cholesterol-sequestering agent digitonin resulted in increased solubility of EhLimA. These results indicate that in addition to cytoskeletal association, EhLimA may also associate with lipid rafts in the parasite plasma membrane and suggest that EhLimA may be part of the molecular system connecting the actin cytoskeleton to membrane rafts.

Cloning and structural analysis of MLP in the silkworm, Bombyx mori

The LIM domain is found in a wide variety of eukaryotic proteins that regulate gene expression and cell differentiation during development. Muscle LIM protein (MLP) gene in Bombyx mori has been cloned by blasting its EST database and PCR test in present report. The resulting sequence covers 2 327 bp of cDNA (GenBank accession No. DQ311195). It has a complete open reading fragment and encodes a 494 amino acid protein. Genomic DNA sequence contains 11 exons and 10 introns, with intron splicing following the GT-AG rule. M.W. and PI of the predicted MLP in Bombyx mori are 53.03 kDa and 8.29 respectively. A single LIM domain linked to a glyscine-rich region is found in a previously deposited LIM protein (AAR23823) in Bombyx mori. MLP identified in this report encodes a protein with five tandem LIM-glycine modules. The two LIM proteins could be produced by alternative splicing and both are probably involved in muscle cell differentiation. This work provides foundation for further research on the in vivo function of MLP.

Molecular characterization of the Caenorhabditis elegans ALP/Enigma gene alp-1.

Members of the ALP/Enigma family of PDZ-LIM proteins play a role in cytoskeletal anchorage and mutations in at least one member of this family are associated with human cardiomyopathy. Here, we describe the analysis of the Caenorhabditis elegans alp-1 gene. alp-1 is predicted to encode the entire nematode ALP/Enigma protein family, consisting of one ALP-related protein with a single LIM domain and three Enigma-like proteins containing four LIM domains. We demonstrate that the ALP-1 proteins are expressed in muscle cells, where they localize to actin anchorage and muscle attachment sites. We show that the PDZ domain of the ALP-1 proteins is sufficient to target the protein to the dense bodies, which are important actin anchorage sites in C. elegans body wall muscle. We demonstrate that the C. elegans ALP/Enigma proteins are also localized to cell-cell junctions and to both epithelial and muscle cell nuclei. These findings suggest new roles for the ALP/Enigma protein family that may lead to the understanding of their involvement in cardiomyopathy.

The LIM-only Proteins FHL2 and FHL3 Interact with α- and β-Subunits of the Muscle α7β1 Integrin Receptor

FHL1, FHL2, and FHL3 are members of the four and one-half LIM domain protein subclass that are expressed in striated muscles. Here we show that FHL2 and FHL3 are novel alpha(7)beta(1) integrin-interacting proteins. They bind both the alpha- and the beta-subunit as well as different splice isoforms. The minimal binding sites for FHL2 and FHL3 on beta(1A)-chain overlap, whereas on alpha(7A) and alpha(7B) subunits they are situated adjacent. Determining the binding sites for integrins on FHL2 or FHL3 revealed that the suprastructure of the whole molecule is important for these associations, rather than any single LIM domain. Immunofluorescence studies with cells expressing full-length FHL proteins or their deletion mutants showed that FHL2 and FHL3 but not FHL1 colocalize with integrins at cell adhesion sites. Further, their recruitment to the membrane results from binding to either the alpha- or the beta-chain of the integrin receptor. The association of FHL2 or FHL3 with integrin receptors neither influences attachment of cells to different substrates nor changes their migration capacity. However, in cardiac and skeletal muscles, FHL2 and FHL3, respectively, are colocalized with alpha(7)beta(1) integrin receptor at the periphery of Z-discs, suggesting a role in mechanical stabilization of muscle cells.

EsMlp, a Muscle-LIM Protein Gene, Is Up-regulated during Cold Exposure in the Freeze-Avoiding Larvae of Epiblema scudderiana

Screening of a cDNA library identified transcripts that were up-regulated by cold (4 or −20°C) exposure in larvae of the freeze-avoiding goldenrod gall moth, Epiblema scudderiana. One clone contained a full-length open reading frame encoding a protein of 94 amino acids. The gene product, with 79.1% of residues identical with the Drosophila LIM protein Mlp60A, was named EsMlp and contained a single LIM domain and consensus sequences characteristic of a LIM protein. Transcript levels rose approx twofold when larvae were shifted from 4 to −20°C and approx threefold over the midwinter months compared with larvae sampled in October or April. EsMlp expression was high in larval head (possibly due to expression in pharyngeal muscles) and body wall but was not detected in fat body. Immunoblotting revealed a three- to fourfold increase in EsMlp protein in midwinter larvae (January-February) compared with November-collected animals and a further rise to eightfold higher than November values in larvae collected in April. Cold up-regulation of EsMlp and the pattern of EsMlp levels in the larvae suggest possible roles for the protein, such as in muscle maintenance over the winter or as a preparative function that could facilitate the rapid resumption of development and metamorphosis when environmental temperatures rise in the spring.

LIM domains of cysteine-rich protein 1 (CRP1) are essential for its zyxin-binding function.

Previous studies have demonstrated that the adhesion-plaque protein, zyxin, interacts specifically with a 23 kDa protein, called the cysteine-rich protein 1 (CRP1), which has been implicated in myogenesis. Primary sequence analyses have revealed that both zyxin and CRP1 exhibit multiple copies of a structural motif called the LIM domain. LIM domains, which are defined by the consensus CX2CX16-23HX2CX2CX2CX16-23CX2-3(C,H,D), are found in a variety of proteins that are involved in cell growth and differentiation. Recent studies have established that LIM domains are zinc-binding structures that mediate specific protein-protein interactions. For example, in the case of the zyxin-CRP1 interaction, one of zyxin's three LIM domains is necessary and sufficient for binding to CRP1. Because the CRP1 molecule is comprised primarily of two LIM domains, we were interested in the possibility that the binding site for zyxin on CRP1 might also be contained within a single LIM domain. Consistent with the hypothesis that the LIM domains of CRP1 are critical for the protein's zyxin-binding function, zinc-depleted CRP1 displays a reduced zyxin-binding activity. However, domain mapping analyses have revealed that neither of the two individual LIM domains of CRP1 can support a wild-type interaction with zyxin. Collectively, our results suggest that the binding site for zyxin on CRP1 is not contained within a single contiguous sequence of amino acids. Instead, the interaction appears to rely on the co-ordinate action of a number of residues that are displayed in both of CRP1's LIM domains.

Nuclear LIM interactor, a rhombotin and LIM homeodomain interacting protein, is expressed early in neuronal development.

LIM domain-containing transcription factors, including the LIM-only rhombotins and LIM-homeodomain proteins, are crucial for cell fate determination of erythroid and neuronal lineages. The zinc-binding LIM domains mediate protein-protein interactions, and interactions between nuclear LIM proteins and transcription factors with restricted expression patterns have been demonstrated. We have isolated a novel protein, nuclear LIM interactor (NLI), that specifically associates with a single LIM domain in all nuclear LIM proteins tested. NLI is expressed in the nuclei of diverse neuronal cell types and is coexpressed with a target interactor islet-1 (Isl1) during the initial stages of motor neuron differentiation, suggesting the mutual involvement of these proteins in the differentiation process. The broad range of interactions between NLI and LIM-containing transcription factors suggests the utilization of a common mechanism to impart unique cell fate instructions.

The pollen-specific LIM protein PLIM-1 from sunflower binds nucleic acids in vitro

The protein PLIM-1 (formerly SF3) from sunflower is expressed exclusively in mature, free pollen. It contains two LIM domains associated with an acidic C-terminus comprising six copies of the pentapeptide motif (A,T,S) (E,D) TQN. We have expressed the pollen protein as well as some of its mutant forms inEscherichia coli and have used the bacterially produced proteins to study interactions with nucleic acids. Our studies show that the protein binds DNA and RNA in vitro to form large complexes, while mutant polypeptides containing either a single LIM domain or a destabilized first or second LIM domain do not. Although these data suggest that the biological function of PLIM-1 involves interactions with nucleic acids, its role in pollen development remains unclear.

Two muscle-specific LIM proteins in Drosophila.

The LIM domain defines a zinc-binding motif found in a growing number of eukaryotic proteins that regulate cell growth and differentiation during development. Members of the cysteine-rich protein (CRP) family of LIM proteins have been implicated in muscle differentiation in vertebrates. Here we report the identification and characterization of cDNA clones encoding two members of the CRP family in Drosophila, referred to as muscle LIM proteins (Mlp). Mlp60A encodes a protein with a single LIM domain linked to a glycine-rich region. Mlp84B encodes a protein with five tandem LIM-glycine modules. In the embryo, Mlp gene expression is spatially restricted to somatic, visceral, and pharyngeal muscles. Within the somatic musculature, Mlp84B transcripts are enriched at the terminal ends of muscle fibers, whereas Mlp60A transcripts are found throughout the muscle fibers. The distributions of the Mlp60A and Mlp84B proteins mirror their respective mRNA localizations, with Mlp84B enrichment occurring at sites of muscle attachment. Northern blot analysis revealed that Mlp gene expression is developmentally regulated, showing a biphasic pattern over the course of the Drosophila life cycle. Peaks of expression occur late in embryogenesis and during metamorphosis, when the musculature is differentiating. Drosophila Mlp60A and Mlp84B, like vertebrate members of the CRP family, have the ability to associate with the actin cytoskeleton when expressed in rat fibroblast cells. The temporal expression and spatial distribution of muscle LIM proteins in Drosophila are consistent with a role for Mlps in myogenesis, late in the differentiation pathway.

Muscle LIM protein, a novel essential regulator of myogenesis, promotes myogenic differentiation

Muscle LIM protein (MLP) is a novel positive regulator of myogenesis. Its expression and that of its Drosophila homolog DMLP1 are enriched in striated muscle and coincide with myogenic differentiation. In the absence of MLP, induced C2 cells express myogenin but fail to exit from the cell cycle and to differentiate. Over-expression of MLP in C2 myoblasts potentiates myogenic differentiation and reduces its sensitivity to TGFβ. Like MLP, single LIM domain deletion mutants of MLP and nonmuscle LIM-only proteins promote myogenic differentiation. In 3T3 fibroblasts, the same LIM proteins prevent phorbol ester-induced inhibition of DNA replication. These results establish MLP as an essential promoter of myogenesis and suggest that LIM-only proteins act via similar mechanisms to regulate aspects of cell differentiation.

Common metal ion coordination in LIM domain proteins.

The LIM motif is a cysteine- and histidine-rich sequence that was first identified in proteins involved in control of gene expression and cell differentiation. In order to characterize structural features of the LIM domain, we have carried out biophysical studies on two polypeptides that display LIM domains: the cysteine-rich intestinal protein (CRIP) and a fragment of the cysteine-rich protein (CRP). Bacterial expression vectors were constructed for the intact CRIP molecule and the C-terminal half of CRP, designated LIM2, such that each expressed protein contained a single LIM domain. Both proteins were recovered as soluble, Zn(II)-containing proteins. The metal coordination properties of these two distinct LIM domain proteins were highly similar, suggesting that a common structural architecture may exist in LIM domain proteins. Both proteins exhibit a maximum of two tetrahedrally bound Zn(II) ions per molecule. Electronic spectroscopy of Co(II) complexes and 113Cd NMR of Cd(II) complexes of CRIP and LIM2 revealed a similar ligand field pattern with one tetrathiolate (S4) site and one S3N1 site for divalent metal ions. The nitrogen ligand was shown to arise from a histidyl imidazole by heteronuclear multiple quantum coherence NMR. The eight conserved residues within the LIM domains of CRIP and LIM2 include seven cysteines and one histidine. It is likely that these conserved residues generate the S4 and S3N1 Zn(II)-binding sites. Metal binding to the two sites within a single LIM domain is sequential, with preferential occupancy of the S4 site. Slow metal ion exchange occurs between sites within an LIM domain, and metal exchange with exogenous metal ions is observed, with exchange at the S3N1 site being kinetically more facile. In the absence of metal binding both proteins appear to be substantially unfolded. Metal binding stabilizes a tertiary fold containing appreciable secondary structural elements. The common metal ion coordination in CRIP and LIM2 suggests that the LIM motif may constitute a structural module with conserved features.