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MLP84B, another muscle LIM protein in consists of 495 amino acids with five copies of the LIM domain, each followed by a glycine rich motif. The five LIM-glycine casettes in MLP84B are separated by linker regions of variable length and composition. The first LIM domain of MLP84B has the sequence CX2CX17HX2CX2CX2CX17CX2C, the same as avian cysteine rich proteins (CRPs) and Drosophila MLP60A. The following four LIM domains of MLP84B display the consensus sequence CX2CX17HX2CX2CX2CX18CX2C; as indicated, they each have one additional residue in the second zinc finger of each LIM domain. Two potential nuclear localization signals are present (Stronach, 1996).

A 23-kD protein interacts with zyxin, a cytoskeletal protein, in vitro. The 23-kD protein has been purified and characterized from avian smooth muscle. It consists of 192 amino acids and exhibits two copies of the LIM motif and belongs to the muscle LIM protein family. The 23-kD protein is 91% identical to the human cysteine-rich protein (hCRP), and is referred to as the chicken cysteine-rich protein (cCRP). cCRP is most prominent in tissues that are enriched in smooth muscle cells, such as gizzard, stomach, and intestine. In primary cell cultures derived from embryonic gizzard, differentiated smooth muscle cells exhibit the most striking staining with anti-cCRP antibodies. The expression of cCRP is developmentally regulated (Crawford, 1994).

Differentiated, quiescent vascular smooth muscle cells assume a dedifferentiated, proliferative phenotype in response to injury, one of the hallmarks of arteriosclerosis. Members of the LIM family of zinc-finger proteins are important in the differentiation of various cells, including striated muscle. The molecular cloning and characterization is described of a developmentally regulated smooth muscle LIM protein, SmLIM, that is expressed preferentially in the rat aorta. This 194-amino acid protein has two LIM domains; comparisons of rat SmLIM to its mouse and human homologs reveal high levels of amino acid sequence conservation (100 and 99%, respectively). SmLIM is a nuclear protein and maps to human chromosome 3. SmLIM mRNA expression is high in aorta but not in striated muscle and low in other smooth muscle tissues such as intestine and uterus. In contrast with arterial tissue, SmLIM mRNA is barely detectable in venous tissue. The presence of SmLIM expression within aortic smooth muscle cells is confirmed by in situ hybridization. In vitro, SmLIM mRNA levels decrease by 80% in response to platelet-derived growth factor-BB in rat aortic smooth muscle cells. In vivo, SmLIM mRNA decreases by 60% in response to vessel wall injury during periods of maximal smooth muscle cell proliferation. The down-regulation of SmLIM by phenotypic change in vascular smooth muscle cells suggests that it may be involved in their growth and differentiation (Jain, 1996).

The muscle LIM protein (MLP) is a muscle-specific LIM-only factor that exhibits a dual subcellular localization, being present in both the nucleus and in the cytoplasm. Overexpression of MLP in C2C12 myoblasts enhances skeletal myogenesis, whereas inhibition of MLP activity blocks terminal differentiation. Thus, MLP functions as a positive developmental regulator, although the mechanism through which MLP promotes terminal differentiation events remains unknown. While examining the distinct roles associated with the nuclear and cytoplasmic forms of MLP, it was found that nuclear MLP functions through a physical interaction with the muscle basic helix-loop-helix (bHLH) transcription factors MyoD, MRF4, and myogenin. This interaction is highly specific since MLP does not associate with nonmuscle bHLH proteins E12 or E47 or with the myocyte enhancer factor-2 (MEF2) protein, which acts cooperatively with the myogenic bHLH proteins to promote myogenesis. The first LIM motif in MLP and the highly conserved bHLH region of MyoD are responsible for mediating the association between these muscle-specific factors. MLP also interacts with MyoD-E47 heterodimers, leading to an increase in the DNA-binding activity associated with this active bHLH complex. Although MLP lacks a functional transcription activation domain, it is proposed that it serves as a cofactor for the myogenic bHLH proteins by increasing their interaction with specific DNA regulatory elements. Thus, the functional complex of MLP-MyoD-E protein reveals a novel mechanism for both initiating and maintaining the myogenic program and suggests a global strategy for how LIM-only proteins may control a variety of developmental pathways (Kong, 1997).

LIM homeodomain and LIM-only (LMO) transcription factors contain two tandemly arranged Zn2+-binding LIM domains capable of mediating protein-protein interactions. These factors have restricted patterns of expression, are found in invertebrates as well as vertebrates, and are required for cell type specification in a variety of developing tissues. A recently identified, widely expressed protein, NLI (Drosophila homolog: Chip), binds with high affinity to the LIM domains of LIM homeodomain and LMO proteins in vitro and in vivo. A 38-amino-acid fragment of NLI is sufficient for the association of NLI with nuclear LIM domains. In addition, NLI forms high affinity homodimers through the amino-terminal 200 amino acids, but dimerization of NLI is not required for association with the LIM homeodomain protein Lmxl. Chemical cross-linking analysis reveals higher-order complexes containing multiple NLI molecules bound to Lmx1, indicating that dimerization of NLI does not interfere with LIM domain interactions. NLI formed complexes with Lmx1 on the rat insulin I promoter and inhibits LIM domain-dependent synergistic transcriptional activation by means of Lmx1 and the basic helix-loop-helix protein E47 from the rat insulin I mini-enhancer. These studies indicate that NLI contains at least two functionally independent domains and may serve as a negative regulator of synergistic transcriptional responses that require direct interaction via LIM domains. Thus, NLI may regulate the transcriptional activity of LIM homeodomain proteins by determining specific partner interactions (Jurata, 1997).

The nuclear LIM domain protein LMO2, a T cell oncoprotein, is essential for embryonic erythropoiesis. LIM-only proteins are presumed to act primarily through protein-protein interactions. A widely expressed protein, Ldb1, has been identified whose C-terminal 76-residues are sufficient to mediate interaction with LMO2. In murine erythroleukemia cells, the endogenous Lbd1 and LMO2 proteins exist in a stable complex, whose binding affinity appears greater than that between LMO2 and the bHLH transcription factor SCL. However, Ldb1, LMO2, and SCL/E12 can assemble as a multiprotein complex on a consensus SCL binding site. Like LMO2, the Ldb1 gene is expressed in fetal liver and erythroid cell lines. Forced expression of Ldb1 in G1ER proerythroblast cells inhibits cellular maturation, a finding compatible with the decrease in Ldb1 gene expression that normally occurs during erythroid differentiation. Overexpression of the LMO2 gene also inhibits erythroid differentiation. These studies demonstrate a function for Ldb1 in hemopoietic cells and suggest that one role of the Ldb1/LMO2 complex is to maintain erythroid precursors in an immature state (Visvader, 1997).

The LIM-only protein Lmo2, activated by chromosomal translocations in T-cell leukemias, is normally expressed in hematopoiesis. It interacts with TAL1 and GATA-1 proteins, but the function of the interaction is unexplained. In erythroid cells Lmo2 forms a novel DNA-binding complex with GATA-1, TAL1, E2A, and the recently identified LIM-binding protein, Ldb1/NLI. This oligomeric complex binds to a unique, bipartite DNA motif comprising an E-box (CAGGTG), followed approximately 9 bp downstream by a GATA site. In vivo assembly of the DNA-binding complex requires interaction of all five proteins and establishes a transcriptional transactivating complex. These data demonstrate one function for the LIM-binding protein Ldb1 and establish a function for the LIM-only protein Lmo2 as an obligatory component of an oligomeric, DNA-binding complex, which may play a role in hematopoiesis (Wadman, 1997).

Several important transcription factors involved in hematopoiesis have been identified from the analysis of chromosome translocation associated with human tumors. One of these genes, LMO2, is associated with the translocations found in childhood T cell acute leukemia. LMO2 (formerly known as RBTN2) encodes a LIM domain protein (in which two zinc-binding LIM domains occur) and is activated after chromosomal translocation by association with either the T cell receptor alpha/beta gene at chromosome 14 (band q11), or the beta gene at chromosome 7 (band q35). As a result of the chromosomal translocations, LMO2 protein is made in the specific T cells that have the translocation, and the consequence of this aberrant expression is thought to be an alteration in the differentiation of the T cell, ultimately resulting in overt leukemia. The evidence of a physiological role for Lmo2 protein in hematopoiesis has come from gene target experiments in which null mutations were introduced into the Lmo2 gene. Null mutant embryonic mice die in utero due to a failure of yolk sac erythropoiesis, showing that Lmo2 is necessary for yolk sac erythropoiesis to take place. Although there is no direct evidence that Lmo2 itself has a DNA binding capacity, in normal expression sites, such as erythroid cells, Lmo2 protein directly interacts with a basic-loop-helix protein, Tal1/Scl, and the GATA DNA-binding protein, Gata-1. An in vitro binding site selection has led to the identification of a complex involving Lmo2 and also including Tal1/Scl, E47, Gata-1, and Ldb1. This erythroid complex binds to a bipartite DNA motif consisting of E box and GATA consensus sequences in which the Tal1/Scl-E47 component binds to the E box and Gata-1 binds to the GATA site. These data strongly support the idea that Lmo2 acts as a bridging molecule bringing together the different DNA binding factors in this erythroid complex. Because Lmo2 can bind also to Gata-2 protein, it is possible that a complex of Lmo2, Gata-2, and other proteins, analogous to that seen in erythroid cells, might occur at earlier times of hematopoiesis when Gata-1 is not expressed (Yamada, 1998 and references).

Lmo2 is required for yolk sac erythropoiesis. The fact that Lmo2 null mutant mice die at embryonic day 9-10 prevents an assessment of a role at other stages of hematopoiesis. The hematopoietic contribution of homozygous mutant Lmo2 -/- mouse embryonic stem cells has been studied and it has been found that Lmo2 -/- cells do not contribute to any hematopoietic lineage in adult chimeric mice, but reintroduction of an Lmo2-expression vector rescues the ability of Lmo2 null embryonic stem cells to contribute to all lineages tested. This disruption of hematopoiesis probably occurs because interaction of Lmo2 protein with factors such as Tal1/Scl is precluded. Thus, Lmo2 is necessary for early stages of hematopoiesis, and the Lmo2 master gene encodes a protein that has a central and crucial role in the hematopoietic development (Yamada, 1998).

Chromosomal translocations in T-cell acute leukemias can activate genes encoding putative transcription factors such as the LIM proteins RBTN1 and RBTN2 (renamed LMO1 and LMO2), and the DNA-binding basic helix-loop-helix transcription factor TAL1 associated with T-cell acute lymphocytic leukemia. While not expressed in normal T cells, RBTN2 and TAL1 are coexpressed in erythroid cells and are both important for erythroid differentiation. The LIM protein RBTN2 is not phosphorylated and is complexed with the TAL1 phosphoprotein in the nucleus of erythroid cells. A complex containing both RBTN1 and TAL1 also occurs in a T-cell acute leukemia cell line. Since both RBTN2 and TAL1 are crucial for normal erythropoiesis, these data have important implications for transcription networks therein. Further, since both proteins can be involved in leukemogenesis, these data provide a direct link between proteins activated by chromosomal translocations in T-cell acute leukemia (Valge-Archer, 1994).

The RBTN1 and RBTN2 genes are activated by distinct translocations involving chromosome 11 in some T cell acute leukemias. The RBTN proteins belong to the LIM family that comprises proteins with one, two or three cysteine-rich LIM domains, sometimes together with homeodomains or protein kinase domains. The RBTN1 and RBTN2 proteins comprise only tandem LIM domains. RBTN1 and RBTN2 proteins are capable of supporting transcriptional transactivation of specific reporter genes in transfection assays. The results, using intact proteins or fusions with the homeodomain of the heterologous protein Isl-1, show that this transcriptional activation ability resides in the NH2-terminal parts of both proteins. The use of yeast assays with RBTN2 shows that RBTN2 forms homodimers and that the NH2-terminal 27 amino acids are sufficient to facilitate transcriptional transactivation. These data expand the functional diversity of the LIM-domain protein family and they augment the previously defined relationship between chromosomal translocations and transcriptional activation (Sanchez-Garcia, 1995).

RBTN2 is a nuclear protein expressed in the erythroid lineage in vivo, and is essential for erythroid development in mice. The homozygous rbtn2 null mutation leads to failure of yolk sac erythropoiesis and embryonic lethality around E10.5. Moreover, in vitro differentiation of yolk sac tissue from homozygous mutant mice and sequentially targeted double-mutant ES cells demonstrates a block to erythroid development. This shows a pivotal role for a LIM domain protein in lineage specification during mammalian development and suggests that RBTN2 and GATA-1 are critical at similar stages of erythroid differentiation (Warren, 1994).

The RBTN2 LIM-domain protein, originally identified as an oncogenic protein in human T-cell leukemia, is essential for erythropoiesis. Direct interaction of the RBTN2 protein during erythropoiesis was observed in vivo and in vitro with the GATA1 or GATA2 zinc-finger transcription factors (See Drosophila Serpent), as well as with the basic helix-loop-helix protein TAL1. By using mammalian two-hybrid analysis, complexes involving RBTN2, TAL1, and GATA1, together with E47, the basic helix-loop-helix heterodimerization partner of TAL1, could be demonstrated. Thus, a molecular link exists between three proteins crucial for erythropoiesis, and the data suggest that variations in amounts of complexes involving RBTN2, TAL1, and GATA1 could be important for erythroid differentiation (Osada, 1995).

Some T cell leukemia patients have chromosomal abnormalities involving both LMO2 (RBTN2) and TAL1, implying that LMO2 and TAL1 act synergistically to promote tumorigenesis after their inappropriate co-expression. To test this hypothesis, transgenic mice were made which co-express Lmo2 and Tal1 genes in T cells. Dimers of Lmo2 and Tal1 proteins are formed in thymocytes of double but not single transgenic mice. Furthermore, thymuses of double transgenic mice are almost completely populated by immature T cells from birth; these mice develop T cell tumours approximately 3 months earlier than those with only the Lmo2 transgene. Thus interaction between these two proteins can alter T cell development and potentiate tumorigenesis. The data also provide formal proof that TAL1 is an oncogene, apparently acting as a tumour promoter in this system (Larson, 1996).

LIM-only proteins KyoT1 and KyoT2

The RBP-J/Su(H) DNA-binding protein (see Drosophila Suppressor of Hairless) plays a key role in transcriptional regulation by targeting to specific promoters the Epstein-Barr virus nuclear antigen 2 (EBNA2) and the intracellular portions of Notch receptors. Using the yeast two-hybrid system, a LIM-only protein, KyoT, has been isolated that physically interacts with RBP-J. Differential splicing gives rise to two transcripts of the KyoT gene, KyoT1 and KyoT2, that encode proteins with four and two LIM domains, respectively. With differential splicing resulting in deletion of an exon, KyoT2 lacks two LIM domains from the C terminus and has a frameshift in the last exon, creating the RBP-J-binding region in the C terminus. KyoT1 has a negligible level of interaction with RBP-J. Strong expression of KyoT mRNAs is detected in skeletal muscle and lung, with a predominance of KyoT1 mRNA. When expressed in F9 embryonal carcinoma cells, KyoT1 and KyoT2 are localized in the cytoplasm and the nucleus, respectively. The binding site of KyoT2 on RBP-J overlaps those of EBNA2 and Notch1 but is distinct from that of Hairless, the negative regulator of RBP-J-mediated transcription in Drosophila. KyoT2 (but not KyoT1) represses the RBP-J-mediated transcriptional activation by EBNA2 and Notch1 by competing with them for binding to RBP-J and by dislocating RBP-J from DNA. KyoT2 is a novel negative regulatory molecule for RBP-J-mediated transcription in mammalian systems (Taniguchi, 1998).

The LIM motif as a protein-protein interaction domain

The POU-type homeodomain protein UNC-86 and the LIM-type homeodomain protein MEC-3, both of which specify neuronal cell fate in the nematode C. elegans, bind cooperatively as a heterodimer to the mec-3 promoter. Heterodimer formation increases DNA binding stability and, therefore, increases DNA binding specificity. The in vivo significance of this heterodimer formation in neuronal differentiation is suggested by (1) a loss-of-function mec-3 mutation whose product in vitro binds DNA well but forms heterodimers with UNC-86 poorly and (2) a mec-3 mutation with wild-type function, whose product binds DNA poorly but forms heterodimers well (Xue, 1993).

A new LIM-domain-binding factor, Ldb1, an novel protein, has been isolated on the basis of its ability to interact with the LIM-HD protein Lhx1 (Lim1). High-affinity binding by Ldb1 requires paired LIM domains and is restricted to the related subgroup of LIM domains found in LIM-HD and LMO proteins. The highly conserved Xenopus Ldb protein XLdb1, interacts with Xlim-1, the Xenopus orthologue of Lhx1. When injected into Xenopus embryos, XLdb1 (or Ldb1) can synergize with Xlim-1 in the formation of partial secondary axes and in activation of the genes encoding goosecoid, chordin, NCAM and XCG7, demonstrating a functional as well as a physical interaction between the two proteins (Agulnick, 1996).

A double MLP construct that accumulates nearly exclusively along actin filaments promotes myogenic differentiation efficiently, arguing for a functional role of cytoskeleton-associated MLP. Binding of MLP to the actin cytoskeleton is specifically attributable to its second LIM motif. An additional LIM motif potentiates binding. Potentiating LIM motifs can be interchanged, resulting in differential targeting of interacting proteins. To analyze LIM-LIM interactions in situ, this property was exploited to develop a hybrid interaction approach based on the relocalization of LIM-containing constructs to the actin cytoskeleton. These experiments reveal the existence of marked selectivity in the interactions of single LIM motifs, and among LIM domains from different LIM-homeo domain and LIM-only proteins. When MLP and RBTN1 are expressed in the same transfected cells, the two LIM-only proteins do not detectably influence their respective subcellular distributions (MLP to nucleus and cytoplasm and RBTN1 to nucleus). A similar outcome was observed in Apterous plus MLP cotransfections. In contrast, cotransfection of RBTN1 with R1-M2 (a hybrid molecule with the first LIM motif in RBTN1 and the second LIM motif of MLP) produces a dramatic redistribution of RBTN1, which now mainly colocalizes with R1-M2 along actin-containing filaments. R1-M2 induces redistribution of R2 but not R1. These findings indicate that the LIM motifs of RBTN1 interact specifically to form heterodimers of R1-R2. Similar expreiments reveal that the LIM motifs of MLP preferentially form M1-M2 heterodimers. Since single LIM motifs of double LIM motif proteins interact with distinct and specific cellular-binding sites, the analysis suggests that the double LIM motif has two interacting interfaces. On the basis of these findings, it is proposed that LIM motifs function as specific adapter elements to promote the assembly and targeting of multiprotein complexes (Arber, 1996).

PINCH is a widely expressed and evolutionarily conserved protein comprising primarily five LIM domains, which are cysteine-rich consensus sequences implicated in mediating protein-protein interactions. PINCH is a binding protein for integrin-linked kinase (ILK), an intracellular serine/threonine protein kinase that plays important roles in the cell adhesion, growth factor, and Wnt signaling pathways. The interaction between ILK and PINCH has been consistently observed under a variety of experimental conditions. They have interacted in yeast two-hybrid assays, in solution, and in solid-phase-based binding assays. Furthermore, ILK, but not vinculin or focal adhesion kinase, has been co-isolated with PINCH from mammalian cells by immunoaffinity chromatography, indicating that PINCH and ILK associate with each other in vivo. The PINCH-ILK interaction is mediated by the N-terminal-most LIM domain (LIM1, residues 1 to 70) of PINCH and multiple ankyrin (ANK) repeats located within the N-terminal domain (residues 1 to 163) of ILK. Additionally, biochemical studies indicate that ILK, through the interaction with PINCH, is capable of forming a ternary complex with Nck-2, an SH2/SH3-containing adapter protein implicated in growth factor receptor kinase and small GTPase signaling pathways. PINCH is concentrated in peripheral ruffles of cells spreading on fibronectin and clusters of PINCH have been detected that are colocalized with the alpha5beta1 integrins. These results demonstrate a specific protein recognition mechanism utilizing a specific LIM domain and multiple ANK repeats and suggest that PINCH functions as an adapter protein connecting ILK and the integrins with components of growth factor receptor kinase and small GTPase signaling pathways (Tu, 1999).

The presence of the LIM domain of Isl-1 inhibits binding of the homeodomain to its DNA target. This in vitro inhibition can be released either by denaturation/renaturation of the protein or by truncation of the LIM domains. A similar inhibition is observed in vivo using reporter constructs. LIM domains in a chimeric protein can inhibit binding of the Ultrabithorax homeodomain to its target. The ability of LIM domains to inhibit DNA binding by the homeodomain provides a possible basis for negative regulation of LIM-homeodomain proteins in vivo (Sanchez-Garcia, 1993).

LIM domains, Cys-rich motifs containing approximately 50 amino acids found in a variety of proteins, are proposed to direct protein-protein interactions. Enigma contains three LIM domains within its carboxyl terminus and LIM3 of Enigma specifically recognizes active but not mutant endocytic codes of the insulin receptor (InsR). Interaction of two random peptide libraries with glutathione S-transferase-LIM3 of Enigma indicates specific binding to Gly-Pro-Hyd-Gly-Pro-Hyd-Tyr-Ala corresponding to the major endocytic code of InsR. Peptide competition demonstrates that both Pro and Tyr residues are required for specific interaction of InsR with Enigma. In contrast to LIM3 of Enigma binding to InsR, LIM2 of Enigma associates specifically with the receptor tyrosine kinase, Ret. Ret is specific for LIM2 of Enigma and does not bind other LIM domains tested. The residues responsible for binding to Enigma are localized to the carboxyl-terminal 61 amino acids of Ret. A peptide corresponding to the carboxyl-terminal 20 amino acids of Ret dissociate Enigma and Ret complexes, while a mutant that changes Asn-Lys-Leu-Tyr in the peptide to Ala-Lys-Leu-Ala or a peptide corresponding to exon16 of InsR fails to disrupt the complexes, indicating the Asn-Lys-Leu-Tyr sequence of Ret is essential to the recognition motif for LIM2 of Enigma. LIM domains of Enigma recognize tyrosine-containing motifs with specificity residing in both the LIM domains and in the target structures (Wu, 1996).

Phenotypic effects of an artificial LIM-domains-only protein

Overexpression of the LIM-domains-only truncated form of the Islet-3 LIM/homeodomain protein of zebrafish induces ocular and cerebellar defects. Islet-3 is expressed specifically in the eyes and the presumptive tectum of the zebrafish. Overexpression of Islet-3 LIM-domains-only protein prevents formation of the optic vesicles and causes abnormal termination of the expression of wnt1, engrailed2 and pax2 in the mesencephalic and metencephalic region between 14 hours and 20 hours post-fertilization. Between 20 and 26 hours such overexpression severely impairs morphogenetic movement in this region, activity which would normally lead to formation of the cerebellar primordium. Such defects are all rescued by simultaneous overexpression of Islet-3, suggesting that the overexpressed LIM domains act as a specific dominant-negative variant of Islet-3. The phenotypic defects of cells overexpressing Islet-3 LIM-domains-only protein can be rescued by forming mosaic embryos (transplanting normal cells in the mesencephalic and metencephalic region). Normal embryos made mosaic by injecting cells overexpressing LIM-domains-only protein show a complete exclusion of overexpressing cells from the optic vesicles, but incorporation of cells in the mesencephalic-metencephalic region. The results show that neighboring normal cells can rescue the phenotypic defect of cells overexpressing the LIM-domains-only protein, suggesting a possibility that the Isl-3 LIM-domains-only protein may interfere with levels of expression of a secreted signaling molecule that is essential for normal differention of these regions but does not impair the reception of this signal. Initiation and maintenance of expression of wnt1, pax2 and eng2 is essential for normal development of the mes and met regions. The zebrafish embryos overexpressing Isl-3 LIM-domains-only protein phenotypically resemble mouse embryos carrying a mild Wnt1 mutant allele that selectively deletes the cerebellum while leaving the midbrain relatively intact in morphology. It is thought that Islet-3 is required for the Wnt1 signal in zebrafish. These data suggest that Islet-3 functions to promote evagination of the optic vesicles and to maintain reciprocal interaction between the mesencephalon and the mesencephalic-metencephalic boundary essential for normal development of this region (Kikuchi, 1997)

Muscle LIM protein at 60A: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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