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1.  The Drosophila Ral Gtpase Regulates Developmental Cell Shape Changes through the Jun Nh2-Terminal Kinase Pathway 
The Journal of Cell Biology  1999;146(2):361-372.
The Ral GTPase is activated by RalGDS, which is one of the effector proteins for Ras. Previous studies have suggested that Ral might function to regulate the cytoskeleton; however, its in vivo function is unknown. We have identified a Drosophila homologue of Ral that is widely expressed during embryogenesis and imaginal disc development. Two mutant Drosophila Ral (DRal) proteins, DRalG20V and DRalS25N, were generated and analyzed for nucleotide binding and GTPase activity. The biochemical analyses demonstrated that DRalG20V and DRalS25N act as constitutively active and dominant negative mutants, respectively. Overexpression of the wild-type DRal did not cause any visible phenotype, whereas DRalG20V and DRalS25N mutants caused defects in the development of various tissues including the cuticular surface, which is covered by parallel arrays of polarized structures such as hairs and sensory bristles. The dominant negative DRal protein caused defects in the development of hairs and bristles. These phenotypes were genetically suppressed by loss of function mutations of hemipterous and basket, encoding Drosophila Jun NH2-terminal kinase kinase (JNKK) and Jun NH2-terminal kinase (JNK), respectively. Expression of the constitutively active DRal protein caused defects in the process of dorsal closure during embryogenesis and inhibited the phosphorylation of JNK in cultured S2 cells. These results indicate that DRal regulates developmental cell shape changes through the JNK pathway.
PMCID: PMC3206575  PMID: 10427090
bristle; dorsal closure; hair; Jun NH2-terminal kinase; Ral
2.  The Patched dependence receptor triggers apoptosis through a DRAL-caspase-9 complex 
Nature Cell Biology  2009;11(6):739-746.
Sonic hedgehog (Shh) and its main receptor Patched (Ptc) are implicated in both neural development and tumorigenesis1, 2. Beside the classic morphogen activity of Shh, Shh is also a survival factor3, 4. Along this line, Ptc has been shown to function as a dependence receptor, inducing apoptosis in the absence of Shh, while its pro-apoptotic activity is blocked in Shh presence5. Here we show that, in the absence of its ligand, Ptc interacts with the adaptor protein DRAL/FHL2. DRAL/FHL2 is required for the pro-apoptotic activity of Ptc both in immortalized cells and during neural tube development in chick embryo. We demonstrate that, in the absence of Shh, Ptc recruits a protein complex that includes DRAL, the CARD containing domain proteins TUCAN or NALP1 and the apical caspase-9. Ptc triggers caspase-9 activation and enhances cell death via a caspase-9-dependent mechanism. Thus, we propose that, upon absence of its ligand Shh, the dependence receptor Ptc serves as the anchor for a caspase-activating complex that includes DRAL, a CARD domain containing protein and caspase-9.
doi:10.1038/ncb1880
PMCID: PMC2844407  PMID: 19465923
Adaptor Proteins, Signal Transducing; genetics; metabolism; Animals; Apoptosis; physiology; Apoptosis Regulatory Proteins; genetics; metabolism; CARD Signaling Adaptor Proteins; genetics; metabolism; Caspase 9; metabolism; Cell Line; Chick Embryo; Hedgehog Proteins; genetics; metabolism; Homeodomain Proteins; genetics; metabolism; Humans; Multiprotein Complexes; metabolism; Muscle Proteins; genetics; metabolism; Neoplasm Proteins; genetics; metabolism; RNA, Small Interfering; genetics; metabolism; Receptors, Cell Surface; genetics; metabolism; Signal Transduction; physiology; Transcription Factors; genetics; metabolism; Two-Hybrid System Techniques
3.  The LIM-Only Protein FHL2 Reduces Vascular Lesion Formation Involving Inhibition of Proliferation and Migration of Smooth Muscle Cells 
PLoS ONE  2014;9(4):e94931.
The LIM-only protein FHL2, also known as DRAL or SLIM3, has a function in fine-tuning multiple physiological processes. FHL2 is expressed in the vessel wall in smooth muscle cells (SMCs) and endothelial cells and conflicting data have been reported on the regulatory function of FHL2 in SMC phenotype transition. At present the function of FHL2 in SMCs in vascular injury is unknown. Therefore, we studied the role of FHL2 in SMC-rich lesion formation. In response to carotid artery ligation FHL2-deficient (FHL2-KO) mice showed accelerated lesion formation with enhanced Ki67 expression compared with wild-type (WT)-mice. Consistent with these findings, cultured SMCs from FHL2-KO mice showed increased proliferation through enhanced phosphorylation of extracellular-regulated kinase-1/2 (ERK1/2) and induction of CyclinD1 expression. Overexpression of FHL2 in SMCs inhibited CyclinD1 expression and CyclinD1-knockdown blocked the enhanced proliferation of FHL2-KO SMCs. We also observed increased CyclinD1 promoter activity in FHL2-KO SMCs, which was reduced upon ERK1/2 inhibition. Furthermore, FHL2-KO SMCs showed enhanced migration compared with WT SMCs. In conclusion, FHL2 deficiency in mice results in exacerbated SMC-rich lesion formation involving increased proliferation and migration of SMCs via enhanced activation of the ERK1/2-CyclinD1 signaling pathway.
doi:10.1371/journal.pone.0094931
PMCID: PMC3988136  PMID: 24736599
4.  The LIM-only protein FHL2 interacts with β-catenin and promotes differentiation of mouse myoblasts 
The Journal of Cell Biology  2002;159(1):113-122.
FHL2 is a LIM-domain protein expressed in myoblasts but down-regulated in malignant rhabdomyosarcoma cells, suggesting an important role of FHL2 in muscle development. To investigate the importance of FHL2 during myoblast differentiation, we performed a yeast two-hybrid screen using a cDNA library derived from myoblasts induced for differentiation. We identified β-catenin as a novel interaction partner of FHL2 and confirmed the specificity of association by direct in vitro binding tests and coimmunoprecipitation assays from cell lysates. Deletion analysis of both proteins revealed that the NH2-terminal part of β-catenin is sufficient for binding in yeast, but addition of the first armadillo repeat is necessary for binding FHL2 in mammalian cells, whereas the presence of all four LIM domains of FHL2 is needed for the interaction. Expression of FHL2 counteracts β-catenin–mediated activation of a TCF/LEF-dependent reporter gene in a dose-dependent and muscle cell–specific manner. After injection into Xenopus embryos, FHL2 inhibited the β-catenin–induced axis duplication. C2C12 mouse myoblasts stably expressing FHL2 show increased myogenic differentiation reflected by accelerated myotube formation and expression of muscle-specific proteins. These data imply that FHL2 is a muscle-specific repressor of LEF/TCF target genes and promotes myogenic differentiation by interacting with β-catenin.
doi:10.1083/jcb.200202075
PMCID: PMC2173499  PMID: 12370240
FHL2; repression of transcription; DRAL; myogenic differentiation; β-catenin–TCF complex
5.  Identification of an FHL1 protein complex containing ACTN1, ACTN4, and PDLIM1 using affinity purifications and MS-based protein–protein interaction analysis† 
Molecular bioSystems  2011;7(4):1185-1196.
Introduction
Four and a half LIM domains protein 1 (FHL1) is the most widely expressed member of the FHL family of proteins, consisting of four and a half highly conserved LIM domains. A multifunctional and integral role for FHL1 has been implicated in muscle development, structural maintenance, and signaling. To date, 27 FHL1 mutations have been identified that result in at least six different X-linked myopathies, with patients often presenting with cardiovascular complications. Since proteins assemble into dynamic complexes within the cell, FHL1 likely mediates its biological functions in conjunction with other proteins. Delineation of FHL1 interactions could provide insight into its regulatory functions.
Methods
We performed tandem affinity purification from human embryonic kidney 293 (HEK-293) cells to purify FHL1 and interacting proteins. To identify the potential interactors of FHL1 we performed a total of 9 different purifications from HEK-293 cells which included 3 experimental replicates for each biological condition: FHL1, tag control (DPYSL3), and negative control (empty vector). Purified samples were analyzed by liquid chromatography mass spectrometry (LC-MS). Potential interactors were then verified by immunoprecipitation from mouse heart ventricles and interactions visualized in adult cardiomyocytes using 3D fluorescence microscopy.
Results
We identified a total of 310 different proteins from all 9 purifications and by applying stringent filtering criteria we eliminated all proteins found in any of the controls and only allowed those that were detected in two or more bait purification. We identified 34 high confidence potential binding partners of FHL1. We then showed that FHL1 exists as part of a complex that binds with PDLIM1, GSN and ACTN1.
doi:10.1039/c0mb00235f
PMCID: PMC3711787  PMID: 21246116 CAMSID: cams3057
6.  An amphioxus LIM-homeobox gene, AmphiLim1/5, expressed early in the invaginating organizer region and later in differentiating cells of the kidney and central nervous system 
A LIM-homeobox gene, AmphiLim1/5, from the Florida amphioxus (Branchiostoma floridae) encodes a protein that phylogenetic analysis positions at the base of a clade comprising vertebrate Lim1 and Lim5. Amphioxus AmphiLim1/5 is expressed in domains that are a composite of those of vertebrate Lim1 and Lim5, which evidently underwent subfunctionalization after duplication of an ancestral protochordate Lim1/5. During amphioxus development, transcription is first detected in the ectoderm of the blastula. Then, in the gastrula, a second expression domain appears in the mesendoderm just within the dorsal lip of the blastopore, a region known to have organizer properties in amphioxus. This mesendodermal expression corresponds to Lim1 expression in the Spemann organizer of vertebrates. At least one of the functions of vertebrate Lim1 in the organizer is to control the transcription of genes involved in cell and tissue movements during gastrulation, and a comparable early function seems likely for AmphiLim1/5 during gastrular invagination of amphioxus. Later embryos and larvae of amphioxus express AmphiLim1/5 in clusters of cells, probably motoneurons, in the anterior part of the central nervous system, in the hindgut, in Hatschek's right diverticulum (a rudiment of the rostral coelom), and in the wall of the first somite on the left side (a precursor of Hatschek's nephridium). In the early larva, expression continues in neural cells, in Hatschek's nephridium, in the wall of the rostral coelom, in the epidermis of the upper lip, and in mesoderm cells near the opening of the second gill slit. The developmental expression in Hatschek's nephridium is especially interesting because it helps support the homology between this amphioxus organ and the vertebrate pronephros.
PMCID: PMC1458433  PMID: 16763670
Spemann organizer; kidney; brain; cephalochordate; lancelet
7.  The Dictyostelium LIM Domain-containing Protein LIM2 Is Essential for Proper Chemotaxis and Morphogenesis 
Molecular Biology of the Cell  2000;11(4):1275-1291.
We have identified limB, a gene encoding a novel LIM domain-containing protein, LIM2, in a screen for genes required for morphogenesis. limB null cells aggregate, although poorly, but they are unable to undergo morphogenesis, and the aggregates arrest at the mound stage. limB null cells exhibit an aberrant actin cytoskeleton and have numerous F-actin–enriched microspikes. The cells exhibit poor adhesion to a substratum and do not form tight cell–cell agglomerates in suspension. Furthermore, limB null cells are unable to properly polarize in chemoattractant gradients and move very poorly. Expression of limB from a prestalk-specific but not a prespore-specific promoter complements the morphogenetic defects of the limB null strain, suggesting that the limB null cell developmental defect results from an inability to properly sort prestalk cells. LIM2 protein is enriched in the cortex of wild-type cells, although it does not colocalize with the actin cytoskeleton. Our analysis indicates that LIM2 is a new regulatory protein that functions to control rearrangements of the actin cytoskeleton and is required for cell motility and chemotaxis. Our findings may be generally applicable to understanding pathways that control cell movement and morphogenesis in all multicellular organisms. Structure function studies on the LIM domains are presented.
PMCID: PMC14846  PMID: 10749929
8.  Regulation of Estrogen-Dependent Transcription by the LIM Cofactors CLIM and RLIM in Breast Cancer 
Cancer research  2009;69(1):128-136.
Mammary oncogenesis is profoundly influenced by signaling pathways controlled by Estrogen Receptor-alpha (ERα). Although it is known that ERα exerts its oncogenic effect by stimulating the proliferation of many human breast cancers through the activation of target genes, our knowledge of the underlying transcriptional mechanisms remains limited. Our published work has shown that the in vivo activity of LIM homeodomain transcription factors (LIM-HDs) is critically regulated by Cofactors of LIM-HD proteins (CLIM) and the ubiquitin ligase RING finger LIM domain interacting protein (RLIM). Here, we identify CLIM and RLIM as novel ERα cofactors that co-localize and interact with ERα in primary human breast tumors. We show that both cofactors associate with estrogen responsive promoters and regulate the expression of endogenous ERα target genes in breast cancer cells. Surprisingly, our results indicate opposing functions of LIM cofactors for ERα and LIM-HDs: whereas CLIM enhances transcriptional activity of LIM-HDs, it inhibits transcriptional activation mediated by ERα on most target genes in vivo. In turn, the ubiquitin ligase RLIM inhibits transcriptional activity of LIM-HDs, but enhances transcriptional activation of endogenous ERα target genes. Results from a human breast cancer tissue microarray (TMA) of 1,335 patients revealed a highly significant correlation of elevated CLIM levels to ER/PR positivity and poor differentiation of tumors. Combined, these results indicate that LIM cofactors CLIM and RLIM regulate the biological activity of ERα during the development of human breast cancer.
doi:10.1158/0008-5472.CAN-08-1630
PMCID: PMC2713826  PMID: 19117995
Breast cancer; mammary gland; transcriptional regulation; cofactors; Estrogen; Estrogen receptor α; LIM cofactors; CLIM; RLIM
9.  Four and a Half LIM Protein 1C (FHL1C): A Binding Partner for Voltage-Gated Potassium Channel Kv1.5 
PLoS ONE  2011;6(10):e26524.
Four-and-a-half LIM domain protein 1 isoform A (FHL1A) is predominantly expressed in skeletal and cardiac muscle. Mutations in the FHL1 gene are causative for several types of hereditary myopathies including X-linked myopathy with postural muscle atrophy (XMPMA). We here studied myoblasts from XMPMA patients. We found that functional FHL1A protein is completely absent in patient myoblasts. In parallel, expression of FHL1C is either unaffected or increased. Furthermore, a decreased proliferation rate of XMPMA myoblasts compared to controls was observed but an increased number of XMPMA myoblasts was found in the G0/G1 phase. Furthermore, low expression of Kv1.5, a voltage-gated potassium channel known to alter myoblast proliferation during the G1 phase and to control repolarization of action potential, was detected. In order to substantiate a possible relation between Kv1.5 and FHL1C, a pull-down assay was performed. A physical and direct interaction of both proteins was observed in vitro. In addition, confocal microscopy revealed substantial colocalization of FHL1C and Kv1.5 within atrial cells, supporting a possible interaction between both proteins in vivo. Two-electrode voltage clamp experiments demonstrated that coexpression of Kv1.5 with FHL1C in Xenopus laevis oocytes markedly reduced K+ currents when compared to oocytes expressing Kv1.5 only. We here present the first evidence on a biological relevance of FHL1C.
doi:10.1371/journal.pone.0026524
PMCID: PMC3203871  PMID: 22053194
10.  Genome-Wide Analysis of LIM Gene Family in Populus trichocarpa, Arabidopsis thaliana, and Oryza sativa 
Abstract
In Eukaryotes, LIM proteins act as developmental regulators in basic cellular processes such as regulating the transcription or organizing the cytoskeleton. The LIM domain protein family in plants has mainly been studied in sunflower and tobacco plants, where several of its members exhibit a specific pattern of expression in pollen. In this paper, we finely characterized in poplar six transcripts encoding these proteins. In Populus trichocarpa genome, the 12 LIM gene models identified all appear to be duplicated genes. In addition, we describe several new LIM domain proteins deduced from Arabidopsis and rice genomes, raising the number of LIM gene models to six for both species. Plant LIM genes have a core structure of four introns with highly conserved coding regions. We also identified new LIM domain proteins in several other species, and a phylogenetic analysis of plant LIM proteins reveals that they have undergone one or several duplication events during the evolution. We gathered several LIM protein members within new monophyletic groups. We propose to classify the plant LIM proteins into four groups: αLIM1, βLIM1, γLIM2, and δLIM2, subdivided according to their specificity to a taxonomic class and/or to their tissue-specific expression. Our investigation of the structure of the LIM domain proteins revealed that they contain many conserved motifs potentially involved in their function.
doi:10.1093/dnares/dsm013
PMCID: PMC2779900  PMID: 17573466
poplar; Arabidopsis; rice; LIM domain protein; tension wood
11.  Inelastic neutron scattering study on bioprotectant systems 
We collected inelastic neutron scattering (INS) spectra of homologous disaccharide (C12H22O11)/H2O mixtures at a very low temperature by using indirect geometry time-of-flight spectrometer TOSCA at the ISIS pulse neutron facility (DRAL, UK). The aim of this work is to investigate the vibrational behaviour of trehalose, maltose and sucrose/H2O mixtures with INS in order to characterize the structural changes induced by these disaccharides on the H2O hydrogen-bonded network. A higher degree of ‘crystallinity’ for the trehalose/H2O system is observed in the vibrational region corresponding to the ice bending modes. This feature could justify the better cryptobiotic action of trehalose compared with maltose and sucrose. On the other hand, the better bioprotective effectiveness could be explained by the higher destructuring effect of trehalose, emphasized by the analysis of the librational modes region.
doi:10.1098/rsif.2005.0059
PMCID: PMC1618500  PMID: 16849211
disaccharides; bioprotectants; water mixtures; vibrational properties; neutron scattering
12.  Biological Potential of Sixteen Legumes in China 
Phenolic acids have been identified in a variety of legumes including lima bean, broad bean, common bean, pea, jack bean, goa bean, adzuki bean, hyacinth bean, chicking vetch, garbanzo bean, dral, cow bean, rice bean, mung bean and soybean. The present study was carried out with the following aims: (1) to identify and quantify the individual phenolic acid and determine the total phenolic content (TPC); (2) to assess their antioxidant activity, inhibition activities of α-glucosidase, tyrosinase, and formation of advanced glycation endproducts; and (3) to investigate correlations among the phytochemicals and biological activity. Common bean possesses the highest antioxidant activity and advanced glycation endproducts formation inhibition activity. Adzuki bean has the highest α-glucosidase inhibition activity, and mung bean has the highest tyrosinase inhibition activity. There are significant differences in phytochemical content and functional activities among the bean species investigated. Selecting beans can help treat diseases such as dermatological hyperpigmentation illness, type 2 diabetes and associated cardiovascular diseases.
doi:10.3390/ijms12107048
PMCID: PMC3211026  PMID: 22072935
legumes; antioxidant; α-glucosidase inhibition; advanced glycation endproducts; tyrosinase inhibition
13.  Concentration dependence of vibrational properties of bioprotectant/water mixtures by inelastic neutron scattering 
Neutron scattering has been demonstrated to be a powerful tool for characterizing the structure and dynamics of biological molecules and for investigating the physical and chemical mechanisms of biophysical processes. The aim of the present work is to investigate by inelastic neutron scattering (INS) the vibrational behaviour of a class of bioprotectant systems, such as homologous disaccharides, trehalose, maltose and sucrose, in water mixtures. INS measurements have been performed on trehalose/H2O, maltose/H2O and sucrose/H2O mixtures at very low temperature as a function of concentration by using the thermal original spectrometer with cylindrical analyzers (TOSCA) spectrometer at the ISIS Facility (DRAL, UK).
The findings allow the analyses of the vibrational features of the INS spectra in order to study the effect of disaccharides on the H2O hydrogen-bonded tetrahedral network. The obtained neutron scattering findings point out that disaccharides, and in particular trehalose, have a destructuring effect on the water tetrahedral network, as emphasized by the analysis of the librational modes region from 50 to 130 meV energy transfer. On the other hand, the analysis of the bending modes region (130–225  meV) shows a locally ordered structure in the disaccharide/H2O mixtures.
Finally, the observed experimental evidences are linked to the different bioprotective effectiveness of disaccharides as a function of concentration.
doi:10.1098/rsif.2006.0162
PMCID: PMC2358967  PMID: 17018423
disaccharides; bioprotection; water mixtures; vibrational properties; neutron scattering
14.  Acetylation of FoxO1 Activates Bim Expression to Induce Apoptosis in Response to Histone Deacetylase Inhibitor Depsipeptide Treatment12 
Neoplasia (New York, N.Y.)  2009;11(4):313-324.
Histone deacetylase (HDAC) inhibitors have been shown to induce cell cycle arrest and apoptosis in cancer cells. However, the mechanisms of HDAC inhibitor induced apoptosis are incompletely understood. In this study, depsipeptide, a novel HDAC inhibitor, was shown to be able to induce significant apoptotic cell death in human lung cancer cells. Further study showed that Bim, a BH3-only proapoptotic protein, was significantly upregulated by depsipeptide in cancer cells, and Bim's function in depsipeptide-induced apoptosis was confirmed by knockdown of Bim with RNAi. In addition, we found that depsipeptide-induced expression of Bim was directly dependent on acetylation of forkhead box class O1 (FoxO1) that is catalyzed by cyclic adenosine monophosphate-responsive element-binding protein-binding protein, and indirectly induced by a decreased four-and-a-half LIM-domain protein 2. Moreover, our results demonstrated that FoxO1 acetylation is required for the depsipeptide-induced activation of Bim and apoptosis, using transfection with a plasmid containing FoxO1 mutated at lysine sites and a luciferase reporter assay. These data show for the first time that an HDAC inhibitor induces apoptosis through the FoxO1 acetylation-Bim pathway.
PMCID: PMC2657887  PMID: 19308286
15.  Regulation of cell migration and survival by focal adhesion targeting of Lasp-1 
The Journal of Cell Biology  2004;165(3):421-432.
Large-scale proteomic and functional analysis of isolated pseudopodia revealed the Lim, actin, and SH3 domain protein (Lasp-1) as a novel protein necessary for cell migration, but not adhesion to, the extracellular matrix (ECM). Lasp-1 is a ubiquitously expressed actin-binding protein with a unique domain configuration containing SH3 and LIM domains, and is overexpressed in 8–12% of human breast cancers. We find that stimulation of nonmotile and quiescent cells with growth factors or ECM proteins facilitates Lasp-1 relocalization from the cell periphery to the leading edge of the pseudopodium, where it associates with nascent focal complexes and areas of actin polymerization. Interestingly, although Lasp-1 dynamics in migratory cells occur independently of c-Abl kinase activity and tyrosine phosphorylation, c-Abl activation by apoptotic agents specifically promotes phosphorylation of Lasp-1 at tyrosine 171, which is associated with the loss of Lasp-1 localization to focal adhesions and induction of cell death. Thus, Lasp-1 is a dynamic focal adhesion protein necessary for cell migration and survival in response to growth factors and ECM proteins.
doi:10.1083/jcb.200311045
PMCID: PMC2172195  PMID: 15138294
cell migration; apoptosis; signal transduction; focal adhesions; Abl tyrosine kinase
16.  The LIM Protein Zyxin Binds CARP-1 and Promotes Apoptosis 
Genes & cancer  2010;1(5):506-515.
Zyxin is a dual-function LIM domain protein that regulates actin dynamics in response to mechanical stress and shuttles between focal adhesions and the cell nucleus. Here we show that zyxin contributes to UV-induced apoptosis. Exposure of wild-type fibroblasts to UV-C irradiation results in apoptotic cell death, whereas cells harboring a homozygous disruption of the zyxin gene display a statistically significant survival advantage. To gain insight into the molecular mechanism by which zyxin promotes apoptotic signaling, we expressed an affinity-tagged zyxin variant in zyxin-null cells and isolated zyxin-associated proteins from cell lysates under physiological conditions. A 130-kDa protein that was co-isolated with zyxin was identified by microsequence analysis as the Cell Cycle and Apoptosis Regulator Protein-1 (CARP-1). CARP-1 associates with the LIM region of zyxin. Zyxin lacking the CARP-1 binding region shows reduced proapoptotic activity in response to UV-C irradiation. We demonstrate that CARP-1 is a nuclear protein. Zyxin is modified by phosphorylation in cells exposed to UV-C irradiation, and nuclear accumulation of zyxin is induced by UV-C exposure. These findings highlight a novel mechanism for modulating the apoptotic response to UV irradiation.
doi:10.1177/1947601910376192
PMCID: PMC2941191  PMID: 20852740
zyxin; CARP-1; apoptosis
17.  The LIM Protein Zyxin Binds CARP-1 and Promotes Apoptosis 
Genes & Cancer  2010;1(5):506-515.
Zyxin is a dual-function LIM domain protein that regulates actin dynamics in response to mechanical stress and shuttles between focal adhesions and the cell nucleus. Here we show that zyxin contributes to UV-induced apoptosis. Exposure of wild-type fibroblasts to UV-C irradiation results in apoptotic cell death, whereas cells harboring a homozygous disruption of the zyxin gene display a statistically significant survival advantage. To gain insight into the molecular mechanism by which zyxin promotes apoptotic signaling, we expressed an affinity-tagged zyxin variant in zyxin-null cells and isolated zyxin-associated proteins from cell lysates under physiological conditions. A 130-kDa protein that was co-isolated with zyxin was identified by microsequence analysis as the Cell Cycle and Apoptosis Regulator Protein-1 (CARP-1). CARP-1 associates with the LIM region of zyxin. Zyxin lacking the CARP-1 binding region shows reduced proapoptotic activity in response to UV-C irradiation. We demonstrate that CARP-1 is a nuclear protein. Zyxin is modified by phosphorylation in cells exposed to UV-C irradiation, and nuclear accumulation of zyxin is induced by UV-C exposure. These findings highlight a novel mechanism for modulating the apoptotic response to UV irradiation.
doi:10.1177/1947601910376192
PMCID: PMC2941191  PMID: 20852740
zyxin; CARP-1; apoptosis
18.  ALP/Enigma PDZ–LIM Domain Proteins in the Heart 
Actinin-associated LIM protein (ALP) and Enigma are two subfamilies of Postsynaptic density 95, discs large and zonula occludens-1 (PDZ)–Lin-11, Isl1 and Mec-3 (LIM) domain containing proteins. ALP family members have one PDZ and one LIM domain, whereas Enigma proteins contain one PDZ and three LIM domains. Four ALP and three Enigma proteins have been identified in mammals, each having multiple splice variants and unique expression patterns. Functionally, these proteins bind through their PDZ domains to α-actinin and bind through their LIM domains or other internal protein interaction domains to other proteins, including signaling molecules. ALP and Enigma proteins have been implicated in cardiac and skeletal muscle structure, function and disease, neuronal function, bipolar disorder, tumor growth, platelet and epithelial cell motility and bone formation. This review will focus on recent advances in the biological roles of ALP/Enigma PDZ–LIM domain proteins in cardiac muscle and provide insights into mechanisms by which mutations in these proteins are related to human cardiac disease.
doi:10.1093/jmcb/mjp038
PMCID: PMC2905065  PMID: 20042479
PDZ; LIM; cypher; ZASP; cardiomyopathy; Z-disc
19.  Molecular Characterization of abLIM, a Novel Actin-binding and Double Zinc Finger Protein  
The Journal of Cell Biology  1997;138(3):575-588.
Molecules that couple the actin-based cytoskeleton to intracellular signaling pathways are central to the processes of cellular morphogenesis and differentiation. We have characterized a novel protein, the actin-binding LIM (abLIM) protein, which could mediate such interactions between actin filaments and cytoplasmic targets. abLIM protein consists of a COOH-terminal cytoskeletal domain that is fused to an NH2-terminal domain consisting of four double zinc finger motifs. The cytoskeletal domain is ∼50% identical to erythrocyte dematin, an actin-bundling protein of the red cell membrane skeleton, while the zinc finger domains conform to the LIM motif consensus sequence. In vitro expression studies demonstrate that abLIM protein can bind to F-actin through the dematin-like domain. Transcripts corresponding to three distinct isoforms have a widespread tissue distribution. However, a polypeptide corresponding to the full-length isoform is found exclusively in the retina and is enriched in biochemical extracts of retinal rod inner segments. abLIM protein also undergoes extensive phosphorylation in light-adapted retinas in vivo, and its developmental expression in the retina coincides with the elaboration of photoreceptor inner and outer segments. Based on the composite primary structure of abLIM protein, actin-binding capacity, potential regulation via phosphorylation, and isoform expression pattern, we speculate that abLIM may play a general role in bridging the actin-based cytoskeleton with an array of potential LIM protein-binding partners. The developmental time course of abLIM expression in the retina suggests that the retina-specific isoform may have a specialized role in the development or elaboration of photoreceptor inner and outer segments.
PMCID: PMC2141644  PMID: 9245787
20.  Ectopic Expression of LIM-nebulette (LASP2) reveals roles in cell migration and spreading 
Cell motility and the cytoskeleton  2008;65(10):827-840.
LIM-nebulette (LASP2) is a small focal adhesion protein and a member of the nebulin family of actin binding proteins. This recently identified splice variant of the nebulette locus is widely expressed and highly enriched in neuronal tissue. Other than the facts that LIM-nebulette is a focal adhesion protein and interacts with zyxin, nothing is known about its function. Given that LIM-nebulette has an identical modular organization and overlapping tissue distributions to that of LASP1, we have analyzed the role of LIM-nebulette in comparison with that of LASP1. We find that LIM-nebulette is a dynamic focal adhesion protein that increases the rate of attachment and spreading of fibroblasts on fibronectin coated surfaces. Additionally, LIM-nebulette is recruited from the cortical cytoskeleton in non-motile cells to focal adhesions at the leading edge of stimulated cells. In confluent cultures of HeLa and NIH3T3 cells, LIM-nebulette co-localizes with α-catenin in putative adherens junctions, whereas LASP1 is devoid of these areas. Interestingly, overexpression of LIM-nebulette in PC6 cells inhibits neurite outgrowth in response to growth factors. Collectively, our data indicate that LIM-nebulette and LASP1 have distinct roles in the actin cytoskeleton.
doi:10.1002/cm.20304
PMCID: PMC2603443  PMID: 18677772
Nebulin; actin binding proteins; focal adhesions; zyxin; neurite outgrowth
21.  JNK1 determines the oncogenic or tumor-suppressive activity of the integrin-linked kinase in human rhabdomyosarcoma 
The Journal of Clinical Investigation  2009;119(6):1558-1570.
Although most reports describe the protein kinase integrin-linked kinase (ILK) as a proto-oncogene, occasional studies detail opposing functions in the regulation of normal and transformed cell proliferation, differentiation, and apoptosis. Here, we demonstrated that ILK functions as an oncogene in the highly aggressive pediatric sarcoma alveolar rhabdomyosarcoma (ARMS) and as a tumor suppressor in the related embryonal rhabdomyosarcoma (ERMS). These opposing functions hinge on signaling through a noncanonical ILK target, JNK1, to the proto-oncogene c-Jun. RNAi-mediated depletion of ILK induced activation of JNK and its target, c-Jun, resulting in growth of ERMS cells, whereas in ARMS cells, it led to loss of JNK/c-Jun signaling and suppression of growth both in vitro and in vivo. Ectopic expression of the fusion gene characteristic of ARMS (paired box 3–forkhead homolog in rhabdomyosarcoma [PAX3-FKHR]) in ERMS cells was sufficient to convert them to an ARMS signaling phenotype and render ILK activity oncogenic. Furthermore, restoration of JNK1 in ARMS reestablished a tumor-suppressive function for ILK. These findings indicate what we believe to be a novel effector pathway regulated by ILK, provide a mechanism for interconversion of oncogenic and tumor-suppressor functions of a single regulatory protein based on the genetic background of the tumor cells, and suggest a rationale for tailored therapy of rhabdomyosarcoma based on the different activities of ILK.
doi:10.1172/JCI37958
PMCID: PMC2689127  PMID: 19478459
22.  Serine and Threonine Phosphorylation of the Paxillin LIM Domains Regulates Paxillin Focal Adhesion Localization and Cell Adhesion to Fibronectin 
Molecular Biology of the Cell  1998;9(7):1803-1816.
We have previously shown that the LIM domains of paxillin operate as the focal adhesion (FA)-targeting motif of this protein. In the current study, we have identified the capacity of paxillin LIM2 and LIM3 to serve as binding sites for, and substrates of serine/threonine kinases. The activities of the LIM2- and LIM3-associated kinases were stimulated after adhesion of CHO.K1 cells to fibronectin; consequently, a role for LIM domain phosphorylation in regulating the subcellular localization of paxillin after adhesion to fibronectin was investigated. An avian paxillin-CHO.K1 model system was used to explore the role of paxillin phosphorylation in paxillin localization to FAs. We found that mutations of paxillin that mimicked LIM domain phosphorylation accelerated fibronectin-induced localization of paxillin to focal contacts. Further, blocking phosphorylation of the LIM domains reduced cell adhesion to fibronectin, whereas constitutive LIM domain phosphorylation significantly increased the capacity of cells to adhere to fibronectin. The potentiation of FA targeting and cell adhesion to fibronectin was specific to LIM domain phosphorylation as mutation of the amino-terminal tyrosine and serine residues of paxillin that are phosphorylated in response to fibronectin adhesion had no effect on the rate of FA localization or cell adhesion. This represents the first demonstration of the regulation of protein localization through LIM domain phosphorylation and suggests a novel mechanism of regulating LIM domain function. Additionally, these results provide the first evidence that paxillin contributes to “inside-out” integrin-mediated signal transduction.
PMCID: PMC25420  PMID: 9658172
23.  FHL2 (SLIM3) Is Not Essential for Cardiac Development and Function 
Molecular and Cellular Biology  2000;20(20):7460-7462.
LIM domain-containing proteins play critical roles in vertebrate development and cellular differentiation. Recently, four members of the four and one-half LIM protein (FHL) family have been identified and cloned. One of these, FHL2, is expressed in a restricted manner in the cardiovascular system throughout development into adulthood, suggesting that FHL2 may play an important role in cardiovascular development and function. Here we describe the generation and analysis of mice carrying a null mutation of the FHL2 gene. FHL2-deficient mice are viable and maintain normal cardiac function both before and after acute mechanical stress induced by aortic constriction. These data suggest that FHL2 is not essential for cardiac development and function.
PMCID: PMC86299  PMID: 11003643
24.  Identification of paxilin domains interacting with β-catenin 
FEBS letters  2012;586(16):2294-2299.
Barrier-protective agonists induce association of focal adhesions (FA) and adherens junctions (AJ) in endothelial cells. Here we identified specific domains of FA protein paxillin interacting with AJ protein and examined regulation of paxillin domain interactions with β-catenin by Rac GTPase. Co-expression of paxillin LD-1,2; LD-3,4; LIM-1,2; and LIM-3,4 domains with β-catenin showed exclusive interaction of LIM-1,2 and LIM-3,4 with β-catenin, which was enhanced by agonist-induced Rac activation or expression of activated Rac mutant. These results demonstrate a novel function of paxillin LIM domains in targeting β-catenin in a Rac-dependent manner, which may play a role in Rac-dependent control of FA-AJ interactions and monolayer integrity.
doi:10.1016/j.febslet.2012.06.016
PMCID: PMC3407333  PMID: 22728435
Focal adhesions; adherens junctions; protein interactions; Rac GTPase
25.  The LIM and SH3 domain protein family: structural proteins or signal transducers or both? 
Molecular Cancer  2008;7:31.
LIM and SH3 Protein 1 (LASP-1) was initially identified from a cDNA library of metastatic axillary lymph nodes (MLN) more than a decade ago. It was found to be overexpressed in human breast and ovarian cancer and became the first member of a newly defined LIM-protein subfamily of the nebulin group characterized by the combined presence of LIM and SH3 domains. LASP2, a novel LASP1-related gene was first identified and characterized in silico. Subsequently it proved to be a splice variant of the Nebulin gene and therefore was also termed LIM/nebulette. LASP-1 and -2 are highly conserved in their LIM, nebulin-like and SH3 domains but differ significantly at their linker regions. Both proteins are ubiquitously expressed and involved in cytoskeletal architecture, especially in the organization of focal adhesions. Here we present the first systematic review to summarize all relevant data concerning their domain organization, expression profiles, regulating factors and function. We compile evidence that both, LASP-1 and LASP-2, are important during early embryo- and fetogenesis and are highly expressed in the central nervous system of the adult. However, only LASP-1 seems to participate significantly in neuronal differentiation and plays an important functional role in migration and proliferation of certain cancer cells while the role of LASP-2 is more structural. The increased expression of LASP-1 in breast tumours correlates with high rates of nodal-metastasis and refers to a possible relevance as a prognostic marker.
doi:10.1186/1476-4598-7-31
PMCID: PMC2359764  PMID: 18419822

Results 1-25 (1070393)