Lin-11, Isl-1, And Mec-3 Research Articles

Tandem LIM domain-containing proteins, LIMK1 and LMO1, directly bind to force-bearing keratin intermediate filaments

The cytoskeleton of the cell is constantly exposed to physical forces that regulate cellular functions. Selected members of the LIM (Lin-11, Isl-1, and Mec-3) domain-containing protein family accumulate along force-bearing actin fibers, with evidence supporting that the LIM domain is solely responsible for this force-induced interaction. However, LIM domain's force-induced interactions are not limited to actin. LIMK1 and LMO1, both containing only two tandem LIM domains, are recruited to force-bearing keratin fibers in epithelial cells. This unique recruitment is mediated by their LIM domains and regulated by the sequences outside the LIM domains. Based on invitro reconstitution of this interaction, LIMK1 and LMO1 directly interact with stretched keratin 8/18 fibers. These results show that LIM domain's mechano-sensing abilities extend to the keratin cytoskeleton, highlighting the diverse role of LIM proteins in force-regulated signaling.

Investigating LIM (Lin-11, Isl-1, and Mec-3) Kinases and Their Correlation with Pathological Events and Microtubule Dynamics in an Experimental Model of Spinal Cord Injury in Rats.

The spinal cord injury (SCI) and the neurodegenerative processes accompanying it follow an intricate pathway with very limited options for treatment strategies until now. Microtubules, essential for the growth and maintenance of neurons, are mostly disorganized and destabilized due to neurodegeneration. Regeneration or plasticity is restricted to the adult central nervous system (CNS) due to several intrinsic and extrinsic mechanisms. Some fundamental or injury-induced expressions of specific molecules can be inhibited or antagonized pharmacologically to protect neurons to a certain extent after neurodegeneration. Accordingly, these molecules offer an excellent target as a therapeutic approach to promote neuroprotection. LIM kinases (LIMKs) are one of these molecules that phosphorylates members of the actin-depolymerizing factor (ADF)/cofilin family of actin-binding and filament-severing proteins. The individual role of LIMKs has not yet been studied in the pathology of SCI. In this study, we targeted LIMK and checked its role in microtubule destabilization in vitro. LIMK1 was found to be upregulated after microtubule depolymerization and inhibition of LIMK with specific inhibitor-protected neurons. Then, we checked the expressions of individual LIMKs throughout different time points across SCI in a rat contusion model, correlating with established pathophysiological markers. The phosphorylated form of LIMK1 was found to be elevated at chronic time points after injury, where scar formation and diminution of neurons prevail. Finally, we targeted the LIMK pathway with its specific inhibitor BMS-5, which showed neuroprotection after SCI. Overall, our results provided a concept concerning how a small-molecule inhibitor of LIMK may offer a strategy to treat SCI-associated neurodegeneration.

HSP90 Contributes to chs3-2D-Mediated Autoimmunity.

Plants employ multi-layered immune system to fight against pathogen infections. Different receptors are able to detect the invasion activities of pathogens, transduce signals to downstream components, and activate defense responses. Among those receptors, nucleotide-binding domain leucine-rich repeat containing proteins (NLRs) are the major intracellular ones. CHILLING SENSITIVE 3 (CHS3) is an Arabidopsis NLR with an additional Lin-11, Isl-1 and Mec-3 (LIM) domain at its C terminus. The gain-of-function mutant, chs3-2D, exhibiting severe dwarfism and constitutively activated defense responses, was selected as a genetic background in this study for a forward genetic screen. A mutant allele of hsp90.2 was isolated as a partial suppressor of chs3-2D, suggesting that HSP90 is required for CHS3-mediated defense signaling. In addition, HSP90 is also required for the autoimmunity of the Dominant Negative (DN)-SNIPER1 and gain-of-function ADR1-L2 D484V transgenic lines, suggesting a broad role for HSP90 in NLR-mediated defense. Overall, our work indicates a larger contribution of HSP90 not only at the sensor, but also the helper NLR levels.

Regulation and Signaling of TGF-β Autoinduction.

Cell signaling is a vital part of biological life. It helps coordinating various cellular processes including cell survival, cell growth, cell death, and cell interaction with the microenvironment and other cells. In general, cell signaling involves the attachment of signaling molecules known as ligands to specific receptors on cell surface, which then activate downstream events that dictate the cell’s response. One of the most studied ligands is transforming growth factor-beta (TGF-β). TGF-β signaling is mainly mediated by suppressor of mothers against decapentaplegic (Smad) proteins, but it also interacts with other pathways such as the Ras and mitogen-activated protein kinase (MAPK) signaling pathways. Furthermore, TGF-β can have a dual role depending on the cellular and microenvironmental context, in which it can act as either a growth promoter or a growth inhibitor. It has been known that TGF-β can self-induce its ligand production, thereby prolonging and amplifying its effect on cells and their microenvironment. The aim of this review is to discuss the regulation and signaling of TGF-β autoinduction, which still remain to be elucidated. Several factors have been found to facilitate TGF-β autoinduction, which include the activator protein-1 (AP1) complex, Smad3-dependent signaling, and non-Smad signaling pathways. On the other hand, the LIM (Lin11, Isl-1 and Mec-3) domain only 7 (LMO7) protein can suppress TGF-β autoinduction by interfering with the activities of AP-1 and Smad3. Since TGF-β autoinduction is implicated in various pathological conditions, better understanding of its regulation and signaling can provide new directions for therapy.

Abstract 101: FHL2 -null Mice Showing Less Cardiac Remodeling

The four and a half Lin11, Isl-1 and Mec-3 (LIM) domain protein 2 (FHL2) is a member of the four and a half LIM domain-only (FHL) gene family, and has been shown to play an important role in cardiac remodeling. Here, we tested the hypothesis that isoproterenol-induced myocardial fibrosis in mice lacking FHL2 is related to resistant to myocardial fibrosis. We compared the cardiac phenotypes of FHL2 -null mice. Extra-cellular matrix remodeling and apoptosis are examined to determine the roles of FHL2 on cardiac fibrosis induced by isoproterenol. Compared to wild type littermates, FHL2 -null mice exhibited lower mortality. Masson’s Trichrome and silver stains showed significantly less severe ventricle fibrosis in FHL2-null mice. In addition, FHL2 -null mice exhibited lower levels of TGF-β, collagen I/III, but higher membrane-type matrix metalloproteinase-1 (MT1-MMP/MMP-14) during cardiac remodeling. This study shows that alternations of FHL2 might be implicated in the pathogenesis of cardiac fibrosis and suggests FHL2 as novel targets for cardiac fibrosis therapy. Further studies are required to define the mechanism by which FHL2 modulates cardiac remodeling.

Autoimmunity conferred by chs3-2D relies on CSA1, its adjacent TNL-encoding neighbour.

Plant innate immunity depends on the function of a large number of intracellular immune receptor proteins, the majority of which are structurally similar to mammalian nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) proteins. CHILLING SENSITIVE 3 (CHS3) encodes an atypical Toll/Interleukin 1 Receptor (TIR)-type NLR protein with an additional Lin-11, Isl-1 and Mec-3 (LIM) domain at its C-terminus. The gain-of-function mutant allele chs3-2D exhibits severe dwarfism and constitutively activated defense responses, including enhanced resistance to virulent pathogens, high defence marker gene expression, and salicylic acid accumulation. To search for novel regulators involved in CHS3-mediated immune signaling, we conducted suppressor screens in the chs3-2D and chs3-2D pad4-1 genetic backgrounds. Alleles of sag101 and eds1-90 were isolated as complete suppressors of chs3-2D, and alleles of sgt1b were isolated as partial suppressors of chs3-2D pad4-1. These mutants suggest that SAG101, EDS1-90, and SGT1b are all positive regulators of CHS3-mediated defense signaling. Additionally, the TIR-type NLR-encoding CSA1 locus located genomically adjacent to CHS3 was found to be fully required for chs3-2D-mediated autoimmunity. CSA1 is located 3.9 kb upstream of CHS3 and is transcribed in the opposite direction. Altogether, these data illustrate the distinct genetic requirements for CHS3-mediated defense signaling.

The Dual Functions of WLIM1a in Cell Elongation and Secondary Wall Formation in Developing Cotton Fibers

LIN-11, Isl1 and MEC-3 (LIM)-domain proteins play pivotal roles in a variety of cellular processes in animals, but plant LIM functions remain largely unexplored. Here, we demonstrate dual roles of the WLIM1a gene in fiber development in upland cotton (Gossypium hirsutum). WLIM1a is preferentially expressed during the elongation and secondary wall synthesis stages in developing fibers. Overexpression of WLIM1a in cotton led to significant changes in fiber length and secondary wall structure. Compared with the wild type, fibers of WLIM1a-overexpressing plants grew longer and formed a thinner and more compact secondary cell wall, which contributed to improved fiber strength and fineness. Functional studies demonstrated that (1) WLIM1a acts as an actin bundler to facilitate elongation of fiber cells and (2) WLIM1a also functions as a transcription factor to activate expression of Phe ammonia lyase-box genes involved in phenylpropanoid biosynthesis to build up the secondary cell wall. WLIM1a localizes in the cytosol and nucleus and moves into the nucleus in response to hydrogen peroxide. Taken together, these results demonstrate that WLIM1a has dual roles in cotton fiber development, elongation, and secondary wall formation. Moreover, our study shows that lignin/lignin-like phenolics may substantially affect cotton fiber quality; this finding may guide cotton breeding for improved fiber traits.

MicroRNA-206 is involved in hypoxia-induced pulmonary hypertension through targeting of the HIF-1α/Fhl-1 pathway

Hypoxia-induced pulmonary hypertension (PH), which is characterized by vasoconstriction and subsequent structural remodeling of blood vessels, is an important event in chronic obstructive pulmonary disease patients and in people living at high altitudes. Hypoxia-inducible factor-1α (HIF-1α) and its regulator four-and-a-half LIM (Lin-11, Isl-1 and Mec-3) domain 1 (Fhl-1) have important roles in hypoxia-induced PH. MicroRNA-206 (miR-206) is critical for myogenesis and related diseases; however, the role of miR-206 in hypoxia-induced PH is unknown. miR-206 expression was evaluated in a hypoxic rat model and in cultured hypoxic pulmonary artery smooth muscle cells (PASMCs) using real-time quantitative PCR (RT-qPCR). HIF-1α and Fhl-1 expression were evaluated using RT-qPCR, western blotting, immunohistochemistry and immunofluorescence. The function of miR-206 was assessed by transfecting miR-206 mimics and inhibitors. Dual-luciferase reporter gene assays and western blotting were performed to validate the target genes of miR-206. siRNA targeted against Fhl-1 was used to investigate the effect of Fhl-1 on miR-206. Flow cytometry was used to detect the cell cycle phase distribution in each group of PASMCs. Significant downregulation of miR-206 in hypoxic lung tissue and PASMCs was identified, whereas HIF-1α and Fhl-1 were upregulated in these samples. The expression of miR-206 in the serum was different from that in the lung tissue. Transfection of pre-miR miR-206 in hypoxic conditions led to increased expression of HIF-1α and Fhl-1 rather than abolishing hypoxia-induced HIF-1α and Fhl-1, as was expected, and promoted the entry of cells into the S phase and enhanced PASMC proliferation. Fhl-1-targeted siRNA in PASMC prevented cell proliferation and led to an increased proportion of cells in the G1 phase without altering miR-206 expression. Bioinformatic analysis and dual-luciferase reporter gene assays revealed direct evidence for miR-206 targeting of HIF-1α. In conclusion, hypoxia-induced downregulation of miR-206 promotes PH by targeting the HIF-1α/Fhl-1 pathway in PASMCs. miR-206 could be a triggering factor of early stage of hypoxia-induced PH.

Deletion of FHL2 Gene Impaired Ischemia-Induced Blood Flow Recovery by Modulating Circulating Proangiogenic Cells

The four and a half Lin11, Isl-1 and Mec-3 (LIM) domain protein 2 (FHL2) is a member of the four and a half LIM domain-only (FHL) gene family, and has been shown to play an important role in inhibiting inflammatory angiogenesis. Here, we tested the hypothesis that impaired ischemia-induced neovascularization in mice lacking FHL2 is related to a defect in proangiogenic cell mobilization and functions in vasculogenesis. Unilateral hindlimb ischemia surgery was conducted in FHL2(-/-) mice and wild-type (FHL2(+/+)) mice. After hindlimb ischemia surgery, expression of FHL2 protein was noted in ischemic tissues of wild-type mice. All FHL2-null mice (100%) suffered from spontaneous foot amputation, but only 20% of wild-type mice had ischemia-induced foot amputation after ischemic surgery. Blood flow recovery was significantly impaired in FHL2(-/-) mice when compared with that in wild-type mice as determined by laser Doppler imaging. Histological analysis revealed that the capillary density in the ischemic limb was increased in wild-type mice, whereas no such increase was noted in FHL2(-/-) mice. Flow cytometry demonstrated that the number of CD34(+) or CD34(+)/Sca-1(+)/Flk-1(+) in peripheral blood after ischemic surgery significantly decreased in FHL2-null mice than those in wild-type mice after hindlimb ischemia surgery. FHL2 deficiency impaired ex vivo angiogenesis in mouse aortic-ring culture assay, which revealed that the mean density of the microvessels was significantly higher in the wild-type aorta than in the FHL2(-/-) aorta. Western blot analysis showed that vascular endothelial growth factor (VEGF), interleukin-6, matrix metalloproteinase-2, matrix metalloproteinase-9, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 levels were significantly downregulated in ischemic muscles in FHL2-null mice compared with wild-type mice. Deletion of FHL2 protein by FHL2 small interfering RNA impaired VEGF production under hypoxia conditions, and also suppressed endothelial progenitor cell angiogenic functions, but these effects could be recovered by administration of VEGF. Deficiency of FHL2 impairs ischemia-induced neovascularization, and these suppressive effects may occur through a reduction in proangiogenic cell mobilization, migration, and vasculogenesis functions.

Screening of LHX2 in patients presenting growth retardation with posterior pituitary and ocular abnormalities

In humans, pituitary hormone deficiency may be part of a syndrome including extra-pituitary defects like ocular abnormalities. Very few genes have been linked to this particular phenotype. In the mouse, Lhx2, which encodes a member of the LIM (Lin-11, Isl-1, and Mec-3) class of homeodomain proteins, was shown to be expressed during early development in the posterior pituitary, eye, and liver, and its expression persists in adulthood in the central nervous system Lhx2(-/-) mice display absence of posterior pituitary and intermediate lobes, malformation of the anterior lobe, anophthalmia, and they die from anemia. We tested the implication of the LHX2 gene in patients presenting pituitary hormone deficiency associated with ectopic or nonvisible posterior pituitary and developmental ocular defects. A cohort of 59 patients, including two familial cases, was studied. Direct sequencing of the LHX2 coding sequence and intron/exon boundaries was performed. LHX2 transcriptional activity on several pituitary promoters (AGSU, PRL, POU1F1, and TSHB) was tested in vitro. Six heterozygous sequence variations were identified, among which two are novel missense changes (p.Ala203Thr and p.Val333Met). In vitro, LHX2 activates transcription of TSHB, PRL, and POU1F1 promoters in the HEK293 cell line. A synergistic action of POU1F1 and LHX2 was also shown on these promoters. The two missense variations were tested and no significant difference was observed, leading to the conclusion that they are not deleterious. These results suggest that if LHX2 is involved in pituitary hormone deficiency associated with posterior pituitary and ocular defects, it would be a rare cause of this disease condition.

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 alpha-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.

PDLIM5 and susceptibility to bipolar disorder: a family-based association study and meta-analysis

The postsynaptic density-95/discs large/zone occludens-1 (PDZ) domain and LIM (Lin-11, Isl-1, and Mec-3) domain 5 (PDLIM5) gene has been analyzed as a candidate gene for both schizophrenia and bipolar disorder (BP) in Japanese samples. We performed a family-based association study to test the hypothesis that variants in PDLIM5 increase susceptibility to BP in European-Americans and a meta-analysis to clarify whether there is a single marker consistently contributing to risk for BP. Five single nucleotide polymorphisms in the PDLIM5 gene were genotyped in 290 European-American BP families. Programs Sibling-Transmission/Disequilibrium Test (sib_tdt) and PDTPHASE were used for allelic and haplotypic association, respectively. We carried out a meta-analysis combing our family-based data and case-control data from two Japanese sample sets and from two genome-wide association (GWA) studies. Our association analysis showed no single nucleotide polymorphism associated with BP. A rare haplotype consisted of rs10008257 and rs2433320 had nominal association (P=0.045), which failed to survive correction for multiple tests. The meta-analysis identified a significant allelic association at rs2433320 in all combined samples (excluding overlapped samples in GWA: overall odds ratio=0.897, 95% confidence interval: 0.838-0.961, adjusted P=0.012) and in all Caucasian samples (excluding overlapped samples in GWA: overall odds ratio=0.905, 95% confidence interval: 0.843-0.971, adjusted P=0.032), but not in the Japanese samples. PDLIM5 may have a minor effect on susceptibility to BP in Caucasians. The findings in Japanese need further confirmation in larger independent samples.

Positive association between PDLIM5 and schizophrenia in the Chinese Han population

The PDZ and LIM domain 5 protein (PDLIM5) contains one PDZ (post-synaptic density-95/discs large/zone occludens-1) domain and three LIM (Lin-11, Isl-1, and Mec-3) domains, and is also known as Enigma homologue LIM domain (ENH) protein or LIM protein. DNA microarray analysis of post-mortem brains of schizophrenics has indicated up-regulation of the mRNA level of PDLIM5, and Horiuchi and colleagues reported two single nucleotide polymorphisms (SNPs) (rs2433320 and rs2433322) in the 5' region of the PDZ and LIM domain 5 gene (PDLIM5) to be significantly associated with schizophrenia in the Japanese population. On the other hand, no association with schizophrenia was observed by Kato and colleagues in a different sample of the Japanese population. In this study, we genotyped six SNPs (including rs2433320 and rs2433322) covering PDLIM5 in 507 schizophrenia patients and 530 normal controls recruited from Jiangxi Province, China. Although rs2433320 was negative in our samples, rs2433322 showed significantly different frequencies between cases and controls (P=0.000010). In addition, high linkage disequilibrium was observed between rs2433320 and rs2433322 (D'=0.880), and haplotypes constructed from the two SNPs were significantly associated with schizophrenia (global P=0.00019, even after strict Bonferroni correction). Our results provide further evidence to support PDLIM5 as a potential susceptible gene for schizophrenia.

Characterization of the REST/NRSF‐interacting LIM domain protein (RILP): localization and interaction with REST/NRSF

We previously identified a nuclear envelope protein repressor element-1 silencing transcription factor (REST)/neuron-restrictive silencer factor (NRSF)-interacting Lin-11, Isl-1 and Mec-3 (LIM) domain protein (RILP) that we proposed functions in the nuclear translocation of the transcriptional repressor REST/NRSF. In this study we assessed the functionality of the prenylation motif, protein kinase A (PKA) phosphorylation sites and nuclear localization sequences (NLSs) of RILP. [(3)H]-mevalonolactone labeled endogenous RILP, showing that RILP is indeed prenylated, while phosphorylation analysis showed that the two PKA sites are phosphorylated. Blocking RILP prenylation, mutating the NLSs or mutating the PKA phosphorylation sites caused RILP to mislocalize to the cytosol. Concurrent with this mislocalization of RILP, REST/NRSF and REST4, which are normally found in the nucleus, co-localized in the cytosol with the RILP mutants. This provides additional evidence that RILP interacts with REST/NRSF and REST4 in vivo, and is involved in the nuclear localization of REST/NRSF and REST4. Reporter gene analysis using the promoter region of the human cholinergic gene locus revealed that these RILP mutants prevented repression of the reporter gene. By trapping REST/NRSF in the cytosol, the RILP mutants prevented translocation to the nucleus where REST/NRSF binds to an RE-1/NRSE element to repress gene transcription. These results show that RILP is required for REST/NRSF nuclear targeting and function.

Minimal features of paxillin that are required for the tyrosine phosphorylation of focal adhesion kinase

Tyrosine phosphorylation of FAK (focal adhesion kinase) regulates signalling that results from the interaction of integrins with extracellular matrix and growth factor receptors. A critical step in this process is the phosphorylation of Tyr397 of FAK, which creates a binding site for Src family kinases, PI3K (phosphoinositide 3-kinase) and Shc (Src homology and collagen homology). An intact Tyr397 site is required for FAK-mediated regulation of cell migration, survival signals and full responsiveness to soluble growth factors. We showed previously that the adaptor protein paxillin is required for the overall tyrosine phosphorylation of FAK in embryonic stem cells [Wade, Bohl and Vande Pol (2002) Oncogene 21, 96-107]. In the present paper, we identify the minimal structural features of paxillin that are required to support overall FAK tyrosine phosphorylation and Tyr397 phosphorylation. Paxillin contains N-terminal leucine-rich LD motifs that bind directly to FAK and four LIM (Lin-11, Isl-1 and Mec-3) domains in the C-terminus. We show that paxillin LIM domains 1, 2 and 3 are each required for FAK tyrosine phosphorylation, while LIM4 is dispensable. In addition to paxillin LIM domains 1, 2 and 3, a single LD motif on paxillin is required to support FAK tyrosine phosphorylation in embryonic stem cells. Both sequence and spatial requirements exist for LD motifs to support FAK tyrosine phosphorylation. Interestingly, synthetic LD motifs that fail to bind FAK in vitro are able to fully support FAK tyrosine phosphorylation, indicating that minimal interactions of LD motifs with FAK suffice. Our results demonstrate at least four distinct structural domains of paxillin support at least three distinct functions that are each required for FAK tyrosine phosphorylation.

αII-Spectrin interacts with Tes and EVL, two actin-binding proteins located at cell contacts

The spectrin-based membrane skeleton, a multi-protein scaffold attached to diverse cellular membranes, is presumed to be involved in the stabilization of membranes, the establishment of membrane domains as well as in vesicle trafficking and nuclear functions. Spectrin tetramers made of alpha- and beta-subunits are linked to actin microfilaments, forming a network that binds a multitude of proteins. The most prevalent alpha-spectrin subunit in non-erythroid cells, alphaII-spectrin, contains two particular spectrin repeats in its central region, alpha9 and alpha10, which host an Src homology 3 domain, a tissue-specific spliced sequence of 20 residues, a calmodulin-binding site and major cleavage sites for caspases and calpains. Using yeast two-hybrid screening of kidney libraries, we identified two partners of the alpha9-alpha10 repeats: the potential tumour suppressor Tes, an actin-binding protein mainly located at focal adhesions; and EVL (Ena/vasodilator-stimulated phosphoprotein-like protein), another actin-binding protein, equally recruited at focal adhesions. Interactions between spectrin and overexpressed Tes and EVL were confirmed by co-immunoprecipitation. In vitro studies showed that the interaction between Tes and spectrin is mediated by a LIM (Lin-11, Isl-1 and Mec3) domain of Tes and by the alpha10 repeat of alphaII-spectrin whereas EVL interacts with the Src homology 3 domain located within the alpha9 repeat. Moreover, we describe an in vitro interaction between Tes and EVL, and a co-localization of these two proteins at focal adhesions. These interactions between alphaII-spectrin, Tes and EVL indicate new functions for spectrin in actin dynamics and focal adhesions.

Roles for the tubulin- and PTP–PEST-binding paxillin LIM domains in cell adhesion and motility

Cell dynamics mediated through cell–extracellular matrix contacts, such as adhesion and motility involve the precise regulation of large complexes of structural and signaling molecules called focal adhesions (FAs). Paxillin is a multi-domain FA adaptor protein containing five amino-terminal paxillin leucine–aspartate repeat (LD) motifs and four carboxyl-terminal Lin-11 Isl-1 and Mec-3 (LIM) domains. The LD motifs support paxillin binding to actopaxin, integrin linked kinase (ILK), FA kinase (FAK), paxillin kinase linker (PKL) and vinculin. Of the LIM domains, LIM2 and 3 comprise the paxillin FA-targeting motif, with phosphorylation of these domains modulating paxillin targeting and cell adhesion to fibronectin (Fn). The identity of the paxillin FA targeting partner remains to be determined; however, the LIM domains mediate interactions with tubulin and the protein-tyrosine phosphatase (PTP)–PEST. PTP–PEST binding requires both LIM3 and 4, whereas, the precise LIM target of tubulin binding is not known. In this report, we demonstrate that the individual paxillin LIM2 and 3 domains support specific binding to tubulin and suggest a potential role for this interaction in the regulation of paxillin sub-cellular compartmentalization. In addition, expression of paxillin molecules with mutations in the tubulin- and PTP–PEST-binding LIM domains differentially impaired Chinese hamster ovary K1 (CHO.K1) cell adhesion and migration to Fn. Perturbation of LIM3 or 4 inhibited adhesion while mutation of LIM2 or 4 decreased cell motility. Interestingly, expression of tandem LIM2–3 inhibited cell adhesion and spreading while LIM3–4 stimulated a well-spread polarized phenotype. These data offer further support for a critical role for paxillin in cell adhesion and motility.

Inhibition of neurite extension by overexpression of individual domains of LIM kinase 1.

Lin-11, Isl-1 and Mec-3 (LIM) kinases are serine/threonine kinases that phosphorylate cofilin, an actin depolymerizing protein. LIM kinases have a highly modular structure composed of two N-terminal LIM domains (LIM 1/2), a PSD-95, Dlg and ZO-1 (PDZ) domain and a C-terminal protein kinase domain. Here, we overexpressed individual domains of mouse LIM kinase 1 (LIMK1) in PC12 cells and investigated their effects on neurite outgrowth. Although none of the LIMK1 domains had an effect on spontaneous neurite outgrowth, the N-terminal LIM 1/2 domains strongly inhibited differentiation of PC12 cells after stimulation with both nerve growth factor (NGF) and the Rho-kinase inhibitor Y-27632. In contrast, the overexpressed PDZ domain reduced neurite outgrowth only when differentiation had been induced by Y-27632, but not by NGF. Our data suggest that the different non-catalytic N-terminal domains of LIMK1 contribute to the regulation of neurite extension by using distinct signal transduction pathways.

Characterization of mouse epithelial protein lost in neoplasm (EPLIN) and comparison of mammalian and zebrafish EPLIN

EPLIN is a cytoskeleton-associated protein that was initially identified as the product of a gene that is transcriptionally down-regulated in cancer cells. In human, there are two known isoforms, EPLIN-α and -β, generated by alternative promoter usage from a single gene. With the exception of a single LIM (lin-11, isl-1, and mec-3) domain, the sequence of EPLIN is unique and does not provide any clues to its function. To identify conserved regions of EPLIN that may be important for its function, we have characterized mouse (m) and zebrafish (zf) EPLIN. As in human, two isoforms, the 593 aa mEPLIN-α (77% identity; 83% similarity) and 753 aa mEPLIN-β (75% identity; 83% similarity), were present in mouse. mEPLIN-α is highly expressed in embryonic tissue and adult lung and spleen, whereas mEPLIN-β is preferentially expressed in kidney, testis, lung and liver. The analysis of mEPLIN gene revealed that the overall organization of the exons in mouse and human are conserved. In zebrafish, there was only one form, the 629 aa zfEPLIN, corresponding to the mammalian EPLIN-β. Like its mammalian counterparts, ectopically expressed zfEPLIN is co-localized to the actin cytoskeleton. While the overall homology between mammalian and zebrafish EPLIN was not striking (37% identity; 50% similarity), there were seven highly conserved regions, which should be useful in structure-function studies of this novel protein.

The Noncatalytic Domain of Protein-tyrosine Phosphatase-PEST Targets Paxillin for Dephosphorylation in Vivo

The noncatalytic domain of protein-tyrosine phosphatase (PTP)-PEST contains a binding site for the focal adhesion-associated protein paxillin. This binding site has been narrowed to a 52-residue sequence that is composed of two nonoverlapping, weak paxillin binding sites. The PTP-PEST binding site on paxillin has been mapped to the two carboxyl-terminal LIM (lin11, isl-1, and mec-3) domains. Transient expression of PTP-PEST reduced tyrosine phosphorylation of p130(cas), as anticipated. A PTP-PEST mutant defective for binding p130(cas) does not cause a reduction in its tyrosine phosphorylation in vivo. Expression of PTP-PEST also caused a reduction of phosphotyrosine on paxillin. Expression of mutants of PTP-PEST with deletions in the paxillin-binding site did not associate with paxillin in vivo and failed to cause a reduction in the phosphotyrosine content of paxillin. These results demonstrate that paxillin can serve as a PTP-PEST substrate in vivo and support the model that a noncatalytic domain interaction recruits paxillin to PTP-PEST to facilitate its dephosphorylation.