LIM kinase 1, a key regulator of actin dynamics, is widely expressed in embryonic and adult tissues

Molecular Genetics of Cancer Division, The Walter and Eliza Hall Institute of Medical Research, PO The Royal Melbourne Hospital, Melbourne, Victoria 3050, Australia.
Experimental Cell Research (Impact Factor: 3.25). 05/2004; 294(2):392-405. DOI: 10.1016/j.yexcr.2003.11.024
Source: PubMed


The expression of endogenous LIM kinase 1 (LIMK1) protein was investigated in embryonic and adult mice using a rat monoclonal antibody (mAb), which recognizes specifically the PDZ domain of LIMK1 and not LIMK2. Immunoblotting analysis revealed widespread expression of LIMK1 existing as a 70-kDa protein in tissues and in cell lines, with a higher mass form (approximately 75 kDa) present in some tissues and cell lines. Smaller isoforms of approximately 50 kDa were also occasionally evident. Immunofluorescence analysis demonstrated LIMK1 subcellular localization at focal adhesions in fibroblasts as revealed by co-staining with actin, paxillin and vinculin in addition to perinuclear (Golgi) and occasional nuclear localization. Furthermore, an association between LIMK1 and paxillin but not vinculin was identified by co-immunoprecipitation analysis. LIMK1 is enriched in both axonal and dendritic growth cones of E18 rat hippocampal pyramidal neurons where it is found in punctae that extend far out into filopodia, as well as in a perinuclear region identified as Golgi. In situ, we identify LIMK1 protein expression in all embryonic and adult tissues examined, albeit at different levels and in different cell populations. The rat monoclonal LIMK1 antibody recognizes proteins of similar size in cell and tissue extracts from numerous species. Thus, LIMK1 is a widely expressed protein that exists as several isoforms.

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    • "Anti-α-tubulin (T5168) and anti-acetyl-tubulin (T6793) were from Sigma. Additional antibodies used were anti-HA (Roche, 11867423001), anti-LIMK1 [(clone 8D5–5-12 [21]], anti-LIMK2 (Abcam, ab45165), TPPP1 [42], anti-p21 (Santa Cruz, sc-397), anti-cofilin (Cytoskeleton, ACFL02) and p53 (gift from Dr Ygal Haupt, Peter MacCallum Cancer Centre, Melbourne, Australia). "
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    ABSTRACT: Drug resistance is a major obstacle for the successful treatment of many malignancies, including neuroblastoma, the most common extracranial solid tumor in childhood. Therefore, current attempts to improve the survival of neuroblastoma patients, as well as those with other cancers, largely depend on strategies to counter cancer cell drug resistance; hence, it is critical to understand the molecular mechanisms that mediate resistance to chemotherapeutics. The levels of LIM-kinase 2 (LIMK2) are increased in neuroblastoma cells selected for their resistance to microtubule-targeted drugs, suggesting that LIMK2 might be a possible target to overcome drug resistance. Here, we report that depletion of LIMK2 sensitizes SHEP neuroblastoma cells to several microtubule-targeted drugs, and that this increased sensitivity correlates with enhanced cell cycle arrest and apoptosis. Furthermore, we show that LIMK2 modulates microtubule acetylation and the levels of tubulin Polymerization Promoting Protein 1 (TPPP1), suggesting that LIMK2 may participate in the mitotic block induced by microtubule-targeted drugs through regulation of the microtubule network. Moreover, LIMK2-depleted cells also show an increased sensitivity to certain DNA-damage agents, suggesting that LIMK2 might act as a general pro-survival factor. Our results highlight the exciting possibility of combining specific LIMK2 inhibitors with anticancer drugs in the treatment of multi-drug resistant cancers.
    PLoS ONE 08/2013; 8(8):e72850. DOI:10.1371/journal.pone.0072850 · 3.23 Impact Factor
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    • "De-epithelialization induced by cadherin-6B or BMP is mediated by the LIM kinase/cofilin pathway Non-canonical BMP signaling has been found to be transduced through several different mediators including LIM kinase 1 (LIMK1), Trb3 and Src, dependent on cellular context (Chan et al., 2007; Foletta et al., 2003; Hassel et al., 2004; Lee-Hoeflich et al., 2004; Miyazono et al., 2010; Wong et al., 2005). LIMK1 is expressed in most tissues of the developing embryo including the neural tube (Foletta et al., 2004). The activity of LIMK1 bound to cytoplasmic tail of BMP Type II receptor is regulated noncanonically by BMP (Foletta et al., 2003; Lee-Hoeflich et al., 2004) and the primary role of LIMK1 is to phosphorylate cofilin in order to regulate actin dynamics (Arber et al., 1998; Yang et al., 1998) as a mechanism to change cell morphology and/or migration . "
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    ABSTRACT: We previously provided evidence that cadherin-6B induces de-epithelialization of the neural crest prior to delamination and is required for the overall epithelial mesenchymal transition (EMT). Furthermore, de-epithelialization induced by cadherin-6B was found to be mediated by BMP receptor signaling independent of BMP. We now find that de-epithelialization is mediated by non-canonical BMP signaling through the BMP type II receptor (BMPRII) and not by canonical Smad dependent signaling through BMP Type I receptor. The LIM kinase/cofilin pathway mediates non-canonical BMPRII induced de-epithelialization, in response to either cadherin-6B or BMP. LIMK1 induces de-epithelialization in the neural tube and dominant negative LIMK1 decreases de-epithelialization induced by either cadherin-6B or BMP. Cofilin is the major known LIMK1 target and a S3A phosphorylation deficient mutated cofilin inhibits de-epithelialization induced by cadherin-6B as well as LIMK1. Importantly, LIMK1 as well as cadherin-6B can trigger ectopic delamination when co-expressed with the competence factor SOX9, showing that this cadherin-6B stimulated signaling pathway can mediate the full EMT in the appropriate context. These findings suggest that the de-epithelialization step of the neural crest EMT by cadherin-6B/BMPRII involves regulation of actin dynamics via LIMK/cofilin.
    Developmental Biology 04/2012; 366(2):232-43. DOI:10.1016/j.ydbio.2012.04.005 · 3.55 Impact Factor
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    • "Predictably, deletion of the NLS from the kinase domain abrogates this effect [15]. Immunohistochemical studies in cultured mammalian cell lines, as well as paraformaldehyde (PFA)-fixed mammalian tissues, indicate that the subcellular compartmentalization of LIMK1 within cells is generally cytoplasmic, although many cell types express moderate to strong nuclear LIMK1, in addition to the cytoplasmic component [16]. Although it is clear that LIMK1 protein is expressed in both the cytoplasm and nucleus, the majority of LIMK1 studies have focused on the role of LIMK1 in regulating actin dynamics within the cytoplasm. "
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    ABSTRACT: LIM kinase 1 (LIMK1) is expressed in both cytoplasmic and nuclear compartments, and is a key regulator of cytoskeletal organization involved in cell migration and proliferation. LIMK1 levels are increased in several human cancers, with LIMK1 over-expression in prostate and breast cancer cells leading to tumor progression. While it has been presumed that the mechanism by which LIMK1 promotes cancer progression is via its cytoplasmic effects, the role of nuclear vs cytoplasmic LIMK1 in the tumorigenic process has not been examined. To determine if cytoplasmic or nuclear LIMK1 expression correlated with breast cancer, we performed immunohistochemical (IHC) analysis of breast tissue microarrays (TMAs), The IHC analysis of breast TMAs revealed that 76% of malignant breast tissue samples strongly expressed LIMK1 in the cytoplasm, with 52% of these specimens also expressing nuclear LIMK1. Only 48% of benign breast samples displayed strong cytoplasmic LIMK1 expression and 27% of these expressed nuclear LIMK1. To investigate the respective roles of cytoplasmic and nuclear LIMK1 in breast cancer progression, we targeted GFP-LIMK1 to cytoplasmic and nuclear subcellular compartments by fusing nuclear export signals (NESs) or nuclear localization sequences (NLS), respectively, to the amino-terminus of GFP-LIMK1. Stable pools of MDA-MB-231 cells were generated by retroviral transduction, and fluorescence microscopy revealed that GFP alone (control) and GFP-LIMK1 were each expressed in both the cytoplasm and nucleus of MDA-MB-231 cells, whereas NLS-GFP-LIMK1 was expressed in the nucleus and NES-GFP-LIMK1 was expressed in the cytoplasm. Western blot analyses revealed equal expression of GFP-LIMK1 and NES-GFP-LIMK1, with NLS-GFP-LIMK1 expression being less but equal to endogenous LIMK1. Also, Western blotting revealed increased levels of phospho-cofilin, phospho-FAK, phospho-paxillin, phospho-Src, phospho-AKT, and phospho-Erk1/2 in cells expressing all GFP-LIMK1 fusions, compared to GFP alone. Invasion assays revealed that all GFP-LIMK1 fusions increased MDA-MB-231 cell invasion ~1.5-fold, compared to GFP-only control cells. Tumor xenograft studies in nude mice revealed that MDA-MB-231 cells stably expressing GFP-LIMK, NLS-GFP-LIMK1 and NES-GFP-LIMK1 enhanced tumor growth 2.5-, 1.6- and 4.7-fold, respectively, compared to GFP-alone. Taken together, these data demonstrate that LIMK1 activity in both the cytoplasmic and nuclear compartments promotes breast cancer progression, underscoring that nuclear LIMK1 contributes to the transforming function of LIMK1.
    Molecular Cancer 06/2011; 10(1):75. DOI:10.1186/1476-4598-10-75 · 4.26 Impact Factor
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