LIM kinase 1, a key regulator of actin dynamics, is widely expressed in embryonic and adult tissues.
ABSTRACT 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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ABSTRACT: Abnormal signal transduction events can impact upon the cytoskeleton, affecting the actin and microtubule networks with direct relevance to Alzheimer's disease (AD). Cytoskeletal anomalies, in turn, promote atypical neuronal responses, with consequences for cellular organization and function. Neuronal cytoskeletal modifications in AD include neurofibrillary tangles, which result from aggregates of hyperphosphorylated tau protein. The latter is a microtubule (MT)-binding protein, whose abnormal phosphorylation leads to MT instability and consequently provokes irregularities in the neuronal trafficking pathways. Early stages of AD are also characterized by synaptic dysfunction and loss of dendritic spines, which correlate with cognitive deficit and impaired brain function. Actin dynamics has a prominent role in maintaining spine plasticity and integrity, thus providing the basis for memory and learning processes. Hence, factors that disrupt both actin and MT network dynamics will compromise neuronal function and survival. The peptide Aβ is the major component of senile plaques and has been described as a pivotal mediator of neuronal dystrophy and synaptic loss in AD. Here, we review Aβ-mediated effects on both MT and actin networks and focus on the relevance of the elicited cytoskeletal signaling events targeted in AD pathology.
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ABSTRACT: LIM kinases (LIMKs) are important cell cytoskeleton regulators that play a prominent role in cancer manifestation and neuronal diseases. The LIMK family consists of two homologues, LIMK1 and LIMK2, which differ from one another in expression profile, intercellular localization, and function. The main substrate of LIMK is cofilin, a member of the actin-depolymerizing factor (ADF) protein family. When phosphorylated by LIMK, cofilin is inactive. LIMKs play a contributory role in several neurodevelopmental disorders and in cancer growth and metastasis. We recently reported the development and validation of a novel LIMK inhibitor, referred to here as T56-LIMKi, using a combination of computational methods and classical biochemistry techniques. Here we report that T56-LIMKi inhibits LIMK2 with high specificity, and shows little or no cross-reactivity with LIMK1. We found that T56-LIMKi decreases phosphorylated cofilin (p-cofilin) levels and thus inhibits growth of several cancerous cell lines, including those of pancreatic cancer, glioma and schwannoma. Because the most promising in-vitro effect of T56-LIMKi was observed in the pancreatic cancer cell line Panc-1, we tested the inhibitor on a nude mouse Panc-1 xenograft model. T56-LIMKi reduced tumor size and p-cofilin levels in the Panc-1 tumors, leading us to propose T56-LIMKi as a candidate drug for cancer therapy.