LIM Kinase Research Articles

Whole-Genome and Poly(A)+Transcriptome Analysis of the Drosophila Mutant agnts3 with Cognitive Dysfunctions.

The temperature-sensitive Drosophila mutant agnts3 exhibits the restoration of learning defects both after heat shock (HS) and under hypomagnetic conditions (HMC). Previously, agnts3 was shown to have an increased level of LIM kinase 1 (LIMK1). However, its limk1 sequence did not significantly differ from that of the wild-type strain Canton-S (CS). Here, we performed whole-genome and poly(A)-enriched transcriptome sequencing of CS and agnts3 males normally, after HMC, and after HS. Several high-effect agnts3-specific mutations were identified, including MED23 (regulation of HS-dependent transcription) and Spn42De, the human orthologs of which are associated with intellectual disorders. Pronounced interstrain differences between the transcription profiles were revealed. Mainly, they included the genes of defense and stress response, long non-coding RNAs, and transposons. After HS, the differences between the transcriptomes became less pronounced. In agnts3, prosalpha1 was the only gene whose expression changed after both HS and HMC. The normal downregulation of prosalpha1 and Spn42De in agnts3 was confirmed by RT-PCR. Analysis of limk1 expression did not reveal any interstrain differences or changes after stress. Thus, behavioral differences between CS and agnts3 both under normal and stressed conditions are not due to differences in limk1 transcription. Instead, MED23, Spn42De, and prosalpha1 are more likely to contribute to the agnts3 phenotype.

LIM kinase 2 activates cardiac fibroblasts and exacerbates postinfarction left ventricular remodeling via crosstalk between the canonical and non-canonical Wnt pathways

Ischemic heart failure rates rise despite decreased acute myocardial infarction (MI) mortality. Excessive myofibroblast activation post-MI leads to adverse remodeling. LIM kinases (LIMK1 and LIMK2) regulate cytoskeleton homeostasis and are pro-fibrotic markers in atrial fibrillation. However, their roles and mechanisms in postinfarction fibrosis and ventricular remodeling remain unclear. This study found that the expression of LIMKs elevated in the border zone (BZ) in mice MI models. LIMK1/2 double knockout (DKO) restrained pathological remodeling and reduced mortality by suppressing myofibroblast activation. By using adeno-associated virus (AAV) with a periostin promoter to overexpress LIMK1 or LIMK2, this study found that myofibroblast-specific LIMK2 overexpression diminished these effects in DKO mice, while LIMK1 did not. LIMK2 kinase activity was critical for myofibroblast proliferation by using AAV overexpressing mutant LIMK2 lack of kinase activity. According to phosphoproteome analysis, functional rescue experiments, co-immunoprecipitation, and protein-protein docking, LIMK2 led to the phosphorylation of β-catenin at Ser 552. LIMK2 nuclear translocation also played a role in myofibroblast proliferation after MI with the help of AAV overexpressing mutant LIMK2 without nuclear location signal. Chromatin immunoprecipitation sequencing identified that LIMK2 bound to Lrp6 promoter region in TGF-β treated cardiac fibroblasts, positively regulating Wnt signaling via Wnt receptor internalization. This study demonstrated that LIMK2 promoted myofibroblast proliferation and adverse cardiac remodeling after MI, by enhancing phospho-β-catenin (Ser552) and Lrp6 signaling. This suggested that LIMK2 could be a target for the treatment of postinfarction injury.

Human T-cell receptor triggering requires inactivation of Lim kinase-1 by Slingshot-1 phosphatase

Actin dynamics control early T-cell receptor (TCR) signalling during T-cell activation. However, the precise regulation of initial actin rearrangements is not completely understood. Here, we have investigated the regulatory role of the phosphatase Slingshot-1 (SSH1) in this process. Our data show that SSH1 rapidly polarises to nascent cognate synaptic contacts and later relocalises to peripheral F-actin networks organised at the mature immunological synapse. Knockdown of SSH1 expression by CRISPR/Cas9-mediated genome editing or small interfering RNA reveal a regulatory role for SSH1 in CD3ε conformational change, allowing Nck binding and proper downstream signalling and immunological synapse organisation. TCR triggering induces SSH1-mediated activation of actin dynamics through a mechanism mediated by Limk-1 inactivation. These data suggest that during early TCR activation, SSH1 is required for rapid F-actin rearrangements that mediate initial conformational changes of the TCR, integrin organisation and proximal signalling events for proper synapse organisation. Therefore, the SSH1 and Limk-1 axis is a key regulatory element for full T cell activation.

The Role of LIM Kinase 1 in Memory Processes

The Role of LIM Kinase 1 in Memory Processes

TAZ is involved in breast cancer cell migration via regulating actin dynamics.

Cancer metastasis is dependent on cell migration. Several mechanisms, including epithelial-to-mesenchymal transition (EMT) and actin fiber formation, could be involved in cancer cell migration. As a downstream effector of the Hippo signaling pathway, transcriptional coactivator with PDZ-binding motif (TAZ) is recognized as a key mediator of the metastatic ability of breast cancer cells. We aimed to examine whether TAZ affects the migration of breast cancer cells through the regulation of EMT or actin cytoskeleton. MCF-7 and MDA-MB-231 cells were treated with siRNA to attenuate TAZ abundance. Transwell migration assay and scratch wound healing assay were performed to study the effects of TAZ knockdown on cancer cell migration. Fluorescence microscopy was conducted to examine the vinculin and phalloidin. Semiquantitative immunoblotting and quantitative real-time PCR were performed to study the expression of small GTPases and kinases. Changes in the expression of genes associated with cell migration were examined through next-generation sequencing. TAZ-siRNA treatment reduced TAZ abundance in MCF-7 and MDA-MB-231 breast cancer cells, which was associated with a significant decrease in cell migration. TAZ knockdown increased the expression of fibronectin, but it did not exhibit the typical pattern of EMT progression. TGF-β treatment in MDA-MB-231 cells resulted in a reduction in TAZ and an increase in fibronectin levels. However, it paradoxically promoted cell migration, suggesting that EMT is unlikely to be involved in the decreased migration of breast cancer cells in response to TAZ suppression. RhoA, a small Rho GTPase protein, was significantly reduced in response to TAZ knockdown. This caused a decrease in the expression of the Rho-dependent downstream pathway, i.e., LIM kinase 1 (LIMK1), phosphorylated LIMK1/2, and phosphorylated cofilin, leading to actin depolymerization. Furthermore, myosin light chain kinase (MLCK) and phosphorylated MLC2 were significantly decreased in MDA-MB-231 cells with TAZ knockdown, inhibiting the assembly of stress fibers and focal adhesions. TAZ knockdown inhibits the migration of breast cancer cells by regulating the intracellular actin cytoskeletal organization. This is achieved, in part, by reducing the abundance of RhoA and Rho-dependent downstream kinase proteins, which results in actin depolymerization and the disassembly of stress fibers and focal adhesions.

Oxidation of the actin cytoskeleton regulator cofilin-1 as a mediator of endothelial stiffening and dysfunction in type 2 diabetes

Vascular disease is a feature of type 2 diabetes (T2D) that is caused in part by oxidative stress and endothelial dysfunction. An emerging instigator of endothelial dysfunction is stiffening of the cell, which is mediated by enhanced polymerization of filamentous actin (F-actin). Actin polymerization is regulated by cofilin-1, an actin severing protein that can be phosphorylated or oxidized, leading to its inactivation and loss of severing capability. Yet, the mechanisms contributing to endothelial stiffening in T2D remain largely unknown. Herein, we tested the hypothesis that endothelial stiffening in T2D is mediated by inactivation of cofilin-1, leading to actin polymerization. In support of this hypothesis, human umbilical vein endothelial cells (HUVECs) treated with plasma from subjects with T2D, and the aortic endothelium of diabetic mice (db/db), demonstrate increased cellular stiffness and F-actin presence. Of note, these findings were not associated with enhanced phosphorylation of cofilin-1, suggesting that its inactivation may instead occur through oxidation. Indeed, we show that cofilin-1 can be oxidized by H2O2, a strong oxidizing agent, and that exposure of HUVECs to H2O2 induces F-actin formation. These findings implicate oxidation and inactivation of cofilin-1 as a putative driver of endothelial F-actin formation and cellular stiffening. In further support of the notion that inactivation of cofilin-1 in endothelial cells promotes cytoskeletal remodeling, we show that genetic silencing or pharmacological inhibition of LIM kinase-1 (LIMK-1), a key enzyme responsible for phosphorylating and thus inactivating cofilin-1, reduces F-actin content and cellular stiffness. Congruently, we show that LIMK inhibition reduces endothelial stiffness in aortic explants from db/db mice. It is conceivable that increased endothelial actin polymerization and stiffening impact vasomotor function. As proof-of-concept, we show that intraluminal exposure of isolated arteries to jasplakinolide, a pharmacological inducer of actin polymerization, impairs endothelial-dependent vasodilation, as assessed via flow-mediated dilation (FMD), while endothelium-independent vasodilation remains unaffected. Conversely, cytochalasin D, an inhibitor of actin polymerization, improves FMD, without influencing endothelium-independent vasodilation. Taken together, these findings suggest the existence of a potential new paradigm of T2D-associated endothelial dysfunction whereby oxidative stress inactivates cofilin-1, contributing to F-actin-dependent endothelial stiffening. Moreover, this work supports the idea that LIMK-1 may be considered a potential target for reducing cofilin-1 inactivation and reversing endothelial stiffening in T2D. Funding: R01HL151384, R01HL153264, R01HL137769, and AHA23PRE1020897. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Loss of nitric oxide bioavailability promotes arterial stiffening in a tissue transglutaminase-dependent manner

Although arterial stiffening undeniably contributes to cardiovascular disease, the cellular and molecular mechanisms leading to increased arterial stiffness are not well understood. Increased arterial stiffness in disease states such as obesity and type 2 diabetes typically coexists with indices of endothelial dysfunction including diminished nitric oxide (NO) bioavailability. In fact, it is commonly assumed that endothelial dysfunction precedes, and causally contributes to, arterial stiffening; yet the molecular underpinnings for this causal relationship remain elusive. We and others previously showed that activation of tissue transglutaminase (TG2), a crosslinking enzyme that also works as a G-protein and cellular scaffold, promotes cytoskeletal actin polymerization and stiffening of vascular smooth muscle cells (VSMC), thus contributing to whole-artery stiffening. It is also reported that NO is an endogenous inhibitor of TG2. Accordingly, herein, we tested the hypothesis that increased exposure of VSMC to NO suppresses TG2 activity and actin polymerization. Conversely, we hypothesized that loss of NO bioavailability promotes arterial stiffening, and that this effect is mediated by TG2 activity and downstream activation of LIM kinase (LIMK), an enzyme that modulates fibrillar (F)-actin severing via cofilin phosphorylation. All differences reported herein are significant at P<0.05. In support of our hypotheses, we show that treatment of cultured human aortic VSMC with the NO donor S-nitrosoglutathione (200μM for 2 hours) reduces TG2 activity (N-epsilon gamma glutamyl Lysine presence) and F-actin formation (phalloidin staining) induced by TG2 activation (dithiothreitol, 200μM). Furthermore, we show that exposure of mouse isolated mesenteric arteries to the NO synthase inhibitor L-NAME (300μM for 18 hours) causes an increase in arterial stiffness ( i.e., increased incremental modulus of elasticity) as determined using pressure myography and videomicroscopy. Of note, L-NAME-induced arterial stiffening is prevented when arteries are co-treated with a TG2 inhibitor (cystamine, 1mM) or LIMK inhibitor (LIMKi, 1μM). Taken together, these findings support the notion that NO is a key downregulator of TG2 activity and demonstrate that reduced NO bioavailability promotes arterial stiffening in a VSMC TG2-LIMK-F-actin dependent manner. As such, this work provides insight into plausible molecular mechanisms by which endothelial dysfunction leads to arterial stiffening. R01HL153264 (to LAML and JP), and MU Research Excellence Program (REP) postdoctoral fellowship (to FIRP). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Does actin polymerization contribute to closure of the avian ductus arteriosus (Gallus gallus)?

The ductus arteriosus (DA) is a cardiovascular embryonic shunt present in all developing land vertebrates. This oxygen-sensitive blood vessel plays the important role of connecting the pulmonary artery to the aorta during development and directs blood flow toward the fetal respiratory organ (placenta in mammals or chorioallantois in birds and reptiles). Permanent vessel closure upon birth or hatch is necessary to establish proper circulation in the neonate. Increased arterial PO2 at birth or hatch stimulates DA closure via smooth muscle contraction. Smooth muscle contraction, such as in the DA, is governed by myosin light chain kinase (MLCK) stimulating increased cross-bridge cycling, and through increases in actin polymerization. The Ras homologous protein family (Rho) of GTPases are involved in calcium sensitization and contraction of vascular smooth muscle but have mostly been studied for their role in cell migration and signaling. Rho GTPases activate the Rho activated kinase (ROCK) pathway, which works in conjunction with MLCK to generate smooth muscle contraction as well as activating LIM kinase which inhibits the actin depolymerizer cofilin. Rac and Cdc42 GTPase pathways increase contractile strength in smooth muscle cells by activating the nucleator Arp2/3, leading to increased actin polymerization. The role of these GTPase pathways during closure of the DA is unknown. We examined the role of the Rho, Rac, and Cdc42 GTPase pathways leading to actin polymerization for their role in the closure of the avian DA in late-term chicken ( Gallus gallus) embryos. We hypothesized that inhibiting the pathways that lead to actin polymerization will inhibit tension development in the DA. Using a Danish Myo Technology wire myograph, DA physiology was examined when exposed to 25% oxygen followed by activators and inhibitors of the Rho, Rac, and Cdc42 GTPase pathways. Activation of Rho A pathways with CN01 (0.2 units/ml) produced contraction of the DA. Blocking ROCK pathways with Y-27632 (10 mM) removed the O2-sensitivity of the vessel, while the vessel was still able to respond to phenylephrine (100 mM). This suggests the Rho pathway is involved in maintaining baseline tension and contractile response to O2. Exposure to inhibitors of Rac (10 uM EHT 1864) and Cdc42 (via ML141) GTPases blocked the ability of vessels to contract in response to 25% oxygen, even after 3 hours of constant O2. Similarly, inhibition of the Arp2/3 complex (100 uM CK666) reduced the ability of the vessel to respond to oxygen. These findings suggest that Rho A, Rac, and Cdc42 pathways play a role in active tension of the chicken DA during development by potentially increasing actin polymerization. This research was funded by Grant R15HL14887 from NIH-NHLBI. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Tetrahydropyridine LIMK inhibitors: Structure activity studies and biological characterization

LIM Kinases, LIMK1 and LIMK2, have become promising targets for the development of inhibitors with potential application for the treatment of several major diseases. LIMKs play crucial roles in cytoskeleton remodeling as downstream effectors of small G proteins of the Rho-GTPase family, and as major regulators of cofilin, an actin depolymerizing factor. In this article we describe the conception, synthesis, and biological evaluation of novel tetrahydropyridine pyrrolopyrimidine LIMK inhibitors. Homology models were first constructed to better understand the binding mode of our preliminary compounds and to explain differences in biological activity. A library of over 60 products was generated and in vitro enzymatic activities were measured in the mid to low nanomolar range. The most promising derivatives were then evaluated in cell on cofilin phosphorylation inhibition which led to the identification of 52 which showed excellent selectivity for LIMKs in a kinase selectivity panel. We also demonstrated that 52 affected the cell cytoskeleton by disturbing actin filaments. Cell migration studies with this derivative using three different cell lines displayed a significant effect on cell motility. Finally, the crystal structure of the kinase domain of LIMK2 complexed with 52 was solved, greatly improving our understanding of the interaction between 52 and LIMK2 active site. The reported data represent a basis for the development of more efficient LIMK inhibitors for future in vivo preclinical validation.

PAK1-Dependent Regulation of Microtubule Organization and Spindle Migration Is Essential for the Metaphase I-Metaphase II Transition in Porcine Oocytes.

P21-activated kinase 1 (PAK1) is a critical downstream target that mediates the effect of small Rho GTPase on the regulation of cytoskeletal kinetics, cell proliferation, and cell migration. PAK1 has been identified as a crucial regulator of spindle assembly during the first meiotic division; however, its roles during the metaphase I (MI) to metaphase II (MII) transition in oocytes remain unclear. In the present study, the potential function of PAK1 in regulating microtubule organization and spindle positioning during the MI-MII transition was addressed in porcine oocytes. The results showed that activated PAK1 was co-localized with α-tubulin, and its expression was increased from the MI to MII stage (p < 0.001). However, inhibiting PAK1 activity with an inhibitor targeting PAK1 activation-3 (IPA-3) at the MI stage decreased the first polar body (PB1) extrusion rate (p < 0.05), with most oocytes arrested at the anaphase-telophase (ATI) stage. IPA-3-treated oocytes displayed a decrease in actin distribution in the plasma membrane (p < 0.001) and an increase in the rate of defects in MII spindle reassembly with abnormal spindle positioning (p < 0.001). Nevertheless, these adverse effects of IPA-3 on oocytes were reversed when the disulfide bond between PAK1 and IPA-3 was reduced by dithiothreitol (DTT). Co-immunoprecipitation revealed that PAK1 could recruit activated Aurora A and transform acidic coiled-coil 3 (TACC3) to regulate spindle assembly and interact with LIM kinase 1 (LIMK1) to facilitate actin filament-mediated spindle migration. Together, PAK1 is essential for microtubule organization and spindle migration during the MI-MII transition in porcine oocytes, which is associated with the activity of p-Aurora A, p-TACC3 and p-LIMK1.

Distinct functional constraints driving conservation of the cofilin N-terminal regulatory tail

Cofilin family proteins have essential roles in remodeling the cytoskeleton through filamentous actin depolymerization and severing. The short, unstructured N-terminal region of cofilin is critical for actin binding and harbors the major site of inhibitory phosphorylation. Atypically for a disordered sequence, the N-terminal region is highly conserved, but specific aspects driving this conservation are unclear. Here, we screen a library of 16,000 human cofilin N-terminal sequence variants for their capacity to support growth in S. cerevisiae in the presence or absence of the upstream regulator LIM kinase. Results from the screen and biochemical analysis of individual variants reveal distinct sequence requirements for actin binding and regulation by LIM kinase. LIM kinase recognition only partly explains sequence constraints on phosphoregulation, which are instead driven to a large extent by the capacity for phosphorylation to inactivate cofilin. We find loose sequence requirements for actin binding and phosphoinhibition, but collectively they restrict the N-terminus to sequences found in natural cofilins. Our results illustrate how a phosphorylation site can balance potentially competing sequence requirements for function and regulation.

FAK-p38 signaling serves as a potential target for reverting matrix stiffness-modulated liver sinusoidal endothelial cell defenestration

Liver sinusoidal endothelial cells (LSECs) are highly specific endothelial cells which play an essential role in the maintenance of liver homeostasis. During the progression of liver fibrosis, matrix stiffening promotes LSEC defenestration, however, the underlying mechanotransduction mechanism remains poorly understood. Here, we applied stiffness-tunable hydrogels to assess the matrix stiffening-induced phenotypic changes in primary mouse LSECs. Results indicated that increased stiffness promoted LSEC defenestration through cytoskeletal reorganization. LSECs sensed the increased matrix stiffness via focal adhesion kinase (FAK), leading to the activation of p38-mitogen activated protein kinase activated protein kinase 2 (MK2) pathway, thereby inducing actin remodeling via LIM Kinase 1 (LIMK1) and Cofilin. Interestingly, inhibition of FAK or p38-MK2 pathway was able to effectively restore the fenestrae to a certain degree in LSECs isolated from early to late stages of liver fibrosis mice. Thus, this study highlights the impact of mechanotransduction in LSEC defenestration, and provides novel insights for potential therapeutic interventions for liver fibrosis.

PAK3 downregulation induces cognitive impairment following cranial irradiation.

Cranial irradiation is used for prophylactic brain radiotherapy as well as the treatment of primary brain tumors. Despite its high efficiency, it often induces unexpected side effects, including cognitive dysfunction. Herein, we observed that mice exposed to cranial irradiation exhibited cognitive dysfunction, including altered spontaneous behavior, decreased spatial memory, and reduced novel object recognition. Analysis of the actin cytoskeleton revealed that ionizing radiation (IR) disrupted the filamentous/globular actin (F/G-actin) ratio and downregulated the actin turnover signaling pathway p21-activated kinase 3 (PAK3)-LIM kinase 1 (LIMK1)-cofilin. Furthermore, we found that IR could upregulate microRNA-206-3 p (miR-206-3 p) targeting PAK3. As the inhibition of miR-206-3 p through antagonist (antagomiR), IR-induced disruption of PAK3 signaling is restored. In addition, intranasal administration of antagomiR-206-3 p recovered IR-induced cognitive impairment in mice. Our results suggest that cranial irradiation-induced cognitive impairment could be ameliorated by regulating PAK3 through antagomiR-206-3 p, thereby affording a promising strategy for protecting cognitive function during cranial irradiation, and promoting quality of life in patients with radiation therapy.

PAK3 downregulation induces cognitive impairment following cranial irradiation

Cranial irradiation is used for prophylactic brain radiotherapy as well as the treatment of primary brain tumors. Despite its high efficiency, it often induces unexpected side effects, including cognitive dysfunction. Herein, we observed that mice exposed to cranial irradiation exhibited cognitive dysfunction, including altered spontaneous behavior, decreased spatial memory, and reduced novel object recognition. Analysis of the actin cytoskeleton revealed that ionizing radiation (IR) disrupted the filamentous/globular actin (F/G-actin) ratio and downregulated the actin turnover signaling pathway p21-activated kinase 3 (PAK3)-LIM kinase 1 (LIMK1)-cofilin. Furthermore, we found that IR could upregulate microRNA-206–3 p (miR-206–3 p) targeting PAK3. As the inhibition of miR-206–3 p through antagonist (antagomiR), IR-induced disruption of PAK3 signaling is restored. In addition, intranasal administration of antagomiR-206–3 p recovered IR-induced cognitive impairment in mice. Our results suggest that cranial irradiation-induced cognitive impairment could be ameliorated by regulating PAK3 through antagomiR-206–3 p, thereby affording a promising strategy for protecting cognitive function during cranial irradiation, and promoting quality of life in patients with radiation therapy.

Caspase-4 has a role in cell division in epithelial cells through actin depolymerization

In addition to its role in pyroptosis and inflammatory cytokine maturation, caspase-4 (CASP4) also contributes to the fusion of phagosomes with lysosomes and cell migration. However, its role in cell division remains elusive. In this study, we demonstrate that CASP4 is indispensable for proper cell division in epithelial cells. Knockout of CASP4 (CASP4 KO) in HepG2 cells led to delayed cell proliferation, increased cell size, and increased multinucleation. In mitosis, CASP4 KO cells showed multipolar spindles, asymmetric spindle positioning, and chromosome segregation errors, ultimately increasing DNA content and chromosome number. We also found that phalloidin, a marker of filamentous actin, increased in CASP4 KO cells owing to suppressed actin depolymerization. Moreover, the levels of actin polymerization-related proteins, including Rho-associated protein kinase1 (ROCK1), LIM kinase1 (LIMK1), and phosphorylated cofilin, significantly increased in CASP4 KO cells. These results suggest that CASP4 contributes to proper cell division through actin depolymerization.

Autoregulation of the LIM kinases by their PDZ domain

LIM domain kinases (LIMK) are important regulators of actin cytoskeletal remodeling. These protein kinases phosphorylate the actin depolymerizing factor cofilin to suppress filament severing, and are key nodes between Rho GTPase cascades and actin. The two mammalian LIMKs, LIMK1 and LIMK2, contain consecutive LIM domains and a PDZ domain upstream of the C-terminal kinase domain. The roles of the N-terminal regions are not fully understood, and the function of the PDZ domain remains elusive. Here, we determine the 2.0 Å crystal structure of the PDZ domain of LIMK2 and reveal features not previously observed in PDZ domains including a core-facing arginine residue located at the second position of the ‘x-Φ-G-Φ’ motif, and that the expected peptide binding cleft is shallow and poorly conserved. We find a distal extended surface to be highly conserved, and when LIMK1 was ectopically expressed in yeast we find targeted mutagenesis of this surface decreases growth, implying increased LIMK activity. PDZ domain LIMK1 mutants expressed in yeast are hyperphosphorylated and show elevated activity in vitro. This surface in both LIMK1 and LIMK2 is critical for autoregulation independent of activation loop phosphorylation. Overall, our study demonstrates the functional importance of the PDZ domain to autoregulation of LIMKs.

Role of LIM-Kinase 1 in Memory Processes

Abstract—According to modern ideas, the basis of intellectual problems in neurological brain damage is active forgetting, regulated by Rac and Rho small GTPases-dependent signal stages of actin remodeling. The key enzyme of these cascades is LIM kinase 1 (LIMK1). Changes in limk1 gene expression lead to neurocognitive pathologies. Rapid screening and testing of targeted therapeutic agents modifying protein-protein interactions of GTPases and components of signaling cascades requires the development and validation of simple animal models. Such an opportunity is provided by Drosophila, the mutant strains of which allow you to identify the nodal moments of intersection of biochemical and neural networks, accompanying active forgetting.

LIMK1 m6A-RNA methylation recognized by YTHDC2 induces 5-FU chemoresistance in colorectal cancer via endoplasmic reticulum stress and stress granule formation

LIM kinase 1 (LIMK1) is a member of the LIMK family that has been considered to be involved in chemoresistance in various tumors, and N6-methyladenosine (m6A) is the most abundant nucleotide modification on mRNA. However, whether elevated expression of LIMK1 leads to chemoresistance due to m6A modification remains to be further studied. The findings of our study indicate that high LIMK1 expression in colorectal cancer (CRC) cells promotes cell proliferation and increases resistance to 5-fluorouracil (5-FU). Moreover, downregulation of YTH domain-containing 2 (YTHDC2), an m6A “reader”, in CRC cells resulted in decreased recognition and binding to the m6A site “GGACA” in LIMK1 mRNA, thereby increasing LIMK1 mRNA stability and expression. Furthermore, the overexpression of LIMK1 facilitated eIF2α phosphorylation, which induced endoplasmic reticulum (ER) stress and promoted stress granule (SG) formation, ultimately leading to 5-FU resistance. This study evaluated the specificity of the YTHDC2/LIMK1/eIF2α signalling axis and the efficacy of related drugs in modulating 5-FU sensitivity in CRC.

Hippocampal LIMK1-mediated Structural Synaptic Plasticity in Neurobehavioral Deficits Induced by a Low-dose Heavy Metal Mixture.

Humans are commonly exposed to the representative neurotoxic heavy metals lead (Pb), cadmium (Cd), and mercury (Hg). These three substances can be detected simultaneously in the blood of the general population. We have previously shown that a low-dose mixture of these heavy metals induces rat learning and memory impairment at human exposure levels, but the pathogenic mechanism is still unclear. LIM kinase 1 (LIMK1) plays a critical role in orchestrating synaptic plasticity during brain function and dysfunction. Hence, we investigated the role of LIMK1 activity in low-dose heavy metal mixture-induced neurobehavioral deficits and structural synaptic plasticity disorders. Our results showed that heavy metal mixture exposure altered rat fear responses and spatial learning at general population exposure levels and that these alterations were accompanied by downregulation of LIMK1 phosphorylation and structural synaptic plasticity dysfunction in rat hippocampal tissues and cultured hippocampal neurons. In addition, upregulation of LIMK1 phosphorylation attenuated heavy metal mixture-induced structural synaptic plasticity, dendritic actin dynamics, and cofilin phosphorylation damage. The potent LIMK1 inhibitor BMS-5 yielded similar results induced by heavy metal mixture exposure and aggravated these impairments. Our findings demonstrate that LIMK1 plays a crucial role in neurobehavioral deficits induced by low-dose heavy metal mixture exposure by suppressing structural synaptic plasticity.

LIM-kinase 1 effects on memory abilities and male courtship song in Drosophila depend on the neuronal type.

The signal pathway of actin remodeling, including LIM-kinase 1 (LIMK1) and its substrate cofilin, regulates multiple processes in neurons of vertebrates and invertebrates. Drosophila melanogaster is widely used as a model object for studying mechanisms of memory formation, storage, retrieval and forgetting. Previously, active forgetting in Drosophila was investigated in the standard Pavlovian olfactory conditioning paradigm. The role of specific dopaminergic neurons (DAN) and components of the actin remodeling pathway in different forms of forgetting was shown. In our research, we investigated the role of LIMK1 in Drosophila memory and forgetting in the conditioned courtship suppression paradigm (CCSP). In the Drosophila brain, LIMK1 and p-cofilin levels appeared to be low in specific neuropil structures, including the mushroom body (MB) lobes and the central complex. At the same time, LIMK1 was observed in cell bodies, such as DAN clusters regulating memory formation in CCSP. We applied GAL4 × UAS binary system to induce limk1 RNA interference in different types of neurons. The hybrid strain with limk1 interference in MB lobes and glia showed an increase in 3-h short-term memory (STM), without significant effects on long-term memory. limk1 interference in cholinergic neurons (CHN) impaired STM, while its interference in DAN and serotoninergic neurons (SRN) also dramatically impaired the flies' learning ability. By contrast, limk1 interference in fruitless neurons (FRN) resulted in increased 15-60 min STM, indicating a possible LIMK1 role in active forgetting. Males with limk1 interference in CHN and FRN also showed the opposite trends of courtship song parameters changes. Thus, LIMK1 effects on the Drosophila male memory and courtship song appeared to depend on the neuronal type or brain structure.