Lemur Tyrosine Kinase-3 Research Articles

LMTK2 switches on canonical TGF-β1 signaling in human bronchial epithelial cells.

Transforming growth factor (TGF-β1) is a critical profibrotic mediator in chronic lung disease, and there are no specific strategies to mitigate its adverse effects. Activation of TGF-β1 signaling is a multipart process involving ligands, transmembrane receptors, and transcription factors. In addition, an intricate network of adaptor proteins fine-tunes the signaling strength, duration, and activity. Namely, Smad7 recruits growth arrest and DNA damage (GADD34) protein that then interacts with the catalytic subunit of phosphoprotein phosphatase 1 (PP1c) to inactivate TGF-β receptor (TβR)-I and downregulate TGF-β1 signaling. Little is known about how TGF-β1 releases TβR-I from the GADD34-PP1c inhibition to activate its signaling. Transmembrane lemur tyrosine kinase 2 (LMTK2) is a PP1c inhibitor, and our published data showed that TGF-β1 recruits LMTK2 to the cell surface. Here, we tested the hypothesis that TGF-β1 recruits LMTK2 to inhibit PP1c, allowing activation of TβR-I. First, LMTK2 interacted with the TGF-β1 pathway in the human bronchial epithelium at multiple checkpoints. Second, TGF-β1 inhibited PP1c by an LMTK2-dependent mechanism. Third, TGF-β1 used LMTK2 to activate canonical Smad3-mediated signaling. We propose a model whereby the LMTK2-PP1c and Smad7-GADD34-PP1c complexes serve as on-and-off switches in the TGF-β1 signaling in human bronchial epithelium.NEW & NOTEWORTHY Activation of the transforming growth factor (TGF)-β1 signaling pathway is complex, involving many ligands, transmembrane receptors, transcription factors, and modulating proteins. The mechanisms of TGF-β1 signaling activation/inactivation are not fully understood. We propose for the first time a model by which transmembrane lemur tyrosine kinase 2 (LMTK2) forms a complex with phosphoprotein phosphatase 1 (PP1c) to activate TGF-β1 signaling and Smad7, growth arrest and DNA damage (GADD34), and PP1C form a complex to inactivate TGF-β1 signaling in human bronchial epithelium.

Chemoinformatics and machine learning techniques to identify novel inhibitors of the lemur tyrosine kinase-3 receptor involved in breast cancer.

Breast cancer is still the largest cause of cancer death in women, and around 70% of primary breast cancer patients are estrogen receptor (ER)-positive, which is the most frequent kind of breast cancer. The lemur tyrosine kinase-3 (LMTK3) receptor has been linked to estrogen responsiveness in breast cancer. However, the function of LMTK3 in reaction to cytotoxic chemotherapy has yet to be studied. Breast cancer therapy research remains tricky due to a paucity of structural investigations on LMTK3. We performed structural investigations on LMTK3 using molecular docking and molecular dynamics (MD) simulations of the LMTK3 receptor in complex with the top three inhibitor molecules along with a control inhibitor. Analysis revealed the top three compounds show the best binding affinities during docking simulations. Interactive analysis of hydrogen bonds inferred hotspot residues Tyr163, Asn138, Asp133, Tyr56, Glu52, Ser132, Asp313, and Asp151. Some other residues in the 5-Å region determined strong alkyl bonds and conventional hydrogen bond linkages. Furthermore, protein dynamics analysis revealed significant modifications among the top complexes and the control system. There was a transition from a loop to a-helix conformation in the protein-top1 complex, and in contrast, in complexes top2 and top3, the formation of a stabilizing sheet in the C chain was observed, which limited significant mobility and increased complex stability. Significant structural alterations were observed in the protein-top complexes, including a shorter helix region and the creation of some loop regions in comparison to the control system. Interestingly, binding free energies, including MMGB/PBSA WaterSwap analysis estimation, reveals that the top1 complex system was more stable than other systems, especially in comparison to the control inhibitor complex system. These results suggest a the plausible mode of action for the novel inhibitors. Therefore, the current investigation contributes to understanding the mechanism of action, serving as a basis for future experimental studies.

Lemur tyrosine kinase 2 has a tumor-inhibition function in human glioblastoma by regulating the RUNX3/Notch pathway

Deregulation of lemur tyrosine kinase 2 (LMTK2) is a vital determinant for the onset and progression of malignancies, yet the relationship between LMTK2 and glioblastoma (GBM) is undetermined. This study was carried out to determine the relevance of LMTK2 in GBM. Initiating investigation by assessing The Cancer Genome Atlas (TCGA) data showed LMTK2 mRNA levels were decreased in GBM tissue. Later examination of clinical specimens confirmed low levels of LMTK2 mRNA and protein in GBM tissue. The downregulated level of LMTK2 in patients with GBM was related to poor overall survival. A suppressive function of LMTK2 on the proliferative capability and metastatic potential of GBM cells was demonstrated by overexpressing LMTK2 in GBM cell lines. Moreover, the restoration of LMTK2 augmented the sensitivity of GBM cells to the chemotherapy drug temozolomide. The mechanistic investigation uncovered LMTK2 as a regulator of the runt-related transcription factor 3 (RUNX3)/Notch signaling pathway. The overexpression of LMTK2 increased the expression of RUNX3 while inhibiting the activation of Notch signaling. The silencing of RUNX3 diminished the regulatory role of LMTK2 on Notch signaling. The inhibition of Notch signaling reversed the LMTK2-silencing-elicited protumor effects. Importantly, LMTK2-overexpressed GBM cells displayed weakened tumorigenicity in xenograft models. Our findings illustrate that LMTK2 has a tumor-inhibition function in GBM by constraining Notch signaling via RUNX3. This work indicates the deregulation of the LMTK2-mediated RUNX3/Notch signaling pathway may be a novel molecular mechanism for the malignant transformation of GBMs. This work highlights the interest in LMTK2-related approaches for treating GBM.

Abstract 5556: Lemur tyrosine kinase 3 serves as a predictor of patient outcome and a target for the treatment of ovarian cancer

Abstract The objective of these in vitro and in vivo studies was to validate Lemur Tyrosine Kinase 3 (LMTK3) as a specific target and predictor of clinical outcome in ovarian cancer. LMTK3 belongs to a family of regulated tyrosine kinases with three structurally related isoforms, LMTK1, LMTK2, and LMTK3. Both nuclear and cytoplasmic LMTK3 expressions correlated with tumor grade and patient survival in cancers such as breast and colorectal cancer. We tested the clinical significance of the LMTK3 gene by immunohistochemistry (IHC) using LMTK3 monoclonal antibody on formalin-fixed paraffin-embedded sections (FFPE) collected from 204 early-stage (stage I-II) ovarian cancer patients. Results from this IHC LMTK3 study revealed a higher cytoplasmic to nuclear localization of LMTK3 correlated with worse overall survival (P<0.01). Further investigation of LMTK3’s prognostic value by screening LMTK3 signaling in 270 stage III-IV ovarian cancer patients is ongoing. We utilized the MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay for testing the killing efficacy of targeting LMTK3 by a monoclonal antibody, siRNA, and specific LMTK3 binding peptides (LMTK3BP) in ovarian cancer cell lines SKOV3, MDAH-2774, A2780, and TOV-21G. Treatment with LMTK3 specific monoclonal antibody, siRNA, and LMTK3BP significantly induced killing of both chemosensitive and chemoresistant ovarian cancer cells without affecting normal cells in vitro. Moreover, we observed this killing as synergistic with both Cisplatin and Taxotere treatment in vitro. Lastly, we used an A2780 cell line derived orthotopic xenograft mouse model of ovarian cancer to test the efficacy of specific LMTK3BP in vivo. Strikingly, LMTK3BP 20 mg/kg IV dose given three times a week for three weeks showed a 35% tumor reduction. Furthermore, in vivo safety studies showed no signs of toxicity of the LMTK3BP, even at a very high dose of 40 mg/kg. Thus, with dose optimization in ongoing experiments, we could expect a higher efficacy. In conclusion, this study highlighted the importance of LMTK3 as a predictor of patient clinical outcome and potential target for treatment in ovarian cancer. Citation Format: Thea K. Kirsch-Mangu, Axel S. Tullberg, Anna Portela, Khalil Helou, Ghassan M. Saed. Lemur tyrosine kinase 3 serves as a predictor of patient outcome and a target for the treatment of ovarian cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5556.

Drug Repurposing of Human LMTK3 from ATP competitive inhibitors for Breast Cancer

Breast cancer is the second most common cancer in the world. The ovarian hormone, estrogen plays its role in stimulating breast cell division, breast growth and development and support of the growth of estrogens-responsive tumors. A recently characterized key molecule to breast cancer cell is Lemur tyrosine kinase-3 (LMTK3). LMTK3, belonging to receptor tyrosine kinase family, has been identified as a key protein involved in breast cancer and endocrine resistance. LMTK3 is involved at molecular level for invasion of cells throughout the surrounding tissue, breaching of tissue barrier and permeation of cancer cells using several mechanism. The computational approach is followed for the designing of specific inhibitors against human LMTK3 towards breast cancer treatment. With the use of 3D model of lemur tyrosine kinase3 domain structure and ATP binding site, ATP competitive inhibitors are used to screen the top inhibitors using the AutoDock Vina and AutoDock. The virtual screening and followed by re-docking methods result in identification of Dabrafenib and Axitinib are potent inhibitors for LMTK3 with effective complex formation. Thus, our results reveal the structural mechanism of the LMTK3 inhibition and proposed inhibitors can be used in breast cancer treatment with further experimental study.

The Inhibitory Properties of a Novel, Selective LMTK3 Kinase Inhibitor

Recently, the oncogenic role of lemur tyrosine kinase 3 (LMTK3) has been well established in different tumor types, highlighting it as a viable therapeutic target. In the present study, using in vitro and cell-based assays coupled with biophysical analyses, we identify a highly selective small molecule LMTK3 inhibitor, namely C36. Biochemical/biophysical and cellular studies revealed that C36 displays a high in vitro selectivity profile and provides notable therapeutic effect when tested in the National Cancer Institute (NCI)-60 cancer cell line panel. We also report the binding affinity between LMTK3 and C36 as demonstrated via microscale thermophoresis (MST). In addition, C36 exhibits a mixed-type inhibition against LMTK3, consistent with the inhibitor overlapping with both the adenosine 5'-triphosphate (ATP)- and substrate-binding sites. Treatment of different breast cancer cell lines with C36 led to decreased proliferation and increased apoptosis, further reinforcing the prospective value of LMTK3 inhibitors for cancer therapy.

LMTK2 inhibits Ab25-35-elicited ferroptosis, oxidative stress and apoptotic damage in PC12 cells through activating Nrf2/ARE signalling pathway.

Alzheimer's disease (AD), the most common contributor to dementia, is a growing global health problem. This study aimed to investigate the role of lemur tyrosine kinase 2 (LMTK2) in AD as well as its relevant mechanism. To establish an in vitro cell model, PC12 cells were challenged with 20 µmol/l Ab 25-35 for 24 h. RT-qPCR and western blot examined LMTK2 mRNA and protein expressions. With the application of CCK-8, TUNEL, iron colorimetric assay kit and DCFH-DA, the viability, apoptosis, Fe 2+ and ROS content in PC12 cells were assessed. Besides, the expressions of oxidative stress-, apoptosis-, ferroptosis- and Nrf2/ARE signalling-related proteins were evaluated with western blot. Moreover, commercial kits examined SOD, MDA and CAT contents. The results manifested that LMTK2 expression was noticeably downregulated in Ab 25-35 -treated PC12 cells. Notably, LMTK2 overexpression exhibited inhibitory effects on oxidative stress, apoptosis and ferroptosis in PC12 cells exposed to Ab 25-35 . The upregulated Nrf2, NQO1 and HO-1 expressions in LMTK2 overexpressed-PC12 cells with Ab 25-35 induction revealed that LMTK2 overexpression could activate the Nrf2/ARE signalling pathway. What is more, a series of cellular experiments further testified that ML385, a specific Nrf2 inhibitor, partly hindered the protective role of LMTK2 overexpression against Ab 25-35 -triggered oxidative stress, apoptosis and ferroptosis in PC12 cells. In conclusion, LMTK2 overexpression alleviated the ferroptosis, oxidant damage and apoptosis in PC12 cells exposed to Ab 25-35 through the activation of the Nrf2/ARE signalling pathway, indicating the potential target of LMTK2 in the treatment of AD.

Lemur tyrosine kinase 2 silencing inhibits the proliferation of gastric cancer cells by regulating GSK-3β phosphorylation and β-catenin nuclear translocation

ABSTRACT Previous studies on the mechanism of proliferation and cell cycle progression of gastric cancer cells have shown promising perspectives for the prevention and treatment of gastric cancer. The aim of the present study was to investigate the role of lemur tyrosine kinase 2 (LMTK2) in gastric cancer cell proliferation and cell cycle progression, as well as in tumor-bearing nude mouse models. The expression levels of LMTK2 were determined in gastric cancer cell lines. In addition, the effects of LMTK2 silencing or overexpression on cell proliferation were measured using Cell Counting Kit-8, BrdU and colony formation assays. Cell cycle progression was analyzed using flow cytometry and western blotting. The expression levels of proteins associated with the β-catenin pathway were assessed using western blot analysis. A tumor-bearing nude mouse model was established by injecting gastric cancer cells, and the effect of LMTK2 knockdown or overexpression on tumor growth was examined. The expression levels of LMTK2 were found to be upregulated in all gastric cancer cell lines. Moreover, LMTK2 knockdown inhibited cell proliferation, colony formation and cell cycle progression. LMTK2 knockdown also inhibited the activation of GSK-3β/β-catenin signaling, as evidenced by reduced GSK-3β phosphorylation and nuclear β-catenin levels. LMTK2 knockdown also suppressed tumor growth, whereas overexpression accelerated this process. In conclusion, LMTK2 silencing can inhibit the proliferation of gastric cancer cells in vitro and tumor growth in vivo by regulating GSK-3β phosphorylation and β-catenin nuclear translocation.

The multifaceted role of lemur tyrosine kinase 3 in health and disease.

In the last decade, LMTK3 (lemur tyrosine kinase 3) has emerged as an important player in breast cancer, contributing to the advancement of disease and the acquisition of resistance to therapy through a strikingly complex set of mechanisms. Although the knowledge of its physiological function is largely limited to receptor trafficking in neurons, there is mounting evidence that LMTK3 promotes oncogenesis in a wide variety of cancers. Recent studies have broadened our understanding of LMTK3 and demonstrated its importance in numerous signalling pathways, culminating in the identification of a potent and selective LMTK3 inhibitor. Here, we review the roles of LMTK3 in health and disease and discuss how this research may be used to develop novel therapeutics to advance cancer treatment.

Differential Gene Expression Analysis Reveals Global LMTK2 Regulatory Network and Its Role in TGF-β1 Signaling.

Lemur tyrosine kinase 2 (LMTK2) is a transmembrane Ser/Thr kinase whose role has been increasingly recognized; however, when compared to other kinases, understanding of the LMTK2 networks and biological functions is still limited. Recent data have shown that transforming growth factor (TGF)-β1 plays a role in modulating LMTK2 function by controlling its endocytic trafficking in human bronchial epithelial cells. Here, we aimed to unveil the LMTK2 regulatory network and elucidate how it affects cellular functions and disease pathways in either TGF-β1 dependent or independent manner. To understand how the LMTK2 and TGF-β1 pathways interconnect, we knocked down (KD) LMTK2 using small(si)RNA-mediated silencing in human bronchial epithelial CFBE41o- cells, treated cells with TGF-β1 or vehicle control, and performed differential gene expression analysis by RNA sequencing (RNAseq). In vehicle-treated cells, LMTK2 KD affected expression of 2,506 genes while it affected 4,162 genes after TGF-β1 stimulation. Bioinformatics analysis shows that LMTK2 is involved in diverse cellular functions and disease pathways, such as cell death and survival, cellular development, and cancer susceptibility. In summary, our study increases current knowledge about the LMTK2 network and its intersection with the TGF-β1 signaling pathway. These findings will serve as basis for future exploration of the predicted LMTK2 interactions and signaling pathways.

LMTK3 promotes tumorigenesis in bladder cancer via the ERK/MAPK pathway.

Lemur tyrosine kinase 3 (LMTK3) is a key member of the serine–threonine tyrosine kinase family. It plays an important role in breast cancer tumorigenesis and progression. However, its biological role in bladder cancer remains elusive. In this study, we demonstrated that LMTK3 was overexpressed in bladder cancer and was positively correlated with bladder cancer malignancy. High LMTK3 expression predicted poor overall survival. Knockdown of LMTK3 in bladder cancer cells triggered cell‐cycle arrest at G2/M phase, suppressed cell growth, and induced cell apoptosis in bladder cancer cells. Furthermore, Transwell assays revealed that reduction of LMTK3 decreased cell migration by regulating the epithelial‐to‐mesenchymal transition pathway. Conversely, LKTM3 overexpression was shown to promote proliferation and migration of bladder cancer cells. We assessed phosphorylation of MEK and ERK1/2 in bladder cancer cells depleted of LMTK3 and demonstrated a reduced phosphorylation status compared with the control group. Using an MAPK signaling‐specific inhibitor, U0126, we could rescue the promotion of proliferation and viability in LMTK3‐overexpressing cells. In conclusion, we extend the status of LMTK3 as an oncogene in bladder cancer and provide evidence for its function via the activation of the ERK/MAPK pathway. Thus, targeting LMTK3 may hold potential as a diagnostic and prognostic biomarker and as a possible future treatment for bladder cancer.

Discovery of cyclic guanidine-linked sulfonamides as inhibitors of LMTK3 kinase

Lemur tyrosine kinase 3 (LMTK3) is oncogenic in various cancers. In breast cancer, LMTK3 phosphorylates and modulates the activity of estrogen receptor-α (ERα) and is essential for the growth of ER-positive cells. LMTK3 is highly expressed in ER-negative breast cancer cells, where it promotes invasion via integrin β1. LMTK3 abundance and/or high nuclear expression have been linked to shorter disease free and overall survival time in a variety of cancers, supporting LMTK3 as a potential target for anticancer drug development. We sought to identify small molecule inhibitors of LMTK3 with the ultimate goal to pharmacologically validate this kinase as a novel target in cancer. We used a homogeneous time resolve fluorescence (HTRF) assay to screen a collection of mixture-based combinatorial chemical libraries containing over 18 million compounds. We identified several cyclic guanidine-linked sulfonamides with sub-micromolar activity and evaluated their binding mode using a 3D homology model of the LMTK3 KD.

TGF-β1 Augments the Apical Membrane Abundance of Lemur Tyrosine Kinase 2 to Inhibit CFTR-Mediated Chloride Transport in Human Bronchial Epithelia.

The most common disease-causing mutation in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, F508del, leads to cystic fibrosis (CF), by arresting CFTR processing and trafficking to the plasma membrane. The FDA-approved modulators partially restore CFTR function and slow down the progression of CF lung disease by increasing processing and delivery to the plasma membrane and improving activity of F508del-CFTR Cl– channels. However, the modulators do not correct compromised membrane stability of rescued F508del-CFTR. Transforming growth factor (TGF)-β1 is a well-established gene modifier of CF associated with worse lung disease in F508del-homozygous patients, by inhibiting CFTR biogenesis and blocking the functional rescue of F508del-CFTR. Lemur tyrosine kinase 2 (LMTK2) is a transmembrane protein localized at the apical and basolateral membrane domain of human bronchial epithelial cells. Phosphorylation of the apical membrane CFTR by LMTK2 triggers its endocytosis and reduces the abundance of membrane-associated CFTR, impairing the CFTR-mediated Cl– transport. We have previously shown that LMTK2 knockdown improves the pharmacologically rescued F508del-CFTR abundance and function. Thus, reducing the LMTK2 recruitment to the plasma membrane may provide a useful strategy to potentiate the pharmacological rescue of F508del-CFTR. Here, we elucidate the mechanism of LMTK2 recruitment to the apical plasma membrane in polarized CFBE41o- cells. TGF-β1 increased LMTK2 abundance selectively at the apical membrane by accelerating its recycling in Rab11-positive vesicles without affecting LMTK2 mRNA levels, protein biosynthesis, or endocytosis. Our data suggest that controlling TGF-β1 signaling may attenuate recruitment of LMTK2 to the apical membrane thereby improving stability of pharmacologically rescued F508del-CFTR.

Lemur Tyrosine Kinase 2 (LMTK2) Level Inversely Correlates with Phospho-Tau in Neuropathological Stages of Alzheimer's Disease.

Alzheimer’s disease (AD) is the most common neurodegenerative dementia. Mapping the pathomechanism and providing novel therapeutic options have paramount significance. Recent studies have proposed the role of LMTK2 in AD. However, its expression pattern and association with the pathognomonic neurofibrillary tangles (NFTs) in different brain regions and neuropathological stages of AD is not clear. We performed chromogenic (CHR) LMTK2 and fluorescent phospho-tau/LMTK2 double-labelling (FDL) immunohistochemistry (IHC) on 10–10 postmortem middle frontal gyrus (MFG) and anterior hippocampus (aHPC) samples with early and late neuropathological Braak tau stages of AD. MFG in early stage was our ‘endogenous control’ region as it is not affected by NFTs. Semiquantitative CHR-IHC intensity scoring revealed significantly higher (p < 0.001) LMTK2 values in this group compared to NFT-affected regions. FDL-IHC demonstrated LMTK2 predominance in the endogenous control region, while phospho-tau overburden and decreased LMTK2 immunolabelling were detected in NFT-affected groups (aHPC in early and both regions in late stage). Spearman’s correlation coefficient showed strong negative correlation between phospho-tau/LMTK2 signals within each group. According to our results, LMTK2 expression is inversely proportionate to the extent of NFT pathology, and decreased LMTK2 level is not a general feature in AD brain, rather it is characteristic of the NFT-affected regions.

Neuropathological characterization of Lemur tyrosine kinase 2 (LMTK2) in Alzheimer\u2019s disease and neocortical Lewy body disease

Alzheimer’s disease (AD) and neocortical Lewy body disease (LBD) are the most common neurodegenerative dementias, with no available curative treatment. Elucidating pathomechanism and identifying novel therapeutic targets are of paramount importance. Lemur tyrosine kinase 2 (LMTK2) is involved in several physiological and pathological cellular processes. Herewith a neuropathological characterization is presented in AD and neocortical LBD samples using chromogenic and fluorescent LMTK2 immunohistochemistry on post-mortem brain tissues and compared them to age-matched controls (CNTs). LMTK2 immunopositivity was limited to the neuronal cytoplasm. Neurons, including tau-positive tangle-bearing ones, showed decreased chromogenic and immunofluorescent labelling in AD in every cortical layer compared to CNT and neocortical LBD. Digital image analysis was performed to measure the average immunopositivity of groups. Mean grey values were calculated for each group after measuring the grey scale LMTK2 signal intensity of each individual neuron. There was significant difference between the mean grey values of CNT vs. AD and neocortical LBD vs. AD. The moderate decrease in neocortical LBD suggests the effect of coexisting AD pathology. We provide neuropathological evidence on decreased neuronal LMTK2 immunolabelling in AD, with implications for pathogenesis.

Overexpression of lemur tyrosine kinase-2 protects neurons from oxygen-glucose deprivation/reoxygenation-induced injury through reinforcement of Nrf2 signaling by modulating GSK-3β phosphorylation

Lemur tyrosine kinase-2 (LMTK2), a newly identified serine/threonine kinase, is a potential regulator of cell survival and apoptosis. However, little is known about its role in regulating neuronal survival during cerebral ischemia/reperfusion injury. The present study aimed to explore the potential function of LMTK2 in regulating neuronal survival using an in vitro model of oxygen-glucose deprivation/reoxygenation (OGD/R)-induced injury. Herein, we found that LMTK2 expression was markedly decreased in neurons following OGD/R exposure. Gain-of-function experiments demonstrated that LMTK2 overexpression significantly improved the viability and reduced apoptosis of neurons with OGD/R-induced injury. Moreover, LMTK2 overexpression reduced the production of reactive oxygen species (ROS) in OGD/R-exposed neurons. Notably, our results elucidated that LMTK2 overexpression reinforced the activation of nuclear factor erythroid 2-related factor (Nrf2)/antioxidant response element (ARE) antioxidant signaling associated with increased glycogen synthase kinase-3β (GSK-3β) phosphorylation. GSK-3β inhibition by its specific inhibitor significantly reversed LMTK2-inhibition-linked apoptosis and ROS production. Additionally, silencing Nrf2 partially reversed the LMTK2-overexpression-mediated neuroprotective effect in OGD/R-injured neurons. Taken together, our results demonstrated that LMTK2 overexpression alleviated OGD/R-induced neuronal apoptosis and oxidative damage by enhancing Nrf2/ARE antioxidant signaling via modulation of GSK-3β phosphorylation. Our study suggests LMTK2 is a potential target for neuroprotection during cerebral ischemia/reperfusion.

Silencing of lemur tyrosine kinase 2 restricts the proliferation and invasion of hepatocellular carcinoma through modulation of GSK-3β/Wnt/β-catenin signaling

Lemur tyrosine kinase 2 (LMTK2) was recently identified as a novel cancer-related gene in several human cancers. However, little is known of its function in hepatocellular carcinoma (HCC). Here we aim to investigate the expression pattern, biological function, and regulatory mechanism of LMTK2 in HCC. We found that LMTK2 was highly expressed in HCC tissues, and patients with high expression of LMTK2 in tumor tissues had shorter survival times. LMTK2 expression was also elevated in HCC cell lines, and LMTK2 silencing markedly repressed the proliferation and invasion of HCC cells. By contrast, LMTK2 overexpression exerted promotion effects on HCC cell proliferation and invasion. Our results demonstrate that LMTK2 silencing decreases the phosphorylation of glycogen synthase kinase-3β (GSK-3β) and the expression of an active β-catenin protein, leading to inhibition of Wnt/β-catenin signaling. Notably, GSK-3β inhibition significantly reversed the LMTK2 silencing-mediated antitumor effect on proliferation, invasion, and Wnt/β-catenin signaling in HCC cells. LMTK2 silencing retarded the tumor growth of HCC cells in an in vivo xenograft tumor model, associated with downregulation of Wnt/β-catenin signaling. In conclusion, our findings suggest that silencing of LMTK2 suppresses the proliferation and invasion of HCC cells through the inhibition of Wnt/β-catenin signaling, via GSK-3β, highlighting the importance of LMTK2/GSK-3β/Wnt/β-catenin signaling in HCC progression.

LMTK2 binds to kinesin light chains to mediate anterograde axonal transport of cdk5/p35 and LMTK2 levels are reduced in Alzheimer\u2019s disease brains

Cyclin dependent kinase-5 (cdk5)/p35 is a neuronal kinase that regulates key axonal and synaptic functions but the mechanisms by which it is transported to these locations are unknown. Lemur tyrosine kinase-2 (LMTK2) is a binding partner for p35 and here we show that LMTK2 also interacts with kinesin-1 light chains (KLC1/2). Binding to KLC1/2 involves a C-terminal tryptophan/aspartate (WD) motif in LMTK2 and the tetratricopeptide repeat (TPR) domains in KLC1/2, and this interaction facilitates axonal transport of LMTK2. Thus, siRNA loss of KLC1 or mutation of the WD motif disrupts axonal transport of LMTK2. We also show that LMTK2 facilitates the formation of a complex containing KLC1 and p35 and that siRNA loss of LMTK2 disrupts axonal transport of both p35 and cdk5. Finally, we show that LMTK2 levels are reduced in Alzheimer’s disease brains. Damage to axonal transport and altered cdk5/p35 are pathogenic features of Alzheimer’s disease. Thus, LMTK2 binds to KLC1 to direct axonal transport of p35 and its loss may contribute to Alzheimer’s disease.

Lemur tyrosine kinase 2 acts as a positive regulator of NF-κB activation and colon cancer cell proliferation.

Lemur tyrosine kinase 2 (LMTK2) belongs to both protein kinase and tyrosine kinase families. LMTK2 is less studied and little is known about its function. Here we demonstrate that LMTK2 modulates NF-κB activity and functions to promote colonic tumorigenesis. We found that LMTK2 protein was abundant in colon cancer cells and LMTK2 knockdown (LMTK2-KD) inhibited proliferation of colon cancer cells through inactivating NF-κB. In unstimulated condition, LMTK2 modulated NF-κB through inhibiting phosphorylation of p65 at Ser468. Mechanistically, LMTK2 phosphorylated protein phosphatase 1A (PP1A) to prevent PP1A from dephosphorylating p-GSK3β(Ser9). The p-GSK3β(Ser9) could not phosphorylate p65 at Ser468, which maintained the basal NF-κB activity. LMTK2 also modulated TNFα-activated NF-κB. LMTK2-KD repressed TNFα-induced IKKβ phosphorylation, IκBα degradation and NF-κB activation, implying that LMTK2 modulates TNFα-activated NF-κB via IKK. These results suggest that LMTK2 modulates basal and TNFα-induced NF-κB activities in different mechanisms. Animal studies show that LMTK2-KD suppressed colon cancer cell xenograft growth, decreased PP1A phosphorylation and increased p-p65(Ser468). Our results reveal the role and underlying mechanism of LMTK2 in colonic tumorigenesis and suggest that LMTK2 may serve as a potential target for chemotherapy of colon cancer.

The lncRNA MIR2052HG regulates ER\u03b1 levels and aromatase inhibitor resistance through LMTK3 by recruiting EGR1

BackgroundOur previous genome-wide association study using the MA.27 aromatase inhibitors adjuvant trial identified SNPs in the long noncoding RNA MIR2052HG associated with breast cancer-free interval. MIR2052HG maintained ERα both by promoting AKT/FOXO3-mediated ESR1 transcription and by limiting ubiquitin-mediated ERα degradation. Our goal was to further elucidate MIR2052HG’s mechanism of action.MethodsRNA-binding protein immunoprecipitation assays were performed to demonstrate that the transcription factor, early growth response protein 1 (EGR1), worked together with MIR2052HG to regulate that lemur tyrosine kinase-3 (LMTK3) transcription in MCF7/AC1 and CAMA-1 cells. The location of EGR1 on the LMTK3 gene locus was mapped using chromatin immunoprecipitation assays. The co-localization of MIR2052HG RNA and the LMTK3 gene locus was determined using RNA-DNA dual fluorescent in situ hybridization. Single-nucleotide polymorphisms (SNP) effects were evaluated using a panel of human lymphoblastoid cell lines.ResultsMIR2052HG depletion in breast cancer cells results in a decrease in LMTK3 expression and cell growth. Mechanistically, MIR2052HG interacts with EGR1 and facilitates its recruitment to the LMTK3 promoter. LMTK3 sustains ERα levels by reducing protein kinase C (PKC) activity, resulting in increased ESR1 transcription mediated through AKT/FOXO3 and reduced ERα degradation mediated by the PKC/MEK/ERK/RSK1 pathway. MIR2052HG regulated LMTK3 in a SNP- and aromatase inhibitor-dependent fashion: the variant SNP increased EGR1 binding to LMTK3 promoter in response to androstenedione, relative to wild-type genotype, a pattern that can be reversed by aromatase inhibitor treatment. Finally, LMTK3 overexpression abolished the effect of MIR2052HG on PKC activity and ERα levels.ConclusionsOur findings support a model in which the MIR2052HG regulates LMTK3 via EGR1, and LMTK3 regulates ERα stability via the PKC/MEK/ERK/RSK1 axis. These results reveal a direct role of MIR2052HG in LMTK3 regulation and raise the possibilities of targeting MIR2052HG or LMTK3 in ERα-positive breast cancer.