Role Of Mixed Lineage Kinase 3 Research Articles

Mixed lineage kinase-3 prevents cardiac dysfunction and structural remodeling with pressure overload.

Myocardial hypertrophy is an independent risk factor for heart failure (HF), yet the mechanisms underlying pathological cardiomyocyte growth are incompletely understood. The c-Jun NH2-terminal kinase (JNK) signaling cascade modulates cardiac hypertrophic remodeling, but the upstream factors regulating myocardial JNK activity remain unclear. In this study, we sought to identify JNK-activating molecules as novel regulators of cardiac remodeling in HF. We investigated mixed lineage kinase-3 (MLK3), a master regulator of upstream JNK-activating kinases, whose role in the remodeling process had not previously been studied. We observed increased MLK3 protein expression in myocardium from patients with nonischemic and hypertrophic cardiomyopathy and in hearts of mice subjected to transverse aortic constriction (TAC). Mice with genetic deletion of MLK3 (MLK3-/-) exhibited baseline cardiac hypertrophy with preserved cardiac function. MLK3-/- mice subjected to chronic left ventricular (LV) pressure overload (TAC, 4 wk) developed worsened cardiac dysfunction and increased LV chamber size compared with MLK3+/+ littermates ( n = 8). LV mass, pathological markers of hypertrophy ( Nppa, Nppb), and cardiomyocyte size were elevated in MLK3-/- TAC hearts. Phosphorylation of JNK, but not other MAPK pathways, was selectively impaired in MLK3-/- TAC hearts. In adult rat cardiomyocytes, pharmacological MLK3 kinase inhibition using URMC-099 blocked JNK phosphorylation induced by neurohormonal agents and oxidants. Sustained URMC-099 exposure induced cardiomyocyte hypertrophy. These data demonstrate that MLK3 prevents adverse cardiac remodeling in the setting of pressure overload. Mechanistically, MLK3 activates JNK, which in turn opposes cardiomyocyte hypertrophy. These results support modulation of MLK3 as a potential therapeutic approach in HF. NEW & NOTEWORTHY Here, we identified a role for mixed lineage kinase-3 (MLK3) as a novel antihypertrophic and antiremodeling molecule in response to cardiac pressure overload. MLK3 regulates phosphorylation of the stress-responsive JNK kinase in response to pressure overload and in cultured cardiomyocytes stimulated with hypertrophic agonists and oxidants. This study reveals MLK3-JNK signaling as a novel cardioprotective signaling axis in the setting of pressure overload.

MLK3 phosphorylation by ERK1/2 is required for oxidative stress-induced invasion of colorectal cancer cells.

Mixed lineage kinase 3 (MLK3) functions in migration and/or invasion of several human cancers; however, the role of MLK3 in colorectal cancer (CRC) invasion is unknown. MLK3 is a mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K) which activates MAPK pathways through either kinase-dependent or -independent mechanisms. Human colorectal tumors display increased levels of reactive oxygen species (ROS) or oxidative stress. ROS, such as H2O2, are important for carcinogenesis and activate MAPK signaling pathways. In human colorectal carcinoma (HCT116) cells treated with H2O2, extracellular signal-regulated kinases 1 and 2 (ERK1/2) were activated and MLK3 exhibited reduced electrophoretic mobility (shift) in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which was eliminated by phosphatase treatment. Pretreatment with the ROS scavenger N- acetyl-L-cysteine (NAC), the ERK1/2 inhibitor UO126, or ERK1/2 siRNA knockdown blocked the H2O2-induced shift of MLK3, while MLK3 inhibition with Cep1347 did not. In co-immunoprecipitation experiments performed on H2O2-treated HCT116 cells, endogenous MLK3 associated with endogenous ERK1/2 and B-Raf. Active ERK1 phosphorylated kinase dead FLAG-MLK3 in vitro, whereas ERK1 phosphorylation of kinase dead FLAG-MLK3-S705A-S758A was reduced. Both MLK3 siRNA knockdown and FLAG-MLK3-S705A-S758A expression decreased ERK1/2 activation in H2O2-treated cells. Prolonged H2O2 treatment activated ERK1/2 and promoted invasion of colon cancer cells, which was attenuated by MLK3 siRNA knockdown. Furthermore, S705A-S758A-FLAG-MLK3 demonstrated decreased oxidative-stress induced colon cancer cell invasion, but increased interaction with GST-B-Raf as compared to wild-type-FLAG-MLK3 in H2O2-treated cells. These results suggest oxidative stress stimulates an ERK1/2-dependent phosphorylation of MLK3 on Ser705 and Ser758, which promotes MLK3-dependent B-Raf and ERK1/2 activation; this positive feedback loop (PFL) enhances the invasion of colon cancer cells.

PKGIα Leucine Zipper Domain Mediates a Novel Interaction with MLK3, a Potential Anti‐Remodeling Substrate in the Heart

BackgroundHeart failure is a significant contributor to cardiovascular mortality. cGMP inhibits cardiac remodeling in part via cGMP‐dependent protein kinase G I α (PKGIα). Knock in mice with a mutant PKGIα Leucine Zipper (LZ) have both decreased cardiac function and JNK activity after transaortic constriction (TAC). JNK is regulated by MAPKs including mixed lineage kinase 3 (MLK3) which contains an LZ interacting motif. We hypothesized PKGIα interacts with MLK3 via the PKGIα LZ domain to regulate JNK.ResultsThe MLK3‐PKGIα interaction was observed in mouse heart by co‐immunoprecipitation of MLK3 and PKGIα (n=3). In Cos1 cells MLK3 co‐precipitated with PKGIα but was prevented by mutation of the PKGIα LZ domain (n=3). In cardiomyocytes JNK stimulation by 8‐Br‐cGMP was prevented in MLK3‐depleted cells (siRNA, n=3). The role of MLK3 in the heart was tested by subjecting MLK3 knockout mice (MLK3‐/‐) to TAC (7 days). Compared to WT littermates MLK3‐/‐ hearts had increased hypertrophy and LV end diastolic pressure indicating advanced cardiac dysfunction (n=8‐10)ConclusionThese data reveal a novel interaction between PKGIα‐MLK3 that requires the LZ domain. In cardiomyocytes MLK3 is necessary for cGMP dependent JNK activation. Loss of MLK3 in vivo promotes adverse cardiac remodeling.This work was supported by the NIH.

Abstract 1829: Regulation of Mixed Lineage Kinase-3 activity by Her2 and its implication in death or survival

Abstract Human Epidermal Growth Factor Receptor 2 (Her2) is a receptor tyrosine kinase that regulates cell growth and differentiation signaling pathways. The protein Her2 is significantly overexpressed in 20-30% of breast cancer. The available clinical data proves that Her2 amplification in breast cancer is associated with worst overall survival. Therefore, Her2 has become a very important candidate for drug targeting and currently Her2 positive patients are treated with humanized monoclonal antibody, Trastuzumab (Herceptin) and a small molecule, Lapatinib that blocks Her2 tyrosine kinase activity. It is reported that Her2 amplification leads to activation of multiple survival pathways, including PI3K-Akt. However, it is not clearly known how Trastuzumab promotes cell death and what pro-apoptotic pathways are activated downstream of Her2 blockage? It is also reported that patients on Trastuzumab develop subsequent resistance to the therapy. Previously we published that in breast cancer tumors, a pro-apoptotic kinase, Mixed Lineage Kinase-3 (MLK-3) was inhibited, specifically in estrogen receptor (ER) positive tumors. The kinase activity of MLK-3 was down regulated by estrogen and this prevented MLK-3-mediated cell death. Since Her2 serves as a drug target in breast cancer, therefore, we asked whether Her2 plays any role in regulating MLK-3 kinase activation and whether MLK-3 plays any role in Trastuzumab resistance. Our results showed that the endogenous MLK-3 kinase activity was up-regulated in SkBr3 cells upon treatment with Trastuzumab and Lapatinib. Whether the activation of MLK-3 upon Her2 blockage leads to cell death or survival is still not known and experiments are underway to investigate these aspects. We also observed that Her3 was the dimerization partner of Her2 in Trastuzumab- or Lapatinib- mediated MLK-3 activation. Not knowing any role of MLK-3 in Trastuzumab resistance, we used Trastuzumab sensitive and resistant BT474 cell model to investigate the role of MLK-3 in Trastuzumab resistance. Our data clearly showed that knockdown of MLK-3 caused spontaneous cell death in resistant cells. The Trastuzumab treatment further amplified cell death in MLK-3 knockdown resistant cells. The Akt1 activation in these cells was also diminished upon MLK-3 knockdown. Taken together, our data demonstrate that like ER, the Her2 also down regulates MLK-3 kinase activity and perhaps promote cell survival. Furthermore, paradoxically, the MLK-3 knockdown promoted cell death in Trastuzumab resistance BT474 cells, suggesting that MLK-3 could also serve as survival factor in resistant cells. Whether MLK-3 serves as a pro-apoptotic kinase or survival kinase in Trastuzumab mediated pathway needs to be extensively examined in near future. Citation Format: Subhasis Das, Gautam Sondarva, Navin Viswakarma, Rakesh Sathish Nair, Clodia Osipo, Basabi Rana, Ajay Rana. Regulation of Mixed Lineage Kinase-3 activity by Her2 and its implication in death or survival. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1829. doi:10.1158/1538-7445.AM2014-1829

Abstract 315: The Role of Mixed Lineage Kinase 3 in Inflammatory Cell-Fibroblast Communication

Mixed lineage kinase 3 (MLK3) is a ubiquitously expressed pro-inflammatory, pro-apoptotic mitogen activated protein kinase kinase kinase (MAP3K). MLK3 is a key regulator of the p38 and c-jun terminal kinase (JNK) pathways and has been studied in cancer and neurodegenerative disease. Although the p38 and JNK pathways have been studied in the cardiac function and disease, little is known regarding the role of MLK3 in the heart. Studies in our laboratory have indicated that MLK3 RNA is highly expressed in macrophages, cardiomyocytes and cardiac fibroblasts, suggesting an important role in cardiac function. Recently published work has indicated that MLK3 plays an important role in inflammatory cell motility, leading us to hypothesize that MLK3 is involved in inflammatory cell-fibroblast communication during cardiac disease. Knockout (KO) or inhibition of MLK3 using the novel small molecule inhibitor URMC-099 does not significantly affect heart rate, mean arterial pressure, systolic pressure, minimum or maximum dp/dt compared to wild type (WT) controls as measured by invasive hemodynamics at 3 months of age. Echocardiographic analysis indicates that MLK3 KO or inhibition does not affect cardiac architecture or cardiac function, indicated by fractional shortening or ejection fraction. However, upon transaortic constriction (TAC), MLK3 KO and URMC-099 treatment results in decreases in Mac-3 positive staining at 3 and 7 days post-TAC as well as decreases in CD-45 staining 7 days post-TAC compared to WT TAC operated, vehicle treated controls, suggesting that MLK3 KO and drug treatment may attenuate the early inflammatory response after TAC. Studies examining the relationship between the MLK3 mediated inflammatory response and subsequent fibrosis and cardiac dysfunction post-TAC are currently ongoing.

Mixed-lineage kinase 3 deficiency promotes neointima formation through increased activation of the RhoA pathway in vascular smooth muscle cells.

Mitogen-activated protein kinase pathways play an important role in neointima formation secondary to vascular injury, in part by promoting proliferation of vascular smooth muscle cells (VSMC). Mixed-lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase that activates multiple mitogen-activated protein kinase pathways and has been implicated in regulating proliferation in several cell types. However, the role of MLK3 in VSMC proliferation and neointima formation is unknown. The aim of this study was to determine the function of MLK3 in the development of neointimal hyperplasia and to elucidate the underlying mechanisms. Neointima formation was analyzed after endothelial denudation of carotid arteries from wild-type and MLK3-deficient mice. MLK3 deficiency promoted injury-induced neointima formation and increased proliferation of primary VSMC derived from aortas isolated from MLK3-deficient mice compared with wild-type mice. Furthermore, MLK3 deficiency increased the activation of p63Rho guanine nucleotide exchange factor, RhoA, and Rho kinase in VSMC, a pathway known to promote neointimal hyperplasia, and reconstitution of MLK3 expression attenuated Rho kinase activation. Furthermore, cJun NH2-terminal kinase activation was decreased in MLK3-deficient VSMC, and proliferation of wild-type but not MLK3 knockout cells treated with a cJun NH2-terminal kinase inhibitor was attenuated. We demonstrate that MLK3 limits RhoA activation and injury-induced neointima formation by binding to and inhibiting the activation of p63Rho guanine nucleotide exchange factor, a RhoA activator. In MLK3-deficient cells, activation of p63Rho guanine nucleotide exchange factor proceeds in an unchecked manner, leading to a net increase in RhoA pathway activation. Reconstitution of MLK3 expression restores MLK3/p63Rho guanine nucleotide exchange factor interaction, which is attenuated by feedback from activated cJun NH2-terminal kinase.

Hepatitis C Virus NS5A Inhibits Mixed Lineage Kinase 3 to Block Apoptosis

Hepatitis C virus (HCV) infection results in the activation of numerous stress responses including oxidative stress, with the potential to induce an apoptotic state. Previously we have shown that HCV attenuates the stress-induced, p38MAPK-mediated up-regulation of the K(+) channel Kv2.1, to maintain the survival of infected cells in the face of cellular stress. We demonstrated that this effect was mediated by HCV non-structural 5A (NS5A) protein, which impaired p38MAPK activity through a polyproline motif-dependent interaction, resulting in reduction of phosphorylation activation of Kv2.1. In this study, we investigated the host cell proteins targeted by NS5A to mediate Kv2.1 inhibition. We screened a phage-display library expressing the entire complement of human SH3 domains for novel NS5A-host cell interactions. This analysis identified mixed lineage kinase 3 (MLK3) as a putative NS5A interacting partner. MLK3 is a serine/threonine protein kinase that is a member of the MAPK kinase kinase (MAP3K) family and activates p38MAPK. An NS5A-MLK3 interaction was confirmed by co-immunoprecipitation and Western blot analysis. We further demonstrate a novel role of MLK3 in the modulation of Kv2.1 activity, whereby MLK3 overexpression leads to the up-regulation of channel activity. Accordingly, coexpression of NS5A suppressed this stimulation. Additionally we demonstrate that overexpression of MLK3 induced apoptosis, which was also counteracted by NS5A. We conclude that NS5A targets MLK3 with multiple downstream consequences for both apoptosis and K(+) homeostasis.

Abstract 301: Targeting Mixed Lineage Kinases in pathological cardiac fibroblast activation and intercellular communication

Cardiovascular disease (CVD) and the final clinical iteration, heart failure (HF), affect more than 82 million Americans yearly, affects >5.7 million). Although the cardiac myocyte has long been the focus of cardiac cellular research, our lab and others have demonstrated that pathological activation of cardiac fibroblasts (CF) after onset of disease or cardiac injury is also a key player in HF pathogenesis. Pathologic activation of CFs causes a release of various paracrine and autocrine factors that target cardiomyocytes (CM), local inflammatory cells and the CFs themselves. Elucidating the mechanisms involved in pathological ‘support cell (CF)’- ‘functional cell (CM)’ communication may hold therapeutic promise. Mixed lineage kinase 3 (MLK3) is a stress activated mitogen-activated protein kinase kinase kinase (MAPKKK) involved in pro-apoptotic pathways. Inhibition of MLK3 in HAND results in the attenuation of microglial cell activation, preservation of neuronal function and synaptic structures. The parity between microglia-neuron communication and CF-CM communication and the effects of pathological activation on the respective support cells for each tissue type suggested a role for MLK3 in aberrant CF-CM cross-talk associated with the development and progression of HF. Preliminary data in mice subjected to a pharmacological model of HF (chronic isoproterenol) that were treated with a small molecule inhibitor (URMC-099) of MLK3 demonstrated a reduction in the development of myocardial fibrosis compared to vehicle treated animals. Additionally, small molecule MLK3 inhibition attenuated the development of myocardial hypertrophy as measured by heart weight: body weight and heart weight: tibia length ratios. Studies in cultured neonatal rat ventricular fibroblasts (NRVF) demonstrated that small molecule MLK3 inhibition attenuated CF activation and transition to a myofibroblast phenotype, including reductions of pathologic CF markers such as α-SMA, IL-6, IL-1β, and others. Our data demonstrate that MLK3 plays an important role in pathologic CF-CM communication and myocardial hypertrophy, and suggest that small molecule inhibition of MLK3 holds therapeutic promise for HF.

Critical Role for Mixed-Lineage Kinase 3 in Acetaminophen-Induced Hepatotoxicity

c-Jun NH(2)-terminal kinase (JNK) activation plays a major role in acetaminophen (APAP)-induced hepatotoxicity. However, the exact mechanism of APAP-induced JNK activation is incompletely understood. It has been established that apoptosis signal-regulating kinase 1 (ASK1) regulates the late phase of APAP-induced JNK activation, but the mitogen-activated protein kinase kinase kinase that mediates the initial phase of APAP-induced JNK activation has not been identified. Oxidative stress produced during APAP metabolism causes JNK activation, which promotes mitochondrial dysfunction and results in the amplification of oxidative stress. Therefore, inhibition of the initial phase of JNK activation may be key to protection against APAP-induced liver injury. The goal of this study was to determine whether mixed-lineage kinase 3 (MLK3) mediates the initial, ASK1-independent phase of APAP-induced JNK activation and thus promotes drug-induced hepatotoxicity. We found that MLK3 was activated by oxidative stress and was required for JNK activation in response to oxidative stress. Loss of MLK3 attenuated APAP-induced JNK activation and hepatocyte death in vitro, independent of receptor-interacting protein 1. Moreover, JNK and glycogen synthase kinase 3β activation was significantly attenuated, and Mcl-1 degradation was inhibited in APAP-treated MLK3-knockout mice. Furthermore, we showed that loss of MLK3 increased expression of glutamate cysteine ligase, accelerated hepatic GSH recovery, and decreased production of reactive oxygen species after APAP treatment. MLK3-deficient mice were significantly protected from APAP-induced liver injury, compared with wild-type mice. Together, these studies establish a novel role for MLK3 in APAP-induced JNK activation and hepatotoxicity, and they suggest MLK3 as a possible target in the treatment of APAP-induced liver injury.

Abstract 2161: MLK3 signals to paxillin in chemokine-induced migration and invasion, and is necessary for breast cancer metastasis

Abstract Breast cancer metastasis is the major cause for mortality of patients with invasive breast cancer. Metastasis involves multiple steps including cancer cell invasion through basement membrane, intravasation, survival in the circulation, extravasation and colonization of distant organs. Identification of the signaling pathways through which prometastatic factors promote cancer cell migration and invasion is key to develop effective therapeutics for breast cancer. Mixed lineage kinase 3 (MLK3) is a MAP3K involved in activating each of the three major MAPK signaling pathways. The MLK3-JNK-AP-1 signaling axis has been shown to be required in breast cancer cell migration and invasion. Herein, we demonstrate that the prometastatic factors, CXCL12 and HGF, signal through MLK3-JNK to induce phosphorylation of the focal adhesion scaffold, paxillin on Ser 178, which is required for migration and invasion of human breast cancer cells. Furthermore, we show that MLK3 regulates the phosphorylation of paxillin at Tyr 118 and promotes the interaction of FAK with paxillin, which can be blocked by inhibition of MLK3. To further understand the role of paxillin phosphorylation at S178, a nonphosphorylatable paxillin mutant S178 A was generated. Expression of S178A blocks Y118 phosphorylation of paxillin in breast cancer cells. Silencing of mlk3, inhibition of JNK, or expression of the nonphosphorylatable paxillin S178A increases focal adhesion numbers and increases Rho activity, suggesting that the MLK3-JNK-paxillin axis limits Rho activity in breast cancer cells. Furthermore, MLK3 silencing is sufficient to prevent formation of metastases in a mouse xenograft model of spontaneous breast cancer metastasis. Our findings have identified a novel role of MLK3 in regulation of focal adhesion dynamics and cytoskeletal remodeling in the process of breast cancer cell migration and suggest that MLK3 may be a potential therapeutic target for metastatic breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2161. doi:1538-7445.AM2012-2161

Abstract 1002: Role of Mixed Lineage Kinase-3 in modulating ß-catenin signaling in cancer cells

Abstract Introduction: ß-catenin is a multifunctional protein that can regulate different cellular functions depending on its sub-cellular localization, and interacting partners. In the membrane, it interacts with E-cadherin and regulates cell adhesion and migration. In the cytoplasm, it interacts with TCF/LEF family of transcription factors and induces transcription of downstream targets (example: Cyclin D1, c-myc), which in turn mediate different cellular events. ß-catenin is a well established human oncogene, with numerous reports describing its over-expression and associated enhanced ß-catenin/TCF transcriptional activity in different tumors. Signaling pathways regulating ß-catenin/TCF axis are thus important targets for developing anticancer therapies particularly to control those with abnormal TCF activation. Paradoxically, ß-catenin can also induce cell death under certain conditions, the detailed mechanism of which is still obscure. It is conceivable that various upstream signaling pathways can modulate ß-catenin-dependent downstream events via post-translational modification. To understand the role of novel signaling pathways in regulating ß-catenin function, we focused on Mixed Lineage Kinase-3 (MLK-3), due to its close involvement with pro-migratory and apoptotic events. MLK3 is a MAP Kinase Kinase Kinase (MAP3K) member that primarily functions via activation of c-Jun-N-terminal Kinase (JNK) pathway. Aims: The present study was designed to determine whether MLK3 can regulate ß-catenin expression and functional activities in cancer cells. Experimental procedures: Cell lines originating from human embryonic kidney (HEK293), cervical carcinoma (HeLa), and prostate cancer (LNCaP) were utilized to determine the effect of MLK3 on ß-catenin expression and downstream events. These studies were performed utilizing Western Blot analysis, Immunoprecipitation, and luciferase assays. Results: Ectopic overexpression of MLK3-wild type (WT) resulted in a significant increase in ß-catenin protein expression in all the cell types tested. This effect requires the kinase activity of MLK3 since the kinase dead mutant MLK3-K/A was unable to induce ß-catenin expression. Immunoprecipitation studies also indicated that MLK3 can interact with ß-catenin in vivo in a kinase-independent manner. Surprisingly, MLK3-mediated increase in ß-catenin protein expression was associated with a reduction in ß-catenin/TCF transcriptional activity, although TCF4-ß-catenin interaction was not reduced under these conditions. Conclusion: The present study reveals that MLK3 can increase ß-catenin protein expression but lead to an inhibition of ß-catenin/TCF transcriptional activity, possibly via recruiting a transcriptional repressor. This pathway can be exploited further for developing MLK3/ß-catenin-based therapeutic strategies to target particularly those with aberrant ß-catenin/TCF signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1002.

Inhibition of MLK3 Decreases Proliferation and Increases Antiproliferative Activity of Epidermal Growth Factor Receptor (EGFR) Inhibitor in pancreatic cancer cell Lines

Pancreatic adenocarcinoma is associated with advanced presentation and poor survival. Currently approved therapies have minimal effect on patient survival. Pancreatic adenocarcinomas have a high incidence of activated K-RAS, which may confer resistance to epidermal growth factor receptor (EGFR) inhibitors. Mixed lineage kinase-3 (MLK3) is a MAP3K that activates multiple MAPK pathways. The role of MLK3 in the pathophysiology and resistance to therapy of pancreatic adenocarcinoma has not been investigated. MLK3 is over expressed in pancreatic cancer cell lines compared to an immortalized pancreatic epithelial cell line. The requirement of MLK3 for cell proliferation and survival of pancreatic cancer cell lines, PANC-1 and MiaPaCa-2, was investigated using RNA interference (siRNA) and MLK inhibitor, K252a, alone or in conjunction with the EGFR inhibitor, Compound 56. Ablation of expression of MLK3 via siRNA-mediated gene silencing and pharmacological inhibition of MLK3 by K252a each decreased cell viability in both pancreatic cancer cell lines, with a concurrent decrease in the activation of ERK, JNK and AKT. Concomitant inhibition of EGFR and MLK3 induced apoptosis, as evidenced by increased cleavage of PARP and caspase-3. These results suggest that MLK3 plays an important role in survival and proliferation of pancreatic cancer cell lines and that inhibition of MLK3 may enhance the therapeutic efficacy of EGFR inhibitors in the treatment of pancreatic cancer.

Novel cell death signaling pathways in neurotoxicity models of dopaminergic degeneration: Relevance to oxidative stress and neuroinflammation in Parkinson's disease

Parkinson's disease (PD) is a common neurodegenerative movement disorder characterized by extensive degeneration of dopaminergic neurons in the nigrostriatal system. Neurochemical and neuropathological analyses clearly indicate that oxidative stress, mitochondrial dysfunction, neuroinflammation and impairment of the ubiquitin-proteasome system (UPS) are major mechanisms of dopaminergic degeneration. Evidence from experimental models and postmortem PD brain tissues demonstrates that apoptotic cell death is the common final pathway responsible for selective and irreversible loss of nigral dopaminergic neurons. Epidemiological studies imply both environmental neurotoxicants and genetic predisposition are risk factors for PD, though the cellular mechanisms underlying selective dopaminergic degeneration remain unclear. Recent progress in signal transduction research is beginning to unravel the complex mechanisms governing dopaminergic degeneration. During the 12th International Neurotoxicology meeting, discussion at one symposium focused on several key signaling pathways of dopaminergic degeneration. This review summarizes two novel signaling pathways of nigral dopaminergic degeneration that have been elucidated using neurotoxicity models of PD. Dr. Anumantha Kanthasamy described a cell death pathway involving the novel protein kinase C delta isoform (PKCδ) in oxidative stress-induced apoptotic cell death in experimental models of PD. Dr. Ajay Rana presented his recent work on the role of mixed lineage kinase-3 (MLK3) in neuroinflammatory processes in neurotoxic cell death. Collectively, PKCδ and MLK3 signaling pathways provide new understanding of neurodegenerative processes in PD, and further exploration of these pathways may translate into effective neuroprotective drugs for the treatment of PD.

A new identity for MLK3 as an NIMA-related, cell cycle-regulated kinase that is localized near centrosomes and influences microtubule organization.

Although conserved counterparts for most proteins involved in the G(2)/M transition of the cell cycle have been found in all eukaryotes, a notable exception is the essential but functionally enigmatic fungal kinase NIMA. While a number of vertebrate kinases have been identified with catalytic domain homology to NIMA, none of these resemble NIMA within its extensive noncatalytic region, a region critical for NIMA function in Aspergillus nidulans. We used a bioinformatics approach to search for proteins with homology to the noncatalytic region of NIMA and identified mixed lineage kinase 3 (MLK3). MLK3 has been proposed to serve as a component in MAP kinase cascades, particularly those resulting in the activation of the c-Jun N-terminal kinase (JNK). Here we describe the first in-depth study of endogenous MLK3 and report that, like NIMA, MLK3 phosphorylation and activity are enhanced during G(2)/M, whereas JNK remains inactive. Coincident with the G(2)/M transition, a period marked by dramatic reorganization of the cytoplasmic microtubule network, endogenous MLK3 transiently disperses away from the centrosome and centrosomal-proximal sites where it is localized during interphase. Furthermore, when overexpressed, MLK3, like NIMA, localizes to the centrosomal region, induces profound disruption of cytoplasmic microtubules and a nuclear distortion phenotype that differs from mitotic chromosome condensation. Cellular depletion of MLK3 protein using siRNA technology results in an increased sensitivity to the microtubule-stabilizing agent taxol. Our studies suggest a new role for MLK3, separable from its function in the JNK pathway, that may contribute to promoting microtubule instability, a hallmark of M phase entry.