Gautam Sondarva

Glycogen Synthase Kinase-3β Induces Neuronal Cell Death via Direct Phosphorylation of Mixed Lineage Kinase 3

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase member that activates the c-Jun N-terminal kinase (JNK) pathway. Aberrant activation of MLK3 has been implicated in neurodegenerative diseases. Similarly, glycogen synthase kinase (GSK)-3β has also been shown to activate JNK and contribute to neuronal apoptosis. Here, we show a functional interaction between MLK3 and GSK-3β during nerve growth factor (NGF) withdrawal-induced cell death in PC-12 cells. The protein kinase activities of GSK-3β, MLK3, and JNK were increased upon NGF withdrawal, which paralleled increased cell death in NGF-deprived PC-12 cells. NGF withdrawal-induced cell death and MLK3 activation were blocked by a GSK-3β-selective inhibitor, kenpaullone. However, the MLK family inhibitor, CEP-11004, although preventing PC-12 cell death, failed to inhibit GSK-3β activation, indicating that induction of GSK-3β lies upstream of MLK3. In GSK-3β-deficient murine embryonic fibroblasts, ultraviolet light was unable to activate MLK3 kinase activity, a defect that was restored upon ectopic expression of GSK-3β. The activation of MLK3 by GSK-3β occurred via phosphorylation of MLK3 on two amino acid residues, Ser789 and Ser793, that are located within the C-terminal regulatory domain of MLK3. Furthermore, the cell death induced by GSK-3β was mediated by MLK3 in a manner dependent on its phosphorylation of the specific residues within the C-terminal domain by GSK-3β. Taken together, our data provide a direct link between GSK-3β and MLK3 activation in a neuronal cell death pathway and identify MLK3 as a direct downstream target of GSK-3β. Inhibition of GSK-3 is thus a potential therapeutic strategy for neurodegenerative diseases caused by trophic factor deprivation.

Mixed Lineage Kinase-3/JNK1 Axis Promotes Migration of Human Gastric Cancer Cells following Gastrin Stimulation

Gastrin is a gastrointestinal peptide hormone, secreted by the gastric G cells and can exist as a fully processed amidated form (G17) or as unprocessed forms. All forms of gastrin possess trophic properties towards the gastrointestinal mucosa. An understanding of the signaling pathways involved is important to design therapeutic approaches to target gastrin-mediated cellular events. The studies described here were designed to identify the signaling pathways by which amidated gastrin (G17) mediates cancer cell migration. These studies indicated a time- and dose-dependent increase in gastric cancer cell migration after G17 stimulation, involving cholecystokinin 2 receptor. G17-induced migration was preceded by activation of MAPK pathways and was antagonized after pretreatment with SP600125, a pharmacological inhibitor of c-Jun-NH(2)-terminal kinase (JNK) pathway. Knockdown of endogenous JNK1 expression via small interference RNA (JNK1-siRNA) inhibited G17-induced phosphorylation of c-Jun and migration, and overexpression of wild-type JNK1 or constitutive active JNK1 promoted G17-induced migration. Studies designed to identify the MAPK kinase kinase member mediating JNK activation indicated the involvement of mixed lineage kinase-3 (MLK3), which was transiently activated upon G17 treatment. Inhibition of MLK3 pathway via a pan-MLK inhibitor or knockdown of MLK3 expression by MLK3-siRNA antagonized G17-induced migration. Incubation with G17 also resulted in an induction of matrix metalloproteinase 7 promoter activity, which is known to mediate migration and invasion pathways in cancer cells. Modulation of MLK3, JNK1, and c-Jun pathways modulated G17-induced matrix metalloproteinase 7 promoter activation. These studies indicate that the MLK3/JNK1 axis mediates G17-induced gastric cancer cell migration, which can be targeted for designing novel therapeutic strategies for treating gastric malignancies.

The Peptidyl-prolyl Isomerase Pin1 Up-regulation and Proapoptotic Function in Dopaminergic Neurons RELEVANCE TO THE PATHOGENESIS OF PARKINSON DISEASE

Background: Pin1 regulates several signaling proteins by isomerizing the cis/trans conformation of the Ser(P)-Pro peptide bond. Results: Pin1 is up-regulated in dopaminergic neurons in cell culture, animal models, and human PD brains. Pin1 inhibition protects dopaminergic neurons in PD models. Conclusion: Pin1 up-regulation plays a proapoptotic function in PD. Significance: Pin1 inhibition may be a viable translational strategy in PD. Parkinson disease (PD) is a chronic neurodegenerative disease characterized by a slow and progressive degeneration of dopaminergic neurons in substantia nigra. The pathophysiological mechanisms underlying PD remain unclear. Pin1, a major peptidyl-prolyl isomerase, has recently been associated with certain diseases. Notably, Ryo et al. (Ryo, A., Togo, T., Nakai, T., Hirai, A., Nishi, M., Yamaguchi, A., Suzuki, K., Hirayasu, Y., Kobayashi, H., Perrem, K., Liou, Y. C., and Aoki, I. (2006) J. Biol. Chem. 281, 4117–4125) implicated Pin1 in PD pathology. Therefore, we sought to systematically characterize the role of Pin1 in PD using cell culture and animal models. To our surprise we observed a dramatic up-regulation of Pin1 mRNA and protein levels in dopaminergic MN9D neuronal cells treated with the parkinsonian toxicant 1-methyl-4-phenylpyridinium (MPP+) as well as in the substantia nigra of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. Notably, a marked expression of Pin1 was also observed in the substantia nigra of human PD brains along with a high co-localization of Pin1 within dopaminergic neurons. In functional studies, siRNA-mediated knockdown of Pin1 almost completely prevented MPP+-induced caspase-3 activation and DNA fragmentation, indicating that Pin1 plays a proapoptotic role. Interestingly, multiple pharmacological Pin1 inhibitors, including juglone, attenuated MPP+-induced Pin1 up-regulation, α-synuclein aggregation, caspase-3 activation, and cell death. Furthermore, juglone treatment in the MPTP mouse model of PD suppressed Pin1 levels and improved locomotor deficits, dopamine depletion, and nigral dopaminergic neuronal loss. Collectively, our findings demonstrate for the first time that Pin1 is up-regulated in PD and has a pathophysiological role in the nigrostriatal dopaminergic system and suggest that modulation of Pin1 levels may be a useful translational therapeutic strategy in PD.

Mixed-lineage kinase 3 phosphorylates prolyl-isomerase Pin1 to regulate its nuclear translocation and cellular function

Nuclear protein peptidyl-prolyl isomerase Pin1-mediated prolyl isomerization is an essential and novel regulatory mechanism for protein phosphorylation. Therefore, tight regulation of Pin1 localization and catalytic activity is crucial for its normal nuclear functions. Pin1 is commonly dysregulated during oncogenesis and likely contributes to these pathologies; however, the mechanism(s) by which Pin1 catalytic activity and nuclear localization are increased is unknown. Here we demonstrate that mixed-lineage kinase 3 (MLK3), a MAP3K family member, phosphorylates Pin1 on a Ser138 site to increase its catalytic activity and nuclear translocation. This phosphorylation event drives the cell cycle and promotes cyclin D1 stability and centrosome amplification. Notably, Pin1 pSer138 is significantly up-regulated in breast tumors and is localized in the nucleus. These findings collectively suggest that the MLK3-Pin1 signaling cascade plays a critical role in regulating the cell cycle, centrosome numbers, and oncogenesis.

Protein 4.1-mediated Membrane Targeting of Human Discs Large in Epithelial Cells

Human discs large (hDlg) protein binds to protein 4.1R via a motif encoded by an alternatively spliced exon located between the SH3 and the C-terminal guanylate kinase-like domains. To evaluate the functional significance of protein 4.1R binding for subcellular localization of hDlg in vivo, we expressed full-length recombinant constructs of two naturally occurring isoforms of hDlg termed hDlg-I2 and hDlg-I3. The hDlg-I3 but not the hDlg-I2 isoform binds to the FERM (Four.1-Ezrin-Radixin-Moesin) domain of protein 4.1R in vitro. Upon transient transfection into subconfluent Madine-Darby canine kidney (MDCK) epithelial cells, the hDlg-I3 fused with the green fluorescent protein accumulated predominantly at the plasma membrane of cell-cell contact sites, whereas the hDlg-I2 fusion protein distributed in the cytoplasm. In contrast, in stably transfected confluent MDCK cells, both hDlg-I2 and -I3 isoforms localized efficiently to the lateral membrane, consistent with the previous notion that the N-terminal domain of hDlg mediates its membrane targeting in polarized epithelial cells. We introduced a double mutation (I38A/I40A) into the N-terminal domain of hDlg, which disrupted its interaction with DLG2, a key event in the membrane targeting of hDlg. Interestingly, the hDlg-I2 isoform harboring the I38A/I40A mutation mislocalized from the membrane into cytoplasm. Importantly, the hDlg-I3 isoform with the same mutation localized efficiently to the membrane of confluent MDCK cells. Together, our results demonstrate that in addition to the N-terminal targeting domain, the alternatively spliced I3 insertion plays a critical role in recruiting hDlg to the lateral membrane in epithelial cells via its interaction with protein 4.1R.

TRAF2-MLK3 interaction is essential for TNF-α-induced MLK3 activation

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase that is activated by tumor necrosis factor-α (TNF-α) and specifically activates c-Jun N-terminal kinase (JNK) on TNF-α stimulation. The mechanism by which TNF-α activates MLK3 is still not known. TNF receptor-associated factors (TRAFs) are adapter molecules that are recruited to cytoplasmic end of TNF receptor and mediate the downstream signaling, including activation of JNK. Here, we report that MLK3 associates with TRAF2, TRAF5 and TRAF6; however only TRAF2 can significantly induce the kinase activity of MLK3. The interaction domain of TRAF2 maps to the TRAF domain and for MLK3 to its C-terminal half (amino acids 511-847). Endogenous TRAF2 and MLK3 associate with each other in response to TNF-α treatment in a time-dependent manner. The association between MLK3 and TRAF2 mediates MLK3 activation and competition with the TRAF2 deletion mutant that binds to MLK3 attenuates MLK3 kinase activity in a dose-dependent manner, on TNF-α treatment. Furthermore the downstream target of MLK3, JNK was activated by TNF-α in a TRAF2-dependent manner. Hence, our data show that the direct interaction between TRAF2 and MLK3 is required for TNF-α-induced activation of MLK3 and its downstream target, JNK.

Estrogen Suppresses MLK3-Mediated Apoptosis Sensitivity in ER+ Breast Cancer Cells

Little knowledge exists about the mechanisms by which estrogen can impede chemotherapy-induced cell death of breast cancer cells. 17beta-Estradiol (E(2)) hinders cytotoxic drug-induced cell death in estrogen receptor-positive (ER(+)) breast cancer cells. We noted that the activity of the proapoptotic mixed lineage kinase 3 (MLK3) kinase was relatively higher in estrogen receptor-negative (ER(-)) breast tumors, suggesting that E(2) might inhibit MLK3 activity. The kinase activities of MLK3 and its downstream target, c-Jun NH(2)-terminal kinase, were rapidly inhibited by E(2) in ER(+) but not in ER(-) cells. Specific knockdown of AKT1/2 prevented MLK3 inhibition by E(2), indicating that AKT mediated this event. Furthermore, MLK3 inhibition by E(2) involved phosphorylation of MLK3 Ser(674) by AKT, attenuating the proapoptotic function of MLK3. We found that a pan-MLK inhibitor (CEP-11004) limited Taxol-induced cell death and that E(2) accentuated this limitation. Taken together, our findings indicate that E(2) inhibits the proapoptotic function of MLK3 as a mechanism to limit cytotoxic drug-induced death of ER(+) breast cancer cells.

Mixed Lineage Kinase 3 Modulates β-Catenin Signaling in Cancer Cells

Background: β-Catenin mediates a wide variety of cellular processes, but the signaling pathways regulating β-catenin downstream events are not fully understood. The role of MLK3 in modulating β-catenin pathway has not been reported earlier. Results: MLK3 can induce β-catenin stabilization but inhibit conventional β-catenin/TCF transcriptional activation. Conclusion: These provide a new mechanism of regulating β-catenin/TCF axis. Significance: MLK3 can be targeted in regulating the growth of β-catenin overexpressing tumors. Expression of β-catenin is strictly regulated in normal cells via the glycogen synthase kinase 3β (GSK3β)- adenomatous polyposis coli-axin-mediated degradation pathway. Mechanisms leading to inactivation of this pathway (example: activation of Wnt/β-catenin signaling or mutations of members of the degradation complex) can result in β-catenin stabilization and activation of β-catenin/T-cell factor (TCF) signaling. β-Catenin-mediated cellular events are diverse and complex. A better understanding of the cellular signaling networks that control β-catenin pathway is important for designing effective therapeutic strategies targeting this axis. To gain more insight, we focused on determining any possible cross-talk between β-catenin and mixed lineage kinase 3 (MLK3), a MAPK kinase kinase member. Our studies indicated that MLK3 can induce β-catenin expression via post-translational stabilization in various cancer cells, including prostate cancer. This function of MLK3 was dependent on its kinase activity. MLK3 can interact with β-catenin and phosphorylate it in vitro. Overexpression of GSK3β-WT or the S9A mutant was unable to antagonize MLK3-induced stabilization, suggesting this to be independent of GSK3β pathway. Surprisingly, despite stabilizing β-catenin, MLK3 inhibited TCF transcriptional activity in the presence of both WT and S37A β-catenin. These resulted in reduced expression of β-catenin/TCF downstream targets Survivin and myc. Immunoprecipitation studies indicated that MLK3 did not decrease β-catenin/TCF interaction but promoted interaction between β-catenin and KLF4, a known repressor of β-catenin/TCF transcriptional activity. In addition, co-expression of MLK3 and β-catenin resulted in significant G2/M arrest. These studies provide a novel insight toward the regulation of β-catenin pathway, which can be targeted to control cancer cell proliferation, particularly those with aberrant activation of β-catenin signaling.

Mixed Lineage Kinase–c-Jun N-Terminal Kinase Axis A Potential Therapeutic Target in Cancer

Mixed lineage kinases (MLKs) are members of the mitogen-activated protein kinase kinase kinase (MAP3K) family and are reported to activate MAP kinase pathways. There have been at least 9 members of the MLK family identified to date, although the physiological functions of all the family members are yet unknown. However, MLKs in general have been implicated in neurodegenerative diseases, including Parkinson and Alzheimer diseases. Recent reports suggest that some of the MLK members could play a role in cancer via modulating cell migration, invasion, cell cycle, and apoptosis. This review article will first describe the biology of MLK members and then discuss the current progress that relates to their functions in cancer.

Human Epidermal Growth Factor Receptor 2 (HER2) Impedes MLK3 Kinase Activity to Support Breast Cancer Cell Survival

Background: Amplification of HER2 suppresses downstream pro-apoptotic pathways for breast cancer cell survival. Results: The pro-apoptotic MLK3 kinase activity is suppressed by HER2. Conclusion: MLK3 mediates HER2-induced breast cancer cell viability. Significance: Understanding the mechanisms of MLK3 suppression by HER2 provides a basis to target MLK3 in HER2 positive breast cancer. Human epidermal growth factor receptor 2 (HER2) is amplified in ∼15–20% of human breast cancer and is important for tumor etiology and therapeutic options of breast cancer. Up-regulation of HER2 oncogene initiates cascades of events cumulating to the stimulation of transforming PI3K/AKT signaling, which also plays a dominant role in supporting cell survival and efficacy of HER2-directed therapies. Although investigating the underlying mechanisms by which HER2 promotes cell survival, we noticed a profound reduction in the kinase activity of a pro-apoptotic mixed lineage kinase 3 (MLK3) in HER2-positive (HER2+) but not in HER2-negative (HER2−) breast cancer tissues, whereas both HER2+ and HER2− tumors expressed a comparable level of MLK3 protein. Furthermore, the kinase activity of MLK3 was inversely correlated with HER2+ tumor grades. Moreover, HER2-directed drugs such as trastuzumab and lapatinib as well as depletion of HER2 or HER3 stimulated MLK3 kinase activity in HER2+ breast cancer cell lines. In addition, the noted inhibitory effect of HER2 on MLK3 kinase activity was mediated via its phosphorylation on Ser674 by AKT and that pharmacological inhibitors of PI3K/AKT prevented trastuzumab- and lapatinib-induced stimulation of MLK3 activity. Consistent with the pro-apoptotic function of MLK3, stable knockdown of MLK3 in the HER2+ cell line blunted the pro-apoptotic effects of trastuzumab and lapatinib. These findings suggest that HER2 activation inhibits the pro-apoptotic function of MLK3, which plays a mechanistic role in mediating anti-tumor activities of HER2-directed therapies. In brief, MLK3 represents a newly recognized integral component of HER2 biology in HER2+ breast tumors.