Tài liệu The regulatory role of mixed lineage kinase 4 beta in mapk signaling and ovarian cancer cell invasion

The University of Toledo The University of Toledo Digital Repository Theses and Dissertations 2013 The regulatory role of mixed lineage kinase 4 beta in MAPK signaling and ovarian cancer cell invasion Widian F. Abi Saab The University of Toledo Follow this and additional works at: http://utdr.utoledo.edu/theses-dissertations Recommended Citation Abi Saab, Widian F., "The regulatory role of mixed lineage kinase 4 beta in MAPK signaling and ovarian cancer cell invasion" (2013). Theses and Dissertations. Paper 2. This Dissertation is brought to you for free and open access by The University of Toledo Digital Repository. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of The University of Toledo Digital Repository. For more information, please see the repository's About page. A Dissertation entitled The Regulatory Role of Mixed Lineage Kinase 4 Beta in MAPK Signaling and Ovarian Cancer Cell Invasion by Widian F. Abi Saab Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biology _________________________________________ Dr. Deborah Chadee, Committee Chair _________________________________________ Dr. Douglas Leaman, Committee Member _________________________________________ Dr. Fan Dong, Committee Member _________________________________________ Dr. John Bellizzi, Committee Member _________________________________________ Dr. Max Funk, Committee Member _________________________________________ Dr. Robert Steven, Committee Member _________________________________________ Dr. William Taylor, Committee Member _________________________________________ Dr. Patricia R. Komuniecki, Dean College of Graduate Studies The University of Toledo May 2013 Copyright 2013, Widian Fouad Abi Saab This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of The Regulatory Role of Mixed Lineage Kinase 4 Beta in MAPK Signaling and Ovarian Cancer Cell Invasion by Widian F. Abi Saab Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biology The University of Toledo May 2013 Mixed lineage kinase 4 (MLK4) is a member of the MLK family of mitogenactivated protein kinase kinase kinases (MAP3Ks). As components of a three-tiered signaling cascade, MAP3Ks promote activation of mitogen-activated protein kinase (MAPK), which in turn regulates different cellular processes including proliferation and invasion. Here, we show that the beta form of MLK4 (MLK4β), unlike its close relative, MLK3, and other known MAP3Ks, negatively regulates the activities of the MAPKs, p38, ERK and JNK, even in response to stimuli such as sorbitol or TNFα. MLK4β also negatively regulates basal, but not TNFα-induced, NF-κB activity. Moreover, MLK4β undergoes autophosphorylation and has kinase activity towards histone H2A, but has no kinase activity towards the MAP2K, MEK4/SEK1, a known substrate for MLK3 and other MAP3Ks. Furthermore, MLK4β interacts with MLK3 and inhibits MLK3 activation. In addition, MLK4 blocks matrix metalloproteinase-9 gelatinase activity and invasion in SKOV3 ovarian cancer cells, both of which are cellular responses that require MLK3. Collectively, our data establish MLK4β as a novel suppressor of MLK3 activation, MAPK signaling and cell invasion. iii This work is dedicated to my dad, Fouad Abi Saab, and mom, Nabila Abi Saab, who sacrificed a lot to provide a good education for my brother and me. I most certainly would not be where I am today if it wasn’t for them. I also dedicate this work to my brother (Rawad), my grandmas (Fayza and Samia), my aunts (Thouraya, Feryal, Noha and Sonia) and all my cousins (Yara, Ziad, Lama, Wahid, Tamara and Faisal). However, a special dedication goes to my beloved Grandma, Fayza Darweesh, who is my role model. She is my inspiration and the source of my strength and has always been my number one supporter. Her words and constant encouragement are my driving force to move forward in life. I would also like to grab this opportunity to thank my dearest friends (Alexis, Ani, Celia, Hadil, Hashem, Meenakshi, Mirella, Nancy and Natalya) who’ve been extremely encouraging and supportive throughout my Ph.D. program. Acknowledgements First, I would like to thank my advisor, Dr. Chadee, who had given me the chance to be here and who taught me most of what I currently know in this field. Dr. Chadee is a very supportive and positive person and creates a very amiable environment for her students. In addition to being successful in her field, she is also extremely compassionate and understanding. She was very supportive especially during hard times and for that I’ll be forever grateful. Not only is Dr. Chadee successful in her career, but she also has an exemplary sense of humanity which makes her a great role model for me. I would also like to thank my committee members Dr. Douglas Leaman, Dr. Fan Dong, Dr. John Bellizzi, Dr. Max Funk, Dr. Robert Steven and Dr. William Taylor for their constant input and guidance. I especially thank Dr. Taylor, Dr. Leaman and Dr. Dong, for their technical support in a number of experiments. I would like to especially thank Cathy (Dr. Yu Zhan) for teaching me most of the techniques in lab and for being a good friend. Special thanks to Natalya Blessing for being a wonderful lab mate and friend. I would also like to thank Meenakshi Bhansali for her amazing friendship and support. Last but not least, I would like to thank Dr. Leah Rider, Jenny, Alan, Peter, Sneha, April and Kyoung for being such good friends and for adding a joyful and pleasant atmosphere to our working environment. v Table of Contents Abstract .............................................................................................................................. iii Acknowledgements ..............................................................................................................v Table of Contents ............................................................................................................... vi List of Figures .................................................................................................................... ix List of Abbreviations ......................................................................................................... xi 1 Introduction………………………………………………………………………..1 1.1 The Mitogen-activated protein kinase signaling cascade ...............................1 1.2 Characteristics and functions of MAPK proteins…………………………….3 1.2.1 The ERK1/2 pathway………………………………………………..3 1.2.2 The JNK pathway…………………………………………………...7 1.2.3 The p38 pathway…………………………………………………...11 1.2.4 The ERK5 pathway………………………………………………...14 1.3 The matrix metalloproteinases……………………………………………….15 1.4 The MAP2Ks………………………………………………………………...18 1.5 The MAP3Ks………………………………………………………………...20 1.5.1 The MEKK group………………………………………………….21 1.5.2 The Raf MAP3Ks………………………………………………….23 1.5.3 The TAK1 MAP3K group…………………………………………25 1.5.4 The TAO/Tpl2 and Mos MAP3K groups………………………….27 1.5.5 The MLK family of MAP3Ks……………………………………..27 vi 1.5.5.1 The DLK subgroup ...........................................................28 1.5.5.2 The ZAK subgroup………………………………………30 1.6 The MLK subfamily…………………………………………………………31 1.6.1 MLK1 and MLK2………………………………………………….32 1.6.2 MLK3 activation…………………………………………………...33 1.7 MLK3 signaling……………………………………………………………...36 1.7.1 MLK3 signaling in cancer…………………………………………38 1.8 MLK4: characteristics and function………………………………………….39 1.9 Significance…………………………………………………………………..40 2 Materials and Methods ...........................................................................................42 2.1 Cell culture…………………………………………………………………...42 2.2 Expression vectors…………………………………………………………...43 2.3 Plasmids and siRNA transfections…………………………………………...43 2.4 Immunoblotting………………………………………………………………45 2.5 Preparation of whole cell extracts and treatments…………………………...47 2.6 Immunoprecipitation…………………………………………………………47 2.7 MLK4β kinase assay…………………………………………………………48 2.8 Cell proliferation assay………………………………………………………49 2.9 Luciferase assay……………………………………………………………...50 2.10 Invasion assay………………………………………………………………50 2.11 Gelatin zymography………………………………………………………...51 3 Results……………………………………………………………………………52 3.1 The role of MLK4β in p38 signaling………………………………………...52 vii 3.1.1 The effect of ectopic expression of MLK4β on p38 activation…...52 3.1.2 The effect of endogenous MLK4 on the activation of p38. ………54 3.2 The effect of MLK4 on MEK3/MEK6 activation….………………………..56 3.3 The role of MLK4β in NF-κB signaling……………………………………..57 3.4 Comparison of the effects of MLK4β and MLK3 on p38 activation ……….60 3.5 The effects of MLK3 and MLK4 on ERK and JNK activation……………..62 3.6 MLK4β is not an upstream activator of MEK4……………………………...65 3.7 MLK4β kinase activity……………………………...……………………….67 3.8 The effect of MLK4β on MLK3 activation………………………………….69 3.9 The correlation between MLK4β expression and active MLK3 in different cell lines……………………………………………………………...……………….72 3.10 MLK4β associates with MLK3……………………………………………..75 3.11 The effect of MLK4 on cell proliferation…………………………………..77 3.12 MLK3 is required for cell invasion in ovarian cancer cells………………...79 3.13 MLK4β inhibits SKOV3 cell invasion……………………………………..81 3.14 MLK3 regulates MMP-2 and MMP-9 enzyme activity…………………….82 3.15 MLK4β reduces MMP-9 activity in SKOV3 cells…………………………85 4 Discussion………………………………………………………………………..86 References……………………………………………………………………………….95 viii List of Figures 1-1 The MAPK signaling cascade ..................................................................................2 1-2 The Ras/Raf/ERK1/2 signaling pathway .................................................................6 1-3 JNK-mediated apoptosis…………………………………………………………10 1-4 The p38 MAPK signaling pathway………………………………………………13 1-5 MMP-2 and -9: structure and activation…………………………………………17 1-6 The NF-κB pathway……………………………………………………………...26 1-7 Signaling of the DLK family of MAP3Ks……………………………………….29 1-8 The structural domains of MLKs………………………………………………...31 1-9 Model mechanism of MLK3 activation by Cdc42………………………………35 3-10 MLK4β expression inhibits basal and stimulus-induced p38 activation ...............54 3-11 Elevated active p38 in MLK4 knockdown cells…………………………………55 3-12 MLK4 negatively regulates MEK3/MEK6 activation…………………………...57 3-13 MLK4β negatively regulates basal NF-κB activation but has no effect on TNFα- induced NF-κB signaling……………………………………..………………………….59 3-14 MLK4β, unlike MLK3, inhibits activation of p38 ………………………………61 3-15 MLK3 promotes the activation of ERK and JNK in SKOV3 and HEY1B cells...62 3-16 MLK4 negatively regulates ERK and JNK activation…………………………...64 3-17 MLK4β does not phosphorylate Thr261 on GST-SEK1-KR……………………66 3-18 MLK4β: autophosphorylation and substrate specificity…………………………68 3-19 MLK4β inhibits induced MLK3 activation……………………………………...69 ix 3-20 MLK4 inhibits the basal activation of MLK3 in SKOV3 cells………………….71 3-21 Correlation between MLK4β expression and active MLK3.. …………...............74 3-22 MLK4β associates with MLK3………………………………………………......76 3-23 MLK4 has no effect on HCT116 cell proliferation……………………………...78 3-24 MLK3 is essential for SKOV3 and HEY1B cell invasion……………………….80 3-25 MLK4β reduces the invasion of SKOV3 cells..…………………………………82 3-26 MLK3 mediates MMP-2 and MMP-9 activation in SKOV3 and HEY1B cells by a mechanism that involves ERK and JNK………………….……………………………..84 3-27 MLK4β reduces MMP-9 activity in SKOV3 cells………………………………85 4-28 Schematic diagram illustrating the role of MLK4β in MAPK signaling………..94 x List of Abbreviations Akt1............................Rac-alpha serine/threonine kinase APS ............................Ammonium persulfate AP-1 ...........................Activator protein 1 ASK............................Apoptosis signal-regulating kinase ATF2 ..........................Activating transcription factor 2 ATM………………..Ataxia telangiectasia ATP ...........................Adenosine triphosphate Bax .............................Bcl2-associated X Bcl-2 ...........................B-cell lymphoma 2 BSA ............................Bovine serum albumin CBD ...........................Collagen binding domain CR ..............................Conserved region CRIB ..........................Cdc42/Rac interactive binding protein DTT ............................Dithiothreitol DLK ...........................Dual leucine zipper-bearing kinase DNA ...........................Deoxyribonucleic acid DSP ............................Dual specificity phosphatases ECM ...........................Extracellular matrix EMT ...........................Epithelial-mesenchymal transition EGF ............................Epidermal growth factor ERK ...........................Extracellular signal-regulated kinase FADD.……….……..Fas-associated death domain protein FasL............................Fas ligand FBS ............................Fetal bovine serum FFA…………………Free fatty acids FGD1……………….FYVE, RhoGEF and PH domain-containing protein 1 GADD45 ....................Growth arrest and DNA damage-inducible 45 GDP………………...Guanosine diphosphate GEF ............................Guanosine nucleotide exchange factor xi Grb2 ...........................Growth factor receptor binding protein 2 GST ............................Glutathione S-transferase GTP ............................Guanosine triphosphate h……………………..hours HBx…………………Hepatitis B x antigen HPK1..........................Hematopoietic protein kinase 1 IκBα ...........................Inhibitor of kappa B alpha IKK ............................IκB kinase IKKK..........................IκB kinase kinase IP ................................Immunoprecipitation JIP1 ............................JNK-interacting protein 1 JNK ...........................c-Jun N-terminal kinase LPS .............................Lipopolysaccharide LZ ...............................Leucine zipper KSR ............................Kinase suppressor of ras MAPK ........................Mitogen activated protein kinase MAPKAP-K...............MAPK-activated protein kinase MAP2K ......................MAPK kinase MAP3K ......................MAPK kinase kinase MEK...........................MAPK/ERK kinase MEKK ........................MEK kinase Met .............................Methionine MLK ...........................Mixed lineage kinase MLTKα ......................Mitogen-activated protein triple kinase alpha MKP ...........................MAPK phosphatase MMP ..........................Matrix metalloproteinase MNK ..........................MAPK interacting kinase MP-1 ..........................MEK partner 1 MSK ...........................Mitogen and stress activated kinase ND………………….Neuro-D NF ..............................Neurofibromatosis NF-κB ........................Nuclear factor kappa-light-chain enhancer of activated B cells NGF............................Neuron growth factor PAK1..........................p21-GTPase activated kinase 1 PARP……………….poly (ADP-ribose) polymerase (PARP) PB1.............................Phox/Bem1P PBS ............................Phosphate buffered saline PHD………………...Plextrin-homology domain xii PMSF .........................Phenylmethylsulphonyl fluoride PP2A ..........................Serine/threonine protein phosphatase 2A PP5 .............................Protein phosphatase 5 Pro ..............................Proline PTP............................. protein tyrosine phosphatase PVDF .........................Immobilon-P Polyvinylidene Flouride ROS………………...Reactive oxygen species RSK ............................p90 ribosomal S6 kinase RTK............................Receptor tyrosine kinase SAP1………………..Sodium-associated protein 1 SAM ...........................Sterile-alpha-motif SAPK .........................Stress-activated protein kinase SCG………………...Superior cervical ganglion Ser ..............................Serine SH ..............................Src homology siRNA ........................small interfering RNA SOS ............................Son of sevenless STAT3........................Signal transducer and activator of transcription 3 TAB1..........................TAK1-binding protein 1 TAK1 .........................Transforming growth factor β-activted protein 1 TAO ...........................Thousand and one amino acid TCR…………………T cell antigen receptor (TCR) TGFβ………………..Transforming growth factor beta TIMP ..........................Tissue inhibitor of metalloproteinases Thr ..............................Threonine TNFα ..........................Tumor necrosis factor alpha TLR4 ..........................Toll-like receptor 4 Tpl2 ............................Tumor progression locus 2 TRAF4 .......................TNF receptor-associated factor 4 Tyr ..............................Tyrosine ZAK ...........................Zipper-sterile-alpha motif kinase xiii Chapter 1 Introduction 1.1 The Mitogen-activated protein kinase signaling cascade The mitogen-activated protein kinase (MAPK) signaling pathway is a three-tiered signaling cascade that is conserved from yeast to higher mammals including humans (Widmann, et al., 1999). The MAPK pathway is activated by a wide range of stimuli such as stress, cytokines and growth factors and leads to different cellular responses including proliferation, inflammation, invasion and apoptosis (Figure 1) (Kyriakis and Avruch, 2001; Pearson, et al., 2001; Uhlik, et al., 2004). The MAPK kinase kinases, or MAP3Ks, form the top tier of the signaling cascade (Dhanasekaran and Johnson, 2007). Once MAP3Ks are activated, they phosphorylate and activate their immediate downstream targets, the MAPK kinases (MAP2Ks or MEKs) that in turn phosphorylate and activate MAPKs, the cascade’s executor kinases (Figure 1) (Johnson and Lapadat, 2002; Kyriakis and Avruch, 2001; Lawler, et al., 1998; Raingeaud, et al., 1996). The mammalian extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38 kinase and ERK5 are four major MAPKs involved in this signaling cascade, which upon stimulation, activate cytosolic or nuclear-localized effectors and thereby translate 1 the stimulus into a corresponding cellular response (Figure 1) (Ben-Levy, et al., 1998; Raingeaud, et al., 1996; Uhlik, et al., 2004). Figure 1. The MAPK signaling cascade. MAP3Ks are activated in response to stress, cytokines or growth factors. Active MAP3Ks phosphorylate and activate MAP2Ks that in turn phosphorylate and activate MAPKs. Active MAPKs activate cytosolic targets or activate transcription factors that regulate the expression of genes that control different cellular processes like proliferation, invasion, apoptosis and inflammation. 2 1.2 Characteristics and functions of MAPK proteins MAPKs are proteins that are ubiquitously expressed in all eukaryotic cell types but yet regulate different cellular responses in a stimulus- and cell type-specific manner (Dhanasekaran and Johnson, 2007; Uhlik, et al., 2004; Widmann, et al., 1999). MAPKs are proline directed serine/threonine kinases that activate nuclear or cytosolic substrates, by phosphorylation at serine or threonine residues found within the Pro-X-Ser/Thr-Pro consensus sequence (Alvarez, et al., 1991; Maeda and Firtel, 1997). MAPK proteins are activated upon dual phosphorylation, by specific MAP2Ks, on both the threonine and tyrosine residues of the Thr-X-Tyr motif present in the activation loop, where the amino acid X varies with different MAPKs (Ahn, et al., 1991; D'Mello, et al., 1993; DiDonato, et al., 1996; Estus, et al., 1994; Faris, et al., 1998; Frandsen and Schousboe, 1990). MAPKs undergo an ordered phosphorylation mechanism, whereby the tyrosine residue of the Thr-X-Tyr motif is phosphorylated first resulting in an increase in the affinity between MAPKs and their specific MAP2Ks, a step that allows the subsequent phosphorylation of the threonine residue and full MAPK activation (Haystead, et al., 1992). Dual phosphorylation of MAPKs triggers a series of conformational changes in the activation loop and surrounding sequences that ultimately result in the activation of these proteins (Canagarajah, et al., 1997). Of the MAPK pathways, ERK, JNK and p38 signaling pathways are the best characterized. 1.2.1 The ERK1/2 pathway ERK1 (44 kDa) and ERK2 (42 kDa), often referred to as ERK1/2, are two main ubiquitously expressed isoforms of ERK that share more than 85% sequence identity 3 (Boulton, et al., 1991; Chen, et al., 2001; Seger and Krebs, 1995). Activation of ERK1/2 occurs upon specific recognition and subsequent phosphorylation of the Thr and Tyr residues in the Thr-X-Tyr motif (Thr183 and Tyr185 in human ERK2 and Thr202 and Tyr204 in human ERK1) by the upstream MAP2Ks, MEK1 and MEK2 (Crews, et al., 1992; Zheng and Guan, 1993). Activity of ERK1/2 is also regulated by phosphatases, including the MAPK phosphatases or MKPs which are dual-specificity phosphatases (DSPs) that dephosphorylate both phospho-tyrosine and phospho-threonine residues (Owens and Keyse, 2007; Raman, et al., 2007). Of the different MKPs, MKP3 shows higher specificity towards ERK1/2 than other MAPKs (Zhang, et al., 2003). The serine/threonine protein phosphatase 2A, or PP2A, also functions as a regulator of ERK1/2 activity by dephosphorylating the threonine residue of the Thr-X-Tyr motif in ERK1/2 (Anderson, et al., 1990). Growth factors and mitogens are the primary activators of the ERK1/2 pathway, however, cytokines, activators of G protein-coupled receptors and different stresses have also been reported to activate this pathway (Johnson and Lapadat, 2002; Yoon and Seger, 2006). Early studies revealed a key role for Ras GTPases and B-Raf in ERK activation (depicted in Figure 2 below). Briefly, receptor tyrosine kinases (RTKs), upon binding to their ligands such as growth factors, undergo dimerization and cytoplasmic domain transphosphorylation. Another transphosphorylation process then follows on specific tyrosine residues in the cytoplasmic region of the RTK, which leads to full activation of the receptor. The phospho-tyrosines create docking sites for the Src homology 2 (SH2) domain of adaptor proteins, such as the growth factor receptor binding protein 2, or Grb2. Grb2 then recruits the guanine nucleotide exchange factor (GEF), son of sevenless (SOS), 4 via its Src homology 3 (SH3) domain. SOS then activates Ras by promoting the switch from an inactive GDP-bound to an active GTP-bound form (Buday and Downward, 2008; Downward, 1996; Wittinghofer, et al., 1997). Upon activation, Ras interacts with and promotes the activation of members of the Raf family of MAP3Ks, Raf-1, B-Raf and A-Raf. Once activated, Rafs phosphorylate and activate MEK1 and MEK2 that in turn activate ERK1/2 (Chadee and Kyriakis, 2004; Dhillon, et al., 2007; Dunn, et al., 2005). Active ERK1/2 will then undergo dimerization and either activate cytoplasmic substrates such as p90 ribosomal S6 kinases (RSKs), mitogen and stress activated kinases (MSKs) and MAPK interacting kinase (MNK), or translocate to the nucleus and regulate the expression of certain genes by directly activating several transcription factors including AP-1, c-Myc and c-Fos (Buday and Downward, 2008; Chen, et al., 2001; Dunn, et al., 2005; Raman, et al., 2007). 5 Figure 2. The Ras/Raf/ERK1/2 signaling pathway. In response to interaction with growth factors (GF), RTKs undergo dimerization and activation. Grb2 binds to the active RTK and recruits SOS which in turn activates Ras. Active Ras interacts with and activates the Raf members. Active Rafs phosphorylate and activate MEK1/2 that in turn phosphorylate and activate ERK1/2. Once activated, ERK1/2 can trigger a cellular response either by activating cytoplasmic targets or by inducing transcriptional activation by translocating to the nucleus and activating transcription factors. 6