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

The University of ToledoThe University of Toledo Digital Repository Theses and Dissertations 2013 The regulatory role of mixed lineage kinase 4 beta in MAPK signaling and ovarian cancer

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 invasionWidian F Abi Saab

The University of Toledo

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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.

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

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 mitogen-activated 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

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

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

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

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

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

3-20 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

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

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

PAK1 p21-GTPase activated kinase 1

PARP……….poly (ADP-ribose) polymerase (PARP)

PB1 Phox/Bem1P

PBS Phosphate buffered saline

PHD……… Plextrin-homology domain

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

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

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

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

(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),

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)

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

ERK1/2 is one of the main regulators of cell proliferation and transformation and the ERK signaling pathway is deregulated at a high frequency in human cancers (Dhillon, et al., 2007) ERK1/2, however, is implicated in numerous other functions including differentiation, cell motility, cell migration, cytoskeletal polymerization and golgi fragmentation (Ishibe, et al., 2004; Jesch, et al., 2001; Reszka, et al., 1995; Yoon and Seger, 2006) The ERKs have different subcellular localizations In addition to the cytoplasm and nucleus, ERK1/2 were found to associate with membrane receptors and transporters, intracellular membrane compartments, microtubules, adherens junctions and focal adhesions (Furuchi and Anderson, 1998; Ishibe, et al., 2004; Jesch, et al., 2001; Reszka, et al., 1995) Moreover, continuous ERK1/2 nucleo-cytoplasmic trafficking is required for proper signaling of ERK1/2 (Costa, et al., 2006; Marchi, et al., 2008)

1.2.2 The JNK pathway

JNK is a proline-directed kinase that is activated by stresses and TNFα (Kyriakis,

et al., 1994) JNK1/SAPKβ, JNK2/SAPKα and JNK3/SAPKγ (ranging between 46 and 54kDa) are three JNK isoforms that share more than 85% sequence identity (Johnson and Lapadat, 2002; Kyriakis and Avruch, 2001) While JNK3 expression is mostly restricted

to the brain and heart, JNK1 and JNK2 are ubiquitously expressed (Dhillon, et al., 2007) JNK is activated upon the dual phosphorylation of the threonine and tyrosine residues of the Thr-X-Tyr motif by the MAP2Ks, MEK4 and MEK7 (Lawler, et al., 1998) The dual specificity phosphatases MKP7 and VH5 also function to regulate JNK activity by dephosphorylating the active JNK sites (Alonso, et al., 2004) The phosphoserine/threonine protein phosphatase 5 (PP5) is yet another phosphatase that

deregulates the JNK pathway PP5, however, does so by acting upstream of JNK, primarily by inactivating MAP3Ks such as the apoptosis signal-regulating kinase 1, or ASK1 (Chinkers, 2001; Zhou, et al., 2004)

The JNK pathway is mainly activated by cytokines and stresses but is also activated to a lesser extent by growth factors and inhibition of DNA and protein synthesis (Kyriakis and Avruch, 2001) Once activated, JNK translocates to the nucleus where it activates numerous transcription factors, including c-Jun Activation of c-Jun by JNK

was found to mediate Ras-induced tumorigenesis and cellular transformation in vitro

(Johnson, et al., 1996; Smeal, et al., 1991) Other transcription factors activated by JNK proteins include p53, STAT3, ATF2, ELK1 and nuclear factor of activated T cells (Chen,

et al., 2001; Ip and Davis, 1998) Through gene expression driven by one or more of these transcription factors, JNK can mediate differentiation, cytokine production, actin reorganization, inflammatory responses and apoptosis (Chen, et al., 2001) Of these different cellular responses, the ability of JNK to promote apoptosis is one of the best characterized

Although the exact mechanism by which JNK triggers cell death is not well known, several lines of evidence support a role for JNK in mitochondrial apoptotic cell death (Dhanasekaran and Reddy, 2008) Mitochondrial apoptosis or the intrinsic apoptotic pathway is characterized by the permeabilization of the mitochondrial membrane, the release of cytochrome c, and the activation of caspases 9 and 3 (Baliga and Kumar, 2003) This process is regulated by the Bcl-2 family of proteins (Kim, 2005) JNK can activate mitochondrial cell death through phosphorylation and inhibition of the anti-apoptotic protein, Bcl-2 (Figure 3) (Dhanasekaran and Reddy, 2008) Bcl-2 normally

sequesters and inactivates the pro-apoptotic protein Bax (Breckenridge and Xue, 2004) Upon phosphorylation, Bcl-2 dissociates from Bax rendering Bax active Bax then undergoes multimer formation that inserts in the mitochondrial membrane, forming channels that allow the release of cytochrome c and other apoptotic proteins leading to caspase-3 dependent cell death (Figure 3) (Breckenridge and Xue, 2004) JNK can also induce apoptosis in a p53-dependent manner p53, upon phosphorylation and activation

by JNK, can induce apoptosis by mediating the expression of pro-apoptotic genes such as Bax (Figure 3) (Miyashita and Reed, 1995) p53 can also induce apoptosis in a transcription-independent manner (Figure 3) (Caelles, et al., 1994) JNK can also trigger apoptosis by the induction of the extrinsic apoptotic pathway (Figure 3) (Dhanasekaran and Reddy, 2008; Tang, et al., 2012) The extrinsic apoptotic pathway is mediated by FasL that upon binding to Fas and the adaptor protein FADD, activates caspase 8 which then triggers apoptosis in a caspase 3-dependent manner (Guicciardi and Gores, 2009; Strasser, et al., 2009) JNK was shown to activate this pathway by inducing the expression of FasL (Figure 3) (Dhanasekaran and Reddy, 2008) For instance, JNK upregulates FasL protein expression in HepG2 cells in response to the hepatitis B virus X protein (HBx) in a mechanism that depends on MLK3 and MEK7 (Tang, et al., 2012)

Figure 3 JNK-mediated apoptosis JNK can induce mitochondrial apoptotic cell death

through the phosphorylation and inactivation of Bcl-2 either directly or through Bad and Bim This leads to the activation of Bax which forms mitochondrial channels through which cytochrome c is released Cytochrome c then activates the caspase cascade JNK can also trigger the expression of pro-apoptotic genes by activating transcription factors such as p53 JNK activates the extrinsic apoptotic pathway by increasing the expression

of the pro-apoptotic FasL gene

Bcl-2 Bax JNK

Bcl-2 P

Bax Bax Bax

Bax Bax Cyt c

1.2.3 The p38 pathway

p38 is a family of MAPK proteins that consists of four main members in vertebrates (α, β, γ and δ) The p38 isoforms are encoded by different genes with variations in the sequence homology (Jiang, et al., 1996; Lechner, et al., 1996) The variations in the sequence homology and in the interactions with other molecules could explain the diverse functions of p38 (Cuenda and Rousseau, 2007) Moreover, of the four p38 isoforms, only p38α and p38β are ubiquitously expressed while the other forms show more limited expression, with p38γ mainly found in skeletal muscle and p38δ in pancreas, kidney and small intestine (Goedert, et al., 1997; Raman, et al., 2007)

Thr-Gly-Tyr is the conserved motif in the activation loop of the p38 family, and dual phosphorylation of the threonine and tyrosine residues lead to p38 activation (Kyriakis and Avruch, 2001) MEK3 and MEK6 are the MAP2Ks that selectively activate the p38 family of MAPKs (Cuenda, et al., 1997; Raingeaud, et al., 1996) The JNK activator MEK4 can also activate p38 (Brancho, et al., 2003) Although p38 is mainly activated by MEKs, activation of p38α can also occur in a MEK-independent manner In this respect, p38α, upon binding to TAB1 (Transforming growth factor β-activated protein 1 (TAK1)-binding protein 1), undergoes autophosphorylation at Thr180 and Tyr182 and subsequent activation (Ge, et al., 2002) Moreover, in T cells, p38α was proposed to be activated by undergoing conformational change and autophosphorylation

at the activation sites (Thr180 and Try182) following the phosphorylation of Tyr323 by tyrosine kinases in a mechanism that is mediated by the T cell antigen receptor (TCR) (Salvador, et al., 2005) Similar to other MAPKs, the activity of p38 is also regulated by multiple phosphatases Some of these phospatases that interact with and inhibit p38

include MKP1, MKP5, MKP7 and MKP8 These phosphatases can also act on JNK but show low affinity towards ERK1/2 (Keyse, 2000; Raman, et al., 2007; Tanoue and Nishida, 2003) MKP1-null mice show enhanced activity of p38 and production of proinflammatory cytokines in response to low exposures to LPS as compared to wild types, suggesting MKP1 as an important regulator of LPS-induced p38 activity (Chi, et al., 2006) The tyrosine phosphatase PTP and the serine/threonine phosphatases PP2C, PP2A and PP2B have also been shown to inactivate p38 (Raman, et al., 2007; Takekawa,

et al., 2000; Takekawa, et al., 1998)

Similar to JNK, p38 is activated by stresses and cytokines, but can also be activated to a much lesser extent by growth factors (Zarubin and Han, 2005) Activated p38 then either activates cytoplasmic substrates or translocates to the nucleus where it activates transcription factors that regulate the expression of genes involved in a number

of cellular responses such as apoptosis, growth, migration, differentiation and inflammation, the deregulation of which can lead to certain pathologies (Figure 4) (Ben-Levy, et al., 1998; Krens, et al., 2006; Zarubin and Han, 2005) The wide range of p38 substrates is responsible for the induction of these different biological functions Although all p38 isoforms have many substrates in common including ATF2, ELK-1, and SAP1, differences in substrate specificity exist between p38α and p38β and between p38γ and p38δ For instance, while MAPKAP-K2 and MAPKAP-K3 are substrates for p38α and p38β, they cannot be activated by p38γ and p38δ (Cuenda, et al., 1997; Goedert, et al., 1997) Some substrates are also specific to either p38γ or p38δ p38γ binds to and phosphorylates many PDZ domain-containing proteins via its KETXL C-terminal sequence that is unique among the p38 isoforms (Hasegawa, et al., 1999; Sabio, et al.,

2005; Sabio, et al., 2004) p38δ has stathmin and tau as substrates, both of which play a role in regulating microtubule dynamics (Feijoo, et al., 2005; Parker, et al., 1998)

Figure 4 The p38 MAPK signaling pathway (adapted from (Cuenda and Rousseau, 2007)) p38 in response to stimulating agents, is activated by MEK3/MEK6, also known

as MKK3/MKK6 Once activated, p38 phosphorylates and activates numerous cytosolic proteins and transcription factors to regulate many biological functions Deregulation of p38 signaling can contribute to the development of several pathologies

p38 has many targets in different subcellular localizations In some cell lines, p38 was found in the nucleus in resting cells and in response to stimuli, p38 was phosphorylated and formed a complex with MAPKAP-K2 or MAPKAP-K5 (Ben-Levy,

et al., 1998; Seternes, et al., 2002) p38-MAPKAP-K2 or p38-MAPKAP-K5 complexes then are exported to the cytoplasm where p38 further activates cytosolic substrtaes (Ben-Levy, et al., 1998; Seternes, et al., 2002) The intracellular localization of p38α can also

be regulated by TAK1 and TAB1 (Lu, et al., 2006) The localization of p38 can also vary with isoforms For instance, in cardiac myocytes, p38α and p38β shuttle between the nucleus and the cytosol and are retained in the nucleus in response to the nuclear export inhibitor, leptomycin B This inhibitor, however, did not affect the localization of p38γ which was mainly associated with cytosolic foci (Court, et al., 2002)

1.2.4 The ERK5 pathway

The ERK5 pathway is the least characterized among the different MAPK pathways ERK5 signaling is triggered in response to EGF and TNFα (Kato, et al., 1998; Weldon, et al., 2002) ERK5 is activated upon phosphorylation of Thr218 and Tyr220 of the Thr-X-Tyr motif by the MAP2K, MEK5 (Weldon, et al., 2002) MEKK2 and MEKK3 are the only MAP3Ks shown to activate MEK5 and hence ERK5 (Sun, et al., 2001) A role for ERK5 in cell proliferation, transformation and metastasis has been demonstrated (Buschbeck, et al., 2005; Kato, et al., 1998; Kesavan, et al., 2004) For instance, ERK5 promoted cell proliferation in response to EGF (Kato, et al., 1998) Moreover, ERK5 was shown to mediate transformation downstream of the oncogene, BCR/Abl (Buschbeck, et al., 2005) In addition to regulating MMP-9 and IL-6, ERK5

was found to be overexpressed in human breast cancer patients with poor prognosis These findings support a role for ERK5 in cancer (Kesavan, et al., 2004; Mehta, et al., 2003; Montero, et al., 2009)

1.3 The matrix metalloproteinases

Matrix metalloproteinases (MMPs) are zinc-dependent proteolytic enzymes that can function as downstream targets of MAPKs (Ispanovic and Haas, 2006; Spallarossa, et al., 2006; Westermarck and Kahari, 1999) MMPs play a key role in cancer cell invasion but are also involved in other biological functions including development, morphogenesis, angiogenesis and inflammation (Hua, et al., 2011) Of the 28 different MMPs, members of the gelatinase group (MMP-2 and MMP-9) are implicated the most

in cancer (Bauvois, 2012) The expression and activation of these MMPs can be mediated

by p38, ERK1/2 and/or JNK (Cho, et al., 2000; Ispanovic and Haas, 2006; Spallarossa, et al., 2006) MMPs can thus execute signals triggered by MAP3Ks MMP-2 and MMP-9 consist of the following structural domains: the N-terminal signal peptide domain, the propeptide region, the catalytic domain containing the highly conserved zinc binding region, and the C-terminus that has a hemopexin-like domain that is connected to the catalytic domain by a small hinge region (Figure 5) (Bauvois, 2012; Nagase and Woessner, 1999) The catalytic domain of gelatinases is characterized by the presence of

a collagen-binding domain (CBD), composed of three fibronectin type II modules that function in enhancing the efficiency of collagen and gelatin degradation (Figure 5) (Morgunova, et al., 1999)

MMPs are first synthesized as inactive pre-proenzymes that undergo two main steps of activation (Benjamin and Khalil, 2012; Overall and Lopez-Otin, 2002) The first step involves the cleavage of the signal peptide during translation to yield proMMPs (Figure 5) (Benjamin and Khalil, 2012) ProMMPs are then maintained inactive by a conserved cysteine residue in the prodomain that acts as a “cysteine switch”, whereby, it forms a coordination bond with the active site zinc ion in the catalytic domain (Figure 5) (Springman, et al., 1990) Full activation of MMPs occurs upon disruption of this bond often by the removal of the propeptide domain by an autoproteolytic event that can also

be induced by chemicals (Figure 5) (Springman, et al., 1990)

MMP-2 and -9 are considered as biomarkers for malignant tumors with poor prognosis (Bauvois, 2012) MMP-2 and/or -9 were reported to be overexpressed in a number of cancers including, breast, colorectal, ovarian, prostate and lung (Roy, et al., 2009; Turpeenniemi-Hujanen, 2005) MMP-2 and -9, through their ability to degrade the basement membrane and different other extracellular-matrix (ECM) components, were found to promote cell invasion in several types of cancer (Hua, et al., 2011) MMP-2 and MMP-9 can also mediate the epithelilal-mesenchymal transition (EMT), a process required for tumor invasion, which further supports a role for these MMPs in cell invasion (Tester, et al., 2007; Thiery, et al., 2009; Xu, et al., 2009)

Figure 5 MMP-2 and -9: structure and activation MMP-2 and MMP-9 generally

contain several structural domains that sequentially are: the signal peptide domain (SPD),

the collagen-binding domain (CBD), a hemopexin-like C-terminus connected to the catalytic domain by a hinge linker Activation of these MMPs is a two-step process that first involves cleavage of the SPD then followed by the proteolytic cleavage of the pro-pepetide domain C stands for cysteine and Zn2+ for zinc

1.4 The MAP2Ks

MAP2Ks, as mentioned earlier, represent the second tier of kinases in the MAPK signaling cascade The MAP2Ks are dual specificity kinases where they act as both tyrosine and serine/threonine kinases, and phosphorylate the tyrosine and threonine residues of the Thr-X-Tyr motif within the MAPKs (Ashworth, et al., 1992) In addition

to the Thr-X-Tyr sequence containing the motif, the tertiary structure of the MAPK protein is also very important for MAP2K binding (Widmann, et al., 1999) Thus, each MAP2K interacts with and regulates a limited number of MAPKs (Widmann, et al., 1999) MAP2Ks are much fewer in number than MAP3Ks (Widmann, et al., 1999) Since numerous MAP3Ks are activated in response to each stimuli, the multiple signals triggered are thus funneled down by the MAP2Ks, which specifically direct those signals

to one or more of the MAPK pathways (Widmann, et al., 1999)

MAP2Ks have two domains, a docking (D) domain and a domain for versatile docking (DVD), that are essential for binding to MAPKs and MAP3Ks, respectively (Sharrocks, et al., 2000; Takekawa, et al., 2005) The DVD domain of MAP2Ks lies 20 residues downstream of the catalytic core (Takekawa, et al., 2005) In addition to the D and DVD domains, MEK5, an activator of ERK5, contains a Phox/Bem1P (PB1) domain that plays a role in protein-protein interactions and associates with other PB1-containing proteins such as the MAP3Ks, MEKK2/MEKK3 (Lamark, et al., 2003; Nakamura, et al., 2006) The N-terminal and C-terminal regions of the PB1 domain of MEK5 also bind to ERK5, allowing MEK5 to act as a scaffold protein that couples MEKK2/MEKK3 to ERK5 (Nakamura, et al., 2006)

Seven members of MAP2Ks, MEK1-7, exist with MEK1 and MEK2 being specific activators of ERK1/2, MEK3 and MEK6 of p38, MEK4 and MEK7 of JNK and MEK5 of ERK5 (Dhanasekaran and Johnson, 2007; Dhillon, et al., 2007; Raman, et al., 2007) MEK4, however, also phosphorylates and activates p38 (Brancho, et al., 2003) While MEK7 was suggested to be a stronger activator of JNK than MEK4, full activation

of JNK was reported to require both MEK4 and MEK7 that preferentially phosphorylate the tyrosine and threonine residues within the Thr-X-Tyr motif of JNK, respectively (Lawler, et al., 1998) Another study reported that both of these residues can be phosphorylated by either of the two MAP2Ks (Chen, et al., 2002) However, JNK activation in the absence of either MEK4 or MEK7 was found to be lower than that in the presence of both MAP2Ks (Chen, et al., 2002) MEK4 and MEK7 were both shown to be activated by stress to trigger the JNK pathway (Wang, et al., 2007) Pro-inflammatory cytokines on the other hand, induce JNK signaling primarily by activating MEK7 (Moriguchi, et al., 1997) MEK4 and MEK7 also exhibit some variations in the MAP3Ks that activate them (Wang, et al., 2007) For instance, while MEKK4 activates MEK4, MEKK1 activates both MEK4 and MEK7 (Takekawa, et al., 2005)

The p38 activators, MEK3, MEK4 and MEK6 are activated in response to stresses, proinflammatory cytokines and UV irradiation (Brancho, et al., 2003) p38α is activated by all three MEKs (Brancho, et al., 2003) p38γ is activated in response to environmental stresses by both MEK3 and MEK6, while activation by TNFα is mainly mediated by MEK6 (Remy, et al., 2010) Similar to p38γ, stress-induced p38β activation

is mediated by both MEK3 and MEK6 (Remy, et al., 2010) The activity of the p38δ, in

response to numerous stimuli, including UV radiation, stress and TNFα is primarily regulated by MEK3 (Remy, et al., 2010)

Mutations in MAP2Ks have also been implicated in cancers For instance, MEK4, though at a low frequency, was reported to be inactivated in numerous types of cancer, including breast, colon and lung cancer (Cunningham, et al., 2006) This might suggest a suppressive role for MEK4 in cancer In contrast, overexpressing MEK4 in breast and pancreatic cancer cells promoted proliferation and invasion which implicates a pro-oncogenic role for MEK4 (Wang, et al., 2004) Moreover, although, knocking out MEK4

in human pancreatic cancer cell lines did not yield any effect on proliferation, metastasis was suppressed (Cunningham, et al., 2006) The differences observed in the cellular responses triggered by MEK4 could be attributed to the degree of activation in response

to stresses and environmental context of cells that favor the induction of either proliferation or apoptosis (Dhillon, et al., 2007)

1.5 The MAP3Ks

The MAP3K are serine/threonine kinases that are encoded by at least 20 genes, and represent the largest and most diverse components of the MAPK cascade MAP3Ks function to direct signals generated by a wide range of stimuli to the MAPKs that ultimately lead to specific functional responses (Cuevas, et al., 2007; Uhlik, et al., 2004) The differences in the domains and motifs between MAP3Ks, allow individual MAP3Ks

to selectively regulate the localization and activation of specific MAP2Ks and MAPKs (Dhanasekaran and Johnson, 2007) Many of the MAP3Ks however, can activate more than one MAP2K and thereby regulate multiple MAPK pathways (Cuevas, et al., 2007)

Based on protein homology, the different MAP3Ks are distributed into several main clusters: the mitogen-activated protein/ERK kinase kinase (MEKKs), Rafs, thousand and one amino acid (TAOs), tumor progression locus 2 (Tpl2) and apoptosis signal regulating kinases (ASKs), TGF-β-activated kinase 1 (TAK1) with MOS, dual leucine zipper kinases (DLKs) and zipper sterile alpha kinase (ZAKs), and the mixed lineage kinases (MLK1-4), (Craig, et al., 2008; Cuevas, et al., 2007) However, this cluster distribution

is different if only the homology in the kinase domain is considered, with ASKs being grouped with MEKKs, Tpl2 with the TAOs, and the DLKs and ZAKs with MLKs (Craig,

et al., 2008) Moreover, MAP3Ks, also have kinase-independent functions that are mediated by protein-protein interactions (Mita, et al., 2002) In addition, MAP3Ks, can regulate other signaling pathways as well, including the NF-κB and Akt/MTOR pathways (Hirano, et al., 1996)

1.5.1 The MEKK group

The MEKK family includes six members, MEKK (1-4) as well as ASK1 and ASK2 Each of the MEKK members can regulate multiple biological functions, some of which are not redundant, as evidenced by the lethal effects of knocking out MEKK3 or MEKK4, but not MEKK1 or MEKK2, on mice embryos (Abell and Johnson, 2005; Cuevas, et al., 2007; Yang, et al., 2000) MEKK1 was the first to be identified and is one

of the best characterized MEKK members MEKK1 promotes cell survival by activating ERK1/2 and JNK pathways in response to cytokines and growth factors (Xia, et al., 2000; Yujiri, et al., 1998) Moreover, MEKK1, upon association with and activation by small G proteins, including RhoA, regulates ERK1/2-mediated cell migration (Cuevas, et al.,

2003; Fanger, et al., 1997) Although MEKK1 is a potent activator of ERK1/2, it can also act to negatively regulate ERK1/2 via ubiquitination and subsequent degradation of ERK1/2 (Lu, et al., 2002) ERK1/2 ubiquitination is mediated by the plextrin homology (PH) domain of MEKK1 that functions as an E3 ubiquitin ligase (Lu, et al., 2002) MEKK1 also regulates AP-1-induced gene expression (Cuevas, et al., 2005) MEKK2 and MEKK3 are the only MAP3Ks known to regulate the unique MAPK, ERK5, by heterodimerization with and subsequent activation of MEK5 (Sun, et al., 2001) Dimerization between MEKK2 or MEKK3 and MEK5 requires the PB1 domain found in these three kinases and is a prerequisite for the regulation of ERK5 signaling (Nakamura and Johnson, 2003)

MEKK2 and MEKK3 share around 95% sequence homology in the kinase domain, however, the overall sequence identity is only approximately 55% (Blank, et al., 1996) Although, MEKK2 and MEKK3 both activate ERK5 and induce AP-1-mediated expression of inflammatory cytokines, MEKK3 also plays a role in regulating the p38 and the NF-κB pathways, while MEKK2 functions to activate the JNK pathway (Garrington, et al., 2000; Huang, et al., 2004; Kesavan, et al., 2004; Uhlik, et al., 2003;

Xu, et al., 2004)

MEKK4, one of the least characterized MEKK, was shown to activate both JNK and p38 via the direct phosphorylation of MEK4/MEK7 and MEK3/MEK6, respectively (Abell, et al., 2005; Chi, et al., 2005) The activity of MEKK4 seems to be modulated by numerous upstream proteins Such proteins include Rac, Cdc42 and TRAF4, all of which trigger JNK signaling by binding to MEKK4 (Abell and Johnson, 2005; Fanger, et al., 1997; Gerwins, et al., 1997) Other proteins also known to bind to and activate MEKK4

are the isoforms α, β, and γ of the small growth arrest and DNA damage-inducible (GADD45) proteins (Takekawa and Saito, 1998)

ASK1, also known as MEKK5, is activated by numerous stimuli that can result in elevated intracellular levels of reactive oxygen species (ROS) such as stresses, cytokines and LPS (Hayakawa, et al., 2006) One of the first characterized functions of ASK1 is the regulation of stress-induced apoptosis (Tobiume, et al., 1997) ASK1 however, similar to MEKK4, activates MEK3, MEK6, MEK4 and MEK7 that in turn activate p38 and JNK (Ichijo, et al., 1997) Moreover, studies focused on the activation of ASK1 by LPS revealed an important role for ASK1 in regulation of the innate immune system (Matsuzawa, et al., 2005) A role for ASK2, or MEKK6, in apoptosis has also been demonstrated, whereby ASK2 can form a heterodimer with ASK1 to trigger apoptosis (Ortner and Moelling, 2007; Takeda, et al., 2007)

1.5.2 The Raf MAP3Ks

As mentioned earlier, the Rafs are MAP3Ks that mainly mediate Ras-induced activation of ERK1/2 by phosphorylating MEK1/MEK2 (Dunn, et al., 2005) Three main Raf proteins, A-Raf, B-Raf and Raf-1 (or c-Raf), have three highly conserved regions (CR1-3) The CR1, which is localized in the N-terminus and harbors the Ras-binding domain and the cysteine rich domain, mediates the binding between the Rafs and Ras The CR2 is serine/threonine rich and regulates Raf activity by undergoing phosphorylation, and the CR3 harbors the C-terminal kinase domain (Mott, et al., 1996; Vojtek, et al., 1993; Wellbrock, et al., 2004) The Raf proteins have distinct expression patterns and are not functionally redundant (Cuevas, et al., 2007) Raf-1, but not A-Raf or

B-Raf, is ubiquitously expressed (Craig, et al., 2008) Both B-Raf and A-Raf are widely expressed, but A-Raf is mainly found in urogenital and gastrointestinal tissues (Luckett,

et al., 2000) The Raf isoforms can also display differences in function For instance, while B-Raf and Raf-1 phosphorylate and activate both MEK1 and MEK2, A-Raf only activates MEK1 (Sridhar, et al., 2005; Wu, et al., 1996) Moreover, B-Raf appears to be involved in cellular differentiation through activation of the MAPK, ERK3 (Coulombe, et al., 2003; Hoeflich, et al., 2006) Raf-1 phosphorylates and inactivates Bad, which suggests that it has a role in regulating apoptosis (Kebache, et al., 2007) A role for Raf-1

in neuronal and cardiac apoptosis was also demonstrated in mice with cardiac-specific deletion of Raf-1 (Kanamoto, et al., 2000; Yamaguchi, et al., 2004) A role for Raf-1 in

-/-mice die at the embryonic stage (Mikula, et al., 2001) Moreover, Raf-1 -/- fibroblasts were shown to undergo apoptosis more readily than the wild-type cells, which further supports

an anti-apoptotic function for Raf-1 (Mikula, et al., 2001) Activation of ERK1/2 by Raf

proteins can be facilitated by several scaffold proteins including β-arrestin, and the

kinase suppressor of ras (KSR) (Morrison and Davis, 2003)

B-Raf is a human oncogene that is mutated at a high frequency (more than 60%)

in melanomas (Davies, et al., 2002) b-raf gene mutations are found in a large number of

cancers, including colon cancer and ovarian cancer (Dhillon, et al., 2007; Wan, et al., 2004) The V600E point mutation in the activation loop of B-Raf is the most common

among b-raf mutations (Wan, et al., 2004) This mutation induces constitutive activation

of B-Raf and ERK signaling (Garnett and Marais, 2004) Interestingly, B-Raf, with mutations that don’t enhance activity, also increases ERK activity (Wan, et al., 2004)

Such mutations were proposed to indirectly promote ERK signaling by activating Raf-1,

a heterodimerization partner of B-Raf (Garnett and Marais, 2004; Garnett, et al., 2005; Rushworth, et al., 2006)

1.5.3 The TAK1 MAP3K group

TAK1 is mainly activated by TGFβ but can also be activated by other proinflammatory stimuli such as IL-1β, TNFα and LPS (Irie, et al., 2000; Takaesu, et al., 2003; Wagner and Siddiqui, 2007) Three isoforms of TAK1, TAK1a, TAK1b and TAK1c, exist with TAK1a being the most predominant (Craig, et al., 2008) TAK1 triggers JNK and p38 signaling by activating MEK4 and MEK6, respectively (Shim, et al., 2005) p38 however, when present in high levels, functions as part of a negative feedback loop to regulate TAK1 activity (Cheung, et al., 2003) TAK1 is essential for vesicular development, most likely by inducing the expression of JNK- and p38-dependent genes involved in angiogenesis, and TAK1 deficiency is lethal to mouse embryos (Jadrich, et al., 2006) The most distinctive role of TAK1 that has been identified is the one involving the activation of the NF-κB pathway to regulate cellular responses including inflammation, apoptosis and cell cycle regulation (Ghosh, 1999; Sakurai, et al., 1998) Activation of the NF-κB by TAK1 requires the formation of a complex between TAK1, TAB2, TAB3 and the E3 ubiquitin ligase/TRAF6 (Wang, et al., 2001) The complex then phosphorylates and activates IKK in a ubiquitin-dependent manner, a step that is essential for IκBα degradation (Wang, et al., 2001) Briefly, the transcription factor NF-κB is sequestered in the cytosol in an inactive form by the inhibitor IκBα (Karin, et al., 2002) Stimulation with proinflammatory cytokines like

TNFα and IL-1, activates the IκB kinase (IKK) kinase (IKKK) that in turn phosphorylates and activates IKK (Figure 6) (Karin, et al., 2002) Once activated, IKK phosphorylates IκBα on two serine residues, Ser32 and Ser36 (Ghosh and Karin, 2002) Phospho-IκBα is then targeted for ubiquitination and proteasomal degradation, thereby allowing NF-κB to translocate to the nucleus and activate the expression of genes involved in numerous cellular responses (Figure 6) (Karin, et al., 2002)

Figure 6 The NF-κB pathway NF-κB is maintained in the cytoplasm by its inhibitor

IκBα Proinflammatory stimuli activate the IKK activator, IKKK, which in turn phosphorylates IKK Active IKK then phosphorylates IκBα on Ser32 and Ser36 Phosphorylation on these residues leads to the ubiquitination and subsequent proteasomal degradation of IκBα which leads to the release of NF-κB NF-κB then translocates to the nucleus where it activates gene expression