Dr. R.A.SIDDIQUE

M.V.Sc., PhD Scholar

National Dairy Research Institute,

Karnal, Haryana, 132001

INDIA

E-mail: riazndri@gmail.com

THE ASK1-MAP KINASE CASCADES IN MAMMALIAN STRESS RESPONSE

STRESS:

It is a medical term for wide range of strong stimuli which can cause a physiological response, first described in 1936 by Hans Selye.

It include three stages:

1. Alarm reaction: body detects the external stimuli.

2. Adaptation : body engages defensive countermeasures against the stressor.

3. Exhaustion: body begins to run out of defences.

Here I will mainly concentrate on oxidative stresses and ER stresses,

MAP Kinase cascade transmit stimuli from outside the cell to the MAP Kinase cascade transmit stimuli from outside the cell to the nucleus.nucleus.

Three MAP Kinase cascades in mammals :Three MAP Kinase cascades in mammals : ERKs, c-Jun N-terminal Kinases(JNKs), p38 MAP Kinases.

Each consists of three classes of Ser/Thr Kinases Each consists of three classes of Ser/Thr Kinases MAP Kinase, MAP Kinase Kinase( MAPKK) - --MEK, MAP Kinase Kinase Kinase( MAPKKK)

MAP KKK Phosphorylate and activate MAP KK and this activated MAP MAP KKK Phosphorylate and activate MAP KK and this activated MAP KK phosphorylate and activate MAP Kinase. KK phosphorylate and activate MAP Kinase.

Stimulus MAPKKK MAPKK MAPK Response

Inflammatory

cytokines

ASK1 MKK3/6

MKK4/7

p38 MAPK

SAPK/JNK

Inflammation,

Apoptosis,

Diffentiation

Different MAP Kinases involved in ASK1 Activation

ERKs activated by Cytokines &growth factors and role in Cell ERKs activated by Cytokines &growth factors and role in Cell growth and Diffrentiationgrowth and Diffrentiation

On the other hand JNKs and p38 MAP Kinases activated by:

chemical and physical stressors:

UV radiation

X-ray

heat shock

osmotic shock

proinflammatory cytokines : TNFα

control : stress adaptation, cell death and survival.

ASK1 (Apoptosis Signal Regulating Kinase1)ASK1 (Apoptosis Signal Regulating Kinase1)

160 KDa Ser/Thr Protein Kinase

Member of MAP KKK family

Activates both JNK and p38 pathway by phosphorylating and activating SEK1(MKK4)/ MKK7 and MKK3/Mkk6.

ASk1 activated with death receptor ligands ( TNFα and Fas ligand)

Also activated by cytotoxic stresses: H2O2, anticancer drugs and growth factor deprivation.

New mechanism by ER stress, calcium signaling, GPCR signaling.

Overexpression of wild types ASK1 causes mitochondria dependent apoptosis.

In this cascade Yamamoto et al. 2000 demonstrated ASK1 mediated JNK activation

Phosphorylates Bcl-2

Reduced Antiapoptotic activity

Note: However the detailed molecular mechanism that link mitochondria dependent apoptosis and ASK1-p38/JNK activation remains unknown.

ASK1 JNK Pathway in Bcl-2 Phosphorylation

Structure of ASK1

Mechanism of ASK1 ActivationMechanism of ASK1 Activation

By Homo-oligomerisation It was demonstrated that synthetic ASK1-ASK1 fusion construct activate JNK

and p38pathway In resting stage ASK1 = Homo- oligomer through its C-terminal coiled-coil

domain.

H2O2 Stimuli

Additional interface created on pre-formed ASK1 oligomer

Autophosphorylation of Thr 845 in Mouse

Activation of ASK1 In Human at Thr838 Thr residue located in activation loop of kinase domain

Oxidative Stress and ASK1 interacting ProteinsOxidative Stress and ASK1 interacting Proteins

ROS --- super oxide and H2O2 produced through cellular processes or derived from exogenous sources play imp role in normal cell-proliferation, survival and immune response.

Excessive Production of ROS causes: Severe damage to cellular components. Loss of cell functions Ultimately apoptosis or necrosis Heart failure, myocardial infarction and neuronal cell death

ASK1 strongly activated in cells exposed to oxidants and involved in oxidative stress induced apoptosis

Negative regulation of oxidative-stress induced apoptosis by Trx (ASK1 repressor Thioredoxin)

Trx directly bind to N-terminal Of ASK1 and inhibits Kinase activity.

Contd…Contd…

Binding of Trx to ASK1 require

Reduced form of disulphide bridge between two residues in catalytic site of TRx, Cys32 and Cys35.

Oxidized Trx could bind or inhibit ASK1

This binding also involved in TNFα signaling

TNFα ROS Dissociate Trx from ASK1

GlutaredoxinGlutaredoxin

Another intracellular redox- signaling molecule

Inhibits Glucose deprivation induced ASK1 activation

PP5 dephosphorylate Thr845 and inactivate kinase activity in vivo and in vitro.

PP5 negative feedback of ASK1 activation In resting stage 14-3-3 protein bind to ASK1 through Ser 967 and reduce

ASK1-induced apoptosis.

Goldman et al. demonstrated :

H2O2 induce Ser 967 dephosphorylation and increased activation

• Necessity of ASK1 activation in ROS induced Apoptosis

Tobiume et al. (2001)

Generated ASK1 null mice

MEFs isolated from ASK1-/- mice resistant to H2O2 apoptosis

JNK and p38 activation also suppressed

So essentiality of ASK1-p38/JNK cascade.

Contd…Contd…

Death Receptor mediated ASK1 activationDeath Receptor mediated ASK1 activation

Death receptor- Fas

Fas assembles as DISC upon activation by FasL or agonistic antibodies.

DISC--- FADD and caspase -8

DISC-----acute execution of apoptosis

Alternate Pathway for JNK activationAlternate Pathway for JNK activation

Activated Fas + Daxx

Daxx binds N-terminal of ASk1

Activate JNK

Apoptosis

Note: Mechanism is still not clear

TNFα regulate immune response, inflammation and apoptosis

TRAF2 couples to TNFα receptor

TRAF2 directly interact to C-terminal domain of ASk1 .

TNFα induces dissociation of Trx .

Activated ASk1 ------- apoptosis

Moreover it has been reported that TRAF2 over expression or treatment leads to the production of ROS

Therefore, Trx negative regulator of both H2O2 induced as well as TNFα induced activator of ASK1.

MEFs from ASK1-/- resistant to TNFα induced apoptosis reduced activation of JNK and p38, indicates ASK1 is required for TNFα –induced apoptosis.

Contd…Contd…

ER Stress-induced Apoptosis and ASK1 activationER Stress-induced Apoptosis and ASK1 activation

Accumulated unfolded protein in lumen of ER UPR

UPR regulated by IRE1, PERK, ATF6. PERK and IRE1: ER resident transmembrane Ser/Thr protein kinase.

PERK and IRE1 phosphorylated and activated in response to UPR. ATF = leucine zipper transcription factor , cleaved and activated in Golgi

apparatus.

Activation of above molecules reduction of nascent protein in ER “Adaptive Response”

If , adaptive response not sufficient apoptosis. IRE1 activate JNK signaling mediated via TRAF2

Contd..Contd..

ER stress induced apoptosis in human pathological cases: Amyloidosis Hypercholesterolemia Neurodegenerative diseases: Huntington’s diseases

Huntington's diseases : expansion of CAG repeats

code for expanded polyglutamine (Poly Q).

Long poly Q ER stress activates IRE1 which recruit TRAF2 , and interacts directly with the ASK1 and activate

SEK1-JNK pathway pathway.

Poly Q dependent ER stress due to proteasome dysfunction.

Bence et al., 2001 and Zhou et al., 2003.

IRE-TRAF2-ASK1 Cascade in Pathogenesis of Poly Q disease

G-protein coupled receptor (GPCR) signaling and ASK1 activation G-protein coupled receptor (GPCR) signaling and ASK1 activation in cardiomyocytesin cardiomyocytes

McDonald et al. first reported

β-arrestin 2 key molecule in rec. desensitization and internalization also functions as a scaffold protein in ASK1-SEK1-JNK3 cascade( related to

angiotensin II-induced JNK3 activation. Physiological role unknown

Recently GPCR mediated ASK1 activation ROS production cardiac dysfunction

ASk1 is essential for angiotensin II- induced cardiac hypertrophy by using ASK1-/- mice

Izumiya et al., 2003 ASk1 seems to be promising therapeutic target for cardiac

dysfunction.

Calcium signaling and ASK1-p38 cascade activationCalcium signaling and ASK1-p38 cascade activation

Ca play role in Neuronal Functions and Ca dependent activation of MAP kinase in Synaptic plasticity.

Enters in neuron via NMDA receptor or voltage-gated Ca channels.

Binds to CaM and activate ERK pathway

In this pathway CaMBPs (Ras-GRF and CaMKIV positively modulate ERK1/2 activation induced by NGF

However relationship between Ca signaling and JNK/p38 activation has not been well defined.

ConclusionConclusion

ASK1 causes apoptosis in response to common proapoptotic stresses,

such as oxidative stress and death rec. ligands.

Moreover pathogenic stress (ER stress, GPCR induced ROS production) also causes apoptosis via ASK1-JNK/p38 cascades in neurons and cardiomyocytes.

ASK1 may be therapeutic target for treatment of Neurodegenerative diseases and cardiac dysfunction.

CaMKII phosphorylates ASK1 and activate ASK1-p38 pathway in neurons and play important role in synaptic plasticity

Further knowledge of its regulatory mechanism –more promising therapeutic target for apoptosis based incurable diseases.

In addition, an understanding of novel physiological roles of ASK1 may shed light on diverse cellular processes regulated by this important Protein Kinase