Transcript the ask1-map kinase cascades in mammalian stress response

THE ASK1-MAP KINASE CASCADES IN
MAMMALIAN STRESS RESPONSE
Dr. R.A.SIDDIQUE
M.V.Sc., PhD Scholar
National Dairy Research Institute,
Karnal, Haryana, 132001
INDIA
E-mail: [email protected]
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
nucleus.
 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
 MAP Kinase,
MAP Kinase Kinase( MAPKK) - --MEK,
 MAP Kinase Kinase Kinase( MAPKKK)
 MAP KKK Phosphorylate and activate MAP KK and this activated MAP
KK phosphorylate and activate MAP Kinase.

Different MAP Kinases involved in ASK1 Activation
Stimulus
MAPKKK
Inflammatory ASK1
cytokines
MAPKK
MAPK
Response
MKK3/6
p38 MAPK
Inflammation,
MKK4/7
SAPK/JNK
Apoptosis,
Diffentiation
 ERKs activated by Cytokines &growth factors and role in Cell
growth 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)
 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 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 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…
 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
Glutaredoxin
 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
 Contd…
• 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.
Death 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 activation
Activated Fas + Daxx
Daxx binds N-terminal of ASk1
Activate JNK
Apoptosis
Note: Mechanism is still not clear
 Contd…
 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.
ER 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
“Adaptive Response”
reduction of nascent protein in ER
 If , adaptive response not sufficient
apoptosis.
 IRE1 activate JNK signaling mediated via TRAF2
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
in 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
cardiac dysfunction
ROS production
 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 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.
Conclusion
 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