Dr. R.A.SIDDIQUE

1
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 riazndri_at_gmail.com
2

• 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,

3

• 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.

4
Different MAP Kinases involved in ASK1 Activation
Stimulus MAPKKK MAPKK MAPK Response Stimulus MAPKKK MAPKK MAPK Response Stimulus MAPKKK MAPKK MAPK Response Stimulus MAPKKK MAPKK MAPK Response Stimulus MAPKKK MAPKK MAPK Response
Inflammatory cytokines ASK1 MKK3/6 MKK4/7 p38 MAPK SAPK/JNK Inflammation, Apoptosis, Diffentiation
5

• 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 TNFa
• control stress adaptation, cell death and
  survival.

6
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 ( TNFa
  and Fas ligand)
• Also activated by cytotoxic stresses H2O2,
  anticancer drugs and growth factor deprivation.
• New mechanism by ER stress, calcium signaling,
  GPCR signaling.

7

• 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.
  

8
ASK1 JNK Pathway in Bcl-2 Phosphorylation
9
Structure of ASK1
10
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

11
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.

12
? 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 TNFa signaling
• TNFa ROS Dissociate Trx from
  ASK1

13
(No Transcript)
14
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

15
? 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.

16
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

17
Alternate Pathway for JNK activation

• Activated Fas Daxx
• Daxx binds N-terminal of ASk1
• Activate JNK
• Apoptosis
• Note Mechanism is still not clear

18
(No Transcript)
19
? Contd

• TNFa regulate immune response, inflammation and
  apoptosis
• TRAF2 couples to TNFa receptor
• TRAF2 directly interact to C-terminal domain of
  ASk1 .
• TNFa 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 TNFa induced activator of
  ASK1.
• MEFs from ASK1-/- resistant to TNFa induced
  apoptosis reduced activation of JNK and p38,
  indicates ASK1 is required for TNFa induced
  apoptosis.

20
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
  reduction of nascent protein in ER
• Adaptive Response
• If , adaptive response not sufficient
  apoptosis.
• IRE1 activate JNK signaling mediated via TRAF2

21
Contd..

• ER stress induced apoptosis in human pathological
  cases
• Amyloidosis
• Hypercholesterolemia
• Neurodegenerative diseases Huntingtons
  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.

22
IRE-TRAF2-ASK1 Cascade in Pathogenesis of Poly Q
disease
23
G-protein coupled receptor (GPCR) signaling and
ASK1 activation in cardiomyocytes

• McDonald et al. first reported
• ß-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.

24
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.

25
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

26

• THANKS