Negative Regulation of JNK Signaling by the Tumor Suppressor CYLD

1
Negative Regulation of JNK Signaling by the
Tumor Suppressor CYLD

• The Journal of Biological Chemistry
• Authors William Reily, Minying Zhang, and
  Shao-Cong Sun
• Presentors
• Ronesha Franklin
• Kimberly Kimbrough

2
Objective of Experiment

• To investigate the function of endogenous CYLD in
  the regulation of cell signaling.

3
What is CYLD?

• CYLD is a tumor suppressor that is mutated in
  familial cylindromatosis.
• Familial cylindromatosis is a genetic syndrome in
  which numerous benign tumors of skin adnexa
  develop, principally on the head and neck. This
  disorder is inherited in an autosomal manner and
  is caused by mutation of CYLD gene on chromosome
  16q12-q13.
• Results in a pile up of cells.

4
CYLD Functions

• CYLD
• Is a deubiquitinating enzyme that negatively
  regulates NFkB activation by TNFR family members.
• Deubiquitinating enzymes remove ubiqutin, a small
  molecule that serves as a tag that signals
  proteins to proteasomes for degradation.
• Inhibits the ubiquitnation of certain signaling
  molecules, including members of the tumor
  necrosis factor receptor-associated factor (TRAF)
  family.

5
TRAF Function

• TRAFs activate downstream signaling cascades and
  lead to activation of IKK and 3 families of
  MAPKs
• JNK
• Extracellular signal responsive kinase
• P38

6
JNK Functions

• JNK functions include regulation of immune and
  inflammatory responses, cell growth, apoptosis,
  and tumor formation.

7
JNK Activation

• Activation of JNK is mediated by a kinase cascade
  involving MAPK kinase and MAPK kinase kinase. Two
  MAPK kinase, MKK4 MKK7 is required for JNK
  activation by inflammatory cytokines, whereas
  MKK4 is more important for JNK activation by
  stress signals.

8
Overview of Materials and Methods

• 293 cells (human embryonic kidney cells) were
  transected with either the empty vector pcDNA or
  an expression vector encoding HA-tagged CYLD.
• About 7 micrograms of protein lysates were
  subjected to IB using anti-CYLD. The transected
  HA-CYLD, endogenous CYLD, and some nonspecific
  protein bands are indicated.

9
Overview of Materials and Methods Continued

• 293 or HeLa cells (cancerous cells) were
  transected with either the control luciferase
  siRNA or CYLD-specific siRNA. About 20
  micrograms of cell lysates were subjected to IB
  using anti-CYLD.
• Assays used in the experiment include
• Immunoblotting (IB)
• In Vitro Kinase assay
• Electrophoresis Mobility Shift Assay
• RNAi assay
• Immunecomplex kinase assay
• Phospho-specific IB assay

10
Result 1

• CYLD is a negative regulator of JNK but not IKK
  in the TNF-alpha Signaling Pathway

11
Result 1 cont.

• To systematically analyze the role of CYLD
  regulation of cell signaling, a CYLD antibody was
  generated.
• A. The Ab detected endogenous and transfected
  CYLD.
• B. The Ab detected endogenous CYLD

12
Result 1 Cont.

• Examined the effect of CYLD knockdown on cell
  signaling stimulated by the proinflammatory
  cytokine TNF-alpha.
• In both 293 HeLa cells, TNF-alpha stimulated
  the catalytic activity of IKK and JNK as
  demonstrated by immunocomplex kinase assays.

13
Result 1 continued

• Their hypothesis
• IF endogenous CYLD serves as negative regulator
  of TNF-alpha stimulated cell signaling, the CYLD
  knockdown should result in hyperactivation of the
  specific kinases under the negative control of
  CYLD.
• Their conclusion
• CYLD did not promote IKK activation in the
  TNF-alpha stimulated 293 cells or HeLa cells.

14
Result 1 continued

• Parallel Analyses using the same cells revealed
  that the CYLD knockdown markedly enhanced the
  activation of JNK.

15
Result 2

• CYLD Knockdown Has No Effect on JNK Activation by
  a Stress Agent

16
Result 2 Cont.

• Activation of JNK by stress stimulus Anisomycin(
  antibiotic that activates stress-activated
  protein kinase)
• Incubation of 293 cells with anisomycin lead to
  strong activation of JNK
• JNK activated by anisomycin was not affected by
  CYLD knockdown
• TNF-? stimulated JNK activation showed
  enhancement of cytokine-kinase JNK response by
  CYLD knockdown.

17
Result 3

• CYLD Negatively Regulates the Activation of MKK7

18
In vitro Kinase Assay

• First lyse the cell
• Immunoprecipitate the kinase using the
  appropriate antibody (IKKB or MAPK)
• Add buffer or protein of interest
• Analyze by autoradiography

19
Result 3 Cont.

• Effect of CYLD knockdown on TNF-? stimulated
  activation of MKK7 and MKK4.
• MKK7 was enhanced in CYLD knockdown cells
• MKK4 was not significantly enhanced in CYLD
  knockdown cells
• MKK7 is and upstream target of CYLD in JNK
  signaling
• Because MKK7 is enhanced by the knockdown CYLD
  must inhibit activation of MKK7

20
Result 4

• CYLD Negatively Regulates JNK Activation by
  Diverse Stimuli

21
Result 4 Cont.

• CD40 cells did not exhibit significant signaling
  activity under unstimulated conditions
• Cross-linking of CD40 with its agonistic antibody
  lead to activation of IKK and JNK
• CYLD knockdown enhanced the the activation of JNK
  in anti-CD40-treated cells

22
Result 4 Cont.

• Anti-CD40 stimulated hyper activation of NF-?B in
  the CYLD knockdown
• Infection with CYLD-small hairpin RNA suppressed
  the expression of CYLD in BAJB B-cells
• CYLD knockdown enhanced JNK activation by LPS and
  IL-1?
• IKK activation was promoted in CYLD knockdown in
  LPS and IL-1?

23
Conclusions

• CYLD Is a Negative Regulator of JNK but Not IKK
  in the TNF-alpha Signaling Pathway
• CYLD Knockdown Has no effect on JNK Activation by
  a stress Agent
• CYLD Negatively Regulates the Activation
• of MKK7
• CYLD Negatively Regulates JNK activation by
  diverse Stimuli

24
Conclusion

• How CYLD differentially regulates IKK and JNK is
  not completely understood, but one potential
  mechanism is attributed to the differential
  requirement of TRAFs in these signaling pathways.
  
• The findings that CYLD negatively regulates JNK
  as well as IKK provides an insight into the tumor
  suppressor function of CYLD.

25
Conclusion

• The IKK/NFkB pathway is well known for it
  involvement in cell survival and oncogenic
  transformation as well as immune responses.
• Evidence suggest that JNK is a critical factor
  involved in tumorgenisis.
• The JNK signaling pathway is activated in various
  tumor cells.
• JNK has been shown to promote cell growth and
  survial.
• CYLD knockdown increases the magnitude of JNK
  transient activation but does not prolong the
  activation.

26
Reference

• http//anisomycin.4mg.com/
• http//www.jbc.org
• http//www.icr.ac.uk/cyld/natgenpap/ng060
• http//www.gtmb.org/volume4/08_Chen_Tan.htm