Transcript kim and ronesha presentation

Negative Regulation of JNK
Signaling by the Tumor
Suppressor CYLD
The Journal of Biological Chemistry
Authors: William Reily, Minying Zhang, and ShaoCong Sun
Presentors:
Ronesha Franklin
Kimberly Kimbrough
Objective of Experiment
► To
investigate the function of endogenous
CYLD in the regulation of cell signaling.
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.
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.
TRAF Function
► TRAFs
activate downstream signaling
cascades and lead to activation of IKK and 3
families of MAPKs:
 JNK
 Extracellular signal responsive kinase
 P38
JNK Functions
► JNK
functions include regulation of immune
and inflammatory responses, cell growth,
apoptosis, and tumor formation.
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.
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.
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
Result # 1
► CYLD
is a negative regulator of JNK but not
IKK in the TNF-alpha Signaling Pathway
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
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.
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.
Result #1 continued
► Parallel
Analyses using the same cells
revealed that the CYLD knockdown
markedly enhanced the activation of JNK.
Result #2
► CYLD
Knockdown Has No Effect on JNK
Activation by a Stress Agent
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.
Result #3
► CYLD
MKK7
Negatively Regulates the Activation of
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
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
Result #4
► CYLD
Negatively Regulates JNK Activation
by Diverse Stimuli
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
►
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
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
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.
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.
Reference
► http://anisomycin.4mg.com/
► http://www.jbc.org
► http://www.icr.ac.uk/cyld/natgenpap/ng060
► http://www.gtmb.org/volume4/08_Chen_Ta
n.htm