Negative Regulation of Negative Regulation of JNK Signaling by the JNK Signaling by the

Tumor Suppressor CYLDTumor Suppressor CYLDThe Journal of Biological Chemistry The Journal of Biological Chemistry

Authors: William Reily, Minying Zhang, and Authors: William Reily, Minying Zhang, and Shao-Cong SunShao-Cong Sun

Presentors:Presentors:Ronesha FranklinRonesha Franklin

Kimberly KimbroughKimberly Kimbrough

Objective of ExperimentObjective of Experiment

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

What is CYLD?What is CYLD?

► CYLD is a tumor CYLD is a tumor suppressor that is suppressor that is mutated in mutated in familial familial cylindromatosiscylindromatosis.. Familial cylindromatosis is Familial cylindromatosis is

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

cells.cells.

CYLD Functions CYLD Functions

►CYLDCYLD Is a Is a deubiquitinating enzymedeubiquitinating enzyme that that

negatively regulates NFkB activation by negatively regulates NFkB activation by TNFR family members.TNFR family members.►Deubiquitinating enzymes remove ubiqutin, a Deubiquitinating enzymes remove ubiqutin, a

small molecule that serves as a tag that signals small molecule that serves as a tag that signals proteins to proteasomes for degradation.proteins to proteasomes for degradation.

Inhibits the ubiquitnation of certain Inhibits the ubiquitnation of certain signaling molecules, including members of signaling molecules, including members of the tumor necrosis factor receptor-the tumor necrosis factor receptor-associated factor (TRAF) family.associated factor (TRAF) family.

TRAF FunctionTRAF Function

►TRAFs activate downstream signaling TRAFs activate downstream signaling cascades and lead to activation of IKK cascades and lead to activation of IKK and 3 families of MAPKs:and 3 families of MAPKs: JNKJNK Extracellular signal responsive kinaseExtracellular signal responsive kinase P38P38

JNK FunctionsJNK Functions

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

JNK ActivationJNK Activation

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

Overview of Materials and Overview of Materials and MethodsMethods

►293 cells (human embryonic kidney 293 cells (human embryonic kidney cells) were transected with either the cells) were transected with either the empty vector pcDNA or an expression empty vector pcDNA or an expression vector encoding HA-tagged CYLD.vector encoding HA-tagged CYLD.

► About 7 micrograms of protein lysates About 7 micrograms of protein lysates were subjected to IB using anti-CYLD. were subjected to IB using anti-CYLD. The transected HA-CYLD, endogenous The transected HA-CYLD, endogenous CYLD, and some nonspecific protein CYLD, and some nonspecific protein bands are indicated. bands are indicated.

Overview of Materials and Overview of Materials and Methods ContinuedMethods Continued

► 293 or HeLa cells (cancerous cells) were 293 or HeLa cells (cancerous cells) were transected with either the control luciferase transected with either the control luciferase siRNA or CYLD-specific siRNA. About 20 siRNA or CYLD-specific siRNA. About 20 micrograms of cell lysates were subjected to micrograms of cell lysates were subjected to IB using anti-CYLD. IB using anti-CYLD.

► Assays used in the experiment include:Assays used in the experiment include: Immunoblotting (IB)Immunoblotting (IB) In Vitro Kinase assayIn Vitro Kinase assay Electrophoresis Mobility Shift AssayElectrophoresis Mobility Shift Assay RNAi assayRNAi assay Immunecomplex kinase assayImmunecomplex kinase assay Phospho-specific IB assayPhospho-specific IB assay

Result # 1Result # 1

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

Result # 1 cont.Result # 1 cont. To systematically To systematically

analyze the role of analyze the role of CYLD regulation of CYLD regulation of cell signaling, a cell signaling, a CYLD antibody was CYLD antibody was generated. generated. ►A.A. The Ab detected The Ab detected

endogenous and endogenous and transfected CYLD.transfected CYLD.

►B. B. The Ab detected The Ab detected endogenous CYLDendogenous CYLD

Result # 1 Cont.Result # 1 Cont.

► Examined the effect of Examined the effect of CYLD knockdown on cell CYLD knockdown on cell signaling stimulated by signaling stimulated by the proinflammatory the proinflammatory cytokine TNF-alpha. cytokine TNF-alpha.

► In both 293 & HeLa In both 293 & HeLa cells, TNF-alpha cells, TNF-alpha stimulated the catalytic stimulated the catalytic activity of IKK and JNK activity of IKK and JNK as demonstrated by as demonstrated by immunocomplex kinase immunocomplex kinase assays. assays.

Result # 1 continuedResult # 1 continued

►Their hypothesisTheir hypothesis IF endogenous CYLD serves as negative IF endogenous CYLD serves as negative

regulator of TNF-alpha stimulated cell regulator of TNF-alpha stimulated cell signaling, the CYLD knockdown should result signaling, the CYLD knockdown should result in hyperactivation of the specific kinases in hyperactivation of the specific kinases under the negative control of CYLD.under the negative control of CYLD.

►Their conclusionTheir conclusion CYLD did not promote IKK activation in the CYLD did not promote IKK activation in the

TNF-alpha stimulated 293 cells or HeLa cells.TNF-alpha stimulated 293 cells or HeLa cells.

Result #1 continuedResult #1 continued

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

Result #2Result #2

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

Result #2 Cont.Result #2 Cont.► Activation of JNK by stress stimulus Activation of JNK by stress stimulus

Anisomycin( antibiotic that activates stress-activated Anisomycin( antibiotic that activates stress-activated protein kinase)protein kinase)

► Incubation of 293 cells with anisomycin lead to strong Incubation of 293 cells with anisomycin lead to strong activation of JNK activation of JNK

► JNK activated by anisomycin was not affected by CYLD JNK activated by anisomycin was not affected by CYLD knockdownknockdown

► TNF-TNF- stimulated JNK activation showed enhancement stimulated JNK activation showed enhancement of cytokine-kinase JNK response by CYLD knockdown.of cytokine-kinase JNK response by CYLD knockdown.

Result #3Result #3

►CYLD Negatively Regulates the CYLD Negatively Regulates the Activation of MKK7Activation of MKK7

In vitro Kinase AssayIn vitro Kinase Assay

► First lyse the cellFirst lyse the cell► Immunoprecipitate Immunoprecipitate

the kinase using the the kinase using the appropriate appropriate antibody (IKKB or antibody (IKKB or MAPK)MAPK)

► Add buffer or Add buffer or protein of interest protein of interest

► Analyze by Analyze by autoradiography autoradiography

Result #3 Cont.Result #3 Cont.

►

► Effect of CYLD knockdown on TNF-Effect of CYLD knockdown on TNF- stimulated stimulated activation of MKK7 and MKK4.activation of MKK7 and MKK4.

► MKK7 was enhanced in CYLD knockdown cellsMKK7 was enhanced in CYLD knockdown cells► MKK4 was not significantly enhanced in CYLD MKK4 was not significantly enhanced in CYLD

knockdown cellsknockdown cells► MKK7 is and upstream target of CYLD in JNK MKK7 is and upstream target of CYLD in JNK

signalingsignaling► Because MKK7 is enhanced by the knockdown Because MKK7 is enhanced by the knockdown

CYLD must inhibit activation of MKK7CYLD must inhibit activation of MKK7

Result #4Result #4

►CYLD Negatively Regulates JNK CYLD Negatively Regulates JNK Activation by Diverse StimuliActivation by Diverse Stimuli

Result #4 Cont.Result #4 Cont.

► CD40 cells did not CD40 cells did not exhibit significant exhibit significant signaling activity under signaling activity under unstimulated unstimulated conditionsconditions

► Cross-linking of CD40 Cross-linking of CD40 with its agonistic with its agonistic antibody lead to antibody lead to activation of IKK and activation of IKK and JNKJNK

► CYLD knockdown CYLD knockdown enhanced the the enhanced the the activation of JNK in activation of JNK in anti-CD40-treated cellsanti-CD40-treated cells

►

Result #4 Cont.Result #4 Cont.

Anti-CD40 stimulated Anti-CD40 stimulated hyper activation of NF-hyper activation of NF-B B in the CYLD knockdownin the CYLD knockdown

Infection with CYLD-small Infection with CYLD-small hairpin RNA suppressed hairpin RNA suppressed the expression of CYLD the expression of CYLD in BAJB B-cells in BAJB B-cells

CYLD knockdown enhanced CYLD knockdown enhanced JNK activation by LPS JNK activation by LPS and IL-1and IL-1

IKK activation was IKK activation was promoted in CYLD promoted in CYLD knockdown in LPS and IL-knockdown in LPS and IL-11

ConclusionsConclusions

►CYLD Is a Negative Regulator of JNK but CYLD Is a Negative Regulator of JNK but Not IKK in the TNF-alpha Signaling Not IKK in the TNF-alpha Signaling PathwayPathway

►CYLD Knockdown Has no effect on JNK CYLD Knockdown Has no effect on JNK Activation by a stress AgentActivation by a stress Agent

►CYLD Negatively Regulates the Activation CYLD Negatively Regulates the Activation ►of MKK7of MKK7►CYLD Negatively Regulates JNK activation CYLD Negatively Regulates JNK activation

by diverse Stimuliby diverse Stimuli

Conclusion Conclusion

►How CYLD differentially regulates IKK How CYLD differentially regulates IKK and JNK is not completely understood, and JNK is not completely understood, but one potential mechanism is but one potential mechanism is attributed to the differential requirement attributed to the differential requirement of TRAFs in these signaling pathways. of TRAFs in these signaling pathways.

►The findings that CYLD negatively The findings that CYLD negatively regulates JNK as well as IKK provides an regulates JNK as well as IKK provides an insight into the tumor suppressor insight into the tumor suppressor function of CYLD. function of CYLD.

ConclusionConclusion

► The IKK/NFkB pathway is well known for it The IKK/NFkB pathway is well known for it involvement in cell survival and oncogenic involvement in cell survival and oncogenic transformation as well as immune responses. transformation as well as immune responses.

► Evidence suggest that JNK is a critical factor Evidence suggest that JNK is a critical factor involved in tumorgenisis. involved in tumorgenisis. The JNK signaling pathway is activated in various tumor The JNK signaling pathway is activated in various tumor

cells. cells. JNK has been shown to promote cell growth and survial.JNK has been shown to promote cell growth and survial. CYLD knockdown increases the magnitude of JNK CYLD knockdown increases the magnitude of JNK

transient activation but does not prolong the transient activation but does not prolong the activation. activation.

ReferenceReference

►http://anisomycin.4mg.com/http://anisomycin.4mg.com/►http://www.jbc.orghttp://www.jbc.org►http://www.icr.ac.uk/cyld/natgenpap/http://www.icr.ac.uk/cyld/natgenpap/

ng060ng060►http://www.gtmb.org/http://www.gtmb.org/

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