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

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 Negatively Regulates the Activation of MKK7

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 cells ►

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.

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