Biomedical subjects

Chee-Kwee Ea

Publications and source records attributed to Chee-Kwee Ea.

6 recordsLinked to original sources

PubMed2006

Activation of IKK by TNFalpha requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO.

The receptor interacting protein kinase 1 (RIP1) is essential for the activation of nuclear factor kappaB (NF-kappaB) by tumor necrosis factor alpha (TNFalpha). Here, we present evidence that TNFalpha induces the polyubiquitination of RIP1 at Lys-377 and that this polyubiquitination is required for the activation of IkappaB kinase (IKK) and NF-kappaB. A point mutation of RIP1 at Lys-377 (K377R) abolishes its polyubiquitination as well as its ability to restore IKK activation in a RIP1-deficient cell line. The K377R mutation of RIP1 also prevents the recruitment of TAK1 and IKK complexes to TNF receptor. Interestingly, polyubiquitinated RIP1 recruits IKK through the binding between the polyubiquitin chains and NEMO, a regulatory subunit of the IKK complex. Mutations of NEMO that disrupt its polyubiquitin binding also abolish IKK activation. These results reveal the biochemical mechanism underlying the essential signaling function of NEMO and provide direct evidence that signal-induced site-specific ubiquitination of RIP1 is required for IKK activation.

Amino Acid Sequence↗

PubMed2005

Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3.

Viral infection triggers host innate immune responses through activation of the transcription factors NF-kappaB and IRF 3, which coordinately regulate the expression of type-I interferons such as interferon-beta (IFN-beta). Herein, we report the identification of a novel protein termed MAVS (mitochondrial antiviral signaling), which mediates the activation of NF-kappaB and IRF 3 in response to viral infection. Silencing of MAVS expression through RNA interference abolishes the activation of NF-kappaB and IRF 3 by viruses, thereby permitting viral replication. Conversely, overexpression of MAVS induces the expression of IFN-beta through activation of NF-kappaB and IRF 3, thus boosting antiviral immunity. Epistasis experiments show that MAVS is required for the phosphorylation of IRF 3 and IkappaB and functions downstream of RIG-I, an intracellular receptor for viral RNA. MAVS contains an N-terminal CARD-like domain and a C-terminal transmembrane domain, both of which are essential for MAVS signaling. The transmembrane domain targets MAVS to the mitochondria, implicating a new role of mitochondria in innate immunity.

Amino Acid Sequence↗

PubMed2004

TIFA activates IkappaB kinase (IKK) by promoting oligomerization and ubiquitination of TRAF6.

TRAF6 (tumor necrosis factor receptor-associated factor 6) is a RING (really interesting new gene) domain ubiquitin (Ub) ligase that mediates the activation of protein kinases, such as transforming growth factor beta-activated kinase (TAK1) and IkappaB kinase (IKK), by catalyzing the formation of a unique polyubiquitin chain linked through Lys-63 of Ub. Here, we present evidence that TIFA (TRAF-interacting protein with a forkhead-associated domain, also known as T2BP) activates IKK by promoting the oligomerization and Ub ligase activity of TRAF6. We show that recombinant TIFA protein, but not TRAF6-binding-defective mutant, can activate IKK in crude cytosolic extracts. Furthermore, TIFA activates IKK in an in vitro reconstitution system consisting of purified proteins, including TRAF6, the TAK1 kinase complex, and Ub-conjugating enzyme complex Ubc13-Uev1A. Interestingly, a fraction of recombinant TIFA protein exists as high-molecular-weight oligomers, and only these oligomeric forms of TIFA can activate IKK. Importantly, TIFA induces the oligomerization and polyubiquitination of TRAF6, which leads to the activation of TAK1 and IKK through a proteasome-independent mechanism.

Adaptor Proteins, Signal Transducing↗

PubMed2004

TAB2 and TAB3 activate the NF-kappaB pathway through binding to polyubiquitin chains.

The activation of NF-kappaB and IKK requires an upstream kinase complex consisting of TAK1 and adaptor proteins such as TAB1, TAB2, or TAB3. TAK1 is in turn activated by TRAF6, a RING domain ubiquitin ligase that facilitates the synthesis of lysine 63-linked polyubiquitin chains. Here we present evidence that TAB2 and TAB3 are receptors that bind preferentially to lysine 63-linked polyubiquitin chains through a highly conserved zinc finger (ZnF) domain. Mutations of the ZnF domain abolish the ability of TAB2 and TAB3 to bind polyubiquitin chains, as well as their ability to activate TAK1 and IKK. Significantly, replacement of the ZnF domain with a heterologous ubiquitin binding domain restored the ability of TAB2 and TAB3 to activate TAK1 and IKK. We also show that TAB2 binds to polyubiquitinated RIP following TNFalpha stimulation. These results indicate that polyubiquitin binding domains represent a new class of signaling domains that regulate protein kinase activity through a nonproteolytic mechanism.

Adaptor Proteins, Signal Transducing↗

PubMed2004

The TRAF6 ubiquitin ligase and TAK1 kinase mediate IKK activation by BCL10 and MALT1 in T lymphocytes.

The CARD domain protein BCL10 and paracaspase MALT1 are essential for the activation of IkappaB kinase (IKK) and NF-kappaB in response to T cell receptor (TCR) stimulation. Here we present evidence that TRAF6 ubiquitin ligase and TAK1 protein kinase mediate IKK activation by BCL10 and MALT1. RNAi-mediated silencing of MALT1, TAK1, TRAF6, and TRAF2 suppressed TCR-dependent IKK activation and interleukin-2 production in T cells. Furthermore, we have reconstituted the pathway from BCL10 to IKK activation in vitro with purified proteins of MALT1, TRAF6, TAK1, and ubiquitination enzymes including Ubc13/Uev1A. We find that a small fraction of BCL10 and MALT1 proteins form high molecular weight oligomers. Strikingly, only these oligomeric forms of BCL10 and MALT1 can activate IKK in vitro. The MALT1 oligomers bind to TRAF6, induce TRAF6 oligomerization, and activate the ligase activity of TRAF6 to polyubiquitinate NEMO. These results reveal an oligomerization --> ubiquitination --> phosphorylation cascade that culminates in NF-kappaB activation in T lymphocytes.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Increase of the resistance of human cervical carcinoma cells to cisplatin by inhibition of the MEK to ERK signaling pathway partly via enhancement of anticancer drug-induced NF kappa B activation.

In this study, we showed that suppression of the MEK-ERK transduction pathway by a selective inhibitor, 2'-amino-3'-methoxyflavone (PD98059), increased drug resistance of SiHa cells to cisplatin, but not to another common anticancer drug, doxorubicin. The downstream mechanism of this discrepant cellular response was investigated. Both cisplatin and doxorubicin activated nuclear ERK2 and nuclear transcription factor kappa B (NF kappa B) of SiHa cells. However, suppression of the MEK-ERK2 pathway by PD98059 resulted in a further enhancement of cisplatin-induced NF kappa B activation, while no further regulation of NF kappa B was noted in doxorubicin-treated cells. The activation of NF kappa B by cisplatin or doxorubicin was not due to the degradation of cytoplasmic I kappa B alpha, as demonstrated by western blotting. Transfection of a dominant negative I kappa B alpha resulted in a markedly diminished PD98059-induced cisplatin resistance in SiHa cells. Our results suggest that the MEK-ERK signaling pathway plays a role in the chemosensitivity of SiHa cells, and suppression of this pathway increases cisplatin resistance partly via an increase of NF kappa B activation. The mechanism responsible for the discrepant effect of PD98059 on NF kappa B activation and hence the chemosensitivity of SiHa cells towards cisplatin and doxorubicin remains to be investigated.

Antineoplastic Agents↗