Epstein-Barr virus-encoded latent membrane protein 1 activates the JNK pathway through its extreme C terminus via a mechanism involving TRADD and TRAF2.
The transforming Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) activates signalling on the NF-kappaB axis through two distinct domains in its cytoplasmic C terminus, namely, CTAR1 (amino acids [aa] 187 to 231) and CTAR2 (aa 351 to 386). The ability of CTAR1 to activate NF-kappaB appears to be attributable to the direct interaction of tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2), while recent work indicates that CTAR2-induced NF-kappaB is mediated through its association with TNF receptor-associated death domain (TRADD). LMP1 expression also results in activation of the c-Jun N-terminal kinase (JNK) (also known as stress-activated protein kinase) cascade, an effect which is mediated exclusively through CTAR2 and can be dissociated from NF-kappaB induction. The organization and signalling components involved in LMP1-induced JNK activation are not known. In this study we have dissected the extreme C terminus of LMP1 and have identified the last 8 aa of the protein (aa 378 to 386) as being important for JNK signalling. Using a series of fine mutants in which single amino acids between codons 379 and 386 were changed to glycine, we have found that mutations of Pro379, Glu381, Ser383, or Tyr384 diminish the ability of LMP1 CTAR2 to engage JNK signalling. Interestingly, this region was also found to be essential for CTAR2-mediated NF-kappaB induction and coincides with the LMP1 amino acid sequences shown to bind TRADD. Furthermore, we have found that LMP1-mediated JNK activation is synergistically augmented by low levels of TRADD expression, suggesting that this adapter protein is critical for LMP1 signalling. TRAF2 is known to associate with TRADD, and expression of a dominant-negative N-terminal deletion TRAF2 mutant was found to partially inhibit LMP1-induced JNK activation in 293 cells. In addition, the TRAF2-interacting protein A20 blocked both LMP1-induced JNK and NF-kappaB activation, further implicating TRAF2 in these phenomena. While expression of a kinase-inactive mutated NF-kappaB-inducing kinase (NIK), a mitogen-activated protein kinase kinase kinase which also associates with TRAF2, impaired LMP1 signalling on the NF-kappaB axis, it did not inhibit LMP1-induced JNK activation, suggesting that these two pathways may bifurcate at the level of TRAF2. These data further define a role for TRADD and TRAF2 in JNK activation and confirm that LMP1 utilizes signalling mechanisms used by the TNF receptor/CD40 family to elicit its pleiotropic activities.
Eliopoulos
A G
AG
CRC Institute for Cancer Studies, The University of Birmingham Medical School, Birmingham B15 2TA, England.
Blake
S M
SM
Floettmann
J E
JE
Rowe
M
M
Young
L S
LS
D000596:Amino Acids; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D002461:Cell Line, Transformed; D004268:DNA-Binding Proteins; D004789:Enzyme Activation; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D016328:NF-kappa B / Q000378:metabolism; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D047990:TNF Receptor-Associated Factor 1; D047992:TNF Receptor-Associated Factor 2; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3; D014763:Viral Matrix Proteins / Q000235:genetics / Q000378:metabolism*; D000097782:NF-kappaB-Inducing Kinase
D000596:Amino Acids; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D016328:NF-kappa B; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases; D011506:Proteins; D047990:TNF Receptor-Associated Factor 1; D047992:TNF Receptor-Associated Factor 2; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3; D014763:Viral Matrix Proteins; D000097782:NF-kappaB-Inducing Kinase; C037662:EBV-associated membrane antigen, Epstein-Barr virus; C076529:TNFAIP3 protein, human