Abstract
Latent membrane protein 1 (LMP1), an Epstein-Barr virus transforming protein, is able to activate NF-kappaB through its carboxyl-terminal activation region 1 (CTAR1) and 2 (CTAR2), but the exact role of each domain is not fully understood. Here we show that LMP1 activates NF-kappaB in different NF-kappaB essential modulator (NEMO)-defective cell lines, but not in cells lacking both IkappaB kinase 1 (IKK1) and 2 (IKK2). Mutational studies reveal that CTAR1, but not CTAR2, mediates NEMO-independent NF-kappaB activation and that this process largely depends on IKK1. Retroviral expression of LMP1 mutants in cells lacking either functional NF-kappaB inducing kinase (NIK), NEMO, IKK1, or IKK2 further illustrates distinct signals from the two activation regions of LMP1 for persistent NF-kappaB activation. One originates in CTAR2, operates through the canonical NEMO-dependent pathway, and induces NFKB2 p100 production; the second signal originates in CTAR1, utilizes NIK and IKK1, and induces the processing of p100. Our results thus help clarify how two functional domains of LMP1 persistently activate NF-kappaB through distinct signaling pathways.
Highlights
Latent membrane protein-1 (LMP1)1 is an oncogenic transmembrane protein encoded by Epstein-Barr virus [1,2,3] that is known to activate NF-B [4, 5], the c-Jun NH2-terminal kinase pathway [6], and its downstream transcription factors such as AP-1 (6 – 8), Janus-activating tyrosine kinase 3 and signal transducer and activator of transcription [9], and p38 mitogenactivated protein kinase [10]
The entire coding region of the nemo cDNA expressed in Rat-1 cells was cloned by reverse transcription-polymerase chain reaction (RT-PCR), using primers corresponding to regions highly conserved between the human and murine nemo genes (Fig. 1A)
Semiquantitative PCR analysis revealed more than 1000 copies of nemo cDNA in the sample prepared from 100 Rat-1 cells, whereas none was detected in 100 5R cells (Fig. 1C)
Summary
Latent membrane protein-1 (LMP1)1 is an oncogenic transmembrane protein encoded by Epstein-Barr virus [1,2,3] that is known to activate NF-B [4, 5], the c-Jun NH2-terminal kinase pathway [6], and its downstream transcription factors such as AP-1 (6 – 8), Janus-activating tyrosine kinase 3 and signal transducer and activator of transcription [9], and p38 mitogenactivated protein kinase [10]. Retroviral expression of LMP1 in Rat-1 and 5R cells caused similar enhancement of IKK1-associated kinase activity determined in vitro on recombinant IB␣ protein, when the IKK complex was immunoprecipitated with an IKK1-specific antibody at 30 h after infection (Fig. 3A, left upper panel).
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