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Epstein-Barr virus latent membrane protein 2 associates with and is a substrate for mitogen-activated protein kinase
1Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pennsylvania 15261, USA.
Abstract:
The latent membrane protein 2 (LMP2) of Epstein-Barr virus interferes with B-lymphocyte signal transduction through the immunoglobulin (Ig) receptor. Two isoforms of LMP2 exist and differ only in that one isoform (LMP2a) contains an N-terminal cytoplasmic domain that the other isoform does not. LMP2a is a phosphoprotein that is phosphorylated on tyrosines and serines in the cytoplasmic domain. GST1-119, a glutathione S-transferase (GST) fusion protein containing the 119 amino acids of the cytoplasmic domain, affinity precipitated serine kinase activity from BJAB cell extracts. The affinity-precipitated kinase phosphorylated LMP2a sequences, and kinase activity was increased following induction. Probing of Western immunoblots of affinity-precipitated proteins showed that the Erk1 form of mitogen-activated protein kinase (MAPK) was present. Purified MAPK phosphorylated GST fusion proteins containing the cytoplasmic domain of LMP2a and mutational analyses were used to identify S15 and S102 as the sites of in vitro phosphorylation. A polyclonal rabbit antiserum was prepared against a maltose binding protein-LMP2a cytoplasmic domain fusion protein (MBP1-119) and used to immunoprecipitate LMP2a from the in vitro-immortalized lymphoblastoid B-cell line B95-8CR. LMP2a immunoprecipitates from B95-8CR contained MAPK as a coprecipitated protein. Cross-linking surface Ig on B95-8CR cells failed to induce MAPK activity within the cells. Treatment of B95-8CR with phorbol myristate acetate (PMA) was able to bypass the Ig receptor block and activate MAPK activity. Phosphorylation of LMP2a on serine residues increased after PMA induction. The possible role for LMP2a serine phosphorylation by MAPK in the control of latency is discussed.
Insights
Epstein-Barr virus latent membrane protein 2a (LMP2a) is phosphorylated by mitogen-activated protein kinase (MAPK). This phosphorylation, particularly at serine residues, may play a role in controlling viral latency in B-lymphocytes.
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- Epstein-Barr virus (EBV) latent membrane protein 2 (LMP2) disrupts B-lymphocyte signaling via the immunoglobulin (Ig) receptor.
- Two LMP2 isoforms exist: LMP2a includes an N-terminal cytoplasmic domain, unlike the other isoform.
- LMP2a is a phosphoprotein, with phosphorylation occurring on tyrosine and serine residues within its cytoplasmic domain.
Purpose of the Study:
- To investigate the kinase activity associated with the LMP2a cytoplasmic domain.
- To identify the specific kinase responsible for LMP2a phosphorylation.
- To explore the role of LMP2a phosphorylation in B-cell signaling and EBV latency.
Main Methods:
- Affinity precipitation of serine kinase activity using a glutathione S-transferase (GST) fusion protein (GST1-119) containing the LMP2a cytoplasmic domain.
- Western blot analysis to identify precipitated proteins, including mitogen-activated protein kinase (MAPK).
- In vitro phosphorylation assays using purified MAPK and mutational analysis to identify phosphorylation sites (S15, S102).
- Immunoprecipitation of LMP2a from B-lymphoblastoid cells (B95-8CR) and analysis of coprecipitated proteins.
- Stimulation of B-cells with phorbol myristate acetate (PMA) to bypass Ig receptor signaling and assess MAPK activation and LMP2a phosphorylation.
Main Results:
- Serine kinase activity was affinity precipitated by GST1-119 from BJAB cell extracts.
- Mitogen-activated protein kinase (MAPK), specifically Erk1, was identified as a kinase that phosphorylates the LMP2a cytoplasmic domain at serine residues S15 and S102.
- MAPK was found to coprecipitate with LMP2a in B95-8CR cells.
- While Ig receptor cross-linking did not activate MAPK, PMA treatment bypassed this block, activating MAPK and increasing serine phosphorylation of LMP2a.
Conclusions:
- Mitogen-activated protein kinase (MAPK) directly phosphorylates the latent membrane protein 2a (LMP2a) of Epstein-Barr virus.
- Serine phosphorylation of LMP2a by MAPK is inducible by stimuli like PMA, suggesting a role in regulating viral latency.
- LMP2a may act as a scaffold or signaling molecule linking viral proteins to cellular kinase pathways.