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Journal of Virology, June 2001, p. 5711-5718, Vol. 75, No. 12
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.12.5711-5718.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
PY Motifs of Epstein-Barr Virus LMP2A Regulate
Protein Stability and Phosphorylation of LMP2A-Associated
Proteins
Masato
Ikeda,
Akiko
Ikeda, and
Richard
Longnecker*
Department of Microbiology-Immunology,
Northwestern University Medical School, Chicago, Illinois 60611
Received 21 December 2000/Accepted 22 March 2001
Latent membrane protein 2A (LMP2A) is expressed in latent
Epstein-Barr virus (EBV) infection. We have demonstrated that Nedd4 family ubiquitin-protein ligases (E3s), AIP4, WWP2/AIP2, and Nedd4, bind specifically to two PY motifs present within the LMP2A
amino-terminal domain. In this study, LMP2A PY motif mutant viruses
were constructed to investigate the role of the LMP2A PY motifs. AIP4
was found to specifically associate with the LMP2A PY motifs in
EBV-transformed lymphoblastoid cell lines (LCLs), extending our
original observation to EBV-infected cells. Mutation of both of the
LMP2A PY motifs resulted in an absence of binding of AIP4 to LMP2A,
which resulted in an increase in the expression of Lyn and the
constitutive hyperphosphorylation of LMP2A and an unknown 120-kDa
protein. In addition, there was a modest increase in the constitutive
phosphorylation of Syk and an unidentified 60-kDa protein. These
results indicate that the PY motifs contained within LMP2A are
important in regulating phosphorylation in EBV-infected LCLs, likely
through the regulation of Lyn activity by specifically targeting the
degradation of Lyn by ubiquination by Nedd4 family E3s. Despite
differences between PY motif mutant LCLs and wild-type LCLs, the PY
motif mutants still exhibited a block in B-cell receptor (BCR) signal
transduction as measured by the induction of tyrosine phosphorylation
and BZLF1 expression following BCR activation. EBV-transformed LCLs
with mutations in the PY motifs were not different from wild-type LCLs
in serum-dependent cell growth. Protein stability of LMP1, which
colocalizes with LMP2A, was not affected by the LMP2A-associated E3s.
*
Corresponding author. Mailing address: Department of
Microbiology-Immunology, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611. Phone: (312) 503-0467. Fax: (312)
503-1339. E-mail: r-longnecker{at}northwestern.edu.
Journal of Virology, June 2001, p. 5711-5718, Vol. 75, No. 12
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.12.5711-5718.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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