PY motifs of Epstein-Barr virus LMP2A regulate protein stability and phosphorylation of LMP2A-Associated proteins

Latent membrane protein 2A (LMP2A) is expressed in latent Epstein-Barr viru s (EBV) infection. We have demonstrated that Nedd4 family ubiquitin-protein ligases (E3s), AIP4, WWP2/AIP2, and Nedd4, bind specifically to two PY mot ifs 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 moti fs in EBV-transformed lymphoblastoid cell lines (LCLs), extending our origi nal observation to EBV-infected cells, Mutation of both of the LMP2A PY mot ifs resulted in an absence of binding of AIP I to LMP2A, which resulted in an increase in the expression of Lyn and the constitutive hyperphosphorylat ion of LMP2A and an unknown 120-kDa protein, In addition, there was a modes t increase in the constitutive phosphorylation of Syk and an unidentified 6 0-kDa protein. These results indicate that the PY motifs contained within L MP2A are important in regulating phosphorylation in EBV-infected LCLs, like ly through the regulation of Lyn activity by specifically targeting the deg radation of Lyn by ubiquination by Nedd4 family E3s, Despite differences be tween PY motif mutant LCLs and wild-type LCLs, the PY motif mutants still e xhibited a block in B-cell receptor (BCR) signal transduction as measured b y the induction of tyrosine phosphorylation and BZLF1 expression following BCR activation, EBV-transformed LCLs with mutations in the PY motifs were n ot different from wild-type LCLs in serum-dependent cell growth, Protein st ability of LMP1, which colocalizes with LMP2A, was not affected by the LMP2 A-associated E3s.