Ikeda M, Ikeda A, Longan L C, Longnecker R
Department of Microbiology-Immunology, Northwestern University Medical School, Chicago, Illinois, 60611, USA.
Virology. 2000 Mar 1;268(1):178-91. doi: 10.1006/viro.1999.0166.
Latent membrane protein 2A (LMP2A) is expressed in latent Epstein-Barr virus (EBV) infection. LMP2A functions to downregulate B-cell signal transduction and viral reactivation from latency in EBV-immortalized B cells in vitro, and acts to provide B cells with both a survival and developmental signal in vivo. Identification of proteins associated with LMP2A is important for elucidation of the mechanism that LMP2A employs to regulate B-cell signal transduction and EBV latency. LMP2A is constitutively tyrosine phosphorylated and is associated with protein tyrosine kinases such as Lyn and Syk when specific LMP2A tyrosines are phosphorylated. The amino-terminal domain of LMP2A includes multiple proline-rich regions, which may provide binding sites for proteins containing SH3 or WW domains. In this study, we demonstrate that four cellular proteins bind specifically to two PPPPY (PY) motifs present within the LMP2A amino-terminal domain. Protein microsequence analysis determined that three of these proteins were AIP4, WWP2/AIP2, and Nedd4. All of these proteins are members of the Nedd4-like ubiquitin-protein ligases family and have conserved domains including the C2, WW, and ubiquitin-protein ligase domain. The mutation of both PY motifs completely abolished binding activity of these proteins to LMP2A and the interaction of AIP4 and WWP2 with LMP2A was confirmed in cell lines expressing LMP2A, WWP2, and AIP4. Furthermore, a reduction in the level of Lyn and the rapid turnover of LMP2A and Lyn were observed in LMP2A-expressing cells. These findings suggest that LMP2A recruits Nedd4-like ubiquitin-protein ligases and B-cell signal transduction molecules, resulting in the degradation of LMP2A and Lyn by a ubiquitin-dependent mechanism. This provides a new means by which LMP2A may modulate B-cell signal transduction.
潜伏膜蛋白2A（LMP2A）在爱泼斯坦-巴尔病毒（EBV）潜伏感染中表达。LMP2A的功能是在体外下调EBV永生化B细胞中的B细胞信号转导和病毒从潜伏期的重新激活，并在体内为B细胞提供生存和发育信号。鉴定与LMP2A相关的蛋白质对于阐明LMP2A用于调节B细胞信号转导和EBV潜伏期的机制很重要。LMP2A持续酪氨酸磷酸化，当特定的LMP2A酪氨酸被磷酸化时，它与蛋白酪氨酸激酶如Lyn和Syk相关。LMP2A的氨基末端结构域包括多个富含脯氨酸的区域，这可能为含有SH3或WW结构域的蛋白质提供结合位点。在本研究中，我们证明四种细胞蛋白特异性结合LMP2A氨基末端结构域内存在的两个PPPPY（PY）基序。蛋白质微序列分析确定其中三种蛋白质是AIP4、WWP2/AIP2和Nedd4。所有这些蛋白质都是Nedd4样泛素-蛋白连接酶家族的成员，并且具有包括C2、WW和泛素-蛋白连接酶结构域在内的保守结构域。两个PY基序的突变完全消除了这些蛋白质与LMP2A的结合活性，并且在表达LMP2A、WWP2和AIP4的细胞系中证实了AIP4和WWP2与LMP2A的相互作用。此外，在表达LMP2A的细胞中观察到Lyn水平的降低以及LMP2A和Lyn的快速周转。这些发现表明LMP2A募集Nedd4样泛素-蛋白连接酶和B细胞信号转导分子，导致LMP2A和Lyn通过泛素依赖性机制降解。这提供了一种LMP2A可能调节B细胞信号转导的新方式。
