While it delayed and dampened the maximum of the response it caused a sustained, long-lasting increase in the cytosolic Ca2+ concentration compared with the relatively transient increase in wild-type cells. expressed in an inducible manner in the absence of some other EBV signaling protein. This allowed us for the first time to monitor LMP2A signaling em in statu nascendi /em as it occurs during the EBV existence cycle in vivo. We display that mere manifestation of LMP2A not only stimulated Rabbit polyclonal to ACSS3 protein tyrosine kinases but also induced phospholipase C-2-mediated Ca2+ oscillations followed by activation of the extracellular signal-regulated kinase (Erk) mitogen-activated protein kinase pathway and induction of the lytic EBV gene em bzlf1 /em . Furthermore, manifestation of the constitutively phosphorylated LMP2A ITAM modulated rather than inhibited BCR-induced Ca2+ mobilization. Summary Our data set up that LMP2A manifestation has a function beyond the putative inhibition of the BCR by generating a ligand-independent cellular activation signal that may provide a molecular switch for different EBV existence cycle stages and most probably contributes to EBV-associated lymphoproliferative disorders. strong class=”kwd-title” Keywords: B Cells, Epstein-Barr disease, LMP2A, B cell antigen receptor, ITAM, tyrosine phosphorylation, Ca2+, latency, lytic replication Background A common feature of herpes viruses is their ability to preserve latent infections during which no virus particles are produced. The oncogenic Epstein-Barr disease (EBV) establishes such a latent illness in human being B cells [1]. At least four different types of EBV latency have been described based on the manifestation patterns of EBV genes including those encoding latent membrane protein (LMP) 1 and 2A [2]. The lipid raft-resident LMP2A consists of 12 transmembrane domains T338C Src-IN-1 and both, the N- and C-terminus face the cytosol. An immunoreceptor tyrosine-based activation motif (ITAM) in the LMP2A N-terminus is definitely constitutively phosphorylated and activates the protein tyrosine kinase (PTK) Syk [3]. This enables LMP2A to support development and maintenance of peripheral B cells in LMP2A transgenic mouse models [4,5]. We have previously demonstrated that for these purposes LMP2A also employs the intracellular adapter protein SLP65 (BLNK or BASH), which is a important effector molecule of the B cell antigen receptor (BCR) [6]. Following engagement of the BCR, SLP65 in conjunction with the adaptor CIN85 nucleates assembly of the Ca2+ initiation complex comprising Bruton’s tyrosine kinase (Btk) and phospholipase C (PLC)-2 [7,8]. So far, the standard model system for biochemical analysis of LMP2A signaling mechanisms was based on EBV-transformed main human being B cells known as lymphoblastoid cell lines (LCL), which communicate, however, several EBV gene products. Although early studies demonstrated the LMP2A ITAM in the context of chimeric transmembrane proteins activates the Ca2+ initiation complex, experiments using LCL suggested that LMP2A functions as inhibitor of BCR-induced activation signals and helps prevent mobilization of Ca2+ ions from intra- T338C Src-IN-1 and extracellular sources [3,9,10]. This observation led to the hypothesis that LMP2A suppresses viral replication which would be induced upon BCR activation of LMP2A-negative cells [11]. However, recent studies showed that constant activation of BCR-regulated signaling pathways – as carried T338C Src-IN-1 out by LMP2A – induces and maintains BCR unresponsiveness resulting in B cell anergy [12,13]. To circumvent this problem and to analyze LMP2A signaling em in statu nascendi /em in non-anergic cells in the absence of some other EBV gene product, we now founded a Cre/loxP-based system to inducibly communicate LMP2A in B cells. We display that manifestation of LMP2A not only triggered PTKs but also the Ca2+ initiation complex resulting in oscillatory Ca2+ fluxes much like those observed after BCR activation. This induced activation of the mitogen-activated protein kinase (MAPK) pathway as well as the manifestation of EBV-encoded BZLF1,.