Multi-transmembrane protein K15 of Kaposi's sarcoma-associated herpesvirus targets Lyn kinase in the membrane raft and induces NFAT/AP1 activities

Abstract

Viral proteins of γ-2 herpesviruses, such as LMP2A of Epstein Barr virus (EBV) and Tip of herpesvirus saimiri (HVS) dysregulate lymphocyte signaling by interacting with Src family kinases. K15 open reading frame of Kaposi's sarcoma associated herpesvirus (KSHV), located at the right end of the viral genome, encodes several splicing variants differing in numbers of transmembrane domains. Previously, we demonstrated that the cytoplasmic tail of the K15 protein interfered with B cell receptor signal transduction to cellular tyrosine phosphorylation and calcium mobilization. However, the detailed mechanism underlying this phenomenon was not understood. In the C-terminal cytoplasmic region of K15, putative binding domains for Src-SH2 and -SH3 were identified. In this study, we attempted to characterize these modular elements and cellular binding protein(s) by GST pull down and co-immunoprecipitation assays. These studies revealed that K15 interacted with the major B cell tyrosine kinase Lyn. In vitro kinase and transient co-expression assays showed that the expression of K15 protein resulted in activation of Lyn kinase activity. In addition, GST pull down assay suggested that the SH2 domain of Lyn alone was necessary for interaction with the C-terminal SH2B (YEEV) of K15, but the addition of Lyn SH3 to the SH2 domain increases the binding affinity to K15 protein. The data from luciferase assays indicate that K15 expression in BJAB cells induced NFAT and AP1 activities. The tyrosine residue in the C-terminal end of K15 required for the Lyn interaction appeared to be essential for NFAT/AP1 activation, highlighting the significance of the C-terminal SH2B of K15 as a modular element in interfering with B lymphocyte signaling through interaction with Lyn kinase.

Role of the Kaposi's Sarcoma-Associated Herpesvirus K15 SH3 Binding Site in Inflammatory Signaling and B-Cell Activation

“…Previous studies have shown that the KSHV K15 transmembrane protein has the ability to interact through its cytoplasmic domain with a range of cellular proteins and thereby triggers signaling cascades (2,3,6,7,10,21,35,38). Several sequence motifs, such as the putative SH2 and SH3 binding motifs, are located in the C-terminal domain, and an involvement in K15-mediated signaling has been shown for some of these.…”

Palmitoylation of MIR2 Is Required for Its Function

“…It is quite likely that other KSHV-encoded proteins are also palmitoylated, and this palmitoylation may be integral to their function. For example, K15, a homologue of LMP2A, is thought to target the major B cell Src kinase Lyn to lipid rafts (3,28). There are many cysteine residues present in K15, and at least one is predicted to be cytosolic.…”

KSHV

“…Several critical tyrosine residues within these motifs are constitutively phosphorylated by cellular Src family tyrosine kinases, which mediate activation of downstream signaling pathways. Pathways activated by K15 signaling include the Ras/MAPK, JNK/SAPK, and NFκB pathways as well as the NFAT/AP1 transcription factors (Brinkmann et al, 2003; Brinkmann et al, 2007; Cho, Choi, & Choi, 2008). This signaling activates transcription of a number of cellular cytokines and chemokines including IL6, IL8, CCL20, CCL2, CXCL3, IL-1α/β, and Cox2 (Brinkmann et al, 2007; Wang et al, 2007).…”

“…K15 can also downregulate signal transduction and intracellular calcium mobilization induced by the BCR, which may help the virus maintain latency (Choi et al, 2000). A potential mechanism by which K15 accomplishes this may be through K15’s interaction with the tyrosine kinase Lyn, which plays a role in the regulation of BCR signaling (Cho et al, 2008). Additionally, the K15 M allele induces cell motility, and this is dependent on K15-mediated upregulation of the human miRNAs miR-21 and -31 (Tsai et al, 2009).…”