Herpesvirus saimiri Tip associates with Lck and downregulates Lck signal transduction. Here we demonstrate that Tip targets a lysosomal protein p80, which consists of an N-terminal WD repeat domain and a C-terminal coiled-coil domain. Interaction of Tip with p80 facilitated lysosomal vesicle formation and subsequent recruitment of Lck into the lysosomes for degradation. Consequently, Tip interactions with Lck and p80 result in downregulation of T cell receptor (TCR) and CD4 surface expression. Remarkably, these actions of Tip are functionally and genetically separable: the N-terminal p80 interaction is responsible for TCR downregulation and the C-terminal Lck interaction is responsible for CD4 downregulation. Thus, lymphotropic herpesvirus has evolved an elaborate mechanism to deregulate lymphocyte receptor expression to disarm host immune control.

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Recognition of phosphorylated serine/threonine-containing motifs by 14-3-3 depends on the dimerization of 14-3-3. However, the molecular cues that control 14-3-3 dimerization are not well understood. In order to identify proteins that control 14-3-3 dimerization, we analyzed proteins that have effects on 14-3-3 dimerization and report that protein kinase A (PKA) phosphorylates 14-3-3f at a specific residue (Ser58). Phosphorylation by PKA leads to modulation of 14-3-3f dimerization and affect its interaction with partner proteins. Substitution of Ser58 to Ala completely abolished phosphorylation of 14-3-3f by PKA. A phospho-mimic mutant of 14-3-3f, Ser58 to Glu substitution, failed to form homodimers, showed reduced interaction with 14-3-3e and p53, and could not enhance transcriptional activity of p53. Moreover, activation of PKA decreases and inhibition of PKA increases the dimerization of 14-3-3f and the functional interaction of 14-3-3f with p53. Therefore, our results suggest that PKA is a new member of protein kinases that can phosphorylate and impair the function of 14-3-3.

Lipid rafts are proposed to function as platforms for both receptor signaling and trafficking. Following interaction with antigenic peptides, the T-cell receptor (TCR) rapidly translocates to lipid rafts, where it transmits signals and subsequently undergoes endocytosis. The Tip protein of herpesvirus saimiri (HVS), which is a T-lymphotropic tumor virus, interacts with cellular Lck tyrosine kinase and p80, a WD domaincontaining endosomal protein. Interaction of Tip with p80 induces enlarged vesicles and recruits Lck and TCR complex into these vesicles for trafficking. We report here that Tip is constitutively present in lipid rafts and that Tip interaction with p80 but not with Lck is necessary for its efficient localization in lipid rafts. The Tip-Lck interaction was required for recruitment of the TCR complex to lipid rafts, and the Tip-p80 interaction was critical for the aggregation and internalization of lipid rafts. These results suggest the potential mechanism for Tip-mediated TCR downregulation: Tip interacts with Lck to recruit TCR complex to lipid rafts, and it subsequently interacts with p80 to initiate the aggregation and internalization of the lipid raft domain and thereby downregulate the TCR complex. Thus, the signaling and targeting functions of HVS Tip rely on two functionally and genetically separable mechanisms that independently target cellular Lck tyrosine kinase and p80 endosomal protein.