Growth Transformation of Human T Cells by Herpesvirus Saimiri Requires Multiple Tip-Lck Interaction Motifs

Abstract

Lymphoma induction and T-cell transformation by herpesvirus saimiri strain C488 depends on two viral oncoproteins, StpC and Tip. The major interaction partner of Tip is the protein tyrosine kinase Lck, a key regulator of T-cell activation. The Lck binding domain (LBD) of Tip comprises two interaction motifs, a proline-rich SH3 domain-binding sequence (SH3B) and a region with homology to the C terminus of Src family kinase domains (CSKH). In addition, biophysical binding analyses with purified Lck-SH2 domain suggest the phosphorylated tyrosine residue 127 of Tip (pY127) as a potential third Lck interaction site. Here, we addressed the relevance of the individual binding motifs, SH3B, CSKH, and pY127, for Tip-Lck interaction and for human T-cell transformation. Both motifs within the LBD displayed Lck binding activities and cooperated to achieve a highly efficient interaction, while pY127, the major tyrosine phosphorylation site of Tip, did not enhance Lck binding in T cells. Herpesvirus saimiri strain C488 recombinants lacking one or both LBD motifs of Tip lost their transforming potential on human cord blood lymphocytes. Recombinant virus expressing Tip with a mutation at position Y127 was still able to transform human T lymphocytes but, in contrast to wild-type virus, was strictly dependent on exogenous interleukin-2. Thus, the strong Lck binding mediated by cooperation of both LBD motifs was essential for the transformation of human T cells by herpesvirus saimiri C488. The major tyrosine phosphorylation site Y127 of Tip was particularly required for transformation in the absence of exogenous interleukin-2, suggesting its involvement in cytokine signaling pathways.

Inhibition of Retromer Activity by Herpesvirus Saimiri Tip Leads to CD4 Downregulation and Efficient T Cell Transformation

“…Previous studies have shown that Tip interacts with multiple cellular proteins, such as Lck, STAT3, and p80, but only Lck has been shown to be required for transformation of HVS-infected T cells (4,9,22,31,39). To search for additional cellular Tip interaction partners that may contribute to in vitro T cell immortalization, the amino acid sequences of Tip proteins in various HVS subgroup C strains were analyzed, and a conserved glutamic acid-rich region at the amino terminus was discovered (Fig.…”

“…While Tip is not required for viral replication in culture, Tip expression is critical for T cell and Stp-C oncogene immortalization (13). Transformation of human T cells with recombinant HVS expressing a mutant Tip that is unable to bind Lck has shown that the Tip-Lck interaction is required for HVS-induced transformation of human T lymphocytes, while binding to STAT3 is not necessary (22,23). Tip also targets p80, a lysosomal protein that consists of an N-terminal WD repeat domain and a C-terminal coiled-coil domain.…”

Lck-dependent Fyn Activation Requires C Terminus-dependent Targeting of Kinase-active Lck to Lipid Rafts

“…Moreover, some data indicate that phosphorylation within the activation loop in trans by other Src or non-Src family members can also occur (22). An additional mode of activation, which does not involve dephosphorylation of the C-terminal tyrosine, is conferred by high affinity interactions of the SH3 domain with its ligands, resulting in the competitive displacement of low affinity intramolecular interactions (19,(23)(24)(25). This results in an alignment of kinase domain residues critical for catalysis and enables productive binding of ATP and substrates (26).…”

Activation of Noncanonical NF-κB Signaling by the Oncoprotein Tio

“…The function of StpC relies on a TRAF2 binding site that mediates NF-B activation (12,13). Tip (tyrosine kinase-interacting protein) was identified as a binding partner of the Src family kinase (SFK) Lck (14), and complex interactions with this kinase are required for viral transformation (15). The herpesvirus ateles oncogene tio substitutes for stpC and tip in the transformation of human T cells (16).…”