ABSTRACT
The Tip protein of Herpesvirus saimiri strain 484C binds to and activates the Lck tyrosine protein kinase. Two sequences in the Tip protein were previously shown to be involved in binding to Lck. A proline-rich region, residues 132-141, binds to the SH3 domain of the Lck protein. We show here that the other Lck-binding domain, residues 104-113, binds to the carboxyl-terminal half of Lck and that this binding does not require the Lck SH3 domain. Mutated Tip containing only one functional Lck-binding domain can bind stably to Lck, although not as strongly as wild-type Tip. Interaction of Tip with Lck through either Lck-binding domain increases the activity of Lck in vivo. Simultaneous binding of both domains is required for maximal activation of Lck. The transient expression of Tip in T cells was found to stimulate both Stat3-dependent and NF-AT-dependent transcription. Mutant forms of Tip lacking one or the other of the two Lck-binding domains retained the ability to stimulate Stat3-dependent transcription. Tip lacking the proline-rich Lck-binding domain exhibited almost wild-type activity in this assay. In contrast, ablation of either Lck-binding domain abolished the ability of Tip to stimulate NF-AT-dependent transcription. Full biological activity of Tip, therefore, appears to require both Lck-binding domains.
Subject(s)
Herpesvirus 2, Saimiriine/physiology , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/metabolism , Phosphoproteins/metabolism , Viral Proteins/metabolism , Binding Sites , Cell Line , DNA-Binding Proteins/metabolism , Enzyme Activation , Humans , Jurkat Cells , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/chemistry , Models, Molecular , Mutation , Phosphoproteins/chemistry , Phosphoproteins/genetics , Protein Binding , Protein Structure, Tertiary , STAT3 Transcription Factor , T-Lymphocytes/metabolism , T-Lymphocytes/virology , Trans-Activators/metabolism , Viral Proteins/chemistry , Viral Proteins/genetics , src Homology DomainsABSTRACT
In T cells, the JNK mitogen-activated protein kinase is activated by simultaneous stimulation of the T-cell receptor and CD28 or by a number of stress stimuli including ultraviolet light, hydrogen peroxide, and anisomycin. Lck, a Src family kinase, is essential for T-cell receptor-mediated activation of JNK. We asked whether Lck was also involved in stress-mediated activation of JNK. JNK was activated by ultraviolet light irradiation in all of the four T-cell lines we examined, but Lck was not. Additionally, JNK activation by ultraviolet light, hydrogen peroxide, and anisomycin was completely normal in T cells lacking Lck. These data suggest that Lck is not activated by ultraviolet light irradiation, nor is it required for JNK activation in T cells by any of the stress stimuli we tested. We also examined JNK activation by ultraviolet light in mouse fibroblasts expressing no known Src kinases. The activation of JNK by ultraviolet light was completely normal in these cells. Finally, treatment of lymphoid and epithelial cells with a Src kinase family inhibitor PP2-reduced tyrosine phosphorylation of cellular proteins markedly without affecting ultraviolet light-induced activation of JNK. These results suggest that Src kinases are not essential for ultraviolet light-induced activation of JNK in a diverse variety of cell types.
