Specific Subdomains of Vav Differentially Affect T Cell and NK Cell Activation

Abstract

The Vav protooncogene is a multidomain protein involved in the regulation of IL-2 gene transcription in T cells and the development of cell-mediated killing by cytotoxic lymphocytes. We have investigated the differential roles that specific protein subdomains within the Vav protooncogene have in the development of these two distinct cellular processes. Interestingly, a calponin homology (CH) domain mutant of Vav (CH−) fails to enhance NF-AT/AP-1-mediated gene transcription but is still able to regulate the development of cell-mediated killing. The inability of the CH− mutant to enhance NF-AT/AP-1-mediated transcription appears to be secondary to defective intracellular calcium, because 1) the CH− mutant has significantly reduced TCR-initiated calcium signaling, and 2) treatment with the calcium ionophore ionomycin or cotransfection with activated calcineurin restores NF-AT/AP-1-mediated gene transcription. The pleckstrin homology (PH) domain of Vav has also been implicated in regulating Vav activation. We found that deletion of the PH domain of Vav yields a protein that can neither enhance gene transcription from the NF-AT/AP-1 reporter nor enhance TCR- or FcR-mediated killing. In contrast, the PH deletion mutant of Vav is able to regulate the development of natural cytotoxicity, indicating a functional dichotomy for the PH domain in the regulation of these two distinct forms of killing. Lastly, mutation of three tyrosines (Y142, Y160, and Y174) within the acidic domain of Vav has revealed a potential negative regulatory site. Replacement of all three tyrosines with phenylalanine results in a hyperactive protein that increases NF-AT/AP-1-mediated gene transcription and enhances cell-mediated cytotoxicity. Taken together, these data highlight the differential roles that specific subdomains of Vav have in controlling distinct cellular functions. More broadly, the data suggest that separate lymphocyte functions can potentially be modulated by domain-specific targeting of Vav and other critical intracellular signaling molecules.