Nguyen, Chau

Abstract T cell development depends on the coordinated interplay between receptor signaling and transcriptional activation/repression. We performed a genetic complementation screen, and identified a novel transcriptional repressor, NKAP. NKAP associates with HDAC3 and is part of a DNA-binding complex, as shown by chromatin immunoprecipitation. NKAP also associates with CIR, a part of the Notch corepressor complex. The expression of NKAP during T cell development inversely correlates with the expression of Notch target genes, implying that NKAP may modulate Notch-mediated transcription. To examine the function of NKAP in T cell development, Lck-cre NKAP conditional knockout mice were generated. Interestingly, loss of NKAP blocks development of alpha/beta but not gamma/delta T cells. In addition, lck-cre NKAP cKO DP T cells express 8- to 20-fold higher levels of Hes1, Deltex1 and CD25, providing in vivo evidence that NKAP functions as a transcriptional repressor, acting at least in part, by repression of the Notch pathway.

Abstract The activation of T cells and the initiation of an immune response is tightly controlled through the crosstalk of both positive and negative regulators. Two adaptors that function as negative regulators of T cell activation are adaptor in lymphocytes of unknown function X (ALX) and linker for activation of X cell (LAX). Previously, we showed that T cells from mice deficient in ALX and LAX display similar hyperresponsiveness, with increased IL-2 production and proliferation upon TCR/CD28 stimulation, and that these adaptors physically associate. In this study, we analyze the nature of the association between ALX and LAX. We demonstrate that this association occurs in the absence of TCR/CD28 signaling via a mechanism independent of both tyrosine phosphorylation of LAX and the SH2 domain of ALX. Cotransfection of ALX with LAX resulted in LAX tyrosine phosphorylation in the absence of TCR/CD28 stimulation. ALX-mediated LAX phosphorylation depends upon the ALX SH2 domain, which functions to recruit Lck to LAX. We also show that LAX, like ALX, can inhibit RE/AP reporter activation. However, in contrast to its inhibition of NFAT, the inhibition of RE/AP by LAX is independent of its tyrosine phosphorylation. Therefore, it can be concluded that inhibition of signaling events involved in T cell activation by LAX occurs through mechanisms both dependent on and independent of its tyrosine phosphorylation.