SLAP-130 is a recently identified substrate of the TCR-activated protein tyrosine kinases. We reported previously that SLAP-130 functions to inhibit augmented IL-2 gene activation mediated by over-expressed SLP-76. We undertook a structure/function analysis of SLAP-130 to characterize T cell signaling events affected by overexpressed SLAP-130. Deletion mutant studies reveal that a 100 amino acid domain containing three tyrosine residues is required both for inducible association with SLP-76 (via the SLP-76 SH2 domain) and for blockade of SLP-76 mediated augmentation of TCR-stimulated IL-2 gene activation. Optimal IL-2 gene transactivation depends upon activity of at least two transcription factors: NFAT and AP-1. We observed that overexpressed SLP-76 enhances AP-1 binding in a reporter gene assay. To ask whether SLAP-130 might block SLP-76 activity through impairment of AP-1 function, we co-expressed SLP-76, SLAP-130, and an AP-1 reporter construct in Jurkat T cells. Interestingly, overexpressed SLAP-130 fails to block SLP-76 mediated augmentation of TCR-induced AP-1 activity. We next asked whether SLAP-130 affects signals leading to NFAT activity. While SLAP-130 overexpresssion has no effect upon TCR-stimulated intracellular calcium flux, it partially blocks TCR- and TCR-plus costimulus-induced transactivation of a reporter gene driven by an NFAT response element derived from either the IL-2 or the CD95 ligand promoter. The CD95 ligand-derived NFAT element, unlike the IL-2 promoter NFAT, does not contain a consensus AP-1 binding site. Our data suggest that SLAP-130 inhibition of SLP-76 activity appears to depend on the SLP-76/SLAP-130 association, but details of the signaling pathways SLAP-130 modulates remain unclear.
|Original language||English (US)|
|State||Published - Mar 20 1998|