Complete sequence, genomic organization, and chromosomal localization of the human gene encoding the SHP2-interacting transmembrane adaptor protein (SIT)

Engagement of the T-cell antigen receptor (TCR) by monoclonal antibodies or antigen/MHC complexes ultimately induces cellular responses such as prolif eration, differentiation., apoptosis, or anergy. Signal propagation from th e TCR to the nucleus depends on a variety of transmembrane and intracellula r proteins. Tyrosine phosphorylations of these molecules by protein tyrosin e kinases (PTKs) of the src and syk families are among the earliest events occurring after TCR engagement and are prerequisites for the coupling of th e TCR to intracellular signalling pathways such as the Ras/Raf/MAPK- or the calcineurin/NF-AT pathways. In addition, TCR-mediated tyrosine phosphoryla tion plays a key role in the translocation of intracellular signalling mole cules (e.g., Grb2, SHP2, SLP-76) from the cytosol to the inner leaflet of t he plasma membrane, a process partially mediated by a recently described gr oup of signalling molecules termed transmembrane adaptor proteins (Schraven et al. 1999). We have recently reported the identification of one of the transmembrane ad aptor proteins named SHP2-interacting transmembrane adaptor protein (SIT) ( Marie-Cardine et al. 1999). Similar to the other transmembrane adaptor prot eins (LAT, TRIM, PAG/cbp), SIT consists of a short extracellular domain and a comparatively long cytoplasmic tail containing several tyrosine-based si gnalling motifs which become rapidly phosphorylated by PTKs upon T-cell act ivation and then serve as docking sites for SH2 domain-containing intracell ular signalling molecules. SIT inducibly interacts with the SH2-containing tyrosine phosphatase 2 (SHP2) via an immunoreceptor tyrosine-based inhibiti on motif as well as with the adaptor protein Grb2 (growth factor receptor b inding protein 2) via two consensus YxN motifs. In addition. SIT seems to i nhibit the induction of the transcriptional activity of the nuclear factor of T cells (NF-AT) upon TCR engagement. This function is mediated via recru itment of a negative regulatory effector molecule, most likely the tyrosine kinase p50csk (Pfrepper et al., 2001). In the present study, we isolated the SIT gene from a human P-1-derived art ificial chromosome (PAC) library and determined the exon-intron junctions. We additionally analyzed a 2.5-kb 5 ' upstream region and identified a TATA -less sequence containing potential binding sites for both ubiquitous and l ymphoid-specific transcription factors (Sp-1, ETF, NF-kappaB, Ikaros, GATA) . We demonstrated promoter activity of this fragment by transient transfect ion analysis and mapped the SIT gene to its genomic locus 9p12-13 by fluore scence in situ hybridization (FISH).