Differential Function of Themis CABIT Domains during T Cell Development

Abstract

Themis (also named Gasp) is a newly identified Grb2-binding protein that is essential for thymocyte positive selection. Despite the possible involvement of Themis in TCR-mediated signal transduction, its function remains unresolved and controversial. Themis contains two functionally uncharacterized regions called CABIT (cysteine-containing, all-β in Themis) domains, a nuclear localization signal (NLS), and a proline-rich sequence (PRS). To elucidate the role of these motifs in Themis’s function in vivo, we established a series of mutant Themis transgenic mice on a Themis−/− background. Deletion of the highly conserved Core motif of CABIT1 or CABIT2 (Core1 or Core2, respectively), the NLS, or the PRS abolished Grb2-association, as well as TCR-dependent tyrosine-phosphorylation and the ability to induce positive selection in the thymus. The NLS and Core1 motifs were required for the nuclear localization of Themis, whereas Core2 and PRS were not. Furthermore, expression of ΔCore1- but not ΔCore2-Themis conferred dominant negative-type inhibition on T cell development. Collectively, our current results indicate that PRS, NLS, CABIT1, and CABIT2 are all required for positive selection, and that each of the CABIT domains exerts distinct functions during positive selection.

THEMIS: Two Models, Different Thresholds

“…GRB2 contains a central Src homology 2 (SH2) domain flanked by two SH3 domains [7]. The THEMIS:GRB2 interaction was shown to require the THEMIS PRS which binds to one of the GRB2 SH3 domains [8–10]. GRB2, via its SH2 domain, recruits THEMIS to the scaffolding transmembrane T cell adapter Linker for Activation of T cells (LAT) upon TCR engagement [8, 9, 11].…”

“…GRB2, via its SH2 domain, recruits THEMIS to the scaffolding transmembrane T cell adapter Linker for Activation of T cells (LAT) upon TCR engagement [8, 9, 11]. The THEMIS:GRB2 interaction is required for THEMIS in vivo activity [9, 10, 12] indicating that recruitment of THEMIS to LAT by GRB2 is essential for its function.…”

“…THEMIS proteins lacking either the PRS [9, 10, 12] or the NLS [10, 12] were unable to rescue T cell maturation in Themis −/− mice. As described in the text, the PRS is required for binding of THEMIS to the cytosolic adapter GRB2.…”

“…THEMIS is detectable in the nucleus of thymocytes [1, 8, 10, 12] and the NLS is required for its nuclear localization [8, 10, 12]; however, the function of THEMIS in the nucleus remains unclear and requires additional investigation. A THEMIS protein containing C/A mutations of the conserved cysteines in CABIT1(Cys153) and CABIT2(Cys413) was capable of rescuing T cell development in Themis −/− mice [12] whereas deletion of the 23–24aa conserved core sequences in either the CABIT1 or CABIT2 module abolished THEMIS function [10]. Interestingly, the developmental block in Themis −/− thymocytes was significantly worsened by expression a transgenic THEMIS protein that lacked the CABIT1 core sequence (ΔCore1), and THEMIS ΔCore1 had a dominant negative effect on T cell maturation when expressed in Themis +/+ mice [10].…”

THEMIS Is Required for Pathogenesis of Cerebral Malaria and Protection against Pulmonary Tuberculosis

“…Though the CABIT structural motifs have been identified by sequence conservation, their structure-function relationships have yet to be clearly defined. Recent studies have shown that deletion of a part of the CABIT-1 domain (⌬150-174) in mouse THEMIS causes a defect in T cell development (45). We show that the I23N mutation in the CABIT-1 domain causes protein instability in primary thymic cells and in transfected cells, suggesting that I23N may cause misfolding of the protein, which is consequently targeted for degradation.…”