In vivo functional mapping of the conserved protein domains within murine Themis1

Abstract

Thymocyte development requires the coordinated input of signals that originate from numerous cell surface molecules. Although the majority of thymocyte signal initiating receptors are lineage-specific, most trigger ‘ubiquitous’ downstream signaling pathways. T-lineage specific receptors are coupled to these signaling pathways by lymphocyte-restricted adapter molecules. We and others recently identified a new putative adapter protein, Themis1, whose expression is largely restricted to the T lineage. Mice lacking Themis1 exhibit a severe block in thymocyte development and a striking paucity of mature T cells revealing a critical role for Themis1 in T cell maturation. Themis1 orthologs contain three conserved domains: a proline rich region (PRR) that binds to the ubiquitous cytosolic adapter Grb2, a nuclear localization sequence (NLS), and two copies of a novel cysteine-containing globular (CABIT) domain. In the present study, we evaluated the functional importance of each of these motifs by retroviral reconstitution of Themis1−/− progenitor cells. The results demonstrate an essential requirement for the PRR and NLS motifs but not the conserved CABIT cysteines for Themis1 function.

THEMIS enhances TCR signaling and enables positive selection by selective inhibition of the phosphatase SHP-1

“…2e ), indicating that the cysteine residues in the CABIT domains are not essential for regulating the PTP activity of SHP-1. This is consistent with previous observations that retrovirally encoded THEMIS-C-A can rescue the DP to SP developmental block in Themis −/− thymocytes 20 . These results demonstrated that the THEMIS CABIT modules directly inhibit SHP-1 PTP activity and that the conserved core cysteine is not required for this function.…”

“…Although THEMIS CABIT modules can bind directly to SHP-1, experimental data strongly suggest that the interaction of THEMIS with GRB2 is important for its in vivo function 12 , 18 , 20 . Co-binding of SHP-1 and THEMIS to GRB2 brings these proteins into close proximity and may facilitate and stabilize their direct interaction.…”

THEMIS: Two Models, Different Thresholds

“…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].…”