Interchangeability of Themis1 and Themis2 in Thymocyte Development Reveals Two Related Proteins with Conserved Molecular Function

Abstract

Themis1, a recently identified T cell protein, has a critical function in the generation of mature CD4+CD8− and CD4−CD8+ (CD4- and CD8- single positive; SP) thymocytes and T cells. Although Themis1 has been shown to bind to the adapter proteins LAT and Grb2, previous studies have yielded conflicting results regarding whether or not thymocytes from Themis−/− mice exhibit TCR-mediated signaling defects. Here, we demonstrate that, in the absence of Themis1, TCR-mediated signaling is selectively impaired in CD4 SP and CD8 SP thymocytes but is not affected in CD4+CD8+ Double Positive (DP) thymocytes despite high expression of Themis1 in DP thymocytes. Like Themis1, Themis2, a related member of the Themis family, which is expressed in B cells and macrophages, contains two conserved cysteine-based domains, a proline rich region (PRR) and a nuclear localization signal (NLS). To determine if Themis1 and Themis2 can perform similar functions in vivo, we analyzed T cell development and TCR-mediated signaling in Themis1−/− mice reconstituted with either Themis1 or Themis2 transgenes. Notably, Themis1 and Themis2 exhibited the same potential to restore T cell development and TCR-mediated signaling in Themis1−/− mice. Both proteins were tyrosine phosphorylated and were recruited within Grb2 signaling complexes to LAT following TCR engagement. These results suggest that conserved molecular features of the Themis1 and Themis2 proteins are important for their biological activity and predict that Themis1 and Themis2 may perform similar functions in T and B cells, respectively.

GRB2-Mediated Recruitment of THEMIS to LAT Is Essential for Thymocyte Development

“…Our data contradict recent reports suggesting that GRB2-SH3N mediates binding to THEMIS (5,21). Although the reason for this discrepancy is unclear, our data do agree with published reports indicating that GRB2-SH3N preferentially binds to motifs conforming to the consensus PxxPxR (15), such as the type II polyproline helix in SOS (24), and displays only negligible affinities toward RxxK motifs (12,15).…”

“…An all-YF mutant except for Y540/541/ 546 was still efficiently tyrosine phosphorylated. In addition, considering conservation of tyrosines both on the interspecies and intraspecies level (i.e., between THEMIS and THEMIS2 [21]; Supplemental Table I) led us to conclude that Y540 and Y541 are major TCR-induced phosphorylation sites. Attempts to detect pY540/pY541-containing tryptic peptide(s) by MS from THEMIS isolated from TCR-stimulated Jurkat cells failed maybe because of inefficient trypsin digestion as previously noted at other phosphorylation sites (22).…”

Themis1 enhances T cell receptor signaling during thymocyte development by promoting Vav1 activity and Grb2 stability

“…Despite the absence of strong biochemical evidence, it was presumed that Themis1 likely functions as a positive regulator of TCR signaling because several defects in Themis1 −/− mice, such as a marked impairment of both positive and negative selection, were consistent with a deficiency in TCR signaling (2)(3)(4)(5)(6). It was subsequently shown that Themis1 interacts with Vav1 and that Vav1 phosphorylation is reduced in Themis1 −/− thymocytes, results that also implied a positive role for Themis1 in TCR signaling (9). Nevertheless, two reports suggested that Themis1 acts primarily, if not exclusively, as an attenuator of TCR signaling, in part through the recruitment of the phosphatase SHP-1 to LAT, and these studies further proposed that Themis1 is required to prevent the induction of strong TCR signals in response to engagement by low-affinity antigens (10,11).…”

An Epistatic Interaction between Themis1 and Vav1 Modulates Regulatory T Cell Function and Inflammatory Bowel Disease Development

“…The ablation of Themis1 in C57BL/6 mice results in impaired T cell development, particularly of the CD4 + lineage (17)(18)(19)(20)(21). After TCR engagement, Themis1 is phosphorylated by Lck and Zap-70, and recruited to the transmembrane adaptor protein LAT (22,23). Themis1 has been shown to interact with Vav1 and positively regulates its phosphorylation (23).…”

“…After TCR engagement, Themis1 is phosphorylated by Lck and Zap-70, and recruited to the transmembrane adaptor protein LAT (22,23). Themis1 has been shown to interact with Vav1 and positively regulates its phosphorylation (23). However, it remains unclear whether Themis1 acts as a positive or negative regulator of TCR signaling (17,(23)(24)(25).…”