T Cell Receptor (TCR)-induced Tyrosine Phosphorylation Dynamics Identifies THEMIS as a New TCR Signalosome Component

Abstract

Stimulation of the T cell antigen receptor (TCR) induces formation of a phosphorylation-dependent signaling network via multiprotein complexes, whose compositions and dynamics are incompletely understood. Using stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics, we investigated the kinetics of signal propagation after TCR-induced protein tyrosine phosphorylation. We confidently assigned 77 proteins (of 758 identified) as a direct or indirect consequence of tyrosine phosphorylation that proceeds in successive “signaling waves” revealing the temporal pace at which tyrosine kinases activate cellular functions. The first wave includes thymocyte-expressed molecule involved in selection (THEMIS), a protein recently implicated in thymocyte development but whose signaling role is unclear. We found that tyrosine phosphorylation of THEMIS depends on the presence of the scaffold proteins Linker for activation of T cells (LAT) and SH2 domain-containing lymphocyte protein of 76 kDa (SLP-76). THEMIS associates with LAT, presumably via the adapter growth factor receptor-bound protein 2 (Grb2) and with phospholipase Cγ1 (PLC-γ1). RNAi-mediated THEMIS knock-down inhibited TCR-induced IL-2 gene expression due to reduced ERK and nuclear factor of activated T cells (NFAT)/activator protein 1 (AP-1) signaling, whereas JNK, p38, or nuclear factor κB (NF-κB) activation were unaffected. Our study reveals the dynamics of TCR-dependent signaling networks and suggests a specific role for THEMIS in early TCR signalosome function.

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

“…In addition, negative selection and CD4 + T cell lineage commitment, which are dependent on intense or sustained TCR signals, respectively, are both impaired in Themis1 −/− mice (2,5). In agreement with these studies of rodent thymocytes, human Themis1 enhances extracellular signal-regulated kinase (ERK) phosphorylation and nuclear factor of activated T cells (NFAT)-activator protein 1 (AP1) luciferase promoter activity in Jurkat cells stimulated with TCR cross-linking antibodies (8). Further biochemical investigation to identify the molecular function of Themis1 has shown that it interacts with two other positive effectors of TCR signaling, phospholipase C-g1 (PLC-g1) (2) and Vav1, a guanine nucleotide exchange factor (GEF) Rho family guanosine triphosphatases (GTPases) (9), in thymocytes, but the importance of these interactions for TCR signaling and T cell development remains obscure.…”

“…It also contains a bipartite nuclear localization signal (KR-X12-KRRPR) and a C-terminal proline-rich region (PRR) that matches a class II Src homology 3 (SH3) recognition motif. Themis1 is constitutively associated with the cytosolic adaptor protein Grb2 (growth factor receptor-bound protein 2) (5-7) and is recruited to the transmembrane adaptor protein LAT (linker of activated T cells) after TCR engagement, where it becomes rapidly phosphorylated by the protein tyrosine kinase Lck (8,9).…”

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

“…In previous work, we identified THEMIS as an early target of tyrosine phosphorylation downstream of TCR (7). TCR-induced tyrosine phosphorylation of THEMIS depended on the adapters linker for activation of T cells (LAT) and Src homology (SH) 2 domain-containing leukocyte protein of 76 kDa (SLP76), and THEMIS appeared to associate to LAT upon TCR stimulation.…”

“…Reactions were stopped by adding reducing SDS NuPAGE sample buffer (Invitrogen) and incubation for 5 min at 95˚C, followed by alkylation with 55 mM iodoactamide (Sigma). Proteins were separated on 4-12% gradient Bis-Tris NuPAGE gels (Invitrogen), gels were washed in distilled water, lightly stained with Colloidal Blue (Invitrogen), and subjected to GeLC-mass spectrometry (MS)/MS as described previously (7).…”

THEMIS and PTPRK in celiac intestinal mucosa: coexpression in disease and after in vitro gliadin challenge

“…5 Although its expression is reduced in differentiated T lymphocytes, 6 it is thought to have a role in TcR activation signaling. 7 In turn, PTPRK performs functions related to the maintenance of cell junctions [8][9][10] and in the modulation of epidermal growth factor receptor (EGFR) activity, resulting in an inhibition of cell proliferation. 11,12 Interestingly, both THEMIS and PTPRK have been implicated in normal CD4 þ T-cell development in rodents, suggesting that they participate in common biological processes.…”