SOCS-6 Negatively Regulates T Cell Activation through Targeting p56 to Proteasomal Degradation

Abstract

The T cell-specific tyrosine kinase, p56 lck , plays crucial roles in T cell receptor (TCR)-mediated T cell activation. Here, we report that SOCS-6 (suppressor of cytokine signaling-6) is a negative regulator of p56 lck . SOCS-6 was identified as a protein binding to the kinase domain of p56 lck through yeast two-hybrid screening. SOCS-6 bound specifically to p56 lck (F505), which mimics the active form of p56 lck , but not to wild type p56 lck . In Jurkat T cells, SOCS-6 binding to p56 lck was detected 1-2 h after TCR stimulation. Confocal microscopy showed that upon APC-T cell conjugation, SOCS-6 was recruited to the immunological synapse and colocalized with the active form of p56 lck . SOCS-6 promoted p56lck ubiquitination and its subsequent targeting to the proteasome. Moreover, SOCS-6 overexpression led to repression of TCR-dependent interleukin-2 promoter activity. These results establish that SOCS-6 acts as a negative regulator of T cell activation by promoting ubiquitin-dependent proteolysis.The T lymphocyte-specific member of the Src-type tyrosine kinase family, p56 lck , plays essential roles in development, antigen-induced T cell activation, and proliferation (1-3). During TCR 4 -mediated T cell activation, p56 lck is activated and transmits a positive signal by interacting with CD4/CD8 (4, 5). Upon engagement of the antigen-major histocompatibility complex with TCR-CD4-CD8 complexes, p56 lck phosphorylates the immunoreceptor tyrosine-based activation motifs of the CD3 -chain and provides the binding site for ZAP-70 tyrosine kinase (6). Subsequent ZAP-70 activation leads to phosphorylation of signaling proteins, including LAT, and amplification of TCR-mediated signaling (7,8). In this process, p56 lck migrates to T cell-APC contact regions known as immunological synapses (IS) and thereby enhances TCR signaling (9, 10). Given the importance of p56 lck function in T cell activation, establishing how p56 lck activity is regulated is essential to understand that activation.The structure of p56 lck is similar to that of other Src family kinases and exhibits the following common domains: N-terminal attachment sites for saturated fatty acid addition, a unique region; an Src homology 3 (SH3) domain; an SH2 domain; a tyrosine kinase domain (SH1); and a C-terminal negative regulatory site, tyrosine 505 (Tyr-505) (11). p56 lck kinase activity is regulated by intramolecular interactions through its regulatory domains and/or phosphorylation (11). For example, phosphorylation of Tyr-394 in the activation loop of the kinase domain increases enzymatic activity, whereas phosphorylation of Tyr-505 renders the enzyme less active. Phosphorylated Tyr-505 interacts with its own SH2 domain, promoting a closed conformation and maintaining the kinase in an inactive state (12). Phenylalanine substitution of the regulatory tyrosine residue (Y505F) results in a constitutively active form of the protein.The status of the regulatory tyrosine is maintained both by a kinase, Csk, which phosphorylates it (13), and by a transmembrane...

Suppressor of Cytokine Signaling 6 (SOCS6) Negatively Regulates Flt3 Signal Transduction through Direct Binding to Phosphorylated Tyrosines 591 and 919 of Flt3

“…lck kinase (25). We also observed an increased ligand-dependent ubiquitination of the Flt3 receptor in SOCS6-expressing cells.…”

The SOCS box—Adapting proteins for ubiquitination and proteasomal degradation

“…Although this interaction depended on the SH2 domain, low levels of ubiquitination were also detected with a mutant SH2 domain, reflecting either the extended SOCS6-SH2 binding interface or additional binding motifs in the N-terminal region (52). SOCS6 has also been shown to interact, via its N-terminal region, with the Tcell-specific Src family kinase Lck to mediate Lck turnover and thus inhibit T-cell receptor signalling (53).…”

Cell Regulation by Phosphotyrosine-Targeted Ubiquitin Ligases

“…It is again unclear if Nck or Crk/CrkL is a substrate for ubiquitination. Finally, using overexpressed proteins, the N-terminal region of SOCS6 was shown to bind to the SH2 kinase region of active (but not inactive) Lck and less well to other Src-related kinases (47).…”