Phosphoprotein Associated with Glycosphingolipid-Enriched Microdomains (Pag), a Novel Ubiquitously Expressed Transmembrane Adaptor Protein, Binds the Protein Tyrosine Kinase Csk and Is Involved in Regulation of T Cell Activation

TLDR

Signal transduction via immunoreceptors is proposed to depend on interactions with glycosphingolipid‑enriched membrane microdomains (GEMs). The study introduces PAG, a novel GEM‑associated transmembrane adaptor protein. PAG consists of a 16‑residue extracellular domain and a 397‑residue cytoplasmic tail with ten tyrosine residues that are phosphorylated by Src family kinases. PAG is constitutively tyrosine‑phosphorylated in resting T cells, binds Csk, and upon activation becomes dephosphorylated and releases Csk; overexpression of PAG recruits Csk, dampens Src activity, impairs Src‑substrate phosphorylation, and suppresses NFAT‑mediated T‑cell receptor signaling, indicating that the PAG–Csk complex maintains T‑cell quiescence.

Abstract

According to a recently proposed hypothesis, initiation of signal transduction via immunoreceptors depends on interactions of the engaged immunoreceptor with glycosphingolipid-enriched membrane microdomains (GEMs). In this study, we describe a novel GEM-associated transmembrane adaptor protein, termed phosphoprotein associated with GEMs (PAG). PAG comprises a short extracellular domain of 16 amino acids and a 397-amino acid cytoplasmic tail containing ten tyrosine residues that are likely phosphorylated by Src family kinases. In lymphoid cell lines and in resting peripheral blood α/β T cells, PAG is expressed as a constitutively tyrosine-phosphorylated protein and binds the major negative regulator of Src kinases, the tyrosine kinase Csk. After activation of peripheral blood α/β T cells, PAG becomes rapidly dephosphorylated and dissociates from Csk. Expression of PAG in COS cells results in recruitment of endogenous Csk, altered Src kinase activity, and impaired phosphorylation of Src-specific substrates. Moreover, overexpression of PAG in Jurkat cells downregulates T cell receptor–mediated activation of the transcription factor nuclear factor of activated T cells. These findings collectively suggest that in the absence of external stimuli, the PAG–Csk complex transmits negative regulatory signals and thus may help to keep resting T cells in a quiescent state.