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.
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
Abbreviations used in this paper: aa, amino acid(s); EST, expressed sequence tag; GEM, glycosphingolipid-enriched microdomain; GST, glutathione S-transferase; ITAM, immunoreceptor tyrosine-based activation motif; LAT, linker for activation of T cells; Nano ES MS/MS, nanoelectrospray tandem mass spectrometry; NF-AT, nuclear factor of activated T cells; PAG, phosphoprotein associated with GEMs; PEP, PEST-containing phosphatase; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; P-Tyr, phosphotyrosine; RACE, rapid amplification of cDNA end; SH, Src-homology; SHP, SH2-containing protein tyrosine phosphatase; SLP, SH2 domain containing leukocyte phosphoprotein; TRIM, T cell receptor–interacting molecule.
Tomás̆ Brdic̆ka, Dagmar Pavlis̆tová, Albrecht Leo, Eddy Bruyns, Vladimír Kor̆ínek, Pavla Angelisová, Jeanette Scherer, Andrej Shevchenko, Anna Shevchenko, Ivan Hilgert, Jan C̆erný, Karel Drbal, Yasuhiro Kuramitsu, Birgit Kornacker, Václav Hor̆ejs̆í, Burkhart Schraven; 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. J Exp Med 1 May 2000; 191 (9): 1591–1604. doi: https://doi.org/10.1084/jem.191.9.1591
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