Y PAG/Cbp, a Lipid Raft-Associated Transmembrane AdaptorDominique Davidson,1 Marcin Bakinowski,1 Matthew L. Thomas,two Vaclav Horejsi,3 and Andre Veillette1,4,5,6,7 Laboratory of Molecular Oncology, IRCM,1 Department of Medicine, University of Montreal,4 and Departments of Biochemistry,five Microbiology and Immunology,six and Medicine,7 McGill University, Montreal, Quebec, Muscle-Specific Kinase (MuSK) Proteins Gene ID Canada; Howard Hughes Health-related Institute, Division of Pathology, Washington University School of Medicine, St. Louis, Missouri2; and Institute of Molecular Genetics, Academy of Sciences from the Czech Republic, Prague, Czech RepublicReceived 30 October 2002/Returned for modification 16 December 2002/Accepted 24 DecemberPAG/Cbp (hereafter named PAG) is really a transmembrane adaptor molecule located in lipid rafts. In resting human T cells, PAG is tyrosine phosphorylated and connected with Csk, an inhibitor of Src-related protein tyrosine kinases. These modifications are quickly lost in response to T-cell receptor (TCR) stimulation. Overexpression of PAG was reported to inhibit TCR-mediated responses in Jurkat T cells. Herein, we’ve got examined the physiological relevance as well as the mechanism of PAG-mediated inhibition in T cells. Our research showed that PAG tyrosine phosphorylation and association with Csk are suppressed in response to activation of standard mouse T cells. By expressing wild-type and phosphorylation-defective (dominant-negative) PAG polypeptides in these cells, we found that the BTNL9 Proteins Recombinant Proteins inhibitory effect of PAG is dependent on its capacity to become tyrosine phosphorylated and to associate with Csk. PAG-mediated inhibition was accompanied by a repression of proximal TCR signaling and was rescued by expression of a constitutively activated Src-related kinase, implying that it really is because of an inactivation of Src kinases by PAG-associated Csk. We also attempted to recognize the protein tyrosine phosphatases (PTPs) responsible for dephosphorylating PAG in T cells. Through cell fractionation research and analyses of genetically modified mice, we established that PTPs including PEP and SHP-1 are unlikely to become involved within the dephosphorylation of PAG in T cells. Nonetheless, the transmembrane PTP CD45 appears to play an essential part within this course of action. Taken collectively, these data present firm evidence that PAG is a bona fide adverse regulator of T-cell activation as a result of its capacity to recruit Csk. Additionally they recommend that the inhibitory function of PAG in T cells is suppressed by CD45. Lastly, they support the concept that dephosphorylation of proteins on tyrosine residues is critical for the initiation of T-cell activation. T-cell activation is initiated by the interaction on the T-cell receptor (TCR) for antigens with antigenic peptides complexed to key histocompatibility complex molecules (37). TCR engagement by antigens triggers the tyrosine phosphorylation of a short sequence, the immunoreceptor tyrosinebased activation motif, present inside the TCR-associated CD3subunits (7, 23). Such immunoreceptor tyrosine-based activation motifs function by orchestrating the sequential activation of the Src-related protein tyrosine kinases (PTKs) Lck and FynT, which initiate TCR signaling, followed by that from the Zap-70/Syk PTKs, which amplify the response (7). These different PTKs induce tyrosine phosphorylation of a number of polypeptides, like the transmembrane adaptor LAT, the adaptor SLP-76, and enzymatic effectors which include phospholipase C (PLC)- (9, 24, 27, 28). Protein tyrosine phosphorylation subsequentl.
