Phosphorylation-dependent regulation of T-cell activation by PAG/Cbp, a lipid raft-associated transmembrane adaptor.
PAG/Cbp (hereafter named PAG) is a transmembrane adaptor molecule found in lipid rafts. In resting human T cells, PAG is tyrosine phosphorylated and associated with Csk, an inhibitor of Src-related protein tyrosine kinases. These modifications are rapidly 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 have examined the physiological relevance and the mechanism of PAG-mediated inhibition in T cells. Our studies showed that PAG tyrosine phosphorylation and association with Csk are suppressed in response to activation of normal mouse T cells. By expressing wild-type and phosphorylation-defective (dominant-negative) PAG polypeptides in these cells, we found that the inhibitory effect of PAG is dependent on its capacity to be 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 is due to an inactivation of Src kinases by PAG-associated Csk. We also attempted to identify the protein tyrosine phosphatases (PTPs) responsible for dephosphorylating PAG in T cells. Through cell fractionation studies and analyses of genetically modified mice, we established that PTPs such as PEP and SHP-1 are unlikely to be involved in the dephosphorylation of PAG in T cells. However, the transmembrane PTP CD45 seems to play an important role in this process. Taken together, these data provide firm evidence that PAG is a bona fide negative regulator of T-cell activation as a result of its capacity to recruit Csk. They also suggest that the inhibitory function of PAG in T cells is suppressed by CD45. Lastly, they support the idea that dephosphorylation of proteins on tyrosine residues is critical for the initiation of T-cell activation.
Davidson
Dominique
D
Laboratory of Molecular Oncology, IRCM, 110 Pine Avenue West, Montréal, Québec, Canada H2W 1R7.
Bakinowski
Marcin
M
Thomas
Matthew L
ML
Horejsi
Vaclav
V
Veillette
André
A
D019943:Amino Acid Substitution; D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D019556:COS Cells; D000081247:CSK Tyrosine-Protein Kinase; D020013:Calcium Signaling; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D036341:Intercellular Signaling Peptides and Proteins; D017493:Leukocyte Common Antigens / Q000502:physiology*; D008213:Lymphocyte Activation / Q000502:physiology*; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008822:Mice, Transgenic; D010750:Phosphoproteins / Q000235:genetics / Q000502:physiology*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000235:genetics; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000502:physiology; D051076:Proto-Oncogene Proteins c-fyn; D011948:Receptors, Antigen, T-Cell / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000502:physiology; D013329:Structure-Activity Relationship; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D019061:src-Family Kinases
D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C408344:Pag protein, mouse; C498709:Pag1 protein, mouse; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; C494960:Fyn protein, mouse; D051076:Proto-Oncogene Proteins c-fyn; D019061:src-Family Kinases; C000631346:CSK protein, human; D017493:Leukocyte Common Antigens