Degradation of ZAP-70 following antigenic stimulation in human T lymphocytes: role of calpain proteolytic pathway.
T cell activation by the specific Ag results in dramatic changes of the T cell phenotype that include a rapid and profound down-regulation and degradation of triggered TCRs. In this work, we investigated the fate of the TCR-associated ZAP-70 kinase in Ag-stimulated T cells. T cells stimulated by peptide-pulsed APCs undergo an Ag dose-dependent decrease of the total cellular content of ZAP-70, as detected by FACS analysis and confocal microscopy on fixed and permeabilized T cell-APC conjugates and by Western blot on total cell lysates. The time course of ZAP-70 consumption overlaps with that of zeta-chain degradation, indicating that ZAP-70 is degraded in parallel with TCR internalization and degradation. Pharmacological activation of protein kinase C (PKC) does not induce ZAP-70 degradation, which, on the contrary, requires activation of protein tyrosine kinases. Two lines of evidence indicate that the Ca2+-dependent cysteine protease calpain plays a major role in initiating ZAP-70 degradation: 1) treatment of T cells with cell-permeating inhibitors of calpain markedly reduces ZAP-70 degradation; 2) ZAP-70 is cleaved in vitro by calpain. Our results show that, in the course of T cell-APC cognate interaction, ZAP-70 is rapidly degraded via a calpain-dependent mechanism.
Penna
D
D
Institute of Biochemistry, University of Lausanne, BIL Research Center, Epalinges, Switzerland.
Müller
S
S
Martinon
F
F
Demotz
S
S
Iwashima
M
M
Valitutti
S
S
D000938:Antigen-Presenting Cells / Q000276:immunology / Q000378:metabolism; D000941:Antigens / Q000276:immunology*; D002154:Calpain / Q000378:metabolism* / Q000502:physiology; D002461:Cell Line, Transformed; D002999:Clone Cells; D015536:Down-Regulation / Q000276:immunology; D006801:Humans; D006868:Hydrolysis; D008213:Lymphocyte Activation / Q000187:drug effects; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000037:antagonists & inhibitors / Q000378:metabolism; D013601:T-Lymphocytes / Q000201:enzymology / Q000276:immunology / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D051746:ZAP-70 Protein-Tyrosine Kinase
D000941:Antigens; D008565:Membrane Proteins; D011948:Receptors, Antigen, T-Cell; C054979:antigen T cell receptor, zeta chain; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; D002154:Calpain; D013755:Tetradecanoylphorbol Acetate