CXCR3-mediated T-cell chemotaxis involves ZAP-70 and is regulated by signalling through the T-cell receptor.
The chemokine receptor CXCR3 is critical for the function of activated T cells. We studied the molecular mechanisms of CXCR3 signalling. The addition of CXCR3 ligands to normal human T cells expressing CXCR3 led to the tyrosine phosphorylation of multiple proteins. Addition of the same ligands to Jurkat T cells engineered to express CXCR3 induced tyrosine phosphorylation of proteins with molecular weights similar to those in normal cells. Immunoblotting with phosphotyrosine-specific antibodies identified Zeta-associated protein of 70,000 molecular weight (ZAP-70), linker for the activation of T cells (LAT), and phospholipase-C-gamma1 (PLCgamma1) to be among the proteins that become phosphorylated upon CXCR3 activation. ZAP-70 was phosphorylated on tyrosine 319, LAT on tyrosines 171 and 191, and PLCgamma1 on tyrosine 783. The ZAP-70 inhibitor piceatannol reduced CXCR3-mediated tyrosine phosphorylation of ZAP-70, LAT, PLCgamma1 and mitogen-activated protein kinase Erk and it reduced CXCL10-mediated chemotaxis of both CXCR3-transfected Jurkat T cells and normal T cells expressing CXCR3. These results are consistent with the involvement of ZAP-70 in CXCR3-mediated protein tyrosine phosphorylation and CXCR3-induced T-cell chemotaxis. Studies with the Lck-deficient Jurkat T-cell line, JCAM1.6, demonstrated that phosphorylation of ZAP-70 after CXCR3 activation is a Lck-dependent process. Finally, stimulating CXCR3-expressing Jurkat T cells and normal T cells expressing CXCR3 through the T-cell receptor attenuated CXCR3-induced tyrosine phosphorylation and CXCR3-mediated T-cell migration, indicating the occurrence of cross-talk between T-cell receptor and CXCR3-signalling pathways. These results shed light on the mechanisms of CXCR3 signalling. Such information could be useful when designing therapeutic strategies to regulate T-cell function.
Dar
Wasim A
WA
Department of Surgery, Division of Transplantation, University of Wisconsin-Madison, Madison, WI 53792-7375, USA. wasim@surgery.wisc.edu
Knechtle
Stuart J
SJ
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D002478:Cells, Cultured; D002634:Chemotaxis, Leukocyte / Q000187:drug effects / Q000276:immunology*; D006801:Humans; D019169:Jurkat Cells; D008024:Ligands; D008213:Lymphocyte Activation / Q000276:immunology; D008565:Membrane Proteins / Q000378:metabolism; D051966:Phospholipase C gamma / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000276:immunology*; D054367:Receptors, CXCR3; D019707:Receptors, Chemokine / Q000276:immunology*; D015398:Signal Transduction / Q000276:immunology; D013267:Stilbenes / Q000494:pharmacology; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000276:immunology* / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; C515187:CXCR3 protein, human; C110810:LAT protein, human; D008024:Ligands; D008565:Membrane Proteins; D011948:Receptors, Antigen, T-Cell; D054367:Receptors, CXCR3; D019707:Receptors, Chemokine; D013267:Stilbenes; C041525:3,3',4,5'-tetrahydroxystilbene; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; D051966:Phospholipase C gamma