ABSTRACT
Chemokines regulate the homeostatic trafficking of lymphocytes and lymphocyte influx into sites of injury and inflammation. The signaling pathways by which chemokine receptors regulate lymphocyte migration remain incompletely characterized. We demonstrate that Jurkat T cells lacking the ZAP-70 tyrosine kinase exhibit reduced migration in response to the CXCR4 ligand CXCL12 when compared with wild-type Jurkat T cells. Expression of wild-type, but not kinase-inactive, ZAP-70 resulted in enhanced migration of ZAP-70-deficient Jurkat T cells. The tyrosine residue at position 292 in the interdomain B region of ZAP-70 exerts a negative regulatory effect on ZAP-70-dependent migration. Stimulation of Jurkat T cells with CXCL12 also resulted in ZAP-70-dependent tyrosine phosphorylation of the Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) adapter protein. Although CXCL12-dependent migration of SLP-76-deficient Jurkat T cells was impaired, re-expression of SLP-76 did not enhance migration. These results suggest a novel function for ZAP-70, but not SLP-76, in CXCR4 chemokine receptor signaling in human T cells.
Subject(s)
Cell Movement/immunology , Protein-Tyrosine Kinases/metabolism , Receptors, CXCR4/physiology , Signal Transduction/immunology , T-Lymphocytes/enzymology , T-Lymphocytes/immunology , Cell Movement/genetics , Chemokine CXCL12 , Chemokines, CXC/metabolism , Chemokines, CXC/physiology , Humans , Jurkat Cells , Ligands , Protein-Tyrosine Kinases/biosynthesis , Protein-Tyrosine Kinases/deficiency , Protein-Tyrosine Kinases/genetics , Receptors, CXCR4/metabolism , T-Lymphocytes/cytology , T-Lymphocytes/metabolism , ZAP-70 Protein-Tyrosine KinaseABSTRACT
Stimulation of the CD3/TCR results within minutes in an increase in T cell adhesion mediated by beta(1) integrins. The biochemical pathways that control CD3-mediated increases in beta(1) integrin-mediated adhesion remain poorly characterized. In this study, the role of the tyrosine kinase ZAP-70 in the regulation of beta(1) integrin activity by the CD3/TCR was investigated. CD3 stimulation did not increase beta(1) integrin-mediated adhesion of the ZAP-70-deficient Jurkat T cell line, P116, to the beta(1) integrin ligand fibronectin. Reintroduction of wild-type ZAP-70, but not a kinase-inactive variant, K369R, corrected the adhesive defect observed in P116 T cells. In addition, the kinase-inactive ZAP-70 mutant inhibited CD3-induced adhesion of primary human T cell blasts. Interestingly, a ZAP-70 mutant with a tyrosine to phenylalanine substitution at position 319 (Y319F) restored the adhesive defect in P116 T cells, even though Y319F ZAP-70 failed to fully reconstitute CD3-initiated NF-AT-dependent transcription and tyrosine phosphorylation of the LAT adapter protein. Finally, expression of mutants of LAT and the SLP-76 adapter protein that modulate CD3-mediated activation of an NF-AT reporter gene failed to block CD3-induced increases in beta(1) integrin-mediated adhesion. These observations support a model in which the tyrosine kinase activity of ZAP-70 kinase is critical for regulation of beta(1) integrin activity by CD3/TCR. However, the signaling events downstream of ZAP-70 that regulate CD3/TCR-mediated activation of beta(1) integrin function exhibit key differences when compared with the signaling pathways that regulate transcriptional events initiated by CD3/TCR stimulation.
Subject(s)
Adaptor Proteins, Signal Transducing , Integrin beta1/physiology , Membrane Proteins , Nuclear Proteins , Protein-Tyrosine Kinases/physiology , Receptor-CD3 Complex, Antigen, T-Cell/physiology , Signal Transduction/immunology , T-Lymphocytes/metabolism , Transcriptional Activation/immunology , Amino Acid Substitution/genetics , Amino Acid Substitution/immunology , Carrier Proteins/genetics , Cell Adhesion/genetics , Cell Adhesion/immunology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Enzyme Activation/genetics , Enzyme Activation/immunology , Fibronectins/physiology , Humans , Integrin beta1/metabolism , Jurkat Cells , Luciferases/genetics , Luciferases/metabolism , Lymphocyte Activation/physiology , NFATC Transcription Factors , Phenylalanine/genetics , Phosphoproteins/genetics , Phosphoproteins/metabolism , Point Mutation , Protein-Tyrosine Kinases/deficiency , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Signal Transduction/genetics , T-Lymphocytes/enzymology , T-Lymphocytes/immunology , T-Lymphocytes/physiology , Transcription Factors/genetics , Transcription Factors/metabolism , Tyrosine/genetics , ZAP-70 Protein-Tyrosine KinaseABSTRACT
The role of integrin-mediated signaling events in T cell function remains incompletely characterized. We report here that alpha4beta1 integrin stimulation of H9 T cells and normal human T cell blasts results in rapid and transient tyrosine phosphorylation of the adapter protein, SH2 domain-containing 76-kDa protein (SLP-76)-associated phosphoprotein of 130 kDa (SLAP-130)/FYB at levels comparable to those observed following TCR stimulation. Stimulation of T cells via the alpha4beta1 integrin enhances the association of tyrosine phosphorylated SLAP-130/FYB with the SH2 domain of the src tyrosine kinase p59fyn. Activation of normal T cells, but not H9 T cells, via alpha4beta1 leads to tyrosine phosphorylation of SLP-76 as well as SLAP-130/FYB. Overexpression of SLAP-130/FYB in normal T cells enhances T cell migration through fibronectin-coated filters in response to the chemokine stromal cell-derived factor (SDF)-1alpha. These results identify SLAP-130/FYB as a new tyrosine phosphorylated substrate in beta1 integrin signaling and suggest a novel function for SLAP-130/FYB in regulating T lymphocyte motility.