Phosphorylation of Tyr319 in ZAP-70 is required for T-cell antigen receptor-dependent phospholipase C-gamma1 and Ras activation.
Accumulating evidence indicates that the interdomain B regions of ZAP-70 and Syk play pivotal roles in the coupling of T-cell antigen receptor (TCR) stimulation to the activation of downstream signaling pathways. The interdomain B region of ZAP-70 contains at least three candidate sites of tyrosine phosphorylation. In this report, we identify Tyr319 as a functionally important phosphorylation site in the ZAP-70 interdomain B region. TCR crosslinkage triggered a rapid increase in the phosphorylation of Tyr319 in Jurkat T cells. Although mutation of Tyr319 to Phe had no effect on the tyrosine kinase activity of ZAP-70, the resulting ZAP(Y319-->F) mutant failed to reconstitute TCR-dependent Ca2+ mobilization, Ras activation, CD69 expression and NFAT-dependent transcription in ZAP-70-deficient Jurkat cells. These defects were correlated with reduced tyrosine phosphorylation of phospholipase C (PLC)-gamma1 and the LAT adapter protein in the ZAP(Y319-->F)-expressing cells. On the other hand, ZAP(Y319-->F)-expressing cells displayed normal increases in SLP-76 phosphorylation and ERK activation during TCR stimulation. Phosphorylation of Tyr319 promoted the association of ZAP-70 with the SH2 domains of two key signaling molecules, Lck and PLC-gamma1. These studies suggest that Tyr319 phosphorylation is required for the assembly of a ZAP-70-containing signaling complex that leads to the activation of the PLC-gamma1- and Ras-dependent signaling cascades in antigen-stimulated T cells.
Williams
B L
BL
Department of Immunology, Department of Medicine, Mayo Clinic, Rochester, MN 55905, USA.
Irvin
B J
BJ
Sutor
S L
SL
Chini
C C
CC
Yacyshyn
E
E
Bubeck Wardenburg
J
J
Dalton
M
M
Chan
A C
AC
Abraham
R T
RT
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D008954:Models, Biological; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D051966:Phospholipase C gamma; D010750:Phosphoproteins / Q000378:metabolism; D017354:Point Mutation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D010738:Type C Phospholipases / Q000737:chemistry / Q000378:metabolism*; D051746:ZAP-70 Protein-Tyrosine Kinase; D018631:ras Proteins / Q000378:metabolism*; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D007527:Isoenzymes; C110810:LAT protein, human; D008565:Membrane Proteins; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011948:Receptors, Antigen, T-Cell; C092947:SLP-76 signal Transducing adaptor proteins; D014157:Transcription Factors; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D018631:ras Proteins; D002118:Calcium