Tyrosine 474 of ZAP-70 is required for association with the Shc adaptor and for T-cell antigen receptor-dependent gene activation.
The protein tyrosine kinase ZAP-70 plays a central role in T-cell activation. Following receptor engagement, ZAP-70 is recruited to the phosphorylated subunits of the T-cell antigen receptor (TCR). This event results in ZAP-70 activation and in association of ZAP-70 with a number of signaling proteins. Among these is the Shc adaptor, which couples the activated TCR to Ras. Shc interaction with ZAP-70 is mediated by the Shc PTB domain. The inhibitory effect of a Shc mutant containing the isolated PTB domain suggests that Shc interaction with ZAP-70 might be required for TCR signaling. Here, we show that a point mutation (Phe474) of the putative Shc binding site on ZAP-70, spanning tyrosine 474, prevented ZAP-70 interaction with Shc and the subsequent binding of Shc to phospho-zeta. Neither ZAP-70 catalytic activity nor the pattern of protein phosphorylation induced by TCR triggering was affected by this mutation. However expression of the Phe474 ZAP-70 mutant resulted in impaired TCR-dependent gene activation. ZAP-70 could effectively phosphorylate Shc in vitro. Only the CH domain, which contains the two Grb2 binding sites on Shc, was phosphorylated by ZAP-70. Both Grb2 binding sites were excellent substrates for ZAP-70. The data show that Tyr474 on ZAP-70 is required for TCR signaling and suggest that Shc association with ZAP-70 and the resulting phosphorylation of Shc might be an obligatory step in linking the activated TCR to the Ras pathway.
Department of Evolutionary Biology, University of Siena, Via Mattioli 4, 53100 Siena, Italy.
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S
Pacini
C
C
Ulivieri
M M
MM
Di Somma
A
A
Isacchi
L
L
Lanfrancone
P G
PG
Pelicci
J L
JL
Telford
C T
CT
Baldari
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D001665:Binding Sites / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D051380:GRB2 Adaptor Protein; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D019169:Jurkat Cells; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D010766:Phosphorylation; D017354:Point Mutation / Q000235:genetics; D011505:Protein-Tyrosine Kinases / Q000378:metabolism* / Q000502:physiology; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000502:physiology*; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction / Q000502:physiology; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection / Q000235:genetics; D014443:Tyrosine / Q000378:metabolism*; D051746:ZAP-70 Protein-Tyrosine Kinase; D018631:ras Proteins / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D004268:DNA-Binding Proteins; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011506:Proteins; D011948:Receptors, Antigen, T-Cell; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014157:Transcription Factors; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; D018631:ras Proteins