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.
Pacini
S
S
Department of Evolutionary Biology, University of Siena, Via Mattioli 4, 53100 Siena, Italy.
Ulivieri
C
C
Di Somma
M M
MM
Isacchi
A
A
Lanfrancone
L
L
Pelicci
P G
PG
Telford
J L
JL
Baldari
C T
CT
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