Phosphorylation of the linker for activation of T-cells by Itk promotes recruitment of Vav

Juan J. Perez-Villar, Gena S. Whitney, Mitchell Sitnick, Robert J. Dunn, Srividhya Venkatesan, Kathleen O'Day, Gary L. Schieven, Tai An Lin, Steven B. Kanner

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38 Citations (Scopus)

Abstract

The linker for activation of T-cells (LAT) is a palmitoylated integral membrane adaptor protein that resides in lipid membrane rafts and contains nine consensus putative tyrosine phosphorylation sites, several of which have been shown to serve as SH2 binding sites. Upon T-cell antigen receptor (TCR/ CD3) engagement, LAT is phosphorylated by protein tyrosine kinases (PTK) and binds to the adaptors Gads and Grb2, as well as to phospholipase Cγ1 (PLCγ1), thereby facilitating the recruitment of key signal transduction components to drive T-cell activation. The LAT tyrosine residues Y 132 , Y 171 , Y 191 , and Y 226 have been shown previously to be critical for binding to Gads, Grb 2 , and PLCγ1. In this report, we show by generation of LAT truncation mutants that the Syk-family kinase ZAP-70 and the Tec-family kinase Itk favor phosphorylation of carboxy-terminal tyrosines in LAT. By direct binding studies using purified recombinant proteins or phosphopeptides and by mutagenesis of individual tyrosines in LAT to phenylalanine residues, we demonstrate that Y 171 and potentially Y 226 are docking sites for the 8Vav guanine nucleotide exchange factor. Further, overexpression of a kinase-deficient mutant of Itk in T-cells reduced both the tyrosine phosphorylation of endogenous LAT and the recruitment of Vav to LAT complexes. These data indicate that kinases from distinct PTK families are likely responsible for LAT phosphorylation following T-cell activation and that Itk kinase activity promotes recruitment of Vav to LAT.

Original languageEnglish
Pages (from-to)10732-10740
Number of pages9
JournalBiochemistry
Volume41
Issue number34
DOIs
StatePublished - 27 Aug 2002

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Phosphorylation
T-cells
Chemical activation
T-Lymphocytes
Tyrosine
Phosphotransferases
Protein-Tyrosine Kinases
ZAP-70 Protein-Tyrosine Kinase
Personnel Selection
Guanine Nucleotide Exchange Factors
Phosphopeptides
Signal transduction
Mutagenesis
Membrane Lipids
T-Cell Antigen Receptor
Phenylalanine
Recombinant Proteins
Signal Transduction
Membrane Proteins

Cite this

Perez-Villar, J. J., Whitney, G. S., Sitnick, M., Dunn, R. J., Venkatesan, S., O'Day, K., ... Kanner, S. B. (2002). Phosphorylation of the linker for activation of T-cells by Itk promotes recruitment of Vav. Biochemistry, 41(34), 10732-10740. https://doi.org/10.1021/bi025554o
Perez-Villar, Juan J. ; Whitney, Gena S. ; Sitnick, Mitchell ; Dunn, Robert J. ; Venkatesan, Srividhya ; O'Day, Kathleen ; Schieven, Gary L. ; Lin, Tai An ; Kanner, Steven B. / Phosphorylation of the linker for activation of T-cells by Itk promotes recruitment of Vav. In: Biochemistry. 2002 ; Vol. 41, No. 34. pp. 10732-10740.
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abstract = "The linker for activation of T-cells (LAT) is a palmitoylated integral membrane adaptor protein that resides in lipid membrane rafts and contains nine consensus putative tyrosine phosphorylation sites, several of which have been shown to serve as SH2 binding sites. Upon T-cell antigen receptor (TCR/ CD3) engagement, LAT is phosphorylated by protein tyrosine kinases (PTK) and binds to the adaptors Gads and Grb2, as well as to phospholipase Cγ1 (PLCγ1), thereby facilitating the recruitment of key signal transduction components to drive T-cell activation. The LAT tyrosine residues Y 132 , Y 171 , Y 191 , and Y 226 have been shown previously to be critical for binding to Gads, Grb 2 , and PLCγ1. In this report, we show by generation of LAT truncation mutants that the Syk-family kinase ZAP-70 and the Tec-family kinase Itk favor phosphorylation of carboxy-terminal tyrosines in LAT. By direct binding studies using purified recombinant proteins or phosphopeptides and by mutagenesis of individual tyrosines in LAT to phenylalanine residues, we demonstrate that Y 171 and potentially Y 226 are docking sites for the 8Vav guanine nucleotide exchange factor. Further, overexpression of a kinase-deficient mutant of Itk in T-cells reduced both the tyrosine phosphorylation of endogenous LAT and the recruitment of Vav to LAT complexes. These data indicate that kinases from distinct PTK families are likely responsible for LAT phosphorylation following T-cell activation and that Itk kinase activity promotes recruitment of Vav to LAT.",
author = "Perez-Villar, {Juan J.} and Whitney, {Gena S.} and Mitchell Sitnick and Dunn, {Robert J.} and Srividhya Venkatesan and Kathleen O'Day and Schieven, {Gary L.} and Lin, {Tai An} and Kanner, {Steven B.}",
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Perez-Villar, JJ, Whitney, GS, Sitnick, M, Dunn, RJ, Venkatesan, S, O'Day, K, Schieven, GL, Lin, TA & Kanner, SB 2002, 'Phosphorylation of the linker for activation of T-cells by Itk promotes recruitment of Vav', Biochemistry, vol. 41, no. 34, pp. 10732-10740. https://doi.org/10.1021/bi025554o

Phosphorylation of the linker for activation of T-cells by Itk promotes recruitment of Vav. / Perez-Villar, Juan J.; Whitney, Gena S.; Sitnick, Mitchell; Dunn, Robert J.; Venkatesan, Srividhya; O'Day, Kathleen; Schieven, Gary L.; Lin, Tai An; Kanner, Steven B.

In: Biochemistry, Vol. 41, No. 34, 27.08.2002, p. 10732-10740.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Phosphorylation of the linker for activation of T-cells by Itk promotes recruitment of Vav

AU - Perez-Villar, Juan J.

AU - Whitney, Gena S.

AU - Sitnick, Mitchell

AU - Dunn, Robert J.

AU - Venkatesan, Srividhya

AU - O'Day, Kathleen

AU - Schieven, Gary L.

AU - Lin, Tai An

AU - Kanner, Steven B.

PY - 2002/8/27

Y1 - 2002/8/27

N2 - The linker for activation of T-cells (LAT) is a palmitoylated integral membrane adaptor protein that resides in lipid membrane rafts and contains nine consensus putative tyrosine phosphorylation sites, several of which have been shown to serve as SH2 binding sites. Upon T-cell antigen receptor (TCR/ CD3) engagement, LAT is phosphorylated by protein tyrosine kinases (PTK) and binds to the adaptors Gads and Grb2, as well as to phospholipase Cγ1 (PLCγ1), thereby facilitating the recruitment of key signal transduction components to drive T-cell activation. The LAT tyrosine residues Y 132 , Y 171 , Y 191 , and Y 226 have been shown previously to be critical for binding to Gads, Grb 2 , and PLCγ1. In this report, we show by generation of LAT truncation mutants that the Syk-family kinase ZAP-70 and the Tec-family kinase Itk favor phosphorylation of carboxy-terminal tyrosines in LAT. By direct binding studies using purified recombinant proteins or phosphopeptides and by mutagenesis of individual tyrosines in LAT to phenylalanine residues, we demonstrate that Y 171 and potentially Y 226 are docking sites for the 8Vav guanine nucleotide exchange factor. Further, overexpression of a kinase-deficient mutant of Itk in T-cells reduced both the tyrosine phosphorylation of endogenous LAT and the recruitment of Vav to LAT complexes. These data indicate that kinases from distinct PTK families are likely responsible for LAT phosphorylation following T-cell activation and that Itk kinase activity promotes recruitment of Vav to LAT.

AB - The linker for activation of T-cells (LAT) is a palmitoylated integral membrane adaptor protein that resides in lipid membrane rafts and contains nine consensus putative tyrosine phosphorylation sites, several of which have been shown to serve as SH2 binding sites. Upon T-cell antigen receptor (TCR/ CD3) engagement, LAT is phosphorylated by protein tyrosine kinases (PTK) and binds to the adaptors Gads and Grb2, as well as to phospholipase Cγ1 (PLCγ1), thereby facilitating the recruitment of key signal transduction components to drive T-cell activation. The LAT tyrosine residues Y 132 , Y 171 , Y 191 , and Y 226 have been shown previously to be critical for binding to Gads, Grb 2 , and PLCγ1. In this report, we show by generation of LAT truncation mutants that the Syk-family kinase ZAP-70 and the Tec-family kinase Itk favor phosphorylation of carboxy-terminal tyrosines in LAT. By direct binding studies using purified recombinant proteins or phosphopeptides and by mutagenesis of individual tyrosines in LAT to phenylalanine residues, we demonstrate that Y 171 and potentially Y 226 are docking sites for the 8Vav guanine nucleotide exchange factor. Further, overexpression of a kinase-deficient mutant of Itk in T-cells reduced both the tyrosine phosphorylation of endogenous LAT and the recruitment of Vav to LAT complexes. These data indicate that kinases from distinct PTK families are likely responsible for LAT phosphorylation following T-cell activation and that Itk kinase activity promotes recruitment of Vav to LAT.

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U2 - 10.1021/bi025554o

DO - 10.1021/bi025554o

M3 - Article

VL - 41

SP - 10732

EP - 10740

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

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ER -

Perez-Villar JJ, Whitney GS, Sitnick M, Dunn RJ, Venkatesan S, O'Day K et al. Phosphorylation of the linker for activation of T-cells by Itk promotes recruitment of Vav. Biochemistry. 2002 Aug 27;41(34):10732-10740. https://doi.org/10.1021/bi025554o