Phosphorylation of the linker for activation of T-cells by Itk promotes recruitment of Vav.
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 Cgamma1 (PLCgamma1), 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, Grb2, and PLCgamma1. 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 Vav 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.
Department of Immunology, Inflammation, and Pulmonology Discovery, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543, USA.
Juan J
JJ
Perez-Villar
Gena S
GS
Whitney
Mitchell T
MT
Sitnick
Robert J
RJ
Dunn
Srividhya
S
Venkatesan
Kathleen
K
O'Day
Gary L
GL
Schieven
Tai-An
TA
Lin
Steven B
SB
Kanner
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D019169:Jurkat Cells; D008213:Lymphocyte Activation; D008565:Membrane Proteins; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D051567:Proto-Oncogene Proteins c-vav; D013601:T-Lymphocytes / Q000378:metabolism; D014162:Transfection; D051746:ZAP-70 Protein-Tyrosine Kinase
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C110810:LAT protein, human; D008565:Membrane Proteins; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D051567:Proto-Oncogene Proteins c-vav; C496618:VAV1 protein, human; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; C080813:emt protein-tyrosine kinase