Regulation of FynT function by dual domain docking on PAG/Cbp.
In resting T-cells, the transmembrane adaptor protein PAG (phosphoprotein associated with glycosphingolipid-enriched microdomains) is constitutively tyrosine-phosphorylated, a state maintained by the Src family kinase FynT. PAG has a role in negative regulation of Src family kinases in T-cells by recruitment of Csk (C-terminal Src kinase) to the membrane via binding to PAG phosphotyrosine 317. The interaction between FynT and PAG is essential for PAG function; however, so far the FynT binding mode has been unknown. Here, we demonstrate that the FynT-PAG complex formation is a dual domain docking process, involving SH2 domain binding to PAG phosphotyrosines as well as an SH3 domain interaction with the first proline-rich region of PAG. This binding mode affects FynT kinase activity, PAG phosphorylation, and recruitment of FynT and Csk, demonstrated in Jurkat TAg cells after antibody stimulation of the T cell receptor. Furthermore, we show that TCR-induced tyrosine phosphorylation is regulated by SH3 domain modulation of the FynT-PAG interaction in human primary T-cells. Although FynT SH3 domain association is shown to be crucial for efficiently initiating PAG phosphorylation, we suggest that engagement of the SH2 domain on PAG renders FynT insensitive to Csk negative regulation. Thus, in T-cells, PAG is involved in positive as well as negative regulation of FynT activity.
Biotechnology Centre of Oslo and Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo, Gaustadalléen 21, Blindern, Oslo, Norway.
Silje Anette
SA
Solheim
Knut Martin
KM
Torgersen
Kjetil
K
Taskén
Torunn
T
Berge
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D002460:Cell Line; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051076:Proto-Oncogene Proteins c-fyn / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D008565:Membrane Proteins; C498710:PAG1 protein, human; D011994:Recombinant Proteins; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn