A novel negative regulatory function of the phosphoprotein associated with glycosphingolipid-enriched microdomains: blocking Ras activation.
In primary human T cells, anergy induction results in enhanced p59Fyn activity. Because Fyn is the kinase primarily responsible for the phosphorylation of PAG (the phosphoprotein associated with glycosphingolipid-enriched microdomains), which negatively regulates Src-kinase activity by recruiting Csk (the C-terminal Src kinase) to the membrane, we investigated whether anergy induction also affects PAG. Analysis of anergic T cells revealed that PAG is hyperphosphorylated at the Csk binding site, leading to enhanced Csk recruitment and inhibitory tyrosine phosphorylation within Fyn. This together with enhanced phosphorylation of a tyrosine within the SH2 domain of Fyn leads to the formation of a hyperactive conformation, thus explaining the enhanced Fyn kinase activity. In addition, we have also identified the formation of a multiprotein complex containing PAG, Fyn, Sam68, and RasGAP in stimulated T cells. We demonstrate that PAG-Fyn overexpression is sufficient to suppress Ras activation in Jurkat T cells and show that this activity is independent of Csk binding. Thus, in addition to negatively regulating Src family kinases by recruiting Csk, PAG also negatively regulates Ras by recruiting RasGAP to the membrane. Finally, by knocking down PAG, we demonstrate both enhanced Src kinase activity and Ras activation, thereby establishing PAG as an important negative regulator of T-cell activation.
Smida
Michal
M
Institute of Immunology, Otto-von-Guericke University, Magdeburg, Germany.
Posevitz-Fejfar
Anita
A
Horejsi
Vaclav
V
Schraven
Burkhart
B
Lindquist
Jonathan A
JA
D048868:Adaptor Proteins, Signal Transducing / Q000276:immunology*; D002455:Cell Division; D017635:Clonal Anergy; D000242:Cyclic AMP / Q000378:metabolism; D004789:Enzyme Activation; D006706:Homeostasis / Q000276:immunology; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000276:immunology*; D018448:Models, Immunological; D010750:Phosphoproteins / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000276:immunology* / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D018631:ras Proteins / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D008565:Membrane Proteins; C498710:PAG1 protein, human; D010750:Phosphoproteins; D000242:Cyclic AMP; D019061:src-Family Kinases; D018631:ras Proteins; D013755:Tetradecanoylphorbol Acetate