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Mapping Pathways Downstream of S1P1 by Differential Chemical Perturbation and Proteomics
Author(s) -
GonzalezCabrera Pedro J.,
Hla Timothy,
Rosen Hugh
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a427-b
Subject(s) - downregulation and upregulation , microbiology and biotechnology , chemistry , ubiquitin , proteasome , biology , biochemistry , gene
S1P 1 agonists induce lymphopenia. Among these is the potent ligand, FTY720‐P, whose mechanism of action has been linked to its induction of S1P 1 downregulation. Use of a selective S1P 1 agonist, SEW2871 (SEW) with in vivo activity has broaden our understanding of S1P 1 function while highlighting differences in agonists' effect on fate, since SEW does not downregulate S1P 1 . The downregulation mechanism was studied in stable S1P 1 ‐GFP 293 cells by comparing signaling and S1P 1 fate with AFD, chiral FTY720‐P analog, SEW and S1P. All ligands internalized S1P 1 to late endosomes and activated GTP binding and pERK to similar maxima, but only AFD downregulated S1P 1 . Downregulation was time/concentration dependent, partially blocked by proteasome inhibition and reversed by S1P 1 antagonism and chloroquine. Ligands also induced S1P 1 ubiquitination; with AFD being more efficacious and potent vs. S1P or SEW. Ubiquitination was blocked by antagonist and chloroquine and stabilized by proteasome inhibition. Proteomic analysis of AFD samples confirmed the co‐migration of ubiquitin peptides with those of S1P 1 and GFP vs. vehicle. We conclude 1) enhanced ubiquitination by AFD targets S1P 1 for lysosomal degradation 2) downregulation is not necessary for acute S1P 1 function and 3) minimizing ubiquitination by physiological‐like ligands, such as SEW, may preserve S1P 1 pools while maintaining signaling. NIH RO1 AI055509 and Kyorin SFP1499