Abstract
Cytohesin binder and regulator (Cybr; also known as CYTIP, CASP, and PSCDBP) is a cytokine-induced gene preferentially expressed in hematopoietic tissues and in T helper 1 cells. Cybr protein associates with members of the cytohesin family, which are known ADP-ribosylation factors-GDP/GTP exchange factors, and its functions appear to regulate lymphocyte adhesion and cell-cell contact. Here we show that Cybr mRNA and protein levels are increased upon T cell receptor engagement. Cybr expression then influences T cell receptor-dependent signaling events, such as nuclear factor of activated T cells and AP-1 transcriptional activity. In addition, expression of Cybr results in increased T cell receptor-mediated activation of the Rho/Rac exchange factor Vav and of the JNK-p38 MAPK signaling pathway. The effects of Cybr on nuclear factor of activated T cells and AP-1 are dependent on MAPK activation, and enhanced activation of this cascade results in cooperation between the two transcription factors in the regulation of gene expression. These findings provide the first evidence that the adaptor protein Cybr not only regulates lymphocyte adhesion and cell-cell interaction but also contributes to the regulation of the signaling cascade and of the genetic program downstream of the T cell receptor.
Highlights
JULY 21, 2006 VOLUME 281 NUMBER 29 tant downstream event after T cell antigen receptor (TCR) engagement
We originally described Cytohesin binder and regulator (Cybr) as a cytokine-inducible gene being rapidly induced in NK cells and peripheral blood mononuclear cells (PBMCs) following IL-2 and IL-12 stimulation [16, 17]
Cybr Is Inducible upon TCR Triggering—We recently identified Cybr as a cytokine-inducible gene preferentially expressed in immune cells
Summary
Plasmids, Antibodies, and Chemical Reagents—The human leukemic T-cell line Jurkat, subclone E6 was maintained in standard growth medium (RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 100 units/ml of penicillin/streptomycin). Cells were lysed in ice-cold lysis buffer containing 0.5% Triton X-100, 50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 2 mM EDTA, 0.4 mM Na3VO4, 10 g/ml leupeptin, 10 g/ml aprotinin, and 2.5 M p-nitrophenyl p-guanidinobenzoate. Cells were washed once in phosphate-buffered saline and suspended in cold lysis buffer containing 1% Nonidet P-40, 50 mM Tris, pH 7.5, and 150 mM NaCl, supplemented with phosphatase inhibitors. Cells were washed three times with Hanks’ balanced salt solution plus 1% fetal calf serum and resuspended in 7 ml of Hepes-buffered saline (137 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 5 mM glucose, 1 mM CaCl2, 0.5 mM MgCl2, 1 g/liter bovine serum albumin, 10 mM Hepes, pH 7.4). Stimulation with the Ca2ϩ ionophore, ionomycin (1 g/ml), was used as measurement of maximal activity
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