Abstract
Stimulation of the T cell antigen receptor (TCR) activates signaling pathways involving protein kinases, phospholipase Cgamma1, and Ras. How these second messengers interact to initiate distal activation events is an area of intense scrutiny. In this report, we confirm that TCR ligation results in phosphorylation of Sos, a guanine nucleotide exchange factor for Ras. This requires expression of both the CD45 tyrosine phosphatase and the Lck protein tyrosine kinase and depends upon signaling via protein kinase C. In contrast to previous studies examining requirements for Sos phosphorylation following insulin and epidermal growth factor receptor engagement, we show that TCR-induced phosphorylation of Sos does not require activation of the mitogen-activated protein kinase/extracellular-signal regulated kinase (MEK/ERK) pathway. However, the basal phosphorylation of Sos in T cells is affected by either MEK or MEK-dependent kinases. Although Sos phosphorylation results in its dissociation from Grb2 following insulin stimulation in Chinese hamster ovary cells, TCR engagement on the Jurkat T cell line fails to elicit a similar effect. These data demonstrate that the kinases responsible for Sos phosphorylation differ following ligation of various cell surface receptors and that the consequences of Sos phosphorylation relies, at least in part, on sites of its phosphorylation.
Highlights
Stimulation of T cells via their antigen receptor (TCR)1 leads to activation of a number of signaling pathways
Stimulation of Jurkat with Anti-TCR or phorbol 12-myristate 13-acetate (PMA) Results in Phosphorylation of Sos—Stimulation of the TCR has been shown to result in protein tyrosine kinases (PTK)-dependent activation of Ras
CD45 and Lck Are Required for TCR-induced Sos Phosphorylation—We have shown previously that surface expression of CD45 is required for the TCR to couple with the PTK signal transduction pathway [2, 4]
Summary
Cells and Cell Culture—The T cells used in these studies include Jurkat, a CD4ϩ human leukemic line, and Jurkat-derived variants. Both Jurkat and Jurkat-derived cell lines were maintained in RPMI 1640 complete medium supplemented with 10% (v/v) fetal calf serum, penicillin (1000 units/ml), streptomycin (1000 units/ml), and glutamine (20 mM). MAb against the FLAG epitope was purchased from Eastman Kodak Co. Horseradish peroxidase-conjugated goat anti-rabbit and goat anti-mouse antibodies were obtained from Bio-Rad. cDNA encoding fusion protein containing glutathione S-transferase and the first 79 amino acids of c-Jun (GST-cJun) cloned into the pGEX bacterial expression vector and the MEK inhibitor PD98059 were a gift of A. The samples were washed three times with phosphate-buffered saline, solubilized in 2 ϫ Laemmli’s sample buffer, subjected to 5–10% SDS-PAGE, and immunoblotted with anti-SOS mAb. JNK Protein Kinase Assays—Experiments were performed as described previously [37]. Cells were cultured for 40 – 48 h before stimulation
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