Effect of rapamycin on PMA/CD28-induced JNK-1 activity.

(A) Upper panel, time course of JNK activation after PMA/CD28 stimulation. Total cellular lysates (35 μg) from peripheral blood T cells treated with PMA plus αCD28 for various periods of times were used in immunocomplex kinase assay for JNK-1 using recombinant GST-c-Jun (1-169) as substrate. The phosphorylated GST-c-Jun is indicated by an arrow. The faster migrating phosphorylated band is the shorter form of GST-c-Jun (1-169). Lower panel, equal amounts of lysates from time course analysis were analyzed by Western blot analysis using anti-JNK antibody. (B) Effect of LY294002 and rapamycin on PMA/CD28-induced JNK activity. Immunocomplex kinase assays for JNK-1 were performed using total cellular lysates (35 μg) from T cells treated with medium alone, PMA/αCD28 in the presence or absence of LY294002 or rapamycin for 1 hour, or PMA/αCD28 in the presence or absence of rapamycin for 20 hours. (C) Differential effect of rapamycin on PMA/CD28- versus PMA/ionomycin-induced JNK activity. Total cellular lysates (35 μg) from T cells treated with medium alone, PMA/αCD28 in the presence or absence of rapamycin, and PMA/ionomycin in the presence or absence of rapamycin for 18 hours were used for kinase assays. (D) Peripheral blood T cells were treated with medium alone, PMA/αCD28, or PMA/αCD28 plus pretreated rapamycin for various periods of times. Total cellular lysates (25 μg) were analyzed by immunoblotting with an antibody specific for the activated form of p38 that is phosphorylated on The-180 and Tyr-182.