FIG. 2.

ERK signaling regulates c-Fos expression and posttranslational modifications. (A) PDGF induction of c-Fos expression in NIH 3T3 cells. Cells were grown overnight in the absence of serum and then stimulated with PDGF (20 ng/ml) for the indicated times. Nuclear fractions were collected, and c-Fos was detected by Western blotting. (B) ERK dependency of c-Fos expression. Cells were serum starved overnight, incubated for 30 min in the absence (c) or presence of U0126 (U), SP600125 (SP), or SB203580 (SB) (10 μM each), and stimulated with PDGF (20 ng/ml; 2 h), and c-Fos expression in nuclear fractions was determined as described for panel A. (C) ERK dependency of c-fos mRNA expression. Cells were serum starved overnight, incubated for 30 min in the absence or presence of U0126, and stimulated with PDGF (20 ng/ml) for the indicated times. c-fos transcript levels were detected by Northern blotting as described in Materials and Methods. (D) ERK dependency of c-Fos posttranslational modifications. Cells were transiently transfected with pCEFL-AU5-c-Fos (1 μg/well) and grown in serum-free medium overnight after transfection. Cells were stimulated with PDGF (20 ng/ml; 30 min), and total lysates were processed for Western blotting using anti-c-Fos antibodies (top panel). Pretreatment with MAPK inhibitors was performed as described above. Endogenous active (middle panel) and total (bottom panel) ERK expression from the same samples served as controls for PDGF and U0126 effects on ERK phosphorylation. (E) c-Fos phosphorylation induced by PDGF. NIH 3T3 cells were transfected with pCEFL-AU5-c-Fos (1 μg/well), cultured in serum-free medium overnight, and stimulated with PDGF for 10 or 30 min. Cell lysates were subjected to PP2A digestion followed by immunoblotting using anti-c-Fos antibodies (see Materials and Methods). (F) SP600125 (SP) and SB203580 (SB) inhibitory action on JNK and p38 activities. HEK-293T cells were transfected with HA-JNK1 or HA-p38α alone or in combination with their upstream activating kinases (MEKK and MEK3EE, respectively). In vitro kinase assays were performed as described in Materials and Methods in the absence or presence of the indicated final concentrations of the inhibitors. ³²P-labeled substrate (P-ATF2) is indicated. NT, nontransfected cells.