Insulin-induced c-Jun phosphorylation is sensitive to the Src kinase inhibitor PP1. (A) Serum-starved A14 cells were treated with 5 or 50 μM PP1 before being stimulated with 1 μM insulin for 15 min, as indicated. The cells were lysed in Ral buffer in the presence of PP1. Half of the sample was taken up in sample buffer and analyzed for c-Jun phosphorylation (top panel) or ERK phosphorylation (second panel). The second half of the lysate was used to isolate RalGTP, as described in the legend to Fig. 2A, using monoclonal anti-RalA (third panel). The bottom panel shows equal protein levels by detection of the RalA protein in whole extracts. (B) Serum-starved A14 cells were treated with 50 μM PP1 before being stimulated with 1 μM insulin or 20 ng of EGF per ml for 15 min, as indicated. The cells were lysed in sample buffer and analyzed for c-Jun phosphorylation (top panel), c-Jun protein levels (middle panel), or ERK phosphorylation (bottom panel).

Insulin and Ral guanine nucleotide exchange activity induce activation of JNK. (A) Activation of JNK activity by insulin and Rlf-CAAX. A14 cells were either transfected with pCD20 and treated with insulin (1 μM) for 30 min (left two lanes) or cotransfected with pCD20 and either pMT2HA (v), pMT2HA-RlfCAAX (wt), or pMT2HA-Rlf-R328E-CAAX (R328E) as indicated (right three lanes). Transfected cells were isolated by MACS. Endogenous JNK was isolated by using GST–c-Jun1–79 precoupled to glutathione beads. JNK activity using GST–c-Jun1–79 as a substrate was assayed directly by adding ATP. Phosphorylation of GST–c-Jun1–79 was monitored by Western blotting using anti-c-Jun phosphoserine 73. (B) Rlf-CAAX does not induce ERK phosphorylation. A14 cells were cotransfected with pCD20 and either pMT2HA (v), pMT2HA-Rlf-CAAX (wt), pMT2HA-Rlf-R328E-CAAX (R328E), or pMT2HA-RasV12 (Ras) as indicated and transfected cells were isolated by magnetic cell sorting. Phosphorylation of ERK was detected by Western blotting using anti-ERK phosphothreonine-202 phosphotyrosine-204 polyclonal antibody. The positions of phosphorylated ERK1 and ERK2 are indicated.

Ral guanine nucleotide exchange activity induces phosphorylation of endogenous c-Jun. (A) Ral activation by Rlf-CAAX is abolished by the R328E mutation in Rlf-CAAX. A14 cells were cotransfected with HA-Ral and HA-Rlf-CAAX or HA-Rlf-R328E-CAAX. The cells were lysed in Ral buffer, and HA-RalGTP levels were monitored after anti-HA (12CA5) Western blotting of affinity-isolated RalGTP as described in Materials and Methods (middle panel). The bottom panel shows the expression of HA-Ral in total lysates. The top panel shows equal expression of the Rlf-CAAX proteins. wt, wild type. (B) RalGEF-induced NH₂-terminal phosphorylation of endogenous c-Jun. A14 cells were cotransfected with pCD20 and either pMT2HA, pMT2HA-Rlf-CAAX, or pMT2HA-Rlf-R328E-CAAX as indicated. At 40 h after transfection, transfected cells were isolated by MACS as described in Materials and Methods. Isolated cells were lysed, protein levels were equalized, and samples were taken up in sample buffer and analyzed for c-Jun phosphorylation on serine 73 (second panel), serine 63 (third panel) or c-Jun protein expression (bottom panel). The top panel is a blot of the same samples using anti-HA (12CA5) to show equal expression of HA-Rlf-CAAX and HA-Rlf-R328E-CAAX. wt, wild type.

Growth factor-induced phosphorylation of endogenous c-Jun requires Ral signaling. A14 or HEK-293 cells were transiently transfected with either empty vector or a construct expressing either RalB-N28, RalBP-ΔGAP, or RalGDS-RBD as indicated. After 24 h, the cells were serum starved for 16 h and then stimulated with insulin (1 μM) or EGF (20 ng/ml) as indicated, and transfected cells were isolated by MACS. (A) RalB-N28 blocks insulin-induced c-Jun phosphorylation. A14 cells transiently expressing empty pSG5 or pSG5-RalB-N28 as indicated were treated with insulin for the times indicated, and transfected cells were isolated. Samples were analyzed for c-Jun phosphorylation on serine 73 (top panel), c-Jun protein expression (middle panel) or RalB-protein expression (bottom panel). The numbers below the panels indicate fold induction compared to the unstimulated cells. (B) RalB-N28 does not block insulin-induced ATF2 or ERK phosphorylation. Equal amounts of whole-cell extracts isolated in panel A were analyzed for increases in ATF2 phosphorylation and for phosphorylation of ERK. ATF2 phosphorylation was detected by Western blotting using an anti-ATF2 phosphothreonine-71 polyclonal antibody (upper panel). Phosphorylation of ERK was monitored by Western blotting using anti-ERK phosphothreonine-202 phosphotyrosine-204 polyclonal antibody (lower panel). Fold induction is indicated below the panels. (C) RalB-N28 blocks EGF-induced c-Jun phosphorylation in HEK-293 cells. HEK-293 cells transiently expressing empty pSG5 or pSG5-RalB-N28 as indicated were treated with EGF for the times indicated, and transfected cells were isolated. Samples were analyzed for c-Jun phosphorylation on serine 73 (top panel), c-Jun protein expression (middle panel), or RalB-protein expression (bottom panel). (D) RalBP-ΔGAP and RalGDS-RBD block insulin-induced phosphorylation of c-Jun. A14 cells transiently expressing empty pMT2HA, pRK5-MYC-RalBP-ΔGAP, or pMT2HA-RalGDS-RBD as indicated were treated with insulin for the times indicated, and transfected cells were isolated. Isolated samples were analyzed for c-Jun phosphorylation on serine 73 (top panel), c-Jun protein expression (second panel), Myc-RalBP-ΔGAP expression (third panel), or HA-RalGDS-RBD expression (bottom panel).

Regulation of the JNK pathway by Ral signaling. (A) Insulin-induced JNK1 phosphorylation at the JNKK-1 sites. Serum-starved, subconfluent A14 cells were treated with insulin for the indicated times and lysed. JNK was immunoprecipitated (IP) using 10 μg of monoclonal anti-JNK1, precoupled to protein G-Sepharose, for 3 h at 4°C. After extensive washing with lysis buffer, 90% of the protein samples were analyzed by Western blotting using anti-JNK phosphothreonine-183 phosphotyrosine-185 (upper panel). Total JNK1 was detected using the remaining 10% of the samples (lower panel). Ig, immunoglobulin G. (B) Cdc42-V12 induces JNK activity independently of Ral. A14 cells were transfected with either empty vector (lane 1), Myc-Cdc42-V12 (lane 2), both empty vector and pSG5-RalB-N28 (lane 3), or Myc-Cdc42-V12 and pSG5-RalB-N28 (lane 4), together with HA-p54-JNK. HA-JNK was immunoprecipitated from cellular lysates, and kinase activity was assayed in vitro using GST–c-Jun1–79 as a substrate. GST-Jun phosphorylation was monitored by autoradiography after SDS-PAGE of kinase assays (top panel). Myc-Cdc42-V12, RalB-N28, and HA-JNK expression, identified by Western blotting, are shown in the lower panels. (C) Cdc42-V12 does not activate Ral. A14 cells were cotransfected with HA-Ral and HA-Rlf-CAAX or Myc-Cdc42-V12. Cells were lysed in Ral buffer, and HA-RalGTP levels were monitored after anti-HA (12CA5) Western blotting of affinity-isolated RalGTP as described in Materials and Methods (top panel). The other panels show the expression of HA-Rlf-CAAX, Myc-Cdc42-V12, and HA-Ral in total lysates.

Insulin treatment induces phosphorylation of c-Jun in a Ras-dependent manner. (A) Insulin treatment induces NH₂-terminal phosphorylation of c-Jun. Cell extracts from subconfluent, serum-starved A14 cells were treated with insulin (1 μM) for the indicated times and analyzed by Western blotting using anti-c-Jun phosphoserine-73 (upper panel) or anti-c-Jun (lower panel). (B) Insulin induces phosphorylation of both serine 63 and serine 73. c-Jun was immunoprecipitated from orthophosphate-labelled serum-starved A14 cells left untreated (middle panel, control) or treated with insulin (1 μM) for 60 min (right panel, ins). Phosphorylated c-Jun was processed for tryptic peptide mapping as described previously (22). The left panel shows the relative position of the tryptic c-Jun phosphopeptides. Phosphopeptides X and Y, indicated by the arrows, contain the phosphorylation sites serine 73 and serine 63, respectively (3). (C) RasN17 blocks insulin-induced c-Jun serine 73 phosphorylation. In the left panel, A14 cells were infected with 20 PFU of vector or His₆-RasN17 vaccinia virus. After insulin treatment for the indicated times, the cells were lysed in sample buffer and analyzed as in panel A. The lower panel shows expression of the His₆-RasN17 protein together with endogenous Ras. In the right panel, cells were transfected with either empty vector (−) or RasV12, lysed in sample buffer, and analyzed as in panel A. Expression of RasV12 was detected by using the anti-Ras antibody. wt, wild type. (D) Pretreatment of A14 cells for 15 min with the MEK inhibitor PD98059 (40 μM) or the PI-3K inhibitor LY294002 (10 μM) blocks insulin-induced ERK phosphorylation and PKB phosphorylation but not c-Jun phosphorylation. Phosphorylation was monitored using phosphospecific antibodies as indicated.