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First published online November 18, 2003
doi: 10.1242/10.1242/jcs.00812

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Elevated ERK-MAP kinase activity protects the FOS family member FRA-1 against proteasomal degradation in colon carcinoma cells

Cancer Research UK Centre for Cell and Molecular Biology, Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK

View larger version (65K): [in a new window]   Fig. 1. ERK-MAPK-dependent expression of c-JUN and FRA-1 in colon carcinoma cells. (A) Immunoblot analysis of ERK2 and phospho-ERK1/2 proteins in HCT-116 and BE cells 2, 4, 8 and 24 hours after the addition of the MEK inhibitor U0126, or the vehicle alone (ethanol). (B) Immunoblot analysis of FRA-1, c-JUN, c-FOS, JUNB and FRA-2 protein expression in HCT-116 and BE cells 2, 4, 8 and 24 hours after addition of the MEK inhibitor U0126. (C) Immunoblot analysis of FRA-1, c-JUN, ERK2 and phospho-ERK1/2 proteins in BE cells, HCT-116 and LS174T colon carcinoma cells. (D) Northern blot analysis of FRA-1, c-JUN and GAPDH mRNA expression in HCT-116 and BE cells 2, 4, 8 and 24 hours after addition of the MEK inhibitor U0126. (E) Immunoblot analysis of FRA-1, c-JUN, ERK2 and phospho-ERK1/2 proteins in BE cells 2, 4, 8 and 24 hours after the addition of 1 µM of the MEK inhibitor PD184352.

View larger version (53K): [in a new window]   Fig. 2. High ERK-MAPK activity prevents proteasome-dependent degradation of FRA-1. (A) Immunoblot analysis of FRA-1, ERK2 and phospho-ERK1/2 protein expression in HCT-116 and BE cells 16 hours after addition of the proteasome inhibitor lactacystin. (B) Immunoblot analysis of FRA-1, RAF:ER, ERK2 and phospho-ERK1/2 proteins in the HCT-116-RAF:ER cell line 2 and 24 hours after activation of the RAF:ER construct with tamoxifen (4-OHT), or ethanol (EtOH) in control experiments. (C) Northern blot analysis of FRA-1 and GAPDH mRNA accumulation in the HCT-116-RAF:ER cell line 2 and 24 hours after activation of the RAF:ER construct with tamoxifen, or ethanol in control experiments.

View larger version (26K): [in a new window]   Fig. 3. FRA-1 is required for the survival of colon carcinoma cells in suspension. (A) Immunoblot analysis of FRA-1 protein expression in HCT-116 and BE cells 48 hours after transfection of the FRA-1 specific or scrambled siRNAs. (B) Cell-cycle distribution in HCT-116 and BE cells after inhibition of the ERK-MAPK pathway with the synthetic MEK inhibitor PD184352 (or DMSO in control experiments) or after transfection of FRA-1 specific or scrambled siRNAs. Cell-cycle analyses were done either with adherent cells or with cells maintained in suspension for 24 hours. (C) Cell-cycle distribution in HCT-116-RAF:ER cells after activation of RAF:ER with 1 µM tamoxifen (4-OHT) for 24 hours, or ethanol in control experiments, and/or after transfection of FRA-1 specific or scrambled siRNAs. Cell-cycle analyses were done with cells maintained in suspension for 24 hours. Experiments were repeated three times; one representative experiment is shown.