Fig. 4. Activation of ERK2 by LPA. Confluent, serum-starved PANC-1 cells were treated with 10 µM LPA. ERK2 activity was determined by using a synthetic MBP-peptide as ERK-substrate in in vitro phosphorylation assays as described in Materials and Methods. (A) For time-course analysis, PANC-1 cells were incubated with 10 µM LPA for the indicated time periods and ERK2 was immunoprecipitated from 1 mg of cell lysate protein. Results of one representative experiment out of four are given in counts per minute (cpm). (B) To determine the influence of activated MEK1 and activated G_i/o-proteins on ERK activity, cells were either pretreated with 25 µM PD98059 for 15 minutes or with 100 ng/ml PTX overnight, and then incubated for 10 minutes with 10 µM LPA, 30 ng/ml EGF, or carrier. Means ± s.e.m. (n=3) of fold stimulation relative to solvent-treated cells are shown. (C) The influence of dominant negative GTPases on LPA-induced ERK2 activity was determined in transient cotransfection studies. PANC-1 cells were cotransfected with 8 µg of pcDNA3/HA-ERK2 and either 8 µg of pEGFP-C1/H-Ras(S17N), pEGFP-C2/K-Ras(S17N), pEGFPC3/Rac1(T17N), or pEGFP-C3/RhoA(T19N). After 24 hours in growth medium, cells were serum-starved for 4 hours in DMEM with 0.1% FCS and then treated for 10 min with 10 µM LPA. HA-ERK2 was immunoprecipitated using anti-HA antibody (12CA5) and the kinase activity was determined by in vitro radioactive phosphorylation of the MBP peptide. The right diagram shows the results of one representative assay of three experiments with the phosphorylation given in cpm. The corresponding immunoblot (upper-left panel, WB: anti-GFP) shows the expression of ectopically expressed EGFP- and EGFP-GTPase-fusion proteins, as detected using an anti-GFP antibody. The lower panel (IP: anti-HA) shows the corresponding anti-ERK2 immunoblot of one-third of the immunoprecipitated HA-ERK2, as detected using an anti-ERK2 antibody. IP, immunoprecipitation; WB, western blot.

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