FETAL BOVINE SERUM UP-REGULATES MKP-3 MESSENGER LEVELS THORUGHT AN ERK-DEPENDENT MECHANISM

COHEN SABBAN J. ; NUDLER S. ; MORI SEQUEIROS GARCÍA M.; GOROSTIZAGA A.; BLANCO H.; MÉNDEZ C.; GONZÁLEZ-CALVAR S; PAZ C.

Rosario

Congreso; L Reunión anual de la Sociedad Argentina de Bioquímica y Biología Molecular.; 2014

Sociedad Argentina de Bioquímica y Biología Molecular

MDA-MB-231 is a human line of breast cancer cells carrying mutations leading to persistent ERK activation. MAPK phosphatases (MKPs) like MKP-1, -2 and -3 are an enzyme family specifically involved in MAPK inactivation. This work analyzed the expression of these MKPs in this cell line and studied the effect of fetal bovine serum (FBS) 10%, a mitogenic stimulus. mRNA levels were analyzed by RT-PCR in cells starved for 24 h (C) or FBS-stimulated for 1h (MKP-1) or 2h (MKP-2 and MKP-3) after starvation (E). Two isoforms of MKP-2 and MKP-3 mRNA (S and L) were detected in both conditions, L being the most abundant, which is a novel finding for MKP-3 in this cell type. Results demonstrate that starvation does not abrogate the basal expression of any of these MKPs, in contrast with results described in other cell types, while FBS stimulation increases mRNA levels of MKP-3 only. This result agrees with the fact that MKP-3 is specific to ERK1/2 and induced only by mitogenic stimuli. Preincubation with PD98059 (PD), a compound that prevents ERK activation, abrogates the effect of FBS on mRNA levels (E=1,43±0,20; E+PD=0,52±0,20, p menor a 0,05). Western blot analyses show high MKP-3 protein levels in cells stimulated for 2h (C=0,30±0,05; E=1,22±0,25, p menor a 0,05) but not for 3h. To conclude, FBS stimulation tightly regulates MKP-3 in MDAMB-231 cells so as to counteract persistent ERK activation.