ERK-SPECIFIC PHOSPHATASE MKP-3 SPLICE VARIANTS DIFFER IN REGULATION AND SUBCELLULAR LOCALIZATION

COHEN SABBAN JM; NUDLER SI; MORI SEQUEIROS GARCIA MM; GOROSTIZAGA AB,; MALOBERTI PM; CASTILLA R., MALOBERTI P., CASTILLO F., DUARTE A., CORNEJO MACIEL F., PAZ C., PODESTÁ EJ.

Córdoba

Congreso; LII Reunión anual de la Sociedad Argentina de Bioquímica y Biología Molecular; 2016

Sociedad Argentina de Bioquímica y Biología Molecular

Cohen Sabban JM, Nudler SI, Mori Sequeiros Garcia MM, Gorostizaga AB, Maloberti PM and Paz C INBIOMED (UBA-CONICET). Dpto. Bioquímica Humana. Facultad de Medicina. Universidad de Buenos Aires.MAP kinase phosphatase 3 (MKP-3) plays an essential role in cell proliferation. It belongs to a subfamily of three cytoplasmic dual-specificity MKPs and, unlike other subfamily members, is an ERK-specific enzyme. In human tissues and cell lines, two splice variants of MKP-3 are expressed in different proportions: the full transcript or isoform L, and isoform S, where exon 2 is skipped. The lack of important regulatory sites in S could thus affect the L/S ratio and, consequently, impact cell biology. This work analyzes and compares different aspects of both isoforms mRNA and protein. In breast cancer cell line MDA-MB-231, mRNA decay evaluated by RT-PCR showed higher stability for the L isoform. Western blot analysis revealed both L and S protein expression, L levels being upregulated by a MEK inhibitor. MKP-3 S and L were cloned for the expression of flag-tagged proteins under a constitutive promoter and the recombinant vectors transfected in HEK293 cells. Both recombinant isoforms displayed activity against P-ERK. Kinetics profile analysis in FBS-stimulated cells showed that flag-S protein was accumulated over time, while flag-L remained constant. Immunofluorescence microscopy showed both proteins in the cytosol and S also in the nucleus. Therefore, S and L variants exhibit different properties which could affect L/S ratio and its consequences in cell behavior.