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

COHEN SABBAN JM; MORI SEQUEIROS GARCIA MM; GOROSTIZAGA AB; PAZ C; NUDLER SI; MALOBERTI PM

Córdoba

Congreso; 52th Annual Meeting Argentine Society for Biochemistry and Molecular Biology; 2016

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

MAP kinase phosphatase 3 (MKP-3) plays an essential role in cell proliferation. It belongs to a subfamily of three cytoplasmic dualspecificityMKPs and, unlike other subfamily members, is an ERK-specific enzyme. In human tissues and cell lines, two splicevariants of MKP-3 are expressed in different proportions: the full transcript or isoform L, and isoform S, where exon 2 is skipped. Thelack of important regulatory sites in S could thus affect the L/S ratio and, consequently, impact cell biology. This work analyzes andcompares different aspects of both isoforms mRNA and protein. In breast cancer cell line MDA-MB-231, mRNA decay evaluated byRT-PCR showed higher stability for the L isoform. Western blot analysis revealed both L and S protein expression, L levels beingupregulated by a MEK inhibitor. MKP-3 S and L were cloned for the expression of flag-tagged proteins under a constitutive promoterand the recombinant vectors transfected in HEK293 cells. Both recombinant isoforms displayed activity against P-ERK. Kineticsprofile analysis in serum-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 flag-S protein also in the nucleus. Therefore, S and Lvariants exhibit different properties which could affect L/S ratio and its consequences in cell behavior.