ALTERNATIVE SPLICING OF MKP-3 (OR DUSP6) GENE PRODUCES TWO ISOFORMS WITH DIFFERENT EFFECTS ON CELL PROLIFERATION

NUDLER S; MALOBERTI PM; COHEN SABBAN JM; GOROSTIZAGA AB; MORI SEQUEIROS GARCÍA MM; PAZ C

Buenos Aires

Congreso; REUNIÓN CONJUNTA DE SOCIEDADES DE BIOCIENCIAS, 2017; 2017

Mitogen-activated protein kinases (MAPK) are activated by dualphosphorylation in response to several stimuli and regulate multiplecellular functions. MKP-3 (or DUSP6) is a MAPK phosphataseinduced only by proliferative stimuli which specifically dephosphorylatesERK1/2. The human MKP-3 gene generates the full lengthtranscript, variant L, and an alternative splice product, or variant S.MKP-3S protein, encoded by the S transcript, is expressed only inhuman cells, mostly of tumoral origin. MKP-3L is a cytosolic proteinregulated by phosphorylation. MKP-3S protein is catalyticallyactive but lacks the nuclear export signal (NES) and a target residuefor ERK phosphorylation involved in MKP-3L stability. This workfocuses on the analysis of potential differences between isoformsand their effects on cellular localization, proliferation and cell cycleprogression. Previous immunocytochemistry studies have shownthat Flag-tagged L isoform is barely detected in the nucleus, whileFlag-tagged S is evenly spread between cytosol and nucleus. Herewe confirmed this distribution by subcellular fractioning and Westernblot in HEK293-transfected cells for Flag-L or Flag-S expression.Cell proliferation was measured as BrdU incorporation (expressedin arbitrary units as mean ± SEM, values relative to control) in cellstransfected with either one of the plasmids encoding flag taggedproteins or the empty vector. Compared to empty vector-transfectedcells, proliferation rates were decreased in L-transfected cells(0.79 ± 0.07 P< 0.05), but not in S-transfected cells (1.01 ± 0.06).Flow cytometry analysis showed a 25% (P < 0.05) reduction in thepercentage of L-transfected but not S-transfected cells in the G2/Mstage regarding empty vector transfection. Cyclin D levels, assayedby RT-PCR, were also different across groups. In conclusion, thesedifferences observed in MKP-3 isoforms suggest that expressing differentL/S ratios could shape cells proliferative behavior.Keywords: MKP-3, Cell Proliferation, ERK, Alternative Splicing