Alternative splicing of MKP-3 (or DUSP6) gene produces two isoforms with different effects on cell proliferation

COHEN SABBAN, JUAN M; NUDLER S; MORI SEQUEIROS GARCIA MM; GOROSTIZAGA, A; MALOBERTI P; PAZ C

Buenos Aires

Congreso; REUNIÓN CONJUNTA DE SOCIEDADES DE BIOCIENCIAS; 2017

SAIC, SAIB, SAI, SAFE, SAA, SAB, SAFIS, SAH, SAP

Mitogen-activated protein kinases (MAPK) are activated by dual phosphorylation in response to several stimuli and regulate multiple cellular functions. MKP-3 (or DUSP6) is a MAPK phosphatase induced only by proliferative stimuli which specifically dephosphorylates ERK1/2. The human MKP-3 gene generates the full length transcript, variant L, and an alternative splice product, or variant S. MKP-3S protein, encoded by the S transcript, is expressed only in human cells, mostly of tumoral origin. MKP-3L is a cytosolic protein regulated by phosphorylation. MKP-3S protein is catalytically active but lacks the nuclear export signal (NES) and a target residue for ERK phosphorylation involved in MKP-3L stability. This work focuses on the analysis of potential differences between isoforms and their effects on cellular localization, proliferation and cell cycle progression. Previous immunocytochemistry studies have shown that Flag-tagged L isoform is barely detected in the nucleus, while Flag-tagged S is evenly spread between cytosol and nucleus. Here we confirmed this distribution by subcellular fractioning and Western blot in HEK293-transfected cells for Flag-L or Flag-S expression. Cell proliferation was measured as BrdU incorporation (expressed in arbitrary units as mean ± SEM, values relative to control) in cells transfected with either one of the plasmids encoding flag tagged proteins or the empty vector. Compared to empty vector-transfected cells, 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 the percentage of L-transfected but not S-transfected cells in the G2/M stage regarding empty vector transfection. Cyclin D levels, assayed by RT-PCR, were also different across groups. In conclusion, these differences observed in MKP-3 isoforms suggest that expressing different L/S ratios could shape cells proliferative behavior.