THE SPLICING VARIANTS OF MAP KINASE PHOSPHATASE- 3 (MKP-3) EXHIBIT DIFFERENT PROPERTIES: IMPACT ON ERK-DEPENDENT EVENTS

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

Mar del Plata, Buenos Aires, Argentina

Congreso; LXI Reunión Científica Anual de la Sociedad Argentina de Investigación Clínica; 2016

Sociedad Argentina de Investigación Clínica

MAP kinase phosphatase-3 (MKP-3) is an enzyme specificfor ERK1/2 and induced exclusively by proliferative stimuli. TwoMKP-3 transcripts are expressed in some human cells, a short form(S) and a long form (L), which are products of alternative splicing.The ratio L/S is variable among different tissues and also betweennormal and tumoral tissues. As MKP-3 S lacks sites that could beinvolved in the regulation of this enzyme, the aim of this work wasto compare the properties of both isoforms. MKP-3 S and L werecloned for the expression of Flag-MKP-3 S or L under a constitutivepromoter; then, HEK293 cells were transiently transfected withthese vectors. The subcellular localization of each recombinantprotein was analyzed by fluorescence microscopy, while P-ERKand flag-MKP-3 S and L levels were evaluated by Western blotusing an anti P-ERK or anti-Flag antibody. The results show thatMKP-3 S is accumulated equally in the nucleus and cytosol, whileMKP-3 L is mainly localized in the cytosol, reflecting the lack ofa nuclear export sequence in the S isoform. The levels of P-ERKreached after 15 min of stimulation with fetal bovine serum (FBS)were lower in cells transfected with either isoform, which indicatesthat these expression products are active. In addition, theisoforms presented differences in post-translational regulation, assimilar levels of flag-MKP-3 L were detected after different timesof FBS stimulation, whereas isoform S was accumulated over thesame period (4.5-fold at 90 minutes, P