Relevance of different ERK1/2 phosphorylation consensus sites in MKP-1 stability in Leydig cells

MORI SEQUEIROS GARCÍA MM; GOMEZ NV; GOROSTIZAGA AB; BRION L; ACQUIER AB; MENDEZ CF; PAZ C

San Luis

Congreso; XLVII Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular.; 2011

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

In MA-10 Leydig cells, hCG/cAMP up-regulate MAP kinasephosphatase -1 (MKP-1), which in turn attenuates the hormonalaction on MAP kinase activity and steroidogenesis. hCG/cAMPlead to MKP-1 accumulation through gene transcription activationand posttranslational modifications including ERK-dependentphosphorylation. Since ERK1/2 phosphorylation in S359 and S364results in MKP-1 stabilization while in S296 and S323 is associatedwith protein degradation, our aim was to determine the role of thesesites in MKP-1 stabilization by cAMP. We also analyzed a possiblelink between MKP-1 acetylation and stabilization. Western blotanalysis performed with an anti-flag antibody showed that, in MA-10 Leydig cells overexpressing flag-MKP-1 wild type (WT), thisprotein reaches a null or weak expression level in unstimulated cellsand a strong and transient expression in 8Br-cAMP-stimulatedcells. Pulse chase experiments showed that, in stimulated cells, theWT protein half life is 120 min, while the double mutants S359AS364Aand S296A-S323A exhibit a shorter (45 min) and longer halflife (>150 min), respectively. Thus, the hormonal stabilization ofMKP-1 could be the net effect of ERK1/2 phosphorylation in thesefour sites. Also, acetylation could contribute to MKP-1stabilization, as cell exposure to a histone deacetylase inhibitorincreased flag-MKP-1.