RELEVANCE OF DIFFERENT ERK1/2 PHOSPHORYLATION CONSENSUS SITES IN MKP-1 STABILITY IN LEYDIG CELLS

MORI SEQUEIROS GARCÍA MM; GÓMEZ NV, ; GOROSTIZAGA AB,; BRION L ; ACQUIER AB; MÉNDEZ CF; PAZ C.

Mendoza

Congreso; XLVII Reunión anual de la Sociedad Argentina de Investigación Bioquímica; 2011

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

 In MA-10 Leydig cells, hCG/cAMP up-regulate MAP kinase phosphatase -1 (MKP-1), which in turn attenuates the hormonal action on MAP kinase activity and steroidogenesis. hCG/cAMP lead to MKP-1 accumulation through gene transcription activation and posttranslational modifications including ERK-dependent phosphorylation. Since ERK1/2 phosphorylation in S359 and S364 results in MKP-1 stabilization while in S296 and S323 is associated with protein degradation, our aim was to determine the role of these sites in MKP-1 stabilization by cAMP. We also analyzed a possible link between MKP-1 acetylation and stabilization. Western blot analysis performed with an anti-flag antibody showed that, in MA-10 Leydig cells overexpressing flag-MKP-1 wild type (WT), this protein reaches a null or weak expression level in unstimulated cells and a strong and transient expression in 8Br-cAMP-stimulated cells. Pulse chase experiments showed that, in stimulated cells, the WT protein half life is 120 min, while the double mutants S359A-S364A and S296A-S323A exhibit a shorter (45 min) and longer half life (>150 min), respectively. Thus, the hormonal stabilization of MKP-1 could be the net effect of ERK1/2 phosphorylation in these four sites. Also, acetylation could contribute to MKP-1 stabilization, as cell exposure to a histone deacetylase inhibitor increased flag-MKP-1.