Mitogen-activated protein kinase (MAPK) phosphatases [MKPs, also known as dual-specificity phosphatases (DUSPs)] regulate MAPKs -key mediators of cellular processes such as proliferation, differentiat Show more
Mitogen-activated protein kinase (MAPK) phosphatases [MKPs, also known as dual-specificity phosphatases (DUSPs)] regulate MAPKs -key mediators of cellular processes such as proliferation, differentiation, and survival- by dephosphorylating the threonine and tyrosine residues required for MAPK activation. MKP-3/DUSP6 is an ERK-selective phosphatase that has also been reported to regulate the transcription factor FOXO1. The full-length MKP-3 transcript has been shown to encode the MKP-3L protein, whereas alternative splicing gives rise to the shorter isoform MKP-3S. However, the available information regarding the functional differences between these variants is limited. By combining biochemical and bioinformatic approaches, we demonstrate that these isoforms differ significantly in subcellular localization and enzymatic activity. Structural analysis and molecular docking reveal that while MKP-3S retains functional binding domains and recognizes ERK2 similarly to the full-length isoform. However, the absence of critical catalytic motifs in MKP-3S leads to a structural uncoupling where the protein retains its ability to bind ERK2 but fails to induce dephosphorylation, suggesting a non-canonical role as a molecular scaffold. The results obtained demonstrate significant variations in subcellular localization, enzymatic activity, and the capacity to modulate FOXO1 transcriptional activity. This, in turn, affects the expression of genes such as p21. In conclusion, the findings indicate that MKP-3 variants exhibit distinct functional behaviours, which may result in differential regulation of a wide range of cellular processes. Show less
MAPK phosphatases (MKP) downregulate the activity of mitogen-activated protein kinases (MAPK), such as ERK1/2, and modulate the processes regulated by these kinases. ERK1/2 participate in a wide range Show more
MAPK phosphatases (MKP) downregulate the activity of mitogen-activated protein kinases (MAPK), such as ERK1/2, and modulate the processes regulated by these kinases. ERK1/2 participate in a wide range of processes including tissue-specific hormone-stimulated steroidogenesis. H295R cells are a suitable model for the study of human adrenal cortex functions, particularly steroid synthesis, and respond to angiotensin II (Ang II) triggering ERK1/2 phosphorylation in a transient fashion. MKP-3 dephosphorylates ERK1/2 and, as recently reported, forkhead box protein 1 (FOXO1). Here, we analyzed MKP-3 expression in H295R cells and its putative regulation by Ang II. Results showed the expression of MKP-3 full length (L) and a short splice variant (S), and the upregulation of both isoforms by Ang II. L and S messenger and protein levels increased 30 min after Ang II stimulation and declined over the next 3 h, a temporal frame compatible with ERK1/2 dephosphorylation. In addition, FOXO1 activation is known to include its dephosphorylation and nuclear translocation. Therefore, we analyzed the effect of Ang II on FOXO1 modulation. Ang II induced FOXO1 transient phosphorylation and translocation and also the induction of p21, a FOXO1-dependent gene, whereas MKP-3 knock-down reduced both FOXO1 translocation and p21 induction. These data suggest that, through MKP-3, Ang II counteracts its own effects on ERK1/2 activity and also triggers the activation of FOXO-1 and the induction of cell cycle inhibitor p21. Taken together, the current findings reveal the participation of MKP-3 not only in turn-off but also in turn-on signals which control important cellular processes. Show less