Post-transcriptional and post-translational regulation of DUSP6, a phosphatase of ERK 1/2 MAP kinases.

Summary MAP kinase phosphatases (MKPs) belong to the Dual-Specificity Phosphatases (DUSP) family and dephosphorylate the threonine and tyrosine residues of activated MAP kinases. DUSP6/MKP-3 is a cytoplasmic phosphatase that dephosphorylates and thus specifically inactivates ERK1/2 MAP kinases. DUSP6 has an important role during development, particularly in the regulation of FGF-induced signaling, and its absence causes major phenotypic effects in Drosophila, chicken, zebrafish and mouse. DUSP6 could also play an important role during tumor formation and development as its expression is altered in various cancers. For these reasons, I was interested in the molecular mechanisms involved in the regulation of its expression, both at the post-transcriptional and post-translational level. Previous data from the laboratory indicated that DUSP6 was phosphorylated and degraded after stimulation of cells with growth factors in a MEK/ERK-dependent manner (Marchetti et al. , 2005). In the first part of my thesis, I studied the role of other signaling pathways in the regulation of DUSP6. We have shown that another signaling pathway, the PI3K/mTOR pathway, is responsible for part of the phosphorylation and degradation of DUSP6 induced by growth factors (Bermudez et al. , 2008). However, basal MEK activity is required for mTOR-induced phosphorylation of DUSP6 to occur. Mutagenesis studies have shown that serine 159 is the residue phosphorylated by mTOR. The DUSP6 phosphatase could therefore be a new point of interaction between two major cell signaling pathways activated by growth factors, the MEK/ERK pathway and the PI3K/mTOR pathway. In the second part of my work, I focused on the regulation of dusp6 at the level of its mRNA. Other teams have shown that the MEK/ERK pathway plays a role in the transcriptional activation of dusp6. We confirmed that MEK/ERK inhibition strongly reduces dusp6 mRNA levels. To investigate the regulation of dusp6 mRNA stability, we cloned into an expression vector a luciferase reporter gene upstream of the 3'UTR non-coding region of dusp6, which contains consensus sites for different factors that destabilize/stabilize mRNAs. We found that the MEK/ERK pathway stabilizes dusp6 mRNA. Furthermore, hypoxic conditions, a characteristic of many tumors in vivo, induce an increase in dusp6 mRNA levels, which is dependent on HIF-1alpha. Finally, we identified two factors that destabilize dusp6 mRNA, TTP (tristetraprolin) and PUM2, a homolog of the Drosophila pumilio gene. The results presented in this thesis therefore show that the MEK/ERK pathway is involved in the regulation of DUSP6 at different levels, from the regulation of its mRNA to the post-translational level, in a feedback loop. The study of DUSP6 regulation provides additional elements for the understanding of the complex mechanisms involved in ERK1/2 activation within the MAPK signaling network, where positive and negative regulations contribute to a subtle control of ERKs MAP Kinases activation in space and time.