ZFP36L1 and ZFP36L2 control LDLR mRNA stability via the ERK-RSK pathway.
Bottom Line: Low-density lipoprotein receptor (LDLR) mRNA is unstable, but is stabilized upon extracellular signal-regulated kinase (ERK) activation, possibly through the binding of certain proteins to the LDLR mRNA 3'-untranslated region (UTR), although the detailed mechanism underlying this stability control is unclear.Here, using a proteomic approach, we show that proteins ZFP36L1 and ZFP36L2 specifically bind to the 3'-UTR of LDLR mRNA and recruit the CCR4-NOT-deadenylase complex, resulting in mRNA destabilization.These results indicate that ZFP36L1 and ZFP36L2 regulate LDLR protein levels downstream of ERK.
Affiliation: Molecular Profiling Research Center for Drug Discovery (molprof), National Institute of Advanced Industrial Science and Technology (AIST), Tokyo 135-0064, Japan Galaxy Pharma Inc., Akita 010-0951, Japan.Show MeSH
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Mentions: Next, we investigated the underlying mechanisms of PMA-ERK-mediated LDLR mRNA stabilization. Given that ERK is a critical kinase in PMA-mediated LDLR mRNA stabilization (4), we examined whether ZFP36L1 is phosphorylated downstream of ERK. We found that PMA treatment induced an electrophoretic mobility shift of ZFP36L1, which could be reversed when cells were treated with PMA and U0126, a specific inhibitor of the ERK pathway (Figure 4A). We also found that the mobility shift of Flag-ZFP36L1, which we immunopurified from Flag-ZFP36L1-overexpressing and PMA-treated 293T cells, could be reversed by treatment with bacterial alkaline phosphatase (Figure 4B). These results indicate that ZFP36L1 is phosphorylated downstream of ERK. We then analyzed the ERK-dependent phosphorylation sites using an iTRAQ-based quantitative MS approach. We immunopurified Flag-ZFP36L1 protein from mock-, PMA- or PMA + U0126-treated 293T cells and determined the ERK-dependent phosphorylation sites. We found that phosphorylation of the C-terminal serine-334 residue of ZFP36L1 and of the C-terminal serine-493 and -495 residues of ZFP36L2 was increased upon PMA treatment, but was reversed by U0126 treatment (Figure 4C, Supplementary Figure S5A). This result indicates that the phosphorylation of these residues is ERK -dependent. We also analyzed the phosphorylation of endogenous ZFP36L1, which we purified from 293T cell lysate using a Flag-tagged LDLR ARE1 region (Supplementary Table S1), and found that the C-terminal serine-334 residue of endogenous ZFP36L1 is also phosphorylated upon PMA treatment (Supplementary Figure S5B and C).
Affiliation: Molecular Profiling Research Center for Drug Discovery (molprof), National Institute of Advanced Industrial Science and Technology (AIST), Tokyo 135-0064, Japan Galaxy Pharma Inc., Akita 010-0951, Japan.