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ZFP36L1 and ZFP36L2 control LDLR mRNA stability via the ERK-RSK pathway.

Adachi S, Homoto M, Tanaka R, Hioki Y, Murakami H, Suga H, Matsumoto M, Nakayama KI, Hatta T, Iemura S, Natsume T - Nucleic Acids Res. (2014)

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.

View Article: PubMed Central - PubMed

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.

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ERK pathway phosphorylates and inhibits ZFP36L2. (A) 293T cells were transfected with Flag-tagged ZFP36L1. Twenty-four hours after transfection, cells were treated with PMA (for 30 min) and U0126 (for 45 min), as indicated. Cell lysates were subjected to western blot analysis using anti-Flag antibody. (B) 293T cells were transfected with Flag-tagged ZFP36L1. Cells were treated with or without PMA (for 30 min) and the lysates were subjected to IP. Immunopurified Flag-ZFP36L1 was treated with or without bacterial acid phosphatase (BAP) and samples were subjected to western blot analysis using anti-Flag antibody. (C) Alignment of C-terminal amino acid sequences in the human ZFP36 family. The C-terminal end of each protein is shown as (*). Identical residues are shown by (/) and similar residues are shown by (+). The serine residues identified as being phosphorylated downstream of ERK are shown in red. (D) 293T cells were transfected with Myc-tagged ZFP36L1 or ZFP36L2 and then treated with or without PMA (for 30 min). Cell lysates were subjected to IP using the Flag-antibody and the indicated bait RNAs. Co-immunoprecipitated proteins were subjected to western blot analysis using anti-MYC (9E10) antibody. (E) 293T cells were transfected with Flag-tagged ZFP36L1, then treated with PMA (for 30 min) and U0126 (for 45 min) as indicated, and lysates were subjected to IP. Co-immunoprecipitated proteins were subjected to western blot analysis using the indicated antibodies. (F) Schematic representation of the constructs used in this experiment. (G) 293T cells were transfected with the indicated constructs. Twenty-four hours after transfection, cells were treated with or without PMA (for 30 min), and lysates were subjected to IP. Co-immunoprecipitated proteins were subjected to western blot analysis using the indicated antibodies. (H) HeLa cells were transfected with RFP-LDLR 3′-UTR (ARE1) or Luc2-β-Actin-UTR along with the indicated ZFP36L1 constructs. Twenty-four hours after transfection, cells were treated with ActD and PMA (PMA treatment commenced 15 min after ActD treatment), as indicated. Two hours after ActD treatment, cells were harvested and total RNA was extracted. Quantitative RT-PCR (qPCR) was performed using RFP and Luc2-specific primers. Results were normalized to the levels of Luc2 mRNA. The data are representative of at least three independent experiments.
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Figure 4: ERK pathway phosphorylates and inhibits ZFP36L2. (A) 293T cells were transfected with Flag-tagged ZFP36L1. Twenty-four hours after transfection, cells were treated with PMA (for 30 min) and U0126 (for 45 min), as indicated. Cell lysates were subjected to western blot analysis using anti-Flag antibody. (B) 293T cells were transfected with Flag-tagged ZFP36L1. Cells were treated with or without PMA (for 30 min) and the lysates were subjected to IP. Immunopurified Flag-ZFP36L1 was treated with or without bacterial acid phosphatase (BAP) and samples were subjected to western blot analysis using anti-Flag antibody. (C) Alignment of C-terminal amino acid sequences in the human ZFP36 family. The C-terminal end of each protein is shown as (*). Identical residues are shown by (/) and similar residues are shown by (+). The serine residues identified as being phosphorylated downstream of ERK are shown in red. (D) 293T cells were transfected with Myc-tagged ZFP36L1 or ZFP36L2 and then treated with or without PMA (for 30 min). Cell lysates were subjected to IP using the Flag-antibody and the indicated bait RNAs. Co-immunoprecipitated proteins were subjected to western blot analysis using anti-MYC (9E10) antibody. (E) 293T cells were transfected with Flag-tagged ZFP36L1, then treated with PMA (for 30 min) and U0126 (for 45 min) as indicated, and lysates were subjected to IP. Co-immunoprecipitated proteins were subjected to western blot analysis using the indicated antibodies. (F) Schematic representation of the constructs used in this experiment. (G) 293T cells were transfected with the indicated constructs. Twenty-four hours after transfection, cells were treated with or without PMA (for 30 min), and lysates were subjected to IP. Co-immunoprecipitated proteins were subjected to western blot analysis using the indicated antibodies. (H) HeLa cells were transfected with RFP-LDLR 3′-UTR (ARE1) or Luc2-β-Actin-UTR along with the indicated ZFP36L1 constructs. Twenty-four hours after transfection, cells were treated with ActD and PMA (PMA treatment commenced 15 min after ActD treatment), as indicated. Two hours after ActD treatment, cells were harvested and total RNA was extracted. Quantitative RT-PCR (qPCR) was performed using RFP and Luc2-specific primers. Results were normalized to the levels of Luc2 mRNA. The data are representative of at least three independent experiments.

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).


ZFP36L1 and ZFP36L2 control LDLR mRNA stability via the ERK-RSK pathway.

Adachi S, Homoto M, Tanaka R, Hioki Y, Murakami H, Suga H, Matsumoto M, Nakayama KI, Hatta T, Iemura S, Natsume T - Nucleic Acids Res. (2014)

ERK pathway phosphorylates and inhibits ZFP36L2. (A) 293T cells were transfected with Flag-tagged ZFP36L1. Twenty-four hours after transfection, cells were treated with PMA (for 30 min) and U0126 (for 45 min), as indicated. Cell lysates were subjected to western blot analysis using anti-Flag antibody. (B) 293T cells were transfected with Flag-tagged ZFP36L1. Cells were treated with or without PMA (for 30 min) and the lysates were subjected to IP. Immunopurified Flag-ZFP36L1 was treated with or without bacterial acid phosphatase (BAP) and samples were subjected to western blot analysis using anti-Flag antibody. (C) Alignment of C-terminal amino acid sequences in the human ZFP36 family. The C-terminal end of each protein is shown as (*). Identical residues are shown by (/) and similar residues are shown by (+). The serine residues identified as being phosphorylated downstream of ERK are shown in red. (D) 293T cells were transfected with Myc-tagged ZFP36L1 or ZFP36L2 and then treated with or without PMA (for 30 min). Cell lysates were subjected to IP using the Flag-antibody and the indicated bait RNAs. Co-immunoprecipitated proteins were subjected to western blot analysis using anti-MYC (9E10) antibody. (E) 293T cells were transfected with Flag-tagged ZFP36L1, then treated with PMA (for 30 min) and U0126 (for 45 min) as indicated, and lysates were subjected to IP. Co-immunoprecipitated proteins were subjected to western blot analysis using the indicated antibodies. (F) Schematic representation of the constructs used in this experiment. (G) 293T cells were transfected with the indicated constructs. Twenty-four hours after transfection, cells were treated with or without PMA (for 30 min), and lysates were subjected to IP. Co-immunoprecipitated proteins were subjected to western blot analysis using the indicated antibodies. (H) HeLa cells were transfected with RFP-LDLR 3′-UTR (ARE1) or Luc2-β-Actin-UTR along with the indicated ZFP36L1 constructs. Twenty-four hours after transfection, cells were treated with ActD and PMA (PMA treatment commenced 15 min after ActD treatment), as indicated. Two hours after ActD treatment, cells were harvested and total RNA was extracted. Quantitative RT-PCR (qPCR) was performed using RFP and Luc2-specific primers. Results were normalized to the levels of Luc2 mRNA. The data are representative of at least three independent experiments.
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Figure 4: ERK pathway phosphorylates and inhibits ZFP36L2. (A) 293T cells were transfected with Flag-tagged ZFP36L1. Twenty-four hours after transfection, cells were treated with PMA (for 30 min) and U0126 (for 45 min), as indicated. Cell lysates were subjected to western blot analysis using anti-Flag antibody. (B) 293T cells were transfected with Flag-tagged ZFP36L1. Cells were treated with or without PMA (for 30 min) and the lysates were subjected to IP. Immunopurified Flag-ZFP36L1 was treated with or without bacterial acid phosphatase (BAP) and samples were subjected to western blot analysis using anti-Flag antibody. (C) Alignment of C-terminal amino acid sequences in the human ZFP36 family. The C-terminal end of each protein is shown as (*). Identical residues are shown by (/) and similar residues are shown by (+). The serine residues identified as being phosphorylated downstream of ERK are shown in red. (D) 293T cells were transfected with Myc-tagged ZFP36L1 or ZFP36L2 and then treated with or without PMA (for 30 min). Cell lysates were subjected to IP using the Flag-antibody and the indicated bait RNAs. Co-immunoprecipitated proteins were subjected to western blot analysis using anti-MYC (9E10) antibody. (E) 293T cells were transfected with Flag-tagged ZFP36L1, then treated with PMA (for 30 min) and U0126 (for 45 min) as indicated, and lysates were subjected to IP. Co-immunoprecipitated proteins were subjected to western blot analysis using the indicated antibodies. (F) Schematic representation of the constructs used in this experiment. (G) 293T cells were transfected with the indicated constructs. Twenty-four hours after transfection, cells were treated with or without PMA (for 30 min), and lysates were subjected to IP. Co-immunoprecipitated proteins were subjected to western blot analysis using the indicated antibodies. (H) HeLa cells were transfected with RFP-LDLR 3′-UTR (ARE1) or Luc2-β-Actin-UTR along with the indicated ZFP36L1 constructs. Twenty-four hours after transfection, cells were treated with ActD and PMA (PMA treatment commenced 15 min after ActD treatment), as indicated. Two hours after ActD treatment, cells were harvested and total RNA was extracted. Quantitative RT-PCR (qPCR) was performed using RFP and Luc2-specific primers. Results were normalized to the levels of Luc2 mRNA. The data are representative of at least three independent experiments.
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).

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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus