Limits...
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.

Show MeSH

Related in: MedlinePlus

RSK1 directly phosphorylates the C-terminus of ZFP36L1. (A) 293T cells were transfected with Flag-tagged ZFP36L1. Twenty-four hours after transfection, cells were treated with PMA (for 30 min) and BI-D1870 or SL0101 (for 45 min) as indicated, and lysates were subjected to IP with the anti-Flag antibody. Co-immunoprecipitated proteins were subjected to western blot analysis using the indicated antibodies. Five percent of the initial amount of cleared 293T lysate was loaded as total lysate. (B) GST-ZFP36L1 protein was incubated with or without active recombinant RSK1 under phosphorylation conditions for 30 min. Phosphorylation of GST-ZFP36L1 C-terminal peptide was analyzed by MS. The MS/MS spectrum used to identify the m/z 681.8604-peak as the LPIFSRLSISDD peptide fragment of ZFP36L1 is shown. (C) MS/MS spectrum used to identify the m/z 721.8421-peak as the serine-8 phosphorylated LPIFSRLSISDD peptide fragment of ZFP36L1. (D) GST-ZFP36L1 protein was incubated with the indicated recombinant kinase under phosphorylation conditions for 30 min, washed, then added to 293T cell lysate and then subjected to pulldown with glutathione-sepharose. Eluted proteins were subjected to western blot analysis using the indicated antibodies and subjected to Coomassie Brilliant Blue (CBB) staining. The asterisk shows a nonspecific band. Five percent of the initial amount of cleared 293T cell lysate was loaded as total lysate. These data are representative of at least three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4150769&req=5

Figure 5: RSK1 directly phosphorylates the C-terminus of ZFP36L1. (A) 293T cells were transfected with Flag-tagged ZFP36L1. Twenty-four hours after transfection, cells were treated with PMA (for 30 min) and BI-D1870 or SL0101 (for 45 min) as indicated, and lysates were subjected to IP with the anti-Flag antibody. Co-immunoprecipitated proteins were subjected to western blot analysis using the indicated antibodies. Five percent of the initial amount of cleared 293T lysate was loaded as total lysate. (B) GST-ZFP36L1 protein was incubated with or without active recombinant RSK1 under phosphorylation conditions for 30 min. Phosphorylation of GST-ZFP36L1 C-terminal peptide was analyzed by MS. The MS/MS spectrum used to identify the m/z 681.8604-peak as the LPIFSRLSISDD peptide fragment of ZFP36L1 is shown. (C) MS/MS spectrum used to identify the m/z 721.8421-peak as the serine-8 phosphorylated LPIFSRLSISDD peptide fragment of ZFP36L1. (D) GST-ZFP36L1 protein was incubated with the indicated recombinant kinase under phosphorylation conditions for 30 min, washed, then added to 293T cell lysate and then subjected to pulldown with glutathione-sepharose. Eluted proteins were subjected to western blot analysis using the indicated antibodies and subjected to Coomassie Brilliant Blue (CBB) staining. The asterisk shows a nonspecific band. Five percent of the initial amount of cleared 293T cell lysate was loaded as total lysate. These data are representative of at least three independent experiments.

Mentions: We showed that the C-terminal serine-334 of ZFP36L1 and the C-terminal serine-493 and -495 of ZFP36L2 are phosphorylated downstream of ERK. However, these sites do not match the consensus MAP kinase recognition motifs (SP or TP), indicating that ERK does not directly phosphorylate ZFP36L1. We then examined the possibility that RSK, a major downstream kinase of ERK, directly phosphorylates the C-terminus of ZFP36L1. We first investigated whether BI-D1870 and SL0101, established RSK inhibitors, can reverse PMA-mediated dissociation between ZFP36L1 and CNOT7 protein. We found that BI-D1870 and SL0101 clearly reversed the effect of PMA (Figure 5A, Supplementary Figure S6A). We then examined whether RSK1 directly phosphorylates the C-terminus of ZFP36L1 using recombinant proteins. We incubated E. coli-expressed GST-ZFP36L1 with or without active recombinant RSK1 protein under phosphorylation conditions, and analyzed the C-terminal phosphorylation of GST-ZFP36L1 by MS. We found that the C-terminal serine-334 of ZFP36L1 is phosphorylated only when we incubated GST-ZFP36L1 with active RSK1 (Figure 5B and C, Supplementary Figure S6B). We also examined whether active RSK1 inhibits the ability of ZFP36L1 to interact with CNOT proteins. We incubated GST-ZFP36L1 protein with mock buffer, active recombinant ERK1 and/or active recombinant RSK1 under phosphorylation conditions, washed out residual kinase, added 293T cell lysate and performed pulldowns with glutathione-sepharose. We found that GST-ZFP36L1 looses its ability to interact with CNOT1 and CNOT7 proteins when incubated with active recombinant RSK1 (Figure 5D). These results indicate that RSK1 directly phosphorylates the C-terminus of ZFP36L1 downstream of ERK, and inhibits the mRNA destabilization activity of ZFP36L1.


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)

RSK1 directly phosphorylates the C-terminus of ZFP36L1. (A) 293T cells were transfected with Flag-tagged ZFP36L1. Twenty-four hours after transfection, cells were treated with PMA (for 30 min) and BI-D1870 or SL0101 (for 45 min) as indicated, and lysates were subjected to IP with the anti-Flag antibody. Co-immunoprecipitated proteins were subjected to western blot analysis using the indicated antibodies. Five percent of the initial amount of cleared 293T lysate was loaded as total lysate. (B) GST-ZFP36L1 protein was incubated with or without active recombinant RSK1 under phosphorylation conditions for 30 min. Phosphorylation of GST-ZFP36L1 C-terminal peptide was analyzed by MS. The MS/MS spectrum used to identify the m/z 681.8604-peak as the LPIFSRLSISDD peptide fragment of ZFP36L1 is shown. (C) MS/MS spectrum used to identify the m/z 721.8421-peak as the serine-8 phosphorylated LPIFSRLSISDD peptide fragment of ZFP36L1. (D) GST-ZFP36L1 protein was incubated with the indicated recombinant kinase under phosphorylation conditions for 30 min, washed, then added to 293T cell lysate and then subjected to pulldown with glutathione-sepharose. Eluted proteins were subjected to western blot analysis using the indicated antibodies and subjected to Coomassie Brilliant Blue (CBB) staining. The asterisk shows a nonspecific band. Five percent of the initial amount of cleared 293T cell lysate was loaded as total lysate. These data are representative of at least three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150769&req=5

Figure 5: RSK1 directly phosphorylates the C-terminus of ZFP36L1. (A) 293T cells were transfected with Flag-tagged ZFP36L1. Twenty-four hours after transfection, cells were treated with PMA (for 30 min) and BI-D1870 or SL0101 (for 45 min) as indicated, and lysates were subjected to IP with the anti-Flag antibody. Co-immunoprecipitated proteins were subjected to western blot analysis using the indicated antibodies. Five percent of the initial amount of cleared 293T lysate was loaded as total lysate. (B) GST-ZFP36L1 protein was incubated with or without active recombinant RSK1 under phosphorylation conditions for 30 min. Phosphorylation of GST-ZFP36L1 C-terminal peptide was analyzed by MS. The MS/MS spectrum used to identify the m/z 681.8604-peak as the LPIFSRLSISDD peptide fragment of ZFP36L1 is shown. (C) MS/MS spectrum used to identify the m/z 721.8421-peak as the serine-8 phosphorylated LPIFSRLSISDD peptide fragment of ZFP36L1. (D) GST-ZFP36L1 protein was incubated with the indicated recombinant kinase under phosphorylation conditions for 30 min, washed, then added to 293T cell lysate and then subjected to pulldown with glutathione-sepharose. Eluted proteins were subjected to western blot analysis using the indicated antibodies and subjected to Coomassie Brilliant Blue (CBB) staining. The asterisk shows a nonspecific band. Five percent of the initial amount of cleared 293T cell lysate was loaded as total lysate. These data are representative of at least three independent experiments.
Mentions: We showed that the C-terminal serine-334 of ZFP36L1 and the C-terminal serine-493 and -495 of ZFP36L2 are phosphorylated downstream of ERK. However, these sites do not match the consensus MAP kinase recognition motifs (SP or TP), indicating that ERK does not directly phosphorylate ZFP36L1. We then examined the possibility that RSK, a major downstream kinase of ERK, directly phosphorylates the C-terminus of ZFP36L1. We first investigated whether BI-D1870 and SL0101, established RSK inhibitors, can reverse PMA-mediated dissociation between ZFP36L1 and CNOT7 protein. We found that BI-D1870 and SL0101 clearly reversed the effect of PMA (Figure 5A, Supplementary Figure S6A). We then examined whether RSK1 directly phosphorylates the C-terminus of ZFP36L1 using recombinant proteins. We incubated E. coli-expressed GST-ZFP36L1 with or without active recombinant RSK1 protein under phosphorylation conditions, and analyzed the C-terminal phosphorylation of GST-ZFP36L1 by MS. We found that the C-terminal serine-334 of ZFP36L1 is phosphorylated only when we incubated GST-ZFP36L1 with active RSK1 (Figure 5B and C, Supplementary Figure S6B). We also examined whether active RSK1 inhibits the ability of ZFP36L1 to interact with CNOT proteins. We incubated GST-ZFP36L1 protein with mock buffer, active recombinant ERK1 and/or active recombinant RSK1 under phosphorylation conditions, washed out residual kinase, added 293T cell lysate and performed pulldowns with glutathione-sepharose. We found that GST-ZFP36L1 looses its ability to interact with CNOT1 and CNOT7 proteins when incubated with active recombinant RSK1 (Figure 5D). These results indicate that RSK1 directly phosphorylates the C-terminus of ZFP36L1 downstream of ERK, and inhibits the mRNA destabilization activity of ZFP36L1.

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