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Glycogen synthase kinase 3 beta (GSK3β) phosphorylates the RNAase III enzyme Drosha at S300 and S302.

Tang X, Li M, Tucker L, Ramratnam B - PLoS ONE (2011)

Bottom Line: Here, we identify GSK3β as the culprit kinase.We demonstrate that Drosha is unable to selectively localize to the nucleus in cells deficient in GSK3β.These findings expand the substrate base of GSK3β to include a central component of the miRNA biogenesis pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Warren Alpert Medical School of Brown University, Providence, Rhode Island, United States of America.

ABSTRACT
The canonical microRNA (miRNA) pathway commences with the enzymatic cleavage of the primary gene transcript (pri-miRNA) by the RNAase III enzyme Drosha in the nucleus into shorter pre-miRNA species that are subsequently exported to the cytoplasm for further processing into shorter, mature miRNA molecules. Using a series of reporter constructs, we have previously demonstrated that phosphorylation of Drosha at Ser 300 and 302 was required for its nuclear localization. Here, we identify GSK3β as the culprit kinase. We demonstrate that Drosha is unable to selectively localize to the nucleus in cells deficient in GSK3β. These findings expand the substrate base of GSK3β to include a central component of the miRNA biogenesis pathway.

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GSK3β phosphorylates Drosha at S300 and S302.In vitro kinase assays were performed using (A) peptide fragments of Drosha or (B) immunoprecipitated Drosha protein. A. A synthetic Drosha peptide (H-RERHRHRDNRRSPSLERSYKKEYKR-CONH2) was incubated with purified recombinant GSK3β enzyme for 1 hour. The reaction mixture (1 µl) was dot blotted to a nitrocellulose membrane and kinase activity was identified by x-ray film exposure. B. Immunoprecipitated GFP-Drosha wt or S300A/S302A was also used for in vitro GSK3β kinase assay with or without purified recombinant GSK3β enzyme. The reaction mixture was resolved by SDS-PAGE gel and transferred to a nitrocellulose membrane by electrophoresis before exposing to an X-ray film. The top panel shows that GFP-Drosha wt, but not GFP-Drosha S300A/S302A, was phosphorylated by GSK3β. The bottom panel shows that the expression levels of GFP-Drosha wt and S300A/S302A mutant were relatively equal in GSK3β knockout MEF cells.
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pone-0020391-g004: GSK3β phosphorylates Drosha at S300 and S302.In vitro kinase assays were performed using (A) peptide fragments of Drosha or (B) immunoprecipitated Drosha protein. A. A synthetic Drosha peptide (H-RERHRHRDNRRSPSLERSYKKEYKR-CONH2) was incubated with purified recombinant GSK3β enzyme for 1 hour. The reaction mixture (1 µl) was dot blotted to a nitrocellulose membrane and kinase activity was identified by x-ray film exposure. B. Immunoprecipitated GFP-Drosha wt or S300A/S302A was also used for in vitro GSK3β kinase assay with or without purified recombinant GSK3β enzyme. The reaction mixture was resolved by SDS-PAGE gel and transferred to a nitrocellulose membrane by electrophoresis before exposing to an X-ray film. The top panel shows that GFP-Drosha wt, but not GFP-Drosha S300A/S302A, was phosphorylated by GSK3β. The bottom panel shows that the expression levels of GFP-Drosha wt and S300A/S302A mutant were relatively equal in GSK3β knockout MEF cells.

Mentions: To experimentally verify our bioinformatic prediction and cell culture results, we first performed an in vitro kinase assay using a synthetic Drosha peptide fragment harboring the putative GSK3β phosphorylation site. Dot blot analysis revealed kinase activity upon co-incubation of peptide fragment and recombinant GSK3β (Figure 4A). Previous reports have identified autophosphphorylation capacity of GSK3β and indeed we saw a faint reaction in the control lane employing enzyme only (Figure 4A). A challenge of working with Drosha is the lack of availability of pure recombinant protein ascribed to its size (∼150 kD) [20]. To circumvent this problem, we transfected GSK3β−/− MEF cells with our Drosha-GFP or S300A/S302A mutant, respectively. A polyclonal antibody to GFP was used to pull down adequate amounts of protein. Importantly, both constructs expressed similar amounts of protein upon cellular transfection. As seen in Figure 4B, Figure S3 and Figure S4, only the WT construct could be phosphorylated in vitro by GSK3β. To further confirm that GSK3β is required for Drosha nuclear localization, a construct encoding HA-GSK3β (Addgene cat.#14753) was co-transfected with GFP-Drosha. Overexpression of GSK3β in GSK3β−/− MEF cells led to complete restoration of phenotype with Drosha nuclear localization (Figure 5).


Glycogen synthase kinase 3 beta (GSK3β) phosphorylates the RNAase III enzyme Drosha at S300 and S302.

Tang X, Li M, Tucker L, Ramratnam B - PLoS ONE (2011)

GSK3β phosphorylates Drosha at S300 and S302.In vitro kinase assays were performed using (A) peptide fragments of Drosha or (B) immunoprecipitated Drosha protein. A. A synthetic Drosha peptide (H-RERHRHRDNRRSPSLERSYKKEYKR-CONH2) was incubated with purified recombinant GSK3β enzyme for 1 hour. The reaction mixture (1 µl) was dot blotted to a nitrocellulose membrane and kinase activity was identified by x-ray film exposure. B. Immunoprecipitated GFP-Drosha wt or S300A/S302A was also used for in vitro GSK3β kinase assay with or without purified recombinant GSK3β enzyme. The reaction mixture was resolved by SDS-PAGE gel and transferred to a nitrocellulose membrane by electrophoresis before exposing to an X-ray film. The top panel shows that GFP-Drosha wt, but not GFP-Drosha S300A/S302A, was phosphorylated by GSK3β. The bottom panel shows that the expression levels of GFP-Drosha wt and S300A/S302A mutant were relatively equal in GSK3β knockout MEF cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3108596&req=5

pone-0020391-g004: GSK3β phosphorylates Drosha at S300 and S302.In vitro kinase assays were performed using (A) peptide fragments of Drosha or (B) immunoprecipitated Drosha protein. A. A synthetic Drosha peptide (H-RERHRHRDNRRSPSLERSYKKEYKR-CONH2) was incubated with purified recombinant GSK3β enzyme for 1 hour. The reaction mixture (1 µl) was dot blotted to a nitrocellulose membrane and kinase activity was identified by x-ray film exposure. B. Immunoprecipitated GFP-Drosha wt or S300A/S302A was also used for in vitro GSK3β kinase assay with or without purified recombinant GSK3β enzyme. The reaction mixture was resolved by SDS-PAGE gel and transferred to a nitrocellulose membrane by electrophoresis before exposing to an X-ray film. The top panel shows that GFP-Drosha wt, but not GFP-Drosha S300A/S302A, was phosphorylated by GSK3β. The bottom panel shows that the expression levels of GFP-Drosha wt and S300A/S302A mutant were relatively equal in GSK3β knockout MEF cells.
Mentions: To experimentally verify our bioinformatic prediction and cell culture results, we first performed an in vitro kinase assay using a synthetic Drosha peptide fragment harboring the putative GSK3β phosphorylation site. Dot blot analysis revealed kinase activity upon co-incubation of peptide fragment and recombinant GSK3β (Figure 4A). Previous reports have identified autophosphphorylation capacity of GSK3β and indeed we saw a faint reaction in the control lane employing enzyme only (Figure 4A). A challenge of working with Drosha is the lack of availability of pure recombinant protein ascribed to its size (∼150 kD) [20]. To circumvent this problem, we transfected GSK3β−/− MEF cells with our Drosha-GFP or S300A/S302A mutant, respectively. A polyclonal antibody to GFP was used to pull down adequate amounts of protein. Importantly, both constructs expressed similar amounts of protein upon cellular transfection. As seen in Figure 4B, Figure S3 and Figure S4, only the WT construct could be phosphorylated in vitro by GSK3β. To further confirm that GSK3β is required for Drosha nuclear localization, a construct encoding HA-GSK3β (Addgene cat.#14753) was co-transfected with GFP-Drosha. Overexpression of GSK3β in GSK3β−/− MEF cells led to complete restoration of phenotype with Drosha nuclear localization (Figure 5).

Bottom Line: Here, we identify GSK3β as the culprit kinase.We demonstrate that Drosha is unable to selectively localize to the nucleus in cells deficient in GSK3β.These findings expand the substrate base of GSK3β to include a central component of the miRNA biogenesis pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Warren Alpert Medical School of Brown University, Providence, Rhode Island, United States of America.

ABSTRACT
The canonical microRNA (miRNA) pathway commences with the enzymatic cleavage of the primary gene transcript (pri-miRNA) by the RNAase III enzyme Drosha in the nucleus into shorter pre-miRNA species that are subsequently exported to the cytoplasm for further processing into shorter, mature miRNA molecules. Using a series of reporter constructs, we have previously demonstrated that phosphorylation of Drosha at Ser 300 and 302 was required for its nuclear localization. Here, we identify GSK3β as the culprit kinase. We demonstrate that Drosha is unable to selectively localize to the nucleus in cells deficient in GSK3β. These findings expand the substrate base of GSK3β to include a central component of the miRNA biogenesis pathway.

Show MeSH
Related in: MedlinePlus