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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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Effects of various kinase inhibitors on Drosha localization.HeLa cells were transfected with GFP-Drosha. Six hours post-transfection, the cells were treated with various kinase inhibitors as indicated for 20 hours. Thirty minutes before performing confocal imaging, 1 µg/ml Hoechst 33342 was added to the cells to stain the nuclei. A. Solvent control; B. JNK inhibitor Sp600125; C. p38 MAPK inhibitor SB 203580; D. CDK5 inhibitor roscovitine; E. MEK inhibitor U0126; F. PKA inhibitor H-89; G. GSK3β inhibitor CHIR99021. Only inhibition of GSK3β disrupted the nuclear localization of Drosha. Bar indicates 10 micrometers.
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pone-0020391-g001: Effects of various kinase inhibitors on Drosha localization.HeLa cells were transfected with GFP-Drosha. Six hours post-transfection, the cells were treated with various kinase inhibitors as indicated for 20 hours. Thirty minutes before performing confocal imaging, 1 µg/ml Hoechst 33342 was added to the cells to stain the nuclei. A. Solvent control; B. JNK inhibitor Sp600125; C. p38 MAPK inhibitor SB 203580; D. CDK5 inhibitor roscovitine; E. MEK inhibitor U0126; F. PKA inhibitor H-89; G. GSK3β inhibitor CHIR99021. Only inhibition of GSK3β disrupted the nuclear localization of Drosha. Bar indicates 10 micrometers.

Mentions: To identify the putative kinase(s) involved in phosphorylating Drosha at Ser 300/302, we first mined Drosha sequence using web based kinase prediction algorithms (e.g. KinasePhos 2.0: http://kinasephos2.mbc.nctu.edu.tw; PPSP system: http://ppsp.biocuckoo.org) and all identified GSK3β as the putative kinase among other candidates. To test the veracity of these bioinformatic predictions we first noted cellular localization patterns of a Drosha-GFP reporter construct in the presence of the various kinase inhibitors. With the exception of CHIR99021 (GSKβ inhibitor), treatment with all other inhibitors did not lead to cytoplasmic localization of Drosha. In contrast, treatment with CHIR99021 led to cytoplasmic localization, thus providing preliminary foundation for further investigation (Figure 1). Our interest in GSK3β was further increased by noting the presence of a classic GSK3β phosphorylation motif in Drosha comprised of Ser-Pro-Ser-Leu-Glu-Arg-Ser (SPSLERS). To validate these observations we performed our experiments using a GSK3β knockout MEF cell line (Figure 2, Figure S1 and Figure S2) (kindly provided by Dr. James R. Woodgett of Samuel Lunenfeld Research Institute Toronto, Ontario, Canada). We made use of the parental Drosha-GFP reporter construct and two mutant constructs in which both Ser 300/302 were mutated to either a phospho-resistant or mimetic moiety. We transfected constructs individually into WT and KO MEF cells and noted their cellular distribution. As expected, WT-Drosha and phospho-mimetic Drosha behaved similarly with both localizing to the nucleus. In contrast, the phospho-resistant construct revealed a diffuse pattern similar to a construct encoding only GFP (Figure 3). These results suggested that GSK3β was indeed involved in correctly localizing Drosha to the nucleus.


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)

Effects of various kinase inhibitors on Drosha localization.HeLa cells were transfected with GFP-Drosha. Six hours post-transfection, the cells were treated with various kinase inhibitors as indicated for 20 hours. Thirty minutes before performing confocal imaging, 1 µg/ml Hoechst 33342 was added to the cells to stain the nuclei. A. Solvent control; B. JNK inhibitor Sp600125; C. p38 MAPK inhibitor SB 203580; D. CDK5 inhibitor roscovitine; E. MEK inhibitor U0126; F. PKA inhibitor H-89; G. GSK3β inhibitor CHIR99021. Only inhibition of GSK3β disrupted the nuclear localization of Drosha. Bar indicates 10 micrometers.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3108596&req=5

pone-0020391-g001: Effects of various kinase inhibitors on Drosha localization.HeLa cells were transfected with GFP-Drosha. Six hours post-transfection, the cells were treated with various kinase inhibitors as indicated for 20 hours. Thirty minutes before performing confocal imaging, 1 µg/ml Hoechst 33342 was added to the cells to stain the nuclei. A. Solvent control; B. JNK inhibitor Sp600125; C. p38 MAPK inhibitor SB 203580; D. CDK5 inhibitor roscovitine; E. MEK inhibitor U0126; F. PKA inhibitor H-89; G. GSK3β inhibitor CHIR99021. Only inhibition of GSK3β disrupted the nuclear localization of Drosha. Bar indicates 10 micrometers.
Mentions: To identify the putative kinase(s) involved in phosphorylating Drosha at Ser 300/302, we first mined Drosha sequence using web based kinase prediction algorithms (e.g. KinasePhos 2.0: http://kinasephos2.mbc.nctu.edu.tw; PPSP system: http://ppsp.biocuckoo.org) and all identified GSK3β as the putative kinase among other candidates. To test the veracity of these bioinformatic predictions we first noted cellular localization patterns of a Drosha-GFP reporter construct in the presence of the various kinase inhibitors. With the exception of CHIR99021 (GSKβ inhibitor), treatment with all other inhibitors did not lead to cytoplasmic localization of Drosha. In contrast, treatment with CHIR99021 led to cytoplasmic localization, thus providing preliminary foundation for further investigation (Figure 1). Our interest in GSK3β was further increased by noting the presence of a classic GSK3β phosphorylation motif in Drosha comprised of Ser-Pro-Ser-Leu-Glu-Arg-Ser (SPSLERS). To validate these observations we performed our experiments using a GSK3β knockout MEF cell line (Figure 2, Figure S1 and Figure S2) (kindly provided by Dr. James R. Woodgett of Samuel Lunenfeld Research Institute Toronto, Ontario, Canada). We made use of the parental Drosha-GFP reporter construct and two mutant constructs in which both Ser 300/302 were mutated to either a phospho-resistant or mimetic moiety. We transfected constructs individually into WT and KO MEF cells and noted their cellular distribution. As expected, WT-Drosha and phospho-mimetic Drosha behaved similarly with both localizing to the nucleus. In contrast, the phospho-resistant construct revealed a diffuse pattern similar to a construct encoding only GFP (Figure 3). These results suggested that GSK3β was indeed involved in correctly localizing Drosha to the nucleus.

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