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Regulation of glycogen synthase kinase 3beta functions by modification of the small ubiquitin-like modifier.

Eun Jeoung L, Sung Hee H, Jaesun C, Sung Hwa S, Kwang Hum Y, Min Kyoung K, Tae Yoon P, Sang Sun K - Open Biochem J (2008)

Bottom Line: In this report, we showed that the SUMOylation of GSK 3beta occurs on its K(292) residue, and this modification promotes its nuclear localization in COS-1.Additionally, our data showed that the GSK 3beta SUMO mutant (K292R) decreased its kinase activity and protein stability, affecting cell death.Therefore, our observations at first time suggested that SUMOylation on the K(292) residue of GSK 3beta might be a GSK 3beta regulation mechanism for its kinase activation, subcellular localization, protein stability, and cell apoptosis.

View Article: PubMed Central - PubMed

Affiliation: School of Science Education, Chungbuk National University, Gaeshin-dong, Heungdok-gu, Cheongju, Chungbuk, 361-763, Republic of Korea.

ABSTRACT
Modification of the Small Ubiquitin-like Modifier (SUMO) (SUMOylation) appears to regulate diverse cellular processes, including nuclear transport, signal transduction, apoptosis, autophagy, cell cycle control, ubiquitin-dependent degradation and gene transcription. Glycogen synthase kinase 3beta (GSK 3beta) is a serine/threonine kinase that is thought to contribute to a variety of biological events, including embryonic development, metabolism, tumorigenesis, and cell death. GSK 3beta is a constitutively active kinase that regulates many intracellular signaling pathways by phosphorylating substrates such as beta-catenin. We noticed that the putative SUMOylation sites are localized on K(292 )residueof (291)FKFPQ(295) in GSK 3beta based on analysis of the SUMOylation consensus sequence. In this report, we showed that the SUMOylation of GSK 3beta occurs on its K(292) residue, and this modification promotes its nuclear localization in COS-1. Additionally, our data showed that the GSK 3beta SUMO mutant (K292R) decreased its kinase activity and protein stability, affecting cell death. Therefore, our observations at first time suggested that SUMOylation on the K(292) residue of GSK 3beta might be a GSK 3beta regulation mechanism for its kinase activation, subcellular localization, protein stability, and cell apoptosis.

No MeSH data available.


Related in: MedlinePlus

Ha –GSK 3β or GSK 3β SUMO mutant (K292R) was transfected into COS-1 cells and the cells treated with cyclohexamide. The GSK 3β proteins were chased for the indicated time periods. Ha–GSK 3β proteins were immunoprecipitated with a polyclonal anti- Ha antibody and subjected to SDS-PAGE followed by western blotting with a monoclonal GSK 3β antibody (A). To monitor the protein amount, an equal amount of cell lysate was subjected to western blotting with an actin antibody. Results shown are one of five repeated experiments. Quantifi-cation of the pulse-chase experiment is shown in (B) by image analysis with the Fuji Image Quant software.
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Figure 5: Ha –GSK 3β or GSK 3β SUMO mutant (K292R) was transfected into COS-1 cells and the cells treated with cyclohexamide. The GSK 3β proteins were chased for the indicated time periods. Ha–GSK 3β proteins were immunoprecipitated with a polyclonal anti- Ha antibody and subjected to SDS-PAGE followed by western blotting with a monoclonal GSK 3β antibody (A). To monitor the protein amount, an equal amount of cell lysate was subjected to western blotting with an actin antibody. Results shown are one of five repeated experiments. Quantifi-cation of the pulse-chase experiment is shown in (B) by image analysis with the Fuji Image Quant software.

Mentions: To evaluate the effect of SUMOylation on GSK 3β protein stability, we performed the pulse-chase experiments as described in the Materials and Methods section. Each Ha -GSK 3β (wt or SUMO mutant) expression vector was transfected into COS-1 cells and immunoprecipitated with Ha monoclonal Ab following cyclohexamide treatment (Fig. 5A). GSK 3β proteins were chased for the indicated time periods (0, 8, 16, 24 hr), and then immunoprecipitated with a polyclonal anti- Ha antibody and subjected to SDS-PAGE followed by western blot with GSK 3β antibody. To control the protein amount, we monitored the actin in each sample by western blotting (Fig. 5A). The quantification of the pulse-chase experiment, as determined by image analysis of the dried SDS-PAGE gel using the Fuji Image Quant software, is shown in Fig. (5B). As shown in Fig. (5), the protein stability of the GSK 3β wt was twice that of the GSK 3β SUMO mutant, suggesting that SUMOylation on the K292 of GSK 3β seems necessary for protein stability.


Regulation of glycogen synthase kinase 3beta functions by modification of the small ubiquitin-like modifier.

Eun Jeoung L, Sung Hee H, Jaesun C, Sung Hwa S, Kwang Hum Y, Min Kyoung K, Tae Yoon P, Sang Sun K - Open Biochem J (2008)

Ha –GSK 3β or GSK 3β SUMO mutant (K292R) was transfected into COS-1 cells and the cells treated with cyclohexamide. The GSK 3β proteins were chased for the indicated time periods. Ha–GSK 3β proteins were immunoprecipitated with a polyclonal anti- Ha antibody and subjected to SDS-PAGE followed by western blotting with a monoclonal GSK 3β antibody (A). To monitor the protein amount, an equal amount of cell lysate was subjected to western blotting with an actin antibody. Results shown are one of five repeated experiments. Quantifi-cation of the pulse-chase experiment is shown in (B) by image analysis with the Fuji Image Quant software.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2570553&req=5

Figure 5: Ha –GSK 3β or GSK 3β SUMO mutant (K292R) was transfected into COS-1 cells and the cells treated with cyclohexamide. The GSK 3β proteins were chased for the indicated time periods. Ha–GSK 3β proteins were immunoprecipitated with a polyclonal anti- Ha antibody and subjected to SDS-PAGE followed by western blotting with a monoclonal GSK 3β antibody (A). To monitor the protein amount, an equal amount of cell lysate was subjected to western blotting with an actin antibody. Results shown are one of five repeated experiments. Quantifi-cation of the pulse-chase experiment is shown in (B) by image analysis with the Fuji Image Quant software.
Mentions: To evaluate the effect of SUMOylation on GSK 3β protein stability, we performed the pulse-chase experiments as described in the Materials and Methods section. Each Ha -GSK 3β (wt or SUMO mutant) expression vector was transfected into COS-1 cells and immunoprecipitated with Ha monoclonal Ab following cyclohexamide treatment (Fig. 5A). GSK 3β proteins were chased for the indicated time periods (0, 8, 16, 24 hr), and then immunoprecipitated with a polyclonal anti- Ha antibody and subjected to SDS-PAGE followed by western blot with GSK 3β antibody. To control the protein amount, we monitored the actin in each sample by western blotting (Fig. 5A). The quantification of the pulse-chase experiment, as determined by image analysis of the dried SDS-PAGE gel using the Fuji Image Quant software, is shown in Fig. (5B). As shown in Fig. (5), the protein stability of the GSK 3β wt was twice that of the GSK 3β SUMO mutant, suggesting that SUMOylation on the K292 of GSK 3β seems necessary for protein stability.

Bottom Line: In this report, we showed that the SUMOylation of GSK 3beta occurs on its K(292) residue, and this modification promotes its nuclear localization in COS-1.Additionally, our data showed that the GSK 3beta SUMO mutant (K292R) decreased its kinase activity and protein stability, affecting cell death.Therefore, our observations at first time suggested that SUMOylation on the K(292) residue of GSK 3beta might be a GSK 3beta regulation mechanism for its kinase activation, subcellular localization, protein stability, and cell apoptosis.

View Article: PubMed Central - PubMed

Affiliation: School of Science Education, Chungbuk National University, Gaeshin-dong, Heungdok-gu, Cheongju, Chungbuk, 361-763, Republic of Korea.

ABSTRACT
Modification of the Small Ubiquitin-like Modifier (SUMO) (SUMOylation) appears to regulate diverse cellular processes, including nuclear transport, signal transduction, apoptosis, autophagy, cell cycle control, ubiquitin-dependent degradation and gene transcription. Glycogen synthase kinase 3beta (GSK 3beta) is a serine/threonine kinase that is thought to contribute to a variety of biological events, including embryonic development, metabolism, tumorigenesis, and cell death. GSK 3beta is a constitutively active kinase that regulates many intracellular signaling pathways by phosphorylating substrates such as beta-catenin. We noticed that the putative SUMOylation sites are localized on K(292 )residueof (291)FKFPQ(295) in GSK 3beta based on analysis of the SUMOylation consensus sequence. In this report, we showed that the SUMOylation of GSK 3beta occurs on its K(292) residue, and this modification promotes its nuclear localization in COS-1. Additionally, our data showed that the GSK 3beta SUMO mutant (K292R) decreased its kinase activity and protein stability, affecting cell death. Therefore, our observations at first time suggested that SUMOylation on the K(292) residue of GSK 3beta might be a GSK 3beta regulation mechanism for its kinase activation, subcellular localization, protein stability, and cell apoptosis.

No MeSH data available.


Related in: MedlinePlus