Limits...
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

The immunopurified Ha -GSK 3β wt or its SUMO mutant (K292R) protein with Ha Ab from COS-1 was immunoblotted with GSK 3β (A) or Anti- GSK 3β Tyr 216 phospho Ab polyclonal antibody (B). The relative optical density (OD), as determined by image analysis with the Fuji Image Quant software, is indicated below. The GSK 3β kinase activity was measured using human Tau protein as a substrate (C). S422 residue phosphorylation of human Tau protein was detected with its specific antibody. The relative GSK 3β activity by image analysis with the Fuji Image Quant software is indicated below. Results shown are one of five repeated experiments
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2570553&req=5

Figure 4: The immunopurified Ha -GSK 3β wt or its SUMO mutant (K292R) protein with Ha Ab from COS-1 was immunoblotted with GSK 3β (A) or Anti- GSK 3β Tyr 216 phospho Ab polyclonal antibody (B). The relative optical density (OD), as determined by image analysis with the Fuji Image Quant software, is indicated below. The GSK 3β kinase activity was measured using human Tau protein as a substrate (C). S422 residue phosphorylation of human Tau protein was detected with its specific antibody. The relative GSK 3β activity by image analysis with the Fuji Image Quant software is indicated below. Results shown are one of five repeated experiments

Mentions: To further define the biological significance of GSK 3β SUMOylation, we compared the kinase activity of GSK 3β with that of the GSK 3β SUMO mutant. Each Ha –GSK 3β wt or its SUMO mutant (K292R) expression vector was transfected into COS-1 cells, and immunoprecipitated with Ha monoclonal Ab. To monitor the expression of Ha -GSK 3β wt or GSK 3β SUMO mutants, western blot was performed with an anti-GSK 3β antibody (Fig. 4A). Because it has been reported that 216 tyrosine residue phosphorylation of GSK 3β is required for its activation [17, 24], we monitored it with its 216 tyrosine phosphorylation specific antibody (Fig. 4B). As shown in Fig. (4B), we observed that 216 tyrosine phosphorylation of the Ha -GSK 3β SUMO mutant was reduced compared to that of Ha –GSK 3β wt (Fig. 3B), even though the expressed protein amount was almost same (Fig. 4A). Next, we measured the kinase activity with its substrate protein Tau, because Ser 422 of human Tau is phosphorylated by GSK 3β [25, 26]. As shown in Fig. (4C), we observed that the kinase activity of the GSK 3β SUMO mutant was reduced to half that of the GSK 3β wt, consistent with its 216 tyrosine phosphorylation result (Fig. 3B). Taken together, our data suggest that SUMOylation of GSK 3β is also required for not only its 216 tyrosine phosphorylation, but also its kinase activity. To eliminate cell line specificity, we performed the experiment using NIH 3T3 cells and obtained the same results (data not shown).


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)

The immunopurified Ha -GSK 3β wt or its SUMO mutant (K292R) protein with Ha Ab from COS-1 was immunoblotted with GSK 3β (A) or Anti- GSK 3β Tyr 216 phospho Ab polyclonal antibody (B). The relative optical density (OD), as determined by image analysis with the Fuji Image Quant software, is indicated below. The GSK 3β kinase activity was measured using human Tau protein as a substrate (C). S422 residue phosphorylation of human Tau protein was detected with its specific antibody. The relative GSK 3β activity by image analysis with the Fuji Image Quant software is indicated below. Results shown are one of five repeated experiments
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: The immunopurified Ha -GSK 3β wt or its SUMO mutant (K292R) protein with Ha Ab from COS-1 was immunoblotted with GSK 3β (A) or Anti- GSK 3β Tyr 216 phospho Ab polyclonal antibody (B). The relative optical density (OD), as determined by image analysis with the Fuji Image Quant software, is indicated below. The GSK 3β kinase activity was measured using human Tau protein as a substrate (C). S422 residue phosphorylation of human Tau protein was detected with its specific antibody. The relative GSK 3β activity by image analysis with the Fuji Image Quant software is indicated below. Results shown are one of five repeated experiments
Mentions: To further define the biological significance of GSK 3β SUMOylation, we compared the kinase activity of GSK 3β with that of the GSK 3β SUMO mutant. Each Ha –GSK 3β wt or its SUMO mutant (K292R) expression vector was transfected into COS-1 cells, and immunoprecipitated with Ha monoclonal Ab. To monitor the expression of Ha -GSK 3β wt or GSK 3β SUMO mutants, western blot was performed with an anti-GSK 3β antibody (Fig. 4A). Because it has been reported that 216 tyrosine residue phosphorylation of GSK 3β is required for its activation [17, 24], we monitored it with its 216 tyrosine phosphorylation specific antibody (Fig. 4B). As shown in Fig. (4B), we observed that 216 tyrosine phosphorylation of the Ha -GSK 3β SUMO mutant was reduced compared to that of Ha –GSK 3β wt (Fig. 3B), even though the expressed protein amount was almost same (Fig. 4A). Next, we measured the kinase activity with its substrate protein Tau, because Ser 422 of human Tau is phosphorylated by GSK 3β [25, 26]. As shown in Fig. (4C), we observed that the kinase activity of the GSK 3β SUMO mutant was reduced to half that of the GSK 3β wt, consistent with its 216 tyrosine phosphorylation result (Fig. 3B). Taken together, our data suggest that SUMOylation of GSK 3β is also required for not only its 216 tyrosine phosphorylation, but also its kinase activity. To eliminate cell line specificity, we performed the experiment using NIH 3T3 cells and obtained the same results (data not shown).

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