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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

The Glycogen synthase kinase 3β (GSK 3β) functional domains (its protein kinase and FRAT/Axin binding domain) and the putative SU-MOylation site (K292 in 291FKFPQ295) is indicated (A). GSK 3β SUMO mutant (K292R) was constructed by site directed mutagenesis. GSK 3β SUMO mutant (K292R) was inserted into GST fusion (for bacteria) and Ha fusion (for cell line) expression vectors. (B) GSK 3β wild type (wt) protein that was purified from E. coli was incubated with a SUMOylation assay kit (See Material and method). For the negative control, the same assay conditions were used without ATP (right lane). A western blot of the same sample was performed with GSK 3β monoclonal antibody to monitor the protein amount in the experiment (at bottom). SUMOylated GSK 3β, as several high molecular weight protein bands, was indicated. (C) A western bolt of the immunopurified GSK 3β from COS-1 was performed using the SUMO-1 specific antibody. SUMOylation of GSK 3β was detected as high molecular weight protein bands, as indicated (left lane). For the negative control, an unrelated mouse antibody was used (right lane). To monitor the total protein amount to be used in the cell lysates, the western blot was per-formed with actin monoclonal antibody (bottom). (D) Confocal microscopic analysis of endogenous GSK 3β wt (green color) and SUMO-1 (red color). GSK 3β was detected in both the cytoplasm and nuclear region. The SUMO-1 modification proteins were mainly detected in the nuclear region (yellow color). All the figures in this article represent results from three experiments repeated independently.
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Figure 1: The Glycogen synthase kinase 3β (GSK 3β) functional domains (its protein kinase and FRAT/Axin binding domain) and the putative SU-MOylation site (K292 in 291FKFPQ295) is indicated (A). GSK 3β SUMO mutant (K292R) was constructed by site directed mutagenesis. GSK 3β SUMO mutant (K292R) was inserted into GST fusion (for bacteria) and Ha fusion (for cell line) expression vectors. (B) GSK 3β wild type (wt) protein that was purified from E. coli was incubated with a SUMOylation assay kit (See Material and method). For the negative control, the same assay conditions were used without ATP (right lane). A western blot of the same sample was performed with GSK 3β monoclonal antibody to monitor the protein amount in the experiment (at bottom). SUMOylated GSK 3β, as several high molecular weight protein bands, was indicated. (C) A western bolt of the immunopurified GSK 3β from COS-1 was performed using the SUMO-1 specific antibody. SUMOylation of GSK 3β was detected as high molecular weight protein bands, as indicated (left lane). For the negative control, an unrelated mouse antibody was used (right lane). To monitor the total protein amount to be used in the cell lysates, the western blot was per-formed with actin monoclonal antibody (bottom). (D) Confocal microscopic analysis of endogenous GSK 3β wt (green color) and SUMO-1 (red color). GSK 3β was detected in both the cytoplasm and nuclear region. The SUMO-1 modification proteins were mainly detected in the nuclear region (yellow color). All the figures in this article represent results from three experiments repeated independently.

Mentions: Using computer analysis of the SUMOylation consensus sequence from GSK 3β, two sites were found in the C-terminal domain (K292 in 291FKFPQ295), which is near by the Axin and FRAT binding domains (262-299aa), as shown in Fig. (1A) [22, 23]. Therefore, we predicted that GSK 3β is one of the SUMO modified (SUMOylation) proteins [3, 13]. To test our prediction, we constructed GSK 3β SUMO mutant (K292R) which is indicated in Fig. (1A) below, by site directed mutagenesis. Both the GSK 3β wild type (wt) and SUMO mutant (K292R) was inserted into Ha tagged expression vector (for eukaryotic cell) or GST fusion expression vector (for prokaryotic cell).


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 Glycogen synthase kinase 3β (GSK 3β) functional domains (its protein kinase and FRAT/Axin binding domain) and the putative SU-MOylation site (K292 in 291FKFPQ295) is indicated (A). GSK 3β SUMO mutant (K292R) was constructed by site directed mutagenesis. GSK 3β SUMO mutant (K292R) was inserted into GST fusion (for bacteria) and Ha fusion (for cell line) expression vectors. (B) GSK 3β wild type (wt) protein that was purified from E. coli was incubated with a SUMOylation assay kit (See Material and method). For the negative control, the same assay conditions were used without ATP (right lane). A western blot of the same sample was performed with GSK 3β monoclonal antibody to monitor the protein amount in the experiment (at bottom). SUMOylated GSK 3β, as several high molecular weight protein bands, was indicated. (C) A western bolt of the immunopurified GSK 3β from COS-1 was performed using the SUMO-1 specific antibody. SUMOylation of GSK 3β was detected as high molecular weight protein bands, as indicated (left lane). For the negative control, an unrelated mouse antibody was used (right lane). To monitor the total protein amount to be used in the cell lysates, the western blot was per-formed with actin monoclonal antibody (bottom). (D) Confocal microscopic analysis of endogenous GSK 3β wt (green color) and SUMO-1 (red color). GSK 3β was detected in both the cytoplasm and nuclear region. The SUMO-1 modification proteins were mainly detected in the nuclear region (yellow color). All the figures in this article represent results from three experiments repeated independently.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: The Glycogen synthase kinase 3β (GSK 3β) functional domains (its protein kinase and FRAT/Axin binding domain) and the putative SU-MOylation site (K292 in 291FKFPQ295) is indicated (A). GSK 3β SUMO mutant (K292R) was constructed by site directed mutagenesis. GSK 3β SUMO mutant (K292R) was inserted into GST fusion (for bacteria) and Ha fusion (for cell line) expression vectors. (B) GSK 3β wild type (wt) protein that was purified from E. coli was incubated with a SUMOylation assay kit (See Material and method). For the negative control, the same assay conditions were used without ATP (right lane). A western blot of the same sample was performed with GSK 3β monoclonal antibody to monitor the protein amount in the experiment (at bottom). SUMOylated GSK 3β, as several high molecular weight protein bands, was indicated. (C) A western bolt of the immunopurified GSK 3β from COS-1 was performed using the SUMO-1 specific antibody. SUMOylation of GSK 3β was detected as high molecular weight protein bands, as indicated (left lane). For the negative control, an unrelated mouse antibody was used (right lane). To monitor the total protein amount to be used in the cell lysates, the western blot was per-formed with actin monoclonal antibody (bottom). (D) Confocal microscopic analysis of endogenous GSK 3β wt (green color) and SUMO-1 (red color). GSK 3β was detected in both the cytoplasm and nuclear region. The SUMO-1 modification proteins were mainly detected in the nuclear region (yellow color). All the figures in this article represent results from three experiments repeated independently.
Mentions: Using computer analysis of the SUMOylation consensus sequence from GSK 3β, two sites were found in the C-terminal domain (K292 in 291FKFPQ295), which is near by the Axin and FRAT binding domains (262-299aa), as shown in Fig. (1A) [22, 23]. Therefore, we predicted that GSK 3β is one of the SUMO modified (SUMOylation) proteins [3, 13]. To test our prediction, we constructed GSK 3β SUMO mutant (K292R) which is indicated in Fig. (1A) below, by site directed mutagenesis. Both the GSK 3β wild type (wt) and SUMO mutant (K292R) was inserted into Ha tagged expression vector (for eukaryotic cell) or GST fusion expression vector (for prokaryotic cell).

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