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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

(A) The purified GST-GSK 3β wt or GST-GSK 3β SUMO mutant (K292R) fusion protein was used as the substrate protein in the SUMOy-lation assay as described in the Materials and Methods section. The SUMOylation of GSK 3β wt was detected as a high molecular weight protein band (left lane), whereas its SUMO mutant was totally inhibited, as shown (right lane). (B) Ha –GSK 3β wt or Ha –GSK 3β SUMO mutant was transfected to COS-1 cells and immunoprecipitated with Ha mouse monoclonal antibody. The immunoprecipitants were sub-jected to the western bolt with SUMO-1, as described in the Materials and Methods section. The SUMOylation of GSK 3β wt was indicated as several high molecular weight protein bands (left lane), whereas its SUMO mutant was totally inhibited (right lane). To monitor the GSK 3β protein expression, the immunoprecipitants were subjected to the western bolt with GSK 3β polyclonal antibody (bottom).
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Figure 2: (A) The purified GST-GSK 3β wt or GST-GSK 3β SUMO mutant (K292R) fusion protein was used as the substrate protein in the SUMOy-lation assay as described in the Materials and Methods section. The SUMOylation of GSK 3β wt was detected as a high molecular weight protein band (left lane), whereas its SUMO mutant was totally inhibited, as shown (right lane). (B) Ha –GSK 3β wt or Ha –GSK 3β SUMO mutant was transfected to COS-1 cells and immunoprecipitated with Ha mouse monoclonal antibody. The immunoprecipitants were sub-jected to the western bolt with SUMO-1, as described in the Materials and Methods section. The SUMOylation of GSK 3β wt was indicated as several high molecular weight protein bands (left lane), whereas its SUMO mutant was totally inhibited (right lane). To monitor the GSK 3β protein expression, the immunoprecipitants were subjected to the western bolt with GSK 3β polyclonal antibody (bottom).

Mentions: As shown in Fig. (1A), we predicted that K292 in (291FKFPQ295) of GSK 3β was the putative SUMOylation sites based on consensus sequence analysis. To test our prediction, we performed the SUMOylation assay in vitro with GSK 3β SUMO mutant (K292R). We used GST fusion GSK 3β (wt) or GSK 3β SUMO mutant, as the substrate protein. Western blotting of the same sample was performed with GST monoclonal antibody to monitor the amount of protein in the experiment (at bottom). We also performed the SUMOylation assay using GST fusion, GSK 3β K292R mutant protein, and did not observe the SUMOylation of each mutant protein (data not shown). As shown in Fig. (2A), the SUMOylation of GSK 3β SUMO mutant was not detected, whereas that of GST fusion GSK 3β (wt) was observed. Therefore, our data suggested that K292 in (291FKFPQ295) of GSK 3β is the putative SUMOylation sites.


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)

(A) The purified GST-GSK 3β wt or GST-GSK 3β SUMO mutant (K292R) fusion protein was used as the substrate protein in the SUMOy-lation assay as described in the Materials and Methods section. The SUMOylation of GSK 3β wt was detected as a high molecular weight protein band (left lane), whereas its SUMO mutant was totally inhibited, as shown (right lane). (B) Ha –GSK 3β wt or Ha –GSK 3β SUMO mutant was transfected to COS-1 cells and immunoprecipitated with Ha mouse monoclonal antibody. The immunoprecipitants were sub-jected to the western bolt with SUMO-1, as described in the Materials and Methods section. The SUMOylation of GSK 3β wt was indicated as several high molecular weight protein bands (left lane), whereas its SUMO mutant was totally inhibited (right lane). To monitor the GSK 3β protein expression, the immunoprecipitants were subjected to the western bolt with GSK 3β polyclonal antibody (bottom).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: (A) The purified GST-GSK 3β wt or GST-GSK 3β SUMO mutant (K292R) fusion protein was used as the substrate protein in the SUMOy-lation assay as described in the Materials and Methods section. The SUMOylation of GSK 3β wt was detected as a high molecular weight protein band (left lane), whereas its SUMO mutant was totally inhibited, as shown (right lane). (B) Ha –GSK 3β wt or Ha –GSK 3β SUMO mutant was transfected to COS-1 cells and immunoprecipitated with Ha mouse monoclonal antibody. The immunoprecipitants were sub-jected to the western bolt with SUMO-1, as described in the Materials and Methods section. The SUMOylation of GSK 3β wt was indicated as several high molecular weight protein bands (left lane), whereas its SUMO mutant was totally inhibited (right lane). To monitor the GSK 3β protein expression, the immunoprecipitants were subjected to the western bolt with GSK 3β polyclonal antibody (bottom).
Mentions: As shown in Fig. (1A), we predicted that K292 in (291FKFPQ295) of GSK 3β was the putative SUMOylation sites based on consensus sequence analysis. To test our prediction, we performed the SUMOylation assay in vitro with GSK 3β SUMO mutant (K292R). We used GST fusion GSK 3β (wt) or GSK 3β SUMO mutant, as the substrate protein. Western blotting of the same sample was performed with GST monoclonal antibody to monitor the amount of protein in the experiment (at bottom). We also performed the SUMOylation assay using GST fusion, GSK 3β K292R mutant protein, and did not observe the SUMOylation of each mutant protein (data not shown). As shown in Fig. (2A), the SUMOylation of GSK 3β SUMO mutant was not detected, whereas that of GST fusion GSK 3β (wt) was observed. Therefore, our data suggested that K292 in (291FKFPQ295) of GSK 3β is the putative SUMOylation sites.

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