| Item type |
デフォルトアイテムタイプ_（フル）(1) |
| 公開日 |
2023-03-18 |
| タイトル |
|
|
タイトル |
Label-free kinase profiling using phosphate affinity polyacrylamide gel electrophoresis |
|
言語 |
en |
| 作成者 |
Kinoshita, Emiko
Aoki, Yuri
Kinoshita, Eiji
Koike, Tohru
|
| アクセス権 |
|
|
アクセス権 |
open access |
|
アクセス権URI |
http://purl.org/coar/access_right/c_abf2 |
| 権利情報 |
|
|
権利情報 |
Copyright (c) 2007 The American Society for Biochemistry and Molecular Biology, Inc. |
| 主題 |
|
|
主題Scheme |
Other |
|
主題 |
label-free |
| 主題 |
|
|
主題Scheme |
Other |
|
主題 |
kinase profiling |
| 主題 |
|
|
主題Scheme |
Other |
|
主題 |
phosphoproteomics |
| 主題 |
|
|
主題Scheme |
Other |
|
主題 |
phosphate-affinity |
| 主題 |
|
|
主題Scheme |
Other |
|
主題 |
electrophoresis |
| 主題 |
|
|
主題Scheme |
Other |
|
主題 |
phosphorylated protein |
| 主題 |
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|
主題Scheme |
Other |
|
主題 |
tau protein |
| 主題 |
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|
主題Scheme |
Other |
|
主題 |
signal transduction |
| 主題 |
|
|
主題Scheme |
NDC |
|
主題 |
490 |
| 内容記述 |
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|
内容記述 |
Herein we describe three applications of label-free kinase profiling using a novel type of phosphate affinity polyacrylamide gel electrophoresis. The phosphate affinity site is a polyacrylamide-bound dinuclear Mn2+ complex that enables the mobility shift detection of phosphorylated proteins from their nonphosphorylated counterpart. The first application is in vitro kinase activity profiling for the analysis of varied phosphoprotein isotypes in phosphorylation status. The activity profiles of six kinds of kinases, glycogen synthase kinase-3ß, cyclin-dependent kinase 5/p35, protein kinase A, mitogen-activated protein kinase (MAPK), casein kinase II, and calmodulin-dependent protein kinase II, were determined using a substrate protein, Tau, which has a number of phosphorylation sites. Each kinase demonstrated characteristic multiple electrophoresis migration bands up-shifted from the nonphosphorylated Tau due to differences in the phosphorylation sites and stoichiometry. The second application is in vivo kinase activity profiling for the analysis of protein phosphorylation involved in intracellular signal transduction. The time course changes in the epidermal growth factor-induced phosphorylation levels of Shc and MAPK in A431 cells were visualized as highly up-shifted migration bands by subsequent immunoblotting with anti-Shc and anti-MAPK antibodies. The third application is in vitro kinase inhibition profiling for the quantitative screening of kinase-specific inhibitors. The inhibition profile of a tyrosine kinase, Abl (a histidine-tagged recombinant mouse Abl kinase), was determined using the substrate Abltide-GST (a fusion protein consisting of a specific substrate peptide for Abl and glutathione S-transferase) and the approved drug Glivec (an ATP competitor). In the kinase assay, the slower migration band, monophosphorylated Abltide-GST, increased time-dependently, whereas the faster migration band, nonphosphorylated Abltide-GST, decreased. The dose-dependent inhibition of Glivec was determined by a change in the ratio of the faster and slower migration bands, which showed an IC50 value of 1.6 µM in the presence of 0.10 mM ATP. |
|
言語 |
en |
| 出版者 |
|
|
出版者 |
The American Society for Biochemistry and Molecular Biology, Inc. |
| 言語 |
|
|
言語 |
eng |
| 資源タイプ |
|
|
資源タイプ識別子 |
http://purl.org/coar/resource_type/c_6501 |
|
資源タイプ |
journal article |
| 出版タイプ |
|
|
出版タイプ |
AO |
|
出版タイプResource |
http://purl.org/coar/version/c_b1a7d7d4d402bcce |
| 関連情報 |
|
|
|
識別子タイプ |
DOI |
|
|
関連識別子 |
10.1074/mcp.T600044-MCP200 |
| 関連情報 |
|
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|
識別子タイプ |
PMID |
|
|
関連識別子 |
17088264 |
| 収録物識別子 |
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|
収録物識別子タイプ |
NCID |
|
収録物識別子 |
AA11789524 |
| 収録物識別子 |
|
|
収録物識別子タイプ |
ISSN |
|
収録物識別子 |
1535-9476 |
| 開始ページ |
|
|
開始ページ |
356 |
| 書誌情報 |
Molecular and Cellular Proteomics
Molecular and Cellular Proteomics
巻 6,
号 2,
p. 356-366,
発行日 2007-02
|
| 旧ID |
17212 |
MCP_6_356.pdf (1.6 MB)
