Document Detail


An efficient method for protein phosphorylation using the artificially introduced of cognate-binding modules into kinases and substrates.
MedLine Citation:
PMID:  17875337     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Protein phosphorylation is a major post-translational modification that regulates cellular signal transduction. The phosphorylation of substrate proteins by kinases requires cognate pairs of substrates and kinases. In addition, phosphorylation is mediated through both indirect and direct interaction between these kinases and substrates, which makes it difficult to effectively prepare large quantities of recombinant phosphorylated proteins. Here, we report a novel protein phosphorylation method involving the artificial introduction of cognate-binding modules into substrates and enzymes. This enhances the local concentration of substrates around enzymes so that the enzymatic reaction proceeds more efficiently. We prepared substrate proteins containing an SH3 domain at their N-terminus, and a kinase containing an SH3-binding motif at its C-terminus. This method was successfully applied to the phosphorylation of CrkII and the Vav DH domain, and we prepared (15)N-labelled phosphorylated CrkII for NMR analysis.
Authors:
Yoshihiro Kobashigawa; Masato Naito; Fuyuhiko Inagaki
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2007-08-14
Journal Detail:
Title:  Journal of biotechnology     Volume:  131     ISSN:  0168-1656     ISO Abbreviation:  J. Biotechnol.     Publication Date:  2007 Sep 
Date Detail:
Created Date:  2007-09-24     Completed Date:  2007-12-14     Revised Date:  2009-11-19    
Medline Journal Info:
Nlm Unique ID:  8411927     Medline TA:  J Biotechnol     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  458-65     Citation Subset:  IM    
Affiliation:
Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Hokkaido, Japan.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Animals
Base Sequence
Biotechnology / methods*
GRB2 Adaptor Protein / metabolism
Genetic Vectors
Humans
Mice
Molecular Sequence Data
Phosphorylation
Phosphotransferases / metabolism*
Proto-Oncogene Proteins c-abl / metabolism
Proto-Oncogene Proteins c-crk / metabolism*
Proto-Oncogene Proteins c-vav / metabolism*
Substrate Specificity
Chemical
Reg. No./Substance:
0/GRB2 Adaptor Protein; 0/Proto-Oncogene Proteins c-crk; 0/Proto-Oncogene Proteins c-vav; EC 2.7.-/Phosphotransferases; EC 2.7.10.2/Proto-Oncogene Proteins c-abl

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