Substrate specificity of Ca(2+)/calmodulin-dependent protein kinase phosphatase: kinetic studies using synthetic phosphopeptides as model substrates

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概要

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• This is a pre-copy-editing, author-produced PDF of an article accepted for publication in Journal of Biochemistry following peer review. The definitive publisher-authenticated version Oxford University Press, Journal of Biochemistry, 129(5), 2001, 745-753 is available online at: http://jb.oxfordjournals.org/cgi/content/abstract/129/5/745.Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKPase) dephosphorylates and regulates multifunctional Ca(2+)/calmodulin-dependent protein kinases. In order to elucidate the mechanism of substrate recognition by CaMKPase, we chemically synthesized a variety of phosphopeptide analogs and carried out kinetic analysis using them as CaMKPase substrates. This is the first report using systematically synthesized phosphopeptides as substrates for kinetic studies on substrate specificities of protein Ser/Thr phosphatases. CaMKPase was shown to be a protein Ser/Thr phosphatase having a strong preference for a phospho-Thr residue. A Pro residue adjacent to the dephosphorylation site on the C-terminal side and acidic clusters around the dephosphorylation site had detrimental effects on dephosphorylation by CaMKPase. Deletion analysis of a model substrate peptide revealed that the minimal length of the substrate peptide was only 2 to 3 amino acid residues including the dephosphorylation site. The residues on the C-terminal side of the dephosphorylation site were not essential for dephosphorylation, whereas the residue adjacent to the dephosphorylation site on the N-terminal side was essential. Ala-scanning analysis suggested that CaMKPase did not recognize a specific motif around the dephosphorylation site. Myosin light chain phosphorylated by protein kinase C and Erk2 phosphorylated by MEK1 were poor substrates for CaMKPase, while a synthetic phosphopeptide corresponding to the sequence around the phosphorylation site of the former was not dephosphorylated by CaMKPase but that of the latter was fairly good substrate. These data suggest that substrate specificity of CaMKPase is determined by higher-order structure of the substrate protein rather than by the primary structure around its dephosphorylation site. Use of phosphopeptide substrates also revealed that poly-L-lysine, an activator for CaMKPase, activated the enzyme mainly through increase in the V(max) values.

• Japanese Biochemical Societyの論文
• 2001-05-01

著者

• 藤澤 仁

  Department Of Biochemistiy Asahikawa Medical College

• TAKEUCHI Masayuki

  Department of Chemistry and Biochemistry, Graduate School of Engineering, Kyushu University

• Tatsu Yoshiro

  National Institute of Advanced Industrial Science and Technology (AIST)

• Yumoto Noboru

  National Institute of Advanced Industrial Science and Technology (AIST)

• ENDO Yasuko

  National Institute of Advanced Industrial Science and Technology (AIST)

• OKUNO Sachiko

  Department of Biochemistry, Asahikawa Medical College

• KITANI Takako

  Department of Biochemistry, Asahikawa Medical College

• FUJISAWA Hitoshi

  Department of Biochemistry, Asahikawa Medical College

• 亀下 勇

  Kagawa Univ. Kagawa Jpn

• Kameshita I

  Department Of Life Sciences Faculty Of Agriculture Kagawa University

• Kameshita Isamu

  Department Of Life Sciences Faculty Of Agriculture Kagawa University

• Kameshita Isamu

  Department Of Biochemistry Asahikawa Medical College

• ISHIDA Atsuhiko

  Department of Biochemistry, Asahikawa Medical College

• SHIGERI Yasushi

  National Institute of Advanced Industrial Science and Technology (AIST)

• 竹内 昌之

  旭川医大・1生化

• Takeuchi Masayuki

  Department Of Biochemistry Asahikawa Medical College

• 藤澤 仁

  旭川医大・生化学

• Endo Yaeta

  National Institute Of Advanced Industrial Science And Technology (aist)

• Endo Yasuko

  National Institute Of Advanced Industrial Science And Technology

• Tatsu Yoshiro

  National Institute Of Advanced Industrial Science And Technology

• Yumoto N

  National Institute Of Advanced Industrial Science And Technology (aist)

• Yumoto Noboru

  National Institute Of Advanced Industrial Science And Technology

• 木谷 隆子

  Department Of Biochemistry Asahikawa Medical College

• Okuno Sachiko

  Department Of Biochemistry Asahikawa Medical College

• Shigeri Yasushi

  National Institute Of Advanced Industrial Science And Technology

• Takeuchi M

  Department Of Biochemistry Asahikawa Medical College

• Fujisawa Hitoshi

  Department Of Biochemistry Asahikawa Medical College

• Kameshita Isamu

  Kagawa Univ. Kagawa Jpn

• Ishida Atsuhiko

  Dep. Of Biochemistry Asahikawa Medical Coll. Jpn

• Sugiyama Yasunori

  Department Of Life Sciences Faculty Of Agriculture Kagawa University

• 石田 敦彦

  Laboratory Of Molecular Brain Science Graduate School Of Integrated Arts And Sciences Hiroshima Univ

• Kitani T

  Department Of Clinical Laboratory Medicine Shiga University Of Medical Science

• Fujisawa H

  Department Of Applied Chemistry Faculty Of Science Science University Of Tokyo

• Endo Y

  National Institute of Advanced Industrial Science and Technology (AIST)

• Okuno S

  Department of Biochemistry, Asahikawa Medical College

• Ishida Atsuhiko

  Department of Biochemistry, Asahikatca Medical College

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