Phosphorylated TandeMBP: A unique protein substrate for protein phosphatase assay

详细信息    查看全文

• 作者：Yasunori Sugiyama^a ; ^{sugiyama@ag.kagawa-u.ac.jp" class="auth_mail" title="E-mail the corresponding author} ; Sho Yamashita^a ; Yuuki Uezato^a ; Yukako Senga^a ; ^b ; Syouichi Katayama^a ; Naoki Goshima^c ; Yasushi Shigeri^d ; Noriyuki Sueyoshi^a ; Isamu Kameshita^a
• 关键词：Phosphatase assay ; Protein phosphatase ; Protein substrate ; Myelin basic protein ; Malachite green assay
• 刊名：Analytical Biochemistry
• 出版年：2016
• 出版时间：15 November 2016
• 年：2016
• 卷：513
• 期：Complete
• 页码：47-53
• 全文大小：1041 K

文摘

To analyze a variety of protein phosphatases, we developed phosphorylated TandeMBP (P-TandeMBP), in which two different mouse myelin basic protein isoforms were fused in tandem, as a protein phosphatase substrate. P-TandeMBP was prepared efficiently in four steps: (1) phosphorylation of TandeMBP by a protein kinase mixture (Ca²⁺/calmodulin-dependent protein kinase Iδ, casein kinase 1δ, and extracellular signal-regulated kinase 2); (2) precipitation of both P-TandeMBP and protein kinases to remove ATP, Pi, and ADP; (3) acid extraction of P-TandeMBP with HCl to remove protein kinases; and (4) neutralization of the solution that contains P-TandeMBP with Tris. In combination with the malachite green assay, P-TandeMBP can be used to detect protein phosphatase activity without using radioactive materials. Moreover, P-TandeMBP served as an efficient substrate for PPM family phosphatases (PPM1A, PPM1B, PPM1D, PPM1F, PPM1G, PPM1H, PPM1K, and PPM1M) and PPP family phosphatase PP5. Various phosphatase activities were also detected with high sensitivity in gel filtration fractions from mouse brain using P-TandeMBP. These results indicate that P-TandeMBP might be a powerful tool for the detection of protein phosphatase activities.