Development of new antihypertensive drug by comprehensive analysis of WNK signal

2021 Fiscal Year Final Research Report

• Project/Area Number: 20K17244
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 53040:Nephrology-related
• Research Institution: Tokyo Medical and Dental University
• Principal Investigator: ISOBE KIYOSHI 東京医科歯科大学, 大学院医歯学総合研究科, 非常勤講師 (80804591)
• Project Period (FY): 2020-04-01 – 2022-03-31
• Keywords: WNK / アクアポリン２ / ミオシン軽鎖キナーゼ / リン酸化プロテオミクス

Outline of Final Research Achievements

Prior studies have implicated myosin light chain kinase (MLCK) in the regulation of aquaporin-2 (AQP2) in the renal collecting duct. To discover signaling targets of MLCK, we used CRISPR-Cas9 to delete the MLCK gene (Mylk) to obtain MLCK-null mpkCCD cells.　The phosphoproteomics experiments revealed that, of the 1,743 phosphopeptides quantified over multiple replicates, 107 were changed in abundance by MLCK deletion (29 decreased and 78 increased). The presence of multiple proteins in the actomyosin category prompted experiments showing that MLCK deletion inhibits the normal effect of vasopressin to depolymerize F-actin, providing a potential explanation for the AQP2 trafficking defect. We conclude that MLCK is part of a multicomponent signaling pathway in both the cytoplasm and nucleus that includes much more than simple regulation of conventional nonmuscle myosins through myosin regulatory light chain phosphorylation.

Free Research Field

腎臓内科学

Academic Significance and Societal Importance of the Research Achievements

今回我々はCRISPR-Cas9ゲノム編集技術とリン酸化プロテオミクスを用いてMYLKの細胞内リン酸化シグナルを詳細に解明することができた。MYLKは従来ミオシン軽鎖をリン酸化しミオシンの機能を調節する機能しか知られていなかったが実際には多岐にわたる機能があることが判明した。細胞内シグナルの解明により新たな創薬につなげていく重要な情報基盤となる。また、この手法は細胞内の特定のリン酸化酵素の役割を詳細に解明することが出来、新たなシグナル伝達を発見することにつながることが証明されたといえる。