PT - JOURNAL ARTICLE AU - Adachi, Jun AU - Kakudo, Akemi AU - Takada, Yoko AU - Isoyama, Junko AU - Ikemoto, Narumi AU - Abe, Yuichi AU - Narumi, Ryohei AU - Muraoka, Satoshi AU - Gunji, Daigo AU - Hara, Yasuhiro AU - Katayama, Ryohei AU - Tomonaga, Takeshi TI - Systematic identification of ALK substrates by integrated phosphoproteome and interactome analysis AID - 10.26508/lsa.202101202 DP - 2022 Aug 01 TA - Life Science Alliance PG - e202101202 VI - 5 IP - 8 4099 - https://www.life-science-alliance.org/content/5/8/e202101202.short 4100 - https://www.life-science-alliance.org/content/5/8/e202101202.full SO - Life Sci. Alliance2022 Aug 01; 5 AB - The sensitivity of phosphorylation site identification by mass spectrometry has improved markedly. However, the lack of kinase–substrate relationship (KSR) data hinders the improvement of the range and accuracy of kinase activity prediction. In this study, we aimed to develop a method for acquiring systematic KSR data on anaplastic lymphoma kinase (ALK) using mass spectrometry and to apply this method to the prediction of kinase activity. Thirty-seven ALK substrate candidates, including 34 phosphorylation sites not annotated in the PhosphoSitePlus database, were identified by integrated analysis of the phosphoproteome and crosslinking interactome of HEK 293 cells with doxycycline-induced ALK overexpression. Furthermore, KSRs of ALK were validated by an in vitro kinase assay. Finally, using phosphoproteomic data from ALK mutant cell lines and patient-derived cells treated with ALK inhibitors, we found that the prediction of ALK activity was improved when the KSRs identified in this study were used instead of the public KSR dataset. Our approach is applicable to other kinases, and future identification of KSRs will facilitate more accurate estimations of kinase activity and elucidation of phosphorylation signals.