OsRLCK185 is a functional kinase, we then examined whether OsRLCK185 phosphorylates OsPUB20 using an in vitro kinase assay, OsRLCK185K108A served as a negative control. The results showed that GST-OsRLCK185, but not GST-OsRLCK185K108A, phosphorylates OsPUB20 in vitro. Using LC-MS/MS analysis after an in vitro phosphorylation reaction with GST-OsRLCK185 and His-OsPUB20, we identified threonine 153 (T153), located between U-box domain and ARM repeats of OsPUB20, as the OsRLCK185 phosphorylation site.