biostudies-literatureBaltussen LLCancer Research UKThe Francis Crick InstituteMedical Research CouncilNIMH NIH HHSNational Institute of Mental HealthWellcome TrustInternational Foundation for CDKL5 Researche99763https://www.ebi.ac.uk/biostudies/studies/S-EPMC6293278biostudies-literatureUnknown37(24)Loss-of-function mutations in CDKL5 kinase cause severe neurodevelopmental delay and early-onset seizures. Identification of CDKL5 substrates is key to understanding its function. Using chemical genetics, we found that CDKL5 phosphorylates three microtubule-associated proteins: MAP1S, EB2 and ARHGEF2, and determined the phosphorylation sites. Substrate phosphorylations are greatly reduced in CDKL5 knockout mice, verifying these as physiological substrates. In CDKL5 knockout mouse neurons, dendritic microtubules have longer EB3-labelled plus-end growth duration and these altered dynamics are rescued by reduction of MAP1S levels through shRNA expression, indicating that CDKL5 regulates microtubule dynamics via phosphorylation of MAP1S. We show that phosphorylation by CDKL5 is required for MAThe EMBO journalChemical genetic identification of CDKL5 substrates reveals its role in neuronal microtubule dynamics.PMC629327820152375F31 MH010771R21MH10771FC001003FC0012011001110015136238710201Ultanir SKClaxton SMuotri ARSnijders APMoeskops MFlynn HRChristodoulou EBaltussen LLNegraes PDSilvestre MfalseChemical genetic identification of CDKL5 substrates reveals its role in neuronal microtubule dynamics.Loss-of-function mutations in CDKL5 kinase cause severe neurodevelopmental delay and early-onset seizures. Identification of CDKL5 substrates is key to understanding its function. Using chemical genetics, we found that CDKL5 phosphorylates three microtubule-associated proteins: MAP1S, EB2 and ARHGEF2, and determined the phosphorylation sites. Substrate phosphorylations are greatly reduced in CDKL5 knockout mice, verifying these as physiological substrates. In CDKL5 knockout mouse neurons, dendritic microtubules have longer EB3-labelled plus-end growth duration and these altered dynamics are rescued by reduction of MAP1S levels through shRNA expression, indicating that CDKL5 regulates microtubule dynamics via phosphorylation of MAP1S. We show that phosphorylation by CDKL5 is required for MA2018-01-01T00:00:00Z2018 Dec2026-05-06T06:15:03.111Z2019-03-27T00:12:31ZS-EPMC62932783026682410.15252/embj.201899763