Figure 6. T‐loop autophosphorylation is critical for CDKL5 activity.

• A, B
  Tyr‐autophosphorylation of CDKL5. Anti‐FLAG precipitates from HEK293 cells transiently expressing FLAG‐tagged CDKL5 (wild type “WT” or mutants K⁴²R kinase‐dead “KD”, Y¹⁶⁸A,Y¹⁷¹A or both) were subjected to immunoblotting with the antibodies indicated before or after incubation of precipitates with Mg²⁺‐ATP for 30 min at 30°C. Each experiment was done three times, and a representative experiment is shown.
• C
  CDKL5 cannot phosphorylate Tyr‐containing synthetic peptides. Anti‐FLAG precipitates from HEK293 cells transiently expressing FLAG‐tagged CDKL5 (wild type “WT” or a K⁴²R kinase‐dead “KD” mutant) were incubated with the synthetic peptides indicated in the presence of [γ‐³²P]‐labelled ATP‐Mg²⁺, and peptide phosphorylation was measured by Cerenkov counting. Data are represented as mean ± SEM from three independent experiments.
• H
  HEK293 cells were co‐transfected with untagged CDKL5 (wild type “WT” or the mutants indicated) and FLAG‐tagged MAP1S [wild type (WT), a Ser⁹⁰⁰Ala mutant (SA)] or empty vector (−). Anti‐FLAG precipitates were subjected to Western blotting with the antibodies indicated. The input extracts were also subjected to immunoblotting (lower panels). Each experiment was done three times, and a representative example is shown.
• I
  Anti‐FLAG precipitates from HEK293 cells transiently expressing FLAG‐tagged CDKL5 (wild type “WT” or the mutants indicated) were incubated with a synthetic peptide corresponding to the sequence around the MAP1S Ser⁹⁰⁰ phosphorylation site, in the presence of [γ‐³²P]‐labelled ATP‐Mg²⁺. Peptide phosphorylation was quantitated in a scintillation counter. Data are represented as mean ± SEM from three independent experiments.

Source data are available online for this figure.