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Updated: Apr 14, 2026

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
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Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Published on: May 3, 2018

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Dynamics of CDKL5 phosphorylation and its regulatory mechanisms in cultured cells

Syouichi Katayama1,2, Miwako Sakota2, Atsushi Morii3

  • 1Division of Informatics, Bioengineering and Bioscience, Faculty of Engineering, Maebashi Institute of Technology, 460-1 Kamisadori, Maebashi, Gunma 371-0816, Japan.

Journal of Biochemistry
|April 12, 2026
PubMed

Insights

Cyclin-dependent kinase-like 5 (CDKL5) phosphorylation is regulated by protein kinase C (PKC) and protein phosphatase 2A (PP2A). Retinoic acid (RA) signaling influences this balance during neuronal differentiation, impacting CDKL5 deficiency disorder (CDD) pathogenesis.

Area of Science:

  • Neuroscience
  • Molecular Biology
  • Biochemistry

Background:

  • Cyclin-dependent kinase-like 5 (CDKL5) is crucial for brain development, and its gene mutations cause CDKL5 deficiency disorder (CDD).
  • The phosphorylation-dependent regulation of CDKL5 function is not well understood, despite increasing knowledge of its substrates.

Purpose of the Study:

  • To investigate the phosphorylation status of CDKL5.
  • To identify the specific kinases and phosphatases regulating CDKL5 phosphorylation.
  • To explore the role of retinoic acid (RA) signaling in modulating CDKL5 phosphorylation during neuronal differentiation.

Main Methods:

  • Investigated exogenous and endogenous CDKL5 phosphorylation in Neuro2a and P19 embryonic carcinoma (P19EC) cells.
  • Utilized okadaic acid to assess the role of protein phosphatase 2A (PP2A) in CDKL5 dephosphorylation.
  • Employed kinase inhibitor screening and in vitro kinase assays to identify contributing kinases, including protein kinase C (PKC) isoforms.

Main Results:

  • Exogenous and endogenous CDKL5 were found to be highly phosphorylated, primarily in the cytoplasm.
  • CDKL5 dephosphorylation occurred during P19EC cell aggregate formation and was inhibited by retinoic acid (RA).
  • PP2A was identified as a key phosphatase in CDKL5 dephosphorylation, while multiple PKC isoforms were found to phosphorylate CDKL5.

Conclusions:

  • CDKL5 phosphorylation is dynamically regulated by the interplay between PKC and PP2A activities.
  • RA signaling modulates CDKL5 phosphorylation during neuronal differentiation, suggesting a link to CDD pathogenesis.
  • Understanding these phosphorylation mechanisms may reveal therapeutic targets for CDD.
Keywords:
Phos-tag SDS-PAGEcyclin-dependent kinase-like 5 (CDKL5)protein kinase C (PKC)protein phosphatase 2A (PP2A)protein phosphorylation

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