Dynamics of CDKL5 phosphorylation and its regulatory mechanisms in cultured cells

Cyclin-dependent kinase-like 5 (CDKL5) is a serine/threonine protein kinase highly expressed in the brain, and mutations in its gene cause CDKL5 deficiency disorder (CDD). Although reports on CDKL5 substrate proteins are increasing, the phosphorylation-dependent regulation of CDKL5 function remains poorly understood. Therefore, in this study, we investigated the phosphorylation states of CDKL5 and explored the kinases and phosphatases involved in its regulation. The C-terminal region of exogenous CDKL5 was highly phosphorylated in Neuro2a neuroblastoma cells; endogenous CDKL5 in P19 embryonic carcinoma (P19EC) cells was also highly phosphorylated, mainly in the cytoplasm. CDKL5 underwent gradual dephosphorylation during aggregate formation in P19EC cells, and this process was suppressed by retinoic acid (RA), an inducer of neuronal differentiation. Okadaic acid treatment indicated that protein phosphatase 2A (PP2A) partially mediates CDKL5 dephosphorylation during aggregate formation. Kinase inhibitor screening and kinase assay in vitro demonstrated that multiple protein kinase C (PKC) isoforms contribute to CDKL5 phosphorylation. These findings suggest that CDKL5 phosphorylation is dynamically regulated by a balance between PKC and PP2A activities, and that RA signaling modulates this process during neuronal differentiation. Such phosphorylation mechanisms of CDKL5 may underlie the pathogenesis of CDD and help identify potential therapeutic targets.