Abstract
The cyclin-dependent kinase-like 5 (CDKL5) gene has been associated with rare neurodevelopmental disorders characterized by the early onset of seizures and intellectual disability. The CDKL5 protein is widely expressed in most tissues and cells with both nuclear and cytoplasmic localization. In post-mitotic neurons CDKL5 is mainly involved in dendritic arborization, axon outgrowth, and spine formation while in proliferating cells its function is still largely unknown. Here, we report that CDKL5 localizes at the centrosome and at the midbody in proliferating cells. Acute inactivation of CDKL5 by RNA interference (RNAi) leads to multipolar spindle formation, cytokinesis failure and centrosome accumulation. At the molecular level, we observed that, among the several midbody components we analyzed, midbodies of CDKL5-depleted cells were devoid of HIPK2 and its cytokinesis target, the extrachromosomal histone H2B phosphorylated at S14. Of relevance, expression of the phosphomimetic mutant H2B-S14D, which is capable of overcoming cytokinesis failure in HIPK2-defective cells, was sufficient to rescue spindle multipolarity in CDKL5-depleted cells. Taken together, these results highlight a hitherto unknown role of CDKL5 in regulating faithful cell division by guaranteeing proper HIPK2/H2B functions at the midbody.
Highlights
cyclin-dependent kinase-like 5 (CDKL5) is a serine-threonine kinase that was identified through a transcriptional mapping study aimed at identifying disease causing genes in the Xp22 region[1]
The duration of prometaphase and cytokinesis is reported in box plot graphs (***p < 0.001, unpaired t test). (b) Still images related to Supplementary Movies S1, S2 and S3, respectively upper, middle and lower panels
10 μm. (c, d) Midbody localization of HIPK2 (c) and H2B phosphorylated at serine 14 (S14) (d; H2B-S14P) was analyzed in the indicated HeLa cells 60 h post-silencing by staining with Abs against α–tubulin and HIPK2/ H2B-S14P and with DAPI
Summary
CDKL5 is a serine-threonine kinase that was identified through a transcriptional mapping study aimed at identifying disease causing genes in the Xp22 region[1]. CDKL5 overexpression induces cell cycle arrest in neuroblastoma cells[11] whereas CDKL5 inhibition, by RNAi or targeted gene disruption, was shown to increase bromodexoyuridine incorporation[11, 12]. These data suggest the involvement of CDKL5 in cell proliferation, no information is available regarding the functions and the subcellular localization of the kinase during the cell cycle. The centrosome serves as the main microtubule-organizing center that contributes to cell adhesion, motility, and polarity in interphase and to bipolar spindle formation and timely mitotic progression in mitosis[15, 16]. In HIPK2-defective cells, expression of a phosphomimetic H2B-S14D mutant overcomes the cytokinesis failure[21]
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