A phylogenetic analysis of the CDKL protein family unravels its evolutionary history and supports the Drosophila model of CDKL5 deficiency disorder

Abstract

The human CDK-like (CDKL) family of serine‒threonine kinases has five members (CDKL1-5), with a conserved N-terminal kinase domain and variable C-termini. Among these, CDKL5 is of particular interest because of its involvement in CDKL5 deficiency disorder (CDD), a rare epileptic encephalopathy with several comorbidities for which there are no specific treatments. Current CDD vertebrate models are seizure resistant, which could be explained by the genetic background, including leaky expression of other CDKLs. Thus, phylogenetic analysis of the protein family would be valuable for understanding current models and developing new ones. Our phylogenetic studies revealed that ancestral CDKLs were present in all major eukaryotic clades and had ciliary/flagellar functions, which may have diversified throughout evolution. The original CDKL, which was likely similar to human CDKL5, gave rise to the remaining family members through successive duplications. In addition, particular clades have undergone further gene duplication and loss, a pattern that suggests some functional redundancy among them. A separate study focusing on the C-terminal tail of CDKL5 suggested that this domain is only functionally relevant in jawed vertebrates. We have developed a model of CDD in Drosophila based on downregulation of the single Cdkl gene by RNAi, which results in phenotypes similar to those of CDD patients, that are rescued by re-expression of fly Cdkl and human CDKL5. CDKL proteins contain a conserved kinase domain, originally involved in ciliary maintenance; therefore, invertebrate model organisms can be used to investigate CDKL functions that involve the aforementioned domain.

Keywords: CDKL5 deficiency disorder; ciliogenesis; cyclin-dependent-kinase like; epileptic encephalopathy; genetic redundancy; phylogeny; protein family.

FIGURE 1

Phylogeny of the kinase domain of CDKL proteins. The aligned sequences of the kinase domain of CDKL proteins were used to construct a phylogenetic tree by the Bayesian method. Branch lengths are proportional to the amount of genetic change measured as substitutions per site; the scale bar is shown below the tree. For each branch, the bootstrap statistical support is indicated as a percentage. For each sequence, the accession number and species are indicated. On the right, we indicate the protein families defined on the basis of the phylogeny.

FIGURE 2

Proposed evolutionary history of the CDKL protein family. (A) CDKL proteins are color-coded according to their class. A red cross indicates the loss of a CDKL class in all species of a branch, and a half cross indicates loss in some species only (see text for more details). Complete lack of CDKL proteins is indicated by the red type. The appearance of new families is marked by a purple arrow. (B) Cartoon of CDKL proteins representative of the main classes color-coded as above, with kinase domains aligned; coordinates indicate the first and last amino acids of the kinase domain, and the total size of the protein; and amino acid identity matrix for the kinase domain. Dmel: D. melanogaster, Cele: C. elegans, Hsap: H. sapiens, Dkin; D. kinnereticum, Amel: A. mellifera.

FIGURE 3

Phylogeny of the C-terminal tail of CDKL5 proteins. The alignment of the C-terminal regions beyond the kinase domain was used to construct a phylogenetic tree by the Bayesian method. The scale bar indicates substitutions per site. For each branch, the bootstrap statistical support is indicated as a percentage. For each sequence, the accession number and species are indicated.

FIGURE 4

Structure and function of the CG7236 (Cdkl) locus. (A, A′) Annotated transcriptional isoforms of the CG7236 gene; exons are indicated by grey boxes, introns are indicated by solid lines, and the region targeted by JF02655 RNAi is indicated by a red box (A). Relative expression level of the four isoforms (A′). (B) Reduction of Cdkl transcript levels by driving the expression of UAS-Cdkl ^RNAi (grey) via ubiquitous Act5C-Gal4 or pan-neuronal elav-Gal4, compared with an irrelevant UAS-luc ^RNAi (white); color codes are maintained throughout the figure. (C) Increased susceptibility to spontaneous and mechanically and thermally induced seizures (number of episodes) in elav>Cdkl ^RNAi individuals. (D) Decrease in negative geotaxis (% flies reaching the 8 cm mark) and flight stabilization (distance until stabilization) in elav>Cdkl ^RNAi individuals. (E) elav>Cdkl ^RNAi flies have a shortened lifespan. (F) Behavioural alterations in elav>Cdkl ^RNAi individuals in a closed arena regarding total distance walked and the proportion of time spent in the centre. Statistics: (A′, B) ANOVA, Šídák’s multiple comparisons test; (C,D,F) Student’s t-test, except Flight and Time in the Centre, Mann‒Whitney; (E) Log-rank (Mantel‒Cox) test; bars represent mean ± SD (**p < 0.01; ***p < 0.005; ****p < 0.0001).

FIGURE 5

Rescue with Cdkl and CDKL5. (A) Effects of elav-Gal4-directed expression of UAS-Cdkl and UAS-CDKL5 on the transcript levels of Cdkl in head extracts compared with the empty attP insertion site. (B) Susceptibility to spontaneous, mechanically and thermally induced seizures upon expression of UAS-Cdkl and UAS-CDKL5. (C) Negative geotaxis in the same genotypes. (D) Comparison of Cdkl transcript levels in control flies (UAS-luc ^RNAi ), UAS-Cdkl ^RNAi , and the rescues with UAS-Cdkl and UAS-CDKL5. (E) Rescue of the seizure phenotypes of elav>Cdkl ^RNAi by coexpressing Cdkl and CDKL5. (F) Rescue of the negative geotaxis phenotypes of elav>Cdkl ^RNAi by coexpressing Cdkl and CDKL5. Statistics: ANOVA in all panels; multiple comparisons tests (A–C) Dunnett’s test and (D–F) Šídák’s test; bars represent mean ± SD (*p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.0001).