The family of Cdc2-like kinases (CLKs) plays a critical role in regulating both constitutive and alternative pre-mRNA splicing. Of particular interest, CLKs exhibit unique thermosensitive properties characterized by increased activity at lower physiological temperatures. In this study, we integrate phylogenetic, protein-interaction, and structural analyses to investigate the evolutionary history and functional adaptation of CLKs across protista, fungi, plants, and metazoans. Our phylogenetic analysis, comprising 149 CLK homologs from 86 species, traces this gene back to the last eukaryotic common ancestor. The results reveal lineage-specific patterns of gene duplication and loss, including complete loss of CLKs in seven protist lineages and in microsporidian fungi. Interolog mapping identified 92 conserved CLK-interacting proteins across diverse species. In metazoans, these conserved interactors are primarily involved in complex splicing regulation, whereas in yeast they are associated with simplified RNA-processing mechanisms. Comparative structural modeling shows strong conservation of the kinase domain throughout eukaryotes, although notable divergence occurs in some Fungal and Protista lineages. Intrinsic disorder in the CLK N terminus emerges as a conserved structural property; however, sequence variability in this region modulates kinase activity and substrate specificity. Structural conservation in the activation segment, the core driver of CLK thermosensitivity, is observed across all eukaryotic kingdoms, though deviations were identified in various protist and plant lineages. Deleterious mutations often occur in this region following a duplication or preceding complete gene loss. Finally, species-specific temperature activity profiles underscore the adaptive evolution of CLKs, enabling organisms to thrive in diverse environmental conditions, including extreme temperatures.
