Ogle, R. A.; Netherton, J. K.; Robinson, B. R.; Heyd, F.; Zhang, X. D.; Baker, M. A.

The family of CDC2-like kinases (CLKs) play a crucial role in regulating alternative splicing (AS), a process fundamental to eukaryotic gene expression and adaptation. Of particular interest, these enzymes exhibit unique responsiveness to minor temperature shifts, enabling them to modulate AS accordingly. Dysregulated CLK expression is linked to a wide variety of human diseases, establishing them as promising therapeutic targets. Despite the importance of CLKs, limited research has explored the genetic and functional diversification of this gene family. This report investigates the evolutionary origins, diversification, and functional implications of CLKs across major eukaryotic lineages through phylogenetic and structural comparisons. Our data demonstrate these kinases are prevalent throughout eukaryotes, with the original gene (which shares orthology to human CLK2), dating back to the Last Eukaryotic Common Ancestor. We identified three key duplication events in vertebrates, highlighting how this gene family has expanded and diversified in complex metazoans. Despite two instances of CLK paralog loss in vertebrate lineages, CLKs remain prevalent throughout metazoans, suggesting they are essential for complex eukaryotic life. Structural comparisons across diverse eukaryotes demonstrate kinase domain conservation, which is in line with their maintained function in AS regulation. While the N-terminal region of CLK family members varies significantly in amino acid sequence, the function of this domain to regulate phosphorylation of AS factors is conserved, albeit in a species-specific manner. CLKs exhibit unique thermo-sensitive properties across diverse species, challenging conventional enzymatic behaviour. This temperature regulation, mediated by their kinase activation segment, is characterised by increased activity at lower physiological temperatures. The conservation of this structure, and a thermo-sensitive amino acid motif within it, suggests this was an ancient adaptation for responding to environmental cues. Species-specific temperature profiles highlight the adaptive evolution of CLKs, enabling organisms to thrive in diverse environmental conditions including extreme temperatures. Our analysis expands the understanding of CLK biology across diverse eukaryotes and connects insights from model organisms to human biology.