The widespread prevalence of CDC2-like kinases (CLKs) and their homologs across eukaryotes strongly suggests they are functionally important genes. The activation segment of these kinases exhibits unique temperature-dependent regulation of kinase activity, which increases as temperature declines. This is relevant in the context of spermatogenesis which is exceptionally temperature sensitive and must occur ~3°C below core body temperature. Testicular hyperthermia, even by an increase of 1-3°C, leads to reduced sperm output and quality. This temperature profile range is consistent with the activity of mammalian CLKs, which increase ~4-fold when the temperature is dropped from 38°C to 35°C. Furthermore, the knockout of the CLK homologs in Drosophila melanogaster or Caenorhabditis elegans results in complete sterility, with testis-specific knockout of DOA further demonstrating its essential function in spermatogenesis.
To understand their diversification and determine whether these kinases serve essential functions in male fertility, we investigated their phylogenetic history, structural conservation, and impact of gene loss. Phylogenetic analysis reveals the expansion of CLK-related genes throughout eukaryotic diversification, with simple organisms including fly, worm and yeast possessing just one gene, whilst complex organisms such as mammals having a total of four homologs. Knockout studies conducted in various model organisms highlighted the essential nature of specific CLKs, and their critical roles in embryonic development, neural tissue formation, and reproductive function in diverse eukaryotes. These models also suggest that this indispensable function may have been partitioned through the mechanism of gene subfunctionalisation. By integrating findings of the phylogenetic analysis, structural comparisons, gene loss, and knockout studies, we can gain insight into essential functions of CLKs to further our understanding of their diverse roles, including male reproductive function.