Early emergence of negative regulation of the tyrosine kinase Src by the C-terminal Src kinase.

Stringent regulation of tyrosine kinase activity is essential for normal cellular function. In humans, the tyrosine kinase Src is inhibited via phosphorylation of its C-terminal tail by another kinase, C-terminal Src kinase (Csk). Although Src and Csk orthologs are present across holozoan organisms, including animals and protists, the Csk-Src negative regulatory mechanism appears to have evolved gradually. For example, in choanoflagellates, Src and Csk are both active, but the negative regulatory mechanism is reportedly absent. In filastereans, a protist clade closely related to choanoflagellates, Src is active, but Csk is apparently inactive. In this study, we use a combination of bioinformatics, kinase assays, and yeast-based growth assays to characterize holozoan Src and Csk orthologs. We show that, despite appreciable differences in domain architecture, Csk from , a highly diverged holozoan marine protist, is active and can inhibit Src. However, in comparison with other Csk orthologs, Csk displays broad substrate specificity and inhibits Src in an activity-independent manner. Furthermore, in contrast to previous studies, we show that Csk from the filasterean is active and that the Csk-Src negative regulatory mechanism is present in Csk and Src proteins from and the choanoflagellate Our results suggest that negative regulation of Src by Csk is more ancient than previously thought and that it might be conserved across all holozoan species.