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

Abstract

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, in vitro 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 Corallochytrium limacisporum, a highly diverged holozoan marine protist, is active and can inhibit Src. However, in comparison with other Csk orthologs, Corallochytrium 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 Capsaspora owczarzaki is active and that the Csk-Src negative regulatory mechanism is present in Csk and Src proteins from C. owczarzaki and the choanoflagellate Monosiga brevicollis 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.

Keywords: inhibition mechanism; molecular biology; non-receptor tyrosine kinase (nRTK); protein evolution; protein phosphorylation.

Figure 1.

A schematic tree summarizing the phylogenetic relationship of holozoan organisms and the occurrence of Src and Csk orthologs. The results of functional characterization of Src and Csk activity as well as the presence of negative regulation of Src activity by Csk from previous studies are shown in comparison with the data presented in this study (12, 20–24).

Figure 2.

C. limacisporum Csk retains key functional residues and has a unique domain architecture. A, alignment of Csk orthologs. Domains are indicated by colored lines, with the SH3 domain shown in green, the SH2 domain in red, and the kinase domain in blue. Residues that are important for kinase activity are marked by black diamonds, whereas residues involved in Src binding are marked by yellow triangles. The lysine that was mutated to arginine to produce the kinase-dead mutant is marked with a pink diamond. Kinase domain secondary structure elements are shown below the sequence (arrow, β-sheet; cylinder, α-helix). B, schematic of the domain architectures of each Csk ortholog. Hs, H. sapiens; Ef, E. fluviatilis; Hv, H. vulgaris; Mb, M. brevicollis; Mo, M. ovata; Co, C. owczarzaki; Mv, M. vibrans; Cl, C. limacisporum.

Figure 3.

C. limacisporum Src and Csk are active tyrosine kinases with intact negative regulation. We measured the kinase activity of purified human (HsSrc and HsCsk) and C. limacisporum (ClSrc and ClCsk) kinases using a luminescence assay. A and B, tyrosine phosphorylation activity of HsSrc and ClSrc (A) and HsCsk and ClCsk (B) using poly(Glu₄Tyr) as a substrate. C, tyrosine phosphorylation activity of HsCsk and ClCsk using kinase-dead Src as a substrate. Points represent the mean of three replicate luminescence assays, and error bars represent the standard deviation. D, optical density of yeast cultures 48 h after induction of kinase expression. Under the no Src and no Csk conditions, yeasts were transformed with empty p416GAL1 or p415GAL1 vectors. Each point represents a replicate growth assay started from a distinct transformant, black bars represent the mean of the replicates, and error bars represent the standard deviation. KD, kinase-dead mutant; tail, C-terminal regulatory tyrosine mutant (supplemental Table S1).

Figure 4.

M. brevicollis and C. owczarzaki Src and Csk are active tyrosine kinases. A–D, we measured the kinase activity of purified M. brevicollis (MbSrc1 and MbCsk) and C. owczarzaki (CoSrc2 and CoCsk) kinases using a luminescence assay. Tyrosine phosphorylation activity of MbSrc1 (A), CoSrc2 (B), MbCsk (C), and CoCsk (D) using poly(Glu₄Tyr) as a substrate is shown. Human kinases (HsSrc and HsCsk) were included in each case as controls. Points represent the mean of three replicate luminescence assays, and error bars represent the standard deviation. KD, kinase-dead mutant (supplemental Table S1).

Figure 5.

Csk from M. brevicollis and C. owczarzaki inhibit Src. We measured the kinase activity of purified M. brevicollis (MbCsk) and C. owczarzaki (CoCsk) kinases using a luminescence assay. A and B, tyrosine phosphorylation activity levels of MbCsk (A) and CoCsk (B) using their respective kinase-dead Src proteins as substrates. Mutants of the Src C-terminal regulatory tyrosine and human kinases (HsSrc and HsCsk) were included in each case as controls. Points represent the mean of three replicate luminescence assays, and error bars represent the standard deviation. C and D, optical density of yeast cultures 48 h after induction of kinase expression is shown for M. brevicollis (C) and C. owczarzaki (D). Under the no Src and no Csk conditions, yeasts were transformed with empty p416GAL1 or p415GAL1 vectors. Each point represents a replicate growth assay started from a distinct transformant, black bars represent the mean of the replicates, and error bars represent the standard deviation. KD, kinase-dead mutant; tail, C-terminal regulatory tyrosine mutant (supplemental Table S1).