Investigation of a potential tyrosine phosphorylation site in the activation loop of a yeast casein kinase 1 protein kinase

Yck1/2 are essential and redundant proteins in Saccharomyces cerevisiae . They are members of the CK1 protein kinase family, which regulates a range of functions from chromosome segregation to circadian rhythm. Mechanisms of regulation in this family are largely unknown. We tested the hypothesis that phosphorylation of tyrosine 225 (Y225) in the Yck2 activation loop inhibits activity. Using site‐directed mutagenesis, Y225 was converted to glutamate (Y225E) and phenylalanine (Y225F) to mimic constitutive phosphorylation and dephosphorylation, respectively. The resulting mutants were analyzed in vivo and in vitro. In vivo growth assays demonstrated that Y225F supports growth comparable to wt Yck2; it rescued both temperature‐sensitive cells and yck1,2Δ cells, whereas the Y225E variant fails to support growth of either strain. The results from in vitro kinase assays are consistent: the Y225F mutant is as effective as wild‐type at phosphorylation of protein and peptide substrates, while Y225E is almost nonfunctional and cannot be rescued by increasing the concentration of ATP or protein substrate. Computer modeling of the protein's solvent accessibility showed a potential alteration in the ATP binding pocket in the Y225E protein, but not the Y225F variant. Although these results are consistent with an effect of phosphorylation of Y225 on Yck2 activity, altering this residue to other residues yielded results less consistent with this hypothesis. We are currently purifying Yck2 protein for direct assessment of phosphorylation sites.