Investigation of a conserved serine residue in a yeast casein kinase 1 protein kinase

Abstract

Yck1/2 are essential and redundant plasma membrane‐associated casein kinase 1 (CK1) protein kinases in budding yeast. In addition to sequence features common to Ser/Thr protein kinases, CK1 proteins share features unique to this subfamily. We identified the peptide EQSRRDD as such a feature and investigated its role in the function of Yck2 (E258‐D263). Based on location of the conserved peptide in a 3‐D model of CK1 from S. pombe, we propose that this sequence is necessary for substrate binding. We focused on S260 within the conserved sequence, generating mutant alleles that were both integrated into the yeast chromosome to allow investigation of growth phenotypes and expressed as beta‐galactosidase fusion proteins for in vitro kinase assays. Deletion of S260 eliminates Yck2 function. S260T and S260A substitutions have little effect on Yck2 localization, in vitro activity on casein, or growth phenotypes of yeast expressing only the mutant allele. However, S260E substitution results in decreased kinase activity as well as significantly slower growth of yeast expressing only the mutant allele. We are testing the possibility that S260E impairs substrate recognition. Supported by NSF grant MCB‐0517204.