Generation of protein kinase Ck1α mutants which discriminate between canonical and non-canonical substrates

Abstract

Protein kinase CK1 denotes a family of pleiotropic serine/threonine protein kinases implicated in a variety of cellular functions. Typically, CK1 acts as a 'phosphate-directed' kinase whose targeting is primed by a single phosphorylated side chain at position n - 3 or n - 4 relative to serine/threonine, but increasing evidence is accumulating that CK1 can also engage some of its substrates at sites that do not conform to this canonical consensus. In the present paper, we show that CK1α phosphorylates with the same efficiency phosphopeptides primed by a phosphoserine residue at either n - 3 [pS(-3)] or n - 4 [pS(-4)] positions. The phosphorylation efficiency of the pS(-4) peptide, and to a lesser extent that of the pS(-3) peptide, is impaired by the triple mutation of the lysine residues in the K229KQK 232 stretch to alanine residues, promoting 40-fold and 6-fold increases of Km respectively. In both cases, the individual mutation of Lys232 is as detrimental as the triple mutation. A ki