Generation of protein kinase Ck1alpha 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 CK1a 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 K229KQK232 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 kinetic alanine-scan analysis with a series of substituted peptide substrates in which the priming phosphoserine residue was effectively replaced by a cluster of four aspartate residues was also consistent with a crucial role of Lys232 in the recognition of the acidic determinant at position n-4. In sharp contrast, the phosphorylation of b-catenin and of a peptide including the non-canonical b-catenin site (Ser45) lacking acidic/phosphorylated determinants upstream is not significantly affected by mutations in the KKQK stretch. These data provide a molecular insight into the structural features that underlie the site specificity of CK1a and disclose the possibility of developing strategies for the preferential targeting of subsets of CK1 substrates.

Figure 1. The triple mutant K229A,K230A,K232A is variably defective in substrate phosphorylation

Phosphorylation of various peptide and protein substrates by CK1α, either wild-type (open bars) or triply mutated in the K²²⁹KQK²³² cluster (closed bars), was performed and evaluated as detailed in the Experimental section. The activity of the mutant is normalized to that of wild-type (=100%). Specific activities of wild-type CK1α with the different substrates were 132, 280, 3, 14 and 45 pmol of phosphate transferred per min to β-casein, peptide D₄, polyE,Y(4:1), β-catenin and peptide β-cat(38–64) respectively. Similar results were obtained using the single mutant K232A.

Figure 2. Relative efficiencies for the phosphorylation of singly substituted peptides by CK1α mutants

The histograms have been constructed with data taken from Table 2. Efficiencies are normalized to that of the parent peptide D₄ (=100%).

Figure 3. The inhibitory efficacy of polyE,Y(4:1) is abrogated by the K²²⁹KQK²³² triple mutation

Phosphorylation of β-casein (5 mg/ml) by wild-type CK1α (●) and triple mutant K229A,K230A,K232A (■) was determined in the presence of increasing polyE,Y(4:1) concentrations, under conditions detailed in the Experimental section. The activities of both wild-type and mutated CK1α were normalized to those observed in the absence of polyE,Y(4:1) which were 161 and 25 pmol/min per μg respectively.