Expression, purification, crystallization, and preliminary x-ray analysis of casein kinase-1 from Schizosaccharomyces pombe.

G Carmel,B Leichus,X Cheng,S.D Patterson,U Mirza,B.T Chait,J Kuret

doi:10.1016/s0021-9258(17)37284-8

G Carmel, B Leichus + Show 5 more

Open Access

https://doi.org/10.1016/s0021-9258(17)37284-8

Copy DOI

Abstract

The catalytic domain of Schizosaccharomyces pombe casein kinase-1 (the product of the cki1 gene) has been overexpressed in Escherichia coli, purified by chromatographic methods, characterized in vitro, and crystallized in the presence and absence of nucleotide substrate. The best crystals belong to the trigonal space group P3(1)21 or its enantiomorph, have unit cell parameters a = b = 79 A, c = 121 A, and diffract x-rays to 2.0-A resolution. Kinetic characterization of the purified catalytic domain and other C-terminal deletion mutants of Cki1 suggests that it is subject to two forms of regulation. One mechanism involves autophosphorylation, and results in a 4-fold decrease in the affinity for protein substrate. In contrast, truncation of intact Cki1 results in a 3-fold activation in its catalytic rate. This activation may arise from the removal of an inhibitory domain present in the intact enzyme.

Highlights

The catalytic domain of Schizosaccharomyces pombe tions -3 or -4 (relative to thpeosition of a phosphorylatable Ser casein kinase-1 has been or Thr residue), substitutionof those carboxylic acid residues overexpressed in Escherichiacoli, purifiedbychrowith phosphoserine yields a dramatically superior substrate matographic methods,characterizedin vitro, and crys- (Flotow et al, 1990; Meggio et al, 1991; Perich et al, 1992; tallized in the presence and absence of nucleotide sub- Umpress et al, 1992)
Casein kinase-1 (CK1) resolution.Kinetic characterizationof the purified catalytic domain and other C-terminal deletion mutants of C k i l suggests that it is subject to two forms of regulation
Once considered a single entity, it is known to consist of subspecies that together comprise a distinct branch of the eukaryotic protein kinase family (Rowles et al, 1991; Wang et al, 1992; Robinson et al, 1992)

Summary

AND DISCUSSION

Tations, was loaded directly onto a 350-ml (2.2 x 91 cm)column of C-terminal Deletions of Ckil-On the basis of its primary. In the IA.Like Ycklp andYckap, it consists of a typical CK1 catalytic morning, a fine white precipitate was removed by centrifugation (20 core (residues 8-298) followed by a 12-residue segment (resimin at000 x g)and the resulting supernatant loaded oan5toml(l.2 x 5 cm) Sepharose-Q fast flow column equilibrated in dialysis buffer. Fraction 4 was loaded onto a n 8 ml (8 x 75-mm) Monos high performance liquid chromatography column equilibrated in Buffer C (10 dues 299-310) that isconserved among Yckp and Cki proteins but not other forms of CK1. CkilA298 elutesa s a series of sharp peaks between 150 and 200 mM NaCl. Individual protein peaks were pooled and concentrated by centrifugal filtration (Centricon-30A; micon, MA). Individual protein peaks were pooled and concentrated by centrifugal filtration (Centricon-30A; micon, MA) These conhydrophilic tether, because it links thecatalytic domain to the C-terminal localization signal (Wang et al, 1992). Centrates were made1m~ dithiothreitol, adjusted to 10mglml protein, To better define the amino acid residues that comprise the and stored at 4 "C for up t o 1month

Enzyme kmt”

Net difference*

Although crystals grown under theseconditions are frequently