The CDK-activating Kinase (Cak1p) from Budding Yeast Has an Unusual ATP-binding Pocket

Deborah A Enke,Philipp Kaldis,Jennifer K Holmes,Mark J Solomon

doi:10.1074/jbc.274.4.1949

Abstract

Cak1p is an essential protein kinase that phosphorylates and thereby activates the major cyclin-dependent kinase in budding yeast, Cdc28p. The sequence of Cak1p differs from other members of the protein kinase superfamily in several conserved regions. Cak1p lacks the highly conserved glycine loop motif (GXGXXG) that is found in the nucleotide binding fold of virtually all protein kinases and also lacks a number of conserved amino acids found at sites throughout the protein kinase core sequence. We have used kinetic and mutagenic analyses to investigate whether these sequence differences affect the nucleotide-binding properties of Cak1p. Although Cak1p differs dramatically from other protein kinases, it binds ATP with a reasonable affinity, with a KM of 4.8 microM. Mutations of the putative invariant lysine in Cak1p (Lys-31), homologous to a residue required for activity in virtually all protein kinases and that interacts with the ATP phosphates, moderately reduced the ability of Cak1p to bind ATP but did not dramatically affect the catalytic rate of the kinase. Similarly, Cak1p is insensitive to the ATP analog 5'-fluorosulfonylbenzoyladenosine, which inhibits most protein kinases through covalent modification of the invariant lysine. We found that Cak1p is tolerant of mutations within its glycine loop region. Remarkably, Cak1p remains functional even following truncation of its first 31 amino acids, including the glycine loop region and the invariant lysine. We conclude that the Cak1p nucleotide-binding pocket differs significantly from those of most other protein kinases and therefore might provide a specific target for an inhibitory drug.

Highlights

Members of the protein kinase superfamily are related by several highly conserved amino acid motifs that make up the catalytic core [1, 2]
The importance of this motif is further underscored by the fact that a mutation of the third glycine residue in the tyrosine kinase domain of the insulin receptor impairs protein kinase activity and has been implicated in one form of human diabetes [16]
Determination of the Basic Kinetic Parameters for Cak1p— Because of the extensive differences between Cak1p and other protein kinases, within the ATP-binding site, we decided to characterize the ability of Cak1p to utilize nucleotides (ATP and GTP) and protein substrate

Summary

EXPERIMENTAL PROCEDURES

Buffers—CAK buffer is composed of 50 mM Tris, pH 7.5, 15 mM MgCl2, 1 mg/ml ovalbumin, 10 mM DTT, 0.5% Tween 20, and 1ϫ protease inhibitors. To determine the effect of FSBA on ATPase activity, 5 ␮l of mix containing 2.9 ␮M baculovirus GST-Cak1p, 100 ␮M FSBA, 10 mM DTT in FSBA buffer or 4 ␮l of mix lacking DTT was preincubated for 30 min at room temperature. 5 ␮l of ATP mix containing 100 ␮M ATP, 5 ␮Ci/␮l [␥-32P]ATP in FSBA buffer was added, and the reactions were incubated for 1 h at room temperature. Thermal Stability—To determine the thermal stability of wild type and mutant GST-Cak1p, 700 nM GST-Cak1p in CAK buffer was incubated at a given temperature for 10 min and stored on ice until assay. 5 ␮l of 1.5 ␮M CDK2, 200 ␮M ATP, 0.5 ␮Ci/␮l [␥-32P]ATP in CAK buffer was added, and the samples were incubated for 10 min at room temperature.

RESULTS

TABLE I

DISCUSSION