We have developed a method to study the primary sequence specificities of protein kinases by using an oriented degenerate peptide library. We report here the substrate specificities of eight protein Ser/Thr kinases. All of the kinases studied selected distinct optimal substrates. The identified substrate specificities of these kinases, together with known crystal structures of protein kinase A, CDK2, Erk2, twitchin, and casein kinase I, provide a structural basis for the substrate recognition of protein Ser/Thr kinases. In particular, the specific selection of amino acids at the ؉1 and ؊3 positions to the substrate serine/threonine can be rationalized on the basis of sequences of protein kinases. The identification of optimal peptide substrates of CDK5, casein kinases I and II, NIMA, calmodulin-dependent kinases, Erk1, and phosphorylase kinase makes it possible to predict the potential in vivo targets of these kinases.The essential role of protein kinases in regulating signal transduction was established with the discovery of cyclic AMPdependent protein kinase (PKA) (12). To respond to different extracellular stimuli, distinct groups of protein kinases have evolved. Each protein kinase is thought to phosphorylate a unique set of targets in the cell. The substrate specificities of protein kinases are therefore crucial for the fidelity of signaling events.The classical approach for studying the specificity of a protein kinase is to compare the phosphorylation kinetics of synthetic peptides on the basis of known sequences phosphorylated by the kinase. This procedure is helpful in identifying the amino acids critical for efficient phosphorylation. However, it is not practical to synthesize and study each of the billions of possible variations of sequences that must be considered. Moreover, it is extremely difficult to apply this approach to study the specificity of a protein kinase with no known substrates. To overcome these problems, we developed a method for determining the primary sequence specificities of protein kinases by using an oriented degenerate peptide library (21). Optimal peptide substrates of a given protein kinase are identified by phosphorylation of a pool of degenerate peptides containing billions of different species. The specificities determined for PKA, CDC2, and CDK2 by using this technique were consistent with known substrates of these kinases. The results also allowed the prediction of in vivo kinase substrates. Synthetic peptides based on predicted optimal motifs were shown to act as low-K m substrates for the kinases studied. Therefore, this method is a useful tool for studying substrate specificities of protein kinases.We present here the specificities of eight additional protein Ser/Thr kinases: CDK5, casein kinase I (CKI) ␦ and ␥, casein kinase II (CKII), NIMA, calmodulin-dependent (Cam) kinase II, Erk1, and phosphorylase kinase. Our findings demonstrate that each of these protein kinases has a distinct optimal peptide substrate. Critical determinants for recognition by the protein kinas...

The rad3 gene of Schizosaccharomyces pombe is required for checkpoint pathways that respond to DNA damage and replication blocks. We report the complete rad3 gene sequence and show that rad3 is the homologue of Saccharomyces cerevisiae ESR1 (MEC1/SAD3) and Drosophila melanogaster mei‐41 checkpoint genes. This establishes Rad3/Mec1 as the only conserved protein which is required for all the DNA structure checkpoints in both yeast model systems. Rad3 is an inessential member of the ‘lipid kinase’ subclass of kinases which includes the ATM protein defective in ataxia telangiectasia patients. Mutational analysis indicates that the kinase domain is required for Rad3 function, and immunoprecipitation of overexpressed Rad3 demonstrates an associated protein kinase activity. The previous observation that rad3 mutations can be rescued by a truncated clone lacking the kinase domain may be due to intragenic complementation. Consistent with this, biochemical data suggest that Rad3 exists in a complex containing multiple copies of Rad3. We have identified a novel human gene (ATR) whose product is closely related to Rad3/Esr1p/Mei‐41. ATR can functionally complement esr1–1 radiation sensitivity in S. cerevisiae. Together, the structural conservation and functional complementation suggest strongly that the mechanisms underlying the DNA structure checkpoints are conserved throughout evolution.

In simple eukaryotes, protein kinases regulate mitotic and meiotic cell cycles, the response to polypeptide pheromones, and the initiation of nuclear DNA synthesis. The protein HRR25 from the budding yeast Saccharomyces cerevisiae was defined by the mutation hrr25-1. This mutation resulted in sensitivity to continuous expression of the HO double-strand endonuclease, to methyl methanesulfonate, and to x-irradiation. Homozygotes of hrr25-1 were unable to sporulate and disruption and deletion of HRR25 interfered with mitotic and meiotic cell division. Sequence analysis revealed two distinctive regions in the protein. The NH2-terminus of HRR25 contains the hallmark features of protein kinases, whereas the COOH-terminus is rich in proline and glutamine. Mutations in HRR25 at conserved residues found in all protein kinases inactivated the gene, and these mutants exhibited the hrr25 null phenotypes. Taken together, the hrr25 mutant phenotypes and the features of the gene product indicate that HRR25 is a distinctive member of the protein kinase superfamily.

Members of the casein kinase-1 family of protein kinases play an essential role in cell regulation and disease pathogenesis. Unlike most protein kinases, they appear to function as constitutively active enzymes. As a result, selective pharmacological inhibitors can play an important role in dissection of casein kinase-1-dependent processes. To address this need, new small molecule inhibitors of casein kinase-1 acting through ATP-competitive and ATP-noncompetitive mechanisms were isolated on the basis of in vitro screening. Here we report the crystal structure of 3-[(2,4,6-trimethoxyphenyl) methylidenyl]-indolin-2-one (IC261), an ATP-competitive inhibitor with differential activity among casein kinase-1 isoforms, in complex with the catalytic domain of fission yeast casein kinase-1 refined to a crystallographic R-factor of 22.4% at 2.8 Å resolution. The structure reveals that IC261 stabilizes casein kinase-1 in a conformation midway between nucleotide substrate liganded and nonliganded conformations. We propose that adoption of this conformation by casein kinase-1 family members stabilizes a delocalized network of side chain interactions and results in a decreased dissociation rate of inhibitor.

Cyclin-dependent kinase 5 (Cdk5) is a multifunctional neuronal protein kinase that is required for neurite outgrowth and cortical lamination and that plays an important role in dopaminergic signaling in the neostriatum through phosphorylation of Thr-75 of DARPP-32 (dopamine and cAMP-regulated phosphoprotein, molecular mass 32 kDa). Casein kinase 1 (CK1) has been implicated in a variety of cellular functions such as DNA repair, circadian rhythm, and intracellular trafficking. In the neostriatum, CK1 has been found to phosphorylate Ser-137 of DARPP-32. However, first messengers for the regulation of Cdk5 or CK1 have remained unknown. Here we report that both Cdk5 and CK1 are regulated by metabotropic glutamate receptors (mGluRs) in neostriatal neurons. (S)-3,5-dihydroxyphenylglycine (DHPG), an agonist for group I mGluRs, increased Cdk5 and CK1 activities in neostriatal slices, leading to the enhanced phosphorylation of Thr-75 and Ser-137 of DARPP-32, respectively. The effect of DHPG on Thr-75, but not on Ser-137, was blocked by a Cdk5-specific inhibitor, butyrolactone. In contrast, the effects of DHPG on both Thr-75 and Ser-137 were blocked by CK1-7 and IC261, specific inhibitors of CK1, suggesting that activation of Cdk5 by mGluRs requires CK1 activity. In support of this possibility, the DHPG-induced increase in Cdk5 activity, measured in extracts of neostriatal slices, was abolished by CK1-7 and IC261. Treatment of acutely dissociated neurons with DHPG enhanced voltage-dependent Ca 2؉ currents. This enhancement was eliminated by either butyrolactone or CK1-7 and was absent in DARPP-32 knockout mice. Together these results indicate that a CK1-Cdk5-DARPP-32 cascade may be involved in the regulation by mGluR agonists of Ca 2؉ channels. C yclin-dependent kinase 5 (Cdk5)͞p35 plays a variety of roles in the developing and adult nervous system (1-3). Studies of mice in which the gene encoding either Cdk5 or p35 has been disrupted have indicated that both mutants exhibit abnormalities in the laminar structure of the cerebral cortex (4, 5). Conversion of p35 to p25 by the action of calpain causes altered localization and prolonged activation of Cdk5 (6-9). Cdk5 phosphorylates DARPP-32 (dopamine-and cAMP-regulated-phosphoprotein, molecular mass 32 kDa) on Thr-75 (10, 11). Phosphorylation at Thr-34 by cAMP-dependent protein kinase (PKA) converts DARPP-32 into an inhibitor of protein phosphatase-1 (PP-1), a process that is critical for the actions of dopamine in the neostriatum (12). Phosphorylation of Thr-75 by Cdk5 converts DARPP-32 into an inhibitor of PKA, reducing phosphorylation of DARPP-32 at Thr-34, and modulating the DARPP-32͞PP-1 cascade (10).Casein kinase 1 (CK1) was one of the first serine͞threonine protein kinases to be isolated and characterized (13). It is a ubiquitous enzyme that can be found in the nucleus and the cytosol and bound to the cytoskeleton and membranes. The CK1 family consists of multiple isoforms encoded by seven distinct genes (CK1␣, ␤, ␥1, ␥2, ␥3, ␦, and ). These isoforms exhibit more than ...