Cak1 is required for Kin28 phosphorylation and activation in vivo.

Complete activation of most cyclin-dependent protein kinases (CDKs) requires phosphorylation by the CDK-activating kinase (CAK). In the budding yeast, Saccharomyces cerevisiae, the major CAK is a 44-kDa protein kinase known as Cak1. Cak1 is required for the phosphorylation and activation of Cdc28, a major CDK involved in cell cycle control. We addressed the possibility that Cak1 is also required for the activation of other yeast CDKs, such as Kin28, Pho85, and Srb10. We generated three new temperature-sensitive cak1 mutant strains, which arrested at the restrictive temperature with nonuniform budding morphology. All three cak1 mutants displayed significant synthetic interactions with loss-of-function mutations in CDC28 and KIN28. Loss of Cak1 function reduced the phosphorylation and activity of both Cdc28 and Kin28 but did not affect the activity of Pho85 or Srb10. In the presence of the Kin28 regulatory subunits Ccl1 and Tfb3, Kin28 was phosphorylated and activated when coexpressed with Cak1 in insect cells. We conclude that Cak1 is required for the activating phosphorylation of Kin28 as well as that of Cdc28.

Exploiting chemical libraries, structure, and genomics in the search for kinase inhibitors.

Selective protein kinase inhibitors were developed on the basis of the unexpected binding mode of 2,6,9-trisubstituted purines to the adenosine triphosphate-binding site of the human cyclin-dependent kinase 2 (CDK2). By iterating chemical library synthesis and biological screening, potent inhibitors of the human CDK2-cyclin A kinase complex and of Saccharomyces cerevisiae Cdc28p were identified. The structural basis for the binding affinity and selectivity was determined by analysis of a three-dimensional crystal structure of a CDK2-inhibitor complex. The cellular effects of these compounds were characterized in mammalian cells and yeast. In the latter case the effects were characterized on a genome-wide scale by monitoring changes in messenger RNA levels in treated cells with high-density oligonucleotide probe arrays. Purine libraries could provide useful tools for analyzing a variety of signaling and regulatory pathways and may lead to the development of new therapeutics.

A cyclin-dependent kinase-activating kinase (CAK) in budding yeast unrelated to vertebrate CAK.

Progress through the cell cycle is governed by the cyclin-dependent kinases (CDKs), the activation of which requires phosphorylation by the CDK-activating kinase (CAK). In vertebrates, CAK is a trimeric enzyme containing CDK7, cyclin H, and MAT1. CAK from the budding yeast Saccharomyces cerevisiae was identified as an unusual 44-kilodalton protein kinase, Cak1, that is only distantly related to CDKs. Cak1 accounted for most CAK activity in yeast cell lysates, and its activity was constant throughout the cell cycle. The CAK1 gene was essential for cell viability. Thus, the major CAK in S. cerevisiae is distinct from the vertebrate enzyme, suggesting that budding yeast and vertebrates may have evolved different mechanisms of CDK activation.

Cell cycle control by a complex of the cyclin HCS26 (PCL1) and the kinase PHO85.

The events of the eukaryotic cell cycle are governed by cyclin-dependent kinases (cdk's), whose activation requires association with cyclin regulatory subunits expressed at specific cell cycle stages. In the budding yeast Saccharomyces cerevisiae, the cell cycle is thought to be controlled by a single cdk, CDC28. Passage through the G1 phase of the cell cycle is regulated by complexes of CDC28 and G1 cyclins (CLN1, CLN2, and CLN3). A putative G1 cyclin, HCS26, has recently been identified. In a/alpha diploid cells lacking CLN1 and CLN2, HCS26 is required for passage through G1. HCS26 does not associate with CDC28, but instead associates with PHO85, a closely related protein kinase. Thus, budding yeast, like higher eukaryotes, use multiple cdk's in the regulation of cell cycle progression.

A new gene of bacteriophage P4 that controls DNA replication.

Bacteriophage P4 replication may result in either a lytic cycle or plasmid maintenance, depending on the presence or absence, respectively, of helper phase P2 genome. Bacteriophage P4 DNA replication depends on the product of gene alpha, which has origin recognition, primase, and helicase activities. An open reading frame with the coding capacity for a protein of 106 amino acids (orf106) is located upstream of the alpha gene. Genes orf106 and alpha are transcriptionally coregulated. Three amber mutations and an internal deletion (del51) were introduced into orf106. All of the amber mutations exhibited a polar effect on transcription of the downstream alpha gene. The P4 del51 mutant was slightly defective in lytic growth and could not be propagated in the plasmid state. In this latter condition, P4 DNA overreplication was observed. Overexpression of Orf106 severely inhibited P4 DNA replication, preventing P4 lytic growth and plasmid maintenance. The inhibitory effect of Orf106 on P4 replication was not observed when both orf106 and alpha were overexpressed. We suggest that orf106 is involved in P4 replication and that a balanced expression of orf106 relative to alpha may be necessary for proper P4 DNA replication. In particular, orf106 appears to be essential for the control of P4 genome replication in the plasmid state. We propose that orf106 be named cnr, for copy number regulation.