Full activation of p34CDC28 histone H1 kinase activity is unable to promote entry into mitosis in checkpoint-arrested cells of the yeast Saccharomyces cerevisiae.

In most cells, mitosis is dependent upon completion of DNA replication. The feedback mechanisms that prevent entry into mitosis by cells with damaged or incompletely replicated DNA have been termed checkpoint controls. Studies with the fission yeast Schizosaccharomyces pombe and Xenopus egg extracts have shown that checkpoint controls prevent activation of the master regulatory protein kinase, p34cdc2, that normally triggers entry into mitosis. This is achieved through inhibitory phosphorylation of the Tyr-15 residue of p34cdc2. However, studies with the budding yeast Saccharomyces cerevisiae have shown that phosphorylation of this residue is not essential for checkpoint controls to prevent mitosis. We have investigated the basis for checkpoint controls in this organism and show that these controls can prevent entry into mitosis even in cells which have fully activated the cyclin B (Clb)-associated forms of the budding yeast homolog of p34cdc2, p34CDC28, as assayed by histone H1 kinase activity. However, the active complexes in checkpoint-arrested cells are smaller than those in cycling cells, suggesting that assembly of mitosis-inducing complexes requires additional steps following histone H1 kinase activation.

Human cDNAs encoding homologs of the small p34Cdc28/Cdc2-associated protein of Saccharomyces cerevisiae and Schizosaccharomyces pombe.

The Cks1 protein is a component of the Cdc28 protein kinase in the budding yeast Saccharomyces cerevisiae. This paper reports the cloning of two homologs of the S. cerevisiae CKS1 gene from human cells. These homologs, CKShs1 and CKShs2, both encode proteins of 79 amino acids that share considerable homology at the amino acid level with the products of CKS1 from S. cerevisiae and suc1+ from the fission yeast Schizosaccharomyces pombe. Both human homologs are capable of rescuing a null mutation of the S. cerevisiae CKS1 gene when expressed from the S. cerevisiae GAL1 promoter. S. pombe suc1+ expressed from the GAL1 promoter is also capable of rescuing a S. cerevisiae cks1 null mutation. Ckshs1 or Ckshs2 protein linked to Sepharose beads can bind the Cdc28/Cdc2 protein kinase from both S. cerevisiae and human cells. The CKShs1 and CKShs2 mRNAs are expressed in different patterns through the cell cycle in HeLa cells, which may reflect specialized roles for the encoded proteins.

Isocitrate dehydrogenase kinase/phosphatase.

In Escherichia coli, isocitrate dehydrogenase (IDH) is regulated by phosphorylation. This phosphorylation cycle is catalyzed by an unusual, bifunctional protein:IDH kinase/phosphatase. IDH kinase/phosphatase is expressed from a single gene, aceK, and both activities are catalyzed by the same polypeptide. The amino acid sequence of IDH kinase/phosphatase does not exhibit the characteristics which are typical of other protein kinases, although it does contain a consensus ATP binding site. The available evidence suggests that the IDH kinase and IDH phosphatase reactions occur at the same active site and that the IDH phosphatase reaction results from the back reaction of IDH kinase tightly coupled to ATP hydrolysis. The function of the IDH phosphorylation cycle is to control the flux of isocitrate through the glyoxylate bypass. This pathway is essential for growth on acetate because it prevents the quantitative loss of the acetate carbons as CO2 in the Krebs' cycle. IDH kinase/phosphatase monitors general metabolism by responding to the levels of a wide variety of metabolites, many of which activate IDH phosphatase and inhibit IDH kinase. The ability of IDH kinase/phosphatase to monitor general metabolism allows. the IDH phosphorylation cycle to compensate for substantial perturbations of the system, such as a 15-fold overproduction of IDH. The significance of the cellular level of IDH kinase/phosphatase has also been evaluated. The level of this protein is in great excess of that required for steady-state growth on acetate. In contrast, IDH kinase/phosphatase is, in some cases, rate-limiting for the dephosphorylation of IDH which results when preferred carbon sources are added to cultures growing on acetate.

Mutation of the predicted ATP binding site inactivates both activities of isocitrate dehydrogenase kinase/phosphatase.

In Escherichia coli, the reversible phosphorylation of isocitrate dehydrogenase (IDH) is catalyzed by a bifunctional protein: IDH kinase/phosphatase. Although both IDH kinase and IDH phosphatase require ATP, the amino acid sequence of IDH kinase/phosphatase contains a single sequence that matches the consensus for ATP binding sites. A mutation that converted the "invariant" lysine (residue 336) of this consensus sequence to a methionine reduced the activities of both IDH kinase and IDH phosphatase by factors of greater than 500, to levels below the detection limits of the assays. The apparent elimination of both IDH kinase and IDH phosphatase by this mutation is consistent with the proposal that these activities share a common ATP binding site and that these reactions may occur at the same active site. Although conversion of Lys336 to a methionine eliminated detectable IDH kinase activity as measured in vitro, the mutant allele retained the ability to complement an aceK deletion mutation, restoring the ability of these cells to grow on minimal acetate medium. Complementation apparently resulted because the mutant protein retained sufficient activity to phosphorylate IDH in vivo. To determine whether the enzymatic assays performed in vitro had correctly reflected the activity of the mutant protein in vivo, we measured the rates at which mutant and wild-type cultures could incorporate [32P]inorganic phosphate into IDH. The wild-type culture achieved maximal incorporation in less than 3 min. In contrast, 32P incorporation was only barely detectable after 30 min in the mutant culture, indicating that the activity of the mutant protein is, indeed, greatly reduced in vivo. The ability of the mutant allele to complement an aceK null mutation thus suggests that IDH kinase/phosphatase levels in wild-type cells are in great excess over what is required for steady-state growth on acetate medium.

The isocitrate dehydrogenase phosphorylation cycle. Identification of the primary rate-limiting step.

In Escherichia coli, the branch point between the Krebs cycle and the glyoxylate bypass is regulated by the phosphorylation of isocitrate dehydrogenase (IDH). Phosphorylation inactivates IDH, forcing isocitrate through the bypass. This bypass is essential for growth on acetate but does not serve a useful function when alternative carbon sources, such as glucose or pyruvate, are also present. When pyruvate or glucose is added to a culture growing on acetate, the cells responded by dephosphorylating IDH and thus inhibiting the flow of isocitrate through the glyoxylate bypass. In an effort to identify the primary rate-limiting step in the response of IDH phosphorylation to alternative carbon sources, we have examined the response rates of congenic strains of E. coli which express different levels of IDH kinase/phosphatase, the bifunctional protein which catalyzes this phosphorylation cycle. The rate of the pyruvate-induced dephosphorylation of IDH was proportional to the level of IDH kinase/phosphatase, indicating that IDH kinase/phosphatase was primarily rate-limiting for dephosphorylation. However, the identity of the primary rate-limiting step appears to depend on the stimulus, since the rate of dephosphorylation of IDH in response to glucose was independent of the level of IDH kinase/phosphatase.

Nucleotide sequence of aceK, the gene encoding isocitrate dehydrogenase kinase/phosphatase.

In Escherichia coli, the phosphorylation and dephosphorylation of isocitrate dehydrogenase (IDH) are catalyzed by a bifunctional protein kinase/phosphatase. We have determined the nucleotide sequence of aceK, the gene encoding IDH kinase/phosphatase. This gene consists of a single open reading frame of 1,734 base pairs preceded by a Shine-Dalgarno ribosome-binding site. Examination of the deduced amino acid sequence of IDH kinase/phosphatase revealed sequences which are similar to the consensus sequence for ATP-binding sites. This protein did not, however, exhibit the extensive sequence homologies which are typical of other protein kinases. Multiple copies of the REP family of repetitive extragenic elements were found within the intergenic region between aceA (encoding isocitrate lyase) and aceK. These elements have the potential for combining to form an exceptionally stable stem-loop structure (delta G = -54 kcal/mol [ca. -226 kJ/mol]) in the mRNA. This structure, which masks the ribosome-binding site and start codon for aceK, may contribute to the downshift in expression observed between aceA and aceK. Another potential stem-loop structure (delta G = -29 kcal/mol [ca. 121 kJ/mol]), unrelated to the REP sequences, was found within aceK.

Isocitrate dehydrogenase kinase/phosphatase exhibits an intrinsic adenosine triphosphatase activity.

In Escherichia coli, isocitrate dehydrogenase (IDH) is regulated by reversible phosphorylation. The bifunctional enzyme which catalyzes this phosphorylation cycle, IDH kinase/phosphatase, also exhibits a specific ATPase activity. Mutant derivatives of this protein which are nearly devoid of IDH phosphatase activity retain both IDH kinase and ATPase activity, indicating that ATP hydrolysis does not result from the cyclic phosphorylation of IDH. However, the IDH kinase and ATPase activities of these mutant proteins differ significantly from those of the wild-type IDH kinase/phosphatase expressed from the parental allele. This observation suggest that IDH kinase and IDH phosphatase do not reside on structurally independent domains. In contrast to many enzymes which catalyze kinetically unfavorable side reactions, the maximum velocity of the ATPase substantially exceeded those of IDH kinase and IDH phosphatase. ATP hydrolysis was only partially inhibited by phospho- and dephospho-IDH, with saturating levels of phospho-IDH decreasing the rate of ATP hydrolysis by a factor of approximately 5. Even in the presence of near-saturating concentrations of phospho-IDH, the rate of ATP hydrolysis was 4-fold greater than the rate of the cyclic phosphorylation of IDH.