Site-Specific Mutagenesis of cdc2+, a Cell Cycle Control Gene of the Fission Yeast Schizosaccharomyces pombe

Booher, Robert; Beach, David

doi:10.1128/mcb.6.10.3523-3530.1986

Cited by 69 publications

(1 citation statement)

References 45 publications

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“…For example, Kamps and Sefton found that neither histidine nor arginine could replace this lysine to support the protein kinase activity or transforming potential of Src (Kamps & Sefton, 1986). Comparable mutants of cyclin-dependent kinases, protein kinase C, MAP/ERK kinase, and other protein kinases have also been shown to be nonfunctional in ViVo (Booher & Beach, 1986;Jove et al, 1987;Ohno et al, 1990;Lange-Carter et al, 1993;Mansour et al, 1994).…”

Section: Discussionmentioning

confidence: 99%

Mutation of Position 52 in ERK2 Creates a Nonproductive Binding Mode for Adenosine 5‘-Triphosphate^,

et al. 1996

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Among the protein kinases, an absolutely conserved lysine in subdomain II is required for high catalytic activity. This lysine is known to interact with the substrate ATP, but otherwise its role is not well understood. We have used biochemical and structural methods to investigate the function of this lysine (K52) in phosphoryl transfer reactions catalyzed by the MAP kinase ERK2. The kinetic properties of activated wild-type ERK2 and K52 mutants were examined using the oncoprotein TAL2, myelin basic protein, and a designed synthetic peptide as substrates. The catalytic activities of K52R and K52A ERK2 were lower than that of wild-type ERK2, primarily as a consequence of reductions in kcat. Further, there was little difference in Km for ATP, but the Km,app for peptide substrate was higher for the K52 mutants. The three-dimensional structure of unphosphorylated K52R ERK2 in the absence and presence of bound ATP was determined and compared with the structure of unphosphorylated wild-type ERK2. ATP adopted a well-defined but distinct binding mode in K52R ERK2 compared to the binding mode in the wild-type enzyme. The structural and kinetic data show that mutation of K52 created a nonproductive binding mode for ATP and suggest that K52 is essential for orienting ATP for catalysis.

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Section: Discussionmentioning

confidence: 99%

Mutation of Position 52 in ERK2 Creates a Nonproductive Binding Mode for Adenosine 5‘-Triphosphate^,

et al. 1996

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Interaction between cdc13+ and cdc2+ in the control of mitosis in fission yeast; dissociation of the G1 and G2 roles of the cdc2+ protein kinase.

1987

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A cold‐sensitive (cs) allele of cdc2, a gene that acts in both the G1 and G2 phases of the fission yeast cell cycle, has been isolated by classical mutagenesis. Further mutagenesis of a cdc2cs strain yielded an extragenic suppressor that rescued the cs cell cycle defect but simultaneously conferred a temperature‐sensitive (ts) cdc phenotype. This suppressor mutation was shown to be an allele of cdc13, a previously identified gene. A variety of allele‐specific interactions between cdc2 and cdc13 were discovered. These included suppression of cdc13ts alleles by introduction of the cdc2+ gene on a multi‐copy plasmid vector. cdc13+ is required in G2 for mitotic initiation and was shown to play no role in the G1 phase of the cell cycle. cdc2+, however, is essential in G1 for DNA replication and in G2 for mitosis. The newly isolated cs allele of cdc2 that is rescued by a ts allele of cdc13 is defective only in its G2 function. cdc13+ cooperates with cdc2+ in the initiation of mitosis but not in the regulation of DNA replication. We propose that the cdc13+ gene product might be a G2‐specific substrate of the cdc2+ protein kinase.

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p13suc1 acts in the fission yeast cell division cycle as a component of the p34cdc2 protein kinase.

1987

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cdc2+ encodes a protein kinase that is required during both G1 and G2 phases of the cell division cycle in fission yeast. suc1+ is an essential gene that was originally identified as a plasmid‐borne sequence that could rescue certain temperature‐sensitive cdc2 mutants. To investigate the role of the suc1+ gene product in the cell cycle p13suc1 has been expressed in Escherichia coli and purified. An immunoaffinity purified anti‐p13suc1 polyclonal serum has been prepared and used to identify p13suc1 in fission yeast. The abundance of this protein did not alter either during the cell cycle or during entry into stationary phase. p13suc1 was found in yeast lysates in a complex with the cdc2+ gene product. Approximately 5% of cellular p34cdc2 was associated with p13suc1, and this fraction of p34cdc2 was active as a protein kinase. The stability of the complex was disrupted in yeast strains carrying temperature‐sensitive alleles of cdc2 that are suppressible by overexpression of suc1+. The level of association between p13suc1 and p34cdc2 was not affected by cell cycle arrest in adverse nutritional conditions. p13suc1 is not a substrate of the p34cdc2 protein kinase. We propose instead that it acts as a regulatory component of p34cdc2 that facilitates interaction with other proteins.

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Site-Specific Mutagenesis of cdc2⁺, a Cell Cycle Control Gene of the Fission Yeast Schizosaccharomyces pombe

Cited by 69 publications

References 45 publications

Mutation of Position 52 in ERK2 Creates a Nonproductive Binding Mode for Adenosine 5‘-Triphosphate^,

Mutation of Position 52 in ERK2 Creates a Nonproductive Binding Mode for Adenosine 5‘-Triphosphate^,

Interaction between cdc13+ and cdc2+ in the control of mitosis in fission yeast; dissociation of the G1 and G2 roles of the cdc2+ protein kinase.

p13suc1 acts in the fission yeast cell division cycle as a component of the p34cdc2 protein kinase.

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Site-Specific Mutagenesis of cdc2+, a Cell Cycle Control Gene of the Fission Yeast Schizosaccharomyces pombe

Cited by 69 publications

References 45 publications

Mutation of Position 52 in ERK2 Creates a Nonproductive Binding Mode for Adenosine 5‘-Triphosphate,

Mutation of Position 52 in ERK2 Creates a Nonproductive Binding Mode for Adenosine 5‘-Triphosphate,

Interaction between cdc13+ and cdc2+ in the control of mitosis in fission yeast; dissociation of the G1 and G2 roles of the cdc2+ protein kinase.

p13suc1 acts in the fission yeast cell division cycle as a component of the p34cdc2 protein kinase.

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Site-Specific Mutagenesis of cdc2⁺, a Cell Cycle Control Gene of the Fission Yeast Schizosaccharomyces pombe

Mutation of Position 52 in ERK2 Creates a Nonproductive Binding Mode for Adenosine 5‘-Triphosphate^,

Mutation of Position 52 in ERK2 Creates a Nonproductive Binding Mode for Adenosine 5‘-Triphosphate^,