Yeast cAMP-dependent protein kinase regulatory subunit mutations display a variety of phenotypes.

Ten spontaneous and four in vitro constructed mutations in the gene encoding the regulatory subunit of cAMP-dependent protein kinase of Saccharomyces cerevisiae display very different phenotypes. The DNA nucleotide sequence of each spontaneous mutation was determined. Mutations were found in both the cAMP-binding domains and proximal to the cAMP-dependent protein kinase phosphorylation site. The latter mutations exhibited dominant traits when gene dosage was increased. The variation of phenotypes of sra1 mutations was examined. Many aspects of growth are affected, including growth on nonfermentable carbon sources, accumulation of glycogen, ability to sporulate, and ability to survive starvation. The null mutations affect all these traits. None of the spontaneous mutations confer the null phenotype. Instead, these mutations can be placed into groups of increasing severity based on the number of traits affected. These traits reflect the functions of the cAMP-dependent protein kinase substrates and ranking of sra1 phenotypes probably reflects a progressive defect in one or more aspects of the regulatory subunit function.

[1]  J. François,et al.  The control of glycogen metabolism in yeast. 1. Interconversion in vivo of glycogen synthase and glycogen phosphorylase induced by glucose, a nitrogen source or uncouplers. , 1988, European journal of biochemistry.

[2]  G. McKnight,et al.  Expression and characterization of mutant forms of the type I regulatory subunit of cAMP-dependent protein kinase. The effect of defective cAMP binding on holoenzyme activation. , 1989, Journal of Biological Chemistry.

[3]  A. Kerlavage,et al.  Covalent modification of an adenosine 3':5'-monophosphate binding site of the regulatory subunit of cAMP-dependent protein kinase II with 8-azidoadenosine 3':5'-monophosphate. Identification of a single modified tyrosine residue. , 1980, The Journal of biological chemistry.

[4]  M. Wigler,et al.  Rigorous feedback control of cAMP levels in Saccharomyces cerevisiae. , 1987, Genes & development.

[5]  J. Szostak,et al.  Multiple, Tandem Plasmid Integration in Saccharomyces cerevisiae , 1983, Molecular and cellular biology.

[6]  P. Gergely,et al.  The control of phosphorylase phosphatase by cAMP‐dependent protein kinase , 1977, FEBS letters.

[7]  E. Krebs,et al.  Identification of an inhibitory region of the heat-stable protein inhibitor of the cAMP-dependent protein kinase. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[8]  A. Constantinou,et al.  The phosphoform of the regulatory subunit RII of cyclic AMP-dependent protein kinase possesses intrinsic topoisomerase activity , 1985, Cell.

[9]  E. Krebs,et al.  Characterization of a cyclic AMP-binding protein from bakers' yeast. Identification as a regulatory subunit of cyclic AMP-dependent protein kinase. , 1980, The Journal of biological chemistry.

[10]  D. Botstein,et al.  Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. , 1979, Gene.

[11]  E. Krebs,et al.  Role of multiple basic residues in determining the substrate specificity of cyclic AMP-dependent protein kinase. , 1977, The Journal of biological chemistry.

[12]  P. Greengard,et al.  Photoaffinity labeling of a protein kinase from bovine brain with 8-azidoadenosine 3',5'-monophosphate. , 1975, Biochemistry.

[13]  S. Taylor,et al.  Deletion mutants as probes for localizing regions of subunit interaction in cAMP-dependent protein kinase. , 1988, The Journal of biological chemistry.

[14]  E. Reimann Conversion of bovine cardiac adenosine cyclic 3',5'-phosphate dependent protein kinase to a heterodimer by removal of 45 residues at the N-terminus of the regulatory subunit. , 1986, Biochemistry.

[15]  M. Wigler,et al.  In yeast, RAS proteins are controlling elements of adenylate cyclase , 1985, Cell.

[16]  J. Gibbs,et al.  Suppressors of the ras2 mutation of Saccharomyces cerevisiae. , 1986, Genetics.

[17]  E. Krebs,et al.  Amino acid sequence of the regulatory subunit of bovine type II adenosine cyclic 3',5'-phosphate dependent protein kinase. , 1984, Biochemistry.

[18]  K. Murata,et al.  Transformation of intact yeast cells treated with alkali cations , 1983 .

[19]  S. Taylor,et al.  Relationships between structural domains and function in the regulatory subunit of cAMP-dependent protein kinases I and II from porcine skeletal muscle. , 1979, The Journal of biological chemistry.

[20]  G. McKnight,et al.  Inhibition of intracellular cAMP-dependent protein kinase using mutant genes of the regulatory type I subunit. , 1987, The Journal of biological chemistry.

[21]  L. C. Robinson,et al.  RAS2 of Saccharomyces cerevisiae is required for gluconeogenic growth and proper response to nutrient limitation. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[22]  K. Matsumoto,et al.  Heat shock response of Saccharomyces cerevisiae mutants altered in cyclic AMP-dependent protein phosphorylation , 1987, Molecular and cellular biology.

[23]  M. Wigler,et al.  Genetic analysis of yeast RAS1 and RAS2 genes , 1984, Cell.

[24]  M. Wigler,et al.  cAMP-independent control of sporulation, glycogen metabolism, and heat shock resistance in S. cerevisiae , 1988, Cell.

[25]  T. Steitz,et al.  Predicted structures of cAMP binding domains of type I and II regulatory subunits of cAMP-dependent protein kinase. , 1990, Biochemistry.

[26]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[27]  B. Haley Photoaffinity labeling of adenosine 3',5'-cyclic monophosphate binding sites of human red cell membranes. , 1975, Biochemistry.

[28]  D. A. Yphantis,et al.  Activation of type I cyclic AMP-dependent protein kinases with defective cyclic AMP-binding sites. , 1987, The Journal of biological chemistry.

[29]  R. Roskoski [1] Assays of protein kinase , 1983 .

[30]  P. Greengard,et al.  Identification, characterization, and quantitative measurement of cyclic AMP receptor proteins in cytosol of various tissues using a photoaffinity ligand. , 1977, The Journal of biological chemistry.

[31]  K. Tatchell,et al.  Characterization of Saccharomyces cerevisiae genes encoding subunits of cyclic AMP-dependent protein kinase , 1987, Molecular and cellular biology.

[32]  M. Wigler,et al.  Cloning and characterization of BCY1, a locus encoding a regulatory subunit of the cyclic AMP-dependent protein kinase in Saccharomyces cerevisiae , 1987, Molecular and cellular biology.

[33]  J. Messing [2] New M13 vectors for cloning , 1983 .

[34]  V. Chester Heritable glycogen-storage deficiency in yeast and its induction by ultra-violet light. , 1968, Journal of general microbiology.

[35]  K. Matsumoto,et al.  Isolation and characterization of yeast mutants deficient in adenylate cyclase and cAMP-dependent protein kinase. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[36]  J. Szostak,et al.  Yeast recombination: the association between double-strand gap repair and crossing-over. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[37]  S. Johnston,et al.  Isolation of the yeast regulatory gene GAL4 and analysis of its dosage effects on the galactose/melibiose regulon. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[38]  S. H. Lillie,et al.  Reserve carbohydrate metabolism in Saccharomyces cerevisiae: responses to nutrient limitation , 1980, Journal of bacteriology.

[39]  J. Pringle,et al.  Methods for monitoring the growth of yeast cultures and for dealing with the clumping problem. , 1975, Methods in cell biology.

[40]  W. Weber,et al.  cAMP-dependent protein kinases I and II: divergent turnover of subunits. , 1986, Biochemistry.

[41]  E. Krebs,et al.  Phosphorylation-dephosphorylation of enzymes. , 1979, Annual review of biochemistry.

[42]  E. Smith,et al.  Two pools of glycogen in Saccharomyces , 1977, Journal of bacteriology.

[43]  D. Øgreid,et al.  Mutations that prevent cyclic nucleotide binding to binding sites A or B of type I cyclic AMP-dependent protein kinase. , 1988, The Journal of biological chemistry.

[44]  Michael Wigler,et al.  Three different genes in S. cerevisiae encode the catalytic subunits of the cAMP-dependent protein kinase , 1987, Cell.

[45]  M. Wigler,et al.  SCH9, a gene of Saccharomyces cerevisiae that encodes a protein distinct from, but functionally and structurally related to, cAMP-dependent protein kinase catalytic subunits. , 1988, Genes & development.

[46]  S. Taylor,et al.  Correlation of photolabeling with occupancy of cAMP binding sites in the regulatory subunit of cAMP-dependent protein kinase I. , 1987, Biochemistry.

[47]  E. Southern Detection of specific sequences among DNA fragments separated by gel electrophoresis. , 1975, Journal of molecular biology.

[48]  D. Fraenkel On ras gene function in yeast. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[49]  E. Scolnick,et al.  Requirement of either of a pair of ras-related genes of Saccharomyces cerevisiae for spore viability , 1984, Nature.

[50]  D. Breviario,et al.  Carbon source regulation of RAS1 expression in Saccharomyces cerevisiae and the phenotypes of ras2- cells. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[51]  A. Feinberg,et al.  A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. , 1983, Analytical biochemistry.

[52]  R. Kunisawa,et al.  Complete nucleotide sequence of the gene encoding the regulatory subunit of 3', 5'-cyclic AMP-dependent protein kinase from the yeast Saccharomyces cerevisiae , 1987, Nucleic Acids Res..

[53]  E. Krebs,et al.  Comparison of adenosine 3':5'-monophosphate-dependent protein kinases from rabbit skeletal and bovine heart muscle. , 1975, The Journal of biological chemistry.

[54]  K. Tatchell,et al.  SRA5 encodes the low-Km cyclic AMP phosphodiesterase of Saccharomyces cerevisiae , 1988, Molecular and cellular biology.

[55]  S. P. Johnson,et al.  Phosphorylation of the Saccharomyces cerevisiae equivalent of ribosomal protein S6 has no detectable effect on growth , 1987, Molecular and cellular biology.

[56]  R. Printz,et al.  Regulatory subunit of cAMP-dependent protein kinase inhibits phosphoprotein phosphatase. , 1985, Biochemical and biophysical research communications.

[57]  M. Wigler,et al.  Guanine nucleotide activation of, and competition between, RAS proteins from Saccharomyces cerevisiae , 1987, Molecular and cellular biology.