Mutant regulatory subunit of 3′,5′-cAMP-dependent protein kinase of yeast Saccharomyces cerevisiae

Yamano, Shigeyuki; Tanaka, Kazuma; Matsumoto, Kunihiro; Toh‐e, Akio

doi:10.1007/bf00327191

Cited by 17 publications

(14 citation statements)

References 18 publications

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“…To define the role of the PPDJ gene in the RAS-cAMP pathway, we isolated a molecular clone of the PPDI gene now renamed IRA]. Like the RAS2Va-l9 and bcyl mutants (4,42), the iral-l mutant was sensitive to heat shock (42). We utilized this phenotype to isolate the IRA] gene.…”

Section: Resultsmentioning

confidence: 99%

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IRA1, an inhibitory regulator of the RAS-cyclic AMP pathway in Saccharomyces cerevisiae.

1989

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In the yeast Saccharomyces cerevisiae, there are two homologs of mammalian ras genes, RASI and RAS2 (7,26), which appear to be potent activators of adenylate cyclase (39). In mammalian cells, however, neither the target nor the regulator of the ras proteins is understood. S. cerevisiae is potentially a good experimental system for understanding ras function. First, like the mammalian ras proteins, the yeast RAS proteins are capable of binding and hydrolyzing guanine nucleotides (9, 36). Second, yeast cells carrying the RAS2VaI-l9 mutation, analogous to the oncogenic variation in human H-rasval12, do not arrest properly at Gl in response to nutrient limitation (16). Finally, human H-ras protein is also a potent activator of yeast adenylate cyclase (3), although the ultimate target of ras may not be an adenylate cyclase in mammalian cells (1). These structural and biochemical homologies between mammalian and yeast ras proteins suggest that the identification of molecules interacting with RAS proteins in S. cerevisiae may facilitate the understanding of the function of mammalian ras proteins. Recently, it was shown that the CDC25 gene product is essential for activation of adenylate cyclase in S. cerevisiae (4,27). From biochemical experiments, Broek et al. (4) proposed that the CDC25 protein may regulate adenylate cyclase by regulating the guanine nucleotide bound to RAS proteins.In S. cerevisiae, cyclic AMP (cAMP) has a crucial role in cell cycle progression via activation of the cAMP-dependent protein kinase (A kinase). cAMP produced by adenylate cyclase encoded by the CYR] (15) gene binds to the kinase regulatory subunit, encoded by the BCYJ (37, 42) gene, to release catalytic subunits encoded by the TPKI, TPK2, and TPK3 genes (38). Free catalytic subunits then phosphorylate unknown target protein(s) whose phosphorylation is essential for cell cycle progression. Mutations that block activation of A kinase, such as cyrl, cdc25, or rasi ras2, prevent entry into the mitotic cycle, whereas mutations that cause high, constitutive levels of cAMP-dependent protein phos-* Corresponding author. phorylation, such as bcyl, ppdl, or RAS2va l9, interfere with sporulation and entry into the resting state. The ppdl mutant has been reported to be deficient in the dephosphorylation of the putative target proteins for A kinase (for a review, see reference 21; 22).Here we further characterized the PPDJ gene at the molecular level. In contrast to the previous report, we found no significant difference in protein phosphatase activity between a wild-type strain and an isogenic strain in which the PPDI gene was disrupted. Moreover, our results suggest that the PPDJ protein regulates the RAS-cAMP pathway. Thus, we introduce the name "IRAI" (inhibitory regulator of the RAS-cAMP pathway) to replace "PPDJI". In contrast to the function of the CDC25 protein, the IRA] protein is required for keeping cAMP at a low level in response to nutritional limitation. MATERIALS AND METHODSStrains, transformations, and culture media. Yeast strains use...

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Section: Resultsmentioning

confidence: 99%

“…cAMP produced by adenylate cyclase encoded by the CYR] (15) gene binds to the kinase regulatory subunit, encoded by the BCYJ (37,42) gene, to release catalytic subunits encoded by the TPKI, TPK2, and TPK3 genes (38). Free catalytic subunits then phosphorylate unknown target protein(s) whose phosphorylation is essential for cell cycle progression.…”

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confidence: 99%

IRA1, an inhibitory regulator of the RAS-cyclic AMP pathway in Saccharomyces cerevisiae.

1989

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“…A multisubunit cAMP-dependent kinase, the product of the BCY1, TPK1, TPK2, and TPK3 genes, monitors the levels of cAMP in response to nutrient limitation (Toda et al 1987a, b;Yamano et al 1987}. MCK1 encodes a protein kinase with at least two known functions: It is required to ensure the fidelity of mitotic chromosome segregation (Shero and Heiter 1991} and to activate transcription of the IME1 gene (Neigebom and Mitchell 1991}.…”

Section: Discussionmentioning

confidence: 99%

A meiosis-specific protein kinase homolog required for chromosome synapsis and recombination.

Rockmill

Roeder²

1991

Genes & Development

144

110

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The mekl (meiotic _kinase) mutant of Saccharomyces cerevisiae was isolated in a screen for sporulation-proficient, meiotic-lethal mutants. Diploids homozygous for a mekl null mutation produce only 13% viable spores, mekl spore inviability is rescued by a spol3 mutation, which causes cells to bypass the meiosis I division. In a mekl null mutant, meiotic recombination is reduced but not completely eliminated. Nuclear spreads of meiotic chromosomes from mekl diploids reveal numerous stretches of synaptonemal complex (SC) that are shorter than wild-type SCs. Analysis of a mekl::lacZ fusion gene and Northern blot hybridization demonstrate that the MEK1 transcript is present only in meiosis. The sequence of the MEK1 gene predicts a 56.8-kD protein with homology to serine-threonine protein kinases. The MEK1 gene maps to chromosome XV, 13 cM proximal to CDC64. Models for the function of the MEK1 gene product are proposed.

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“…As discussed above, previous purification of C1(Ala241) was complicated by the instability of the holoenzyme. With the addition of R(Asn'4Ala145) (65), a more stable complex was formed and the C1(Ala241) subunit was purified to approximately 85% (as compared with the previous purity of approximately 30%). The value reported here for the inhibition by R subunit is therefore more accurate.…”

mentioning

confidence: 95%

“…Mutants C1(Asp241), C1(Glu241), and C1(Ser241) were purified from strains LL24D, LL24E, and LL24S, respectively. These strains overexpress only the altered C1 subunit and a tightly binding mutant R subunit, R(Asn1"Ala145) (65). The other C subunits with lowered affinities did not form a sufficiently stable holoenzyme to allow purification by anti-R-subunit immunoaffinity chromatography.…”

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confidence: 99%

Association of catalytic and regulatory subunits of cyclic AMP-dependent protein kinase requires a negatively charged side group at a conserved threonine.

Levin¹,

Zoller²

1990

Mol. Cell. Biol.

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In Saccharomyces cerevisiae, as in higher eucaryotes, cyclic AMP (cAMP)-dependent protein kinase is a tetramer composed of two catalytic (C) subunits and two regulatory (R) subunits. In the absence of cAMP, the phosphotransferase activity of the C subunit is inhibited by the tight association with R. Mutation of to Ala in the C1 subunit of S. cerevisiae reduces the affinity of this subunit for the R subunit approximately 30-fold and results in a monomeric cAMP-independent C subunit. The analogous residue in the mammalian C subunit is known to be phosphorylated. Peptide maps of in vivo 32P-labeled wild-type C1 and mutant CI(Ala241) suggest that Thr-241 is phosphorylated in yeast cells. Substituting Thr-241 with either aspartate or glutamate partially restored affinity for the R subunit. Uncharged and positively charged residues substituted at this site resulted in C subunits that failed to associate with the R subunit. Replacement with the phosphorylatable residue serine resulted in a C subunit with wild-type affinity for the R subunit. Analysis of this protein revealed that it appears to be phosphorylated on Ser-241 in vivo. These data suggest that the interaction between R and C involves a negatively charged phosphothreonine at position 241 of yeast Cl, which can be mimicked by either aspartate, glutamate, or phosphoserine.The catalytic activity of many serine/threonine protein kinases is regulated by a pseudosubstrate sequence, located within a regulatory domain, which blocks the active site of the catalytic domain (for a review, see reference 18). The pseudosubstrate site resembles recognition sequences found in protein kinase substrates, and peptides derived from it act as competitive inhibitors of protein substrates in vitro (18,26). Experimental evidence for this model has been demonstrated for the cyclic nucleotide-dependent protein kinases cyclic (cAMP)-dependent protein kinase (cAdPK) (9,11,64) and cyclic cGMP-dependent protein kinase (cGdPK) (33,53), smooth muscle myosin light-chain kinase (26, 39), skeletal muscle myosin light-chain kinase (27, 53), protein kinase C (20, 38), and Ca2+-calmodulin-dependent protein kinase 11 (17,24,32,46). Although much is known about the role of the pseudosubstrate sequence in the regulatory domain, little is known about regions in the catalytic domain that are involved. cAdPK is the best structurally and biochemically characterized protein kinase (56). The porcine catalytic subunit of cAdPK has been crystallized (51), and data collection is still in progress. Group-specific chemical modification and affinity labeling studies have identified a number of amino acid residues thought to be involved in either catalytic activity, substrate binding, or association with the regulatory subunit (2, 5, 56).The cAdPK holoenzyme from the yeast Saccharomyces cerevisiae, as well as from higher eucaryotes, is a tetramer composed of two catalytic (C) and two regulatory (R) subunits. In S. cerevisiae, a single gene encoding the R subunit (BCYI) (7,22,28,59) subunits (TPKI, TPK2, a...

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Mutant regulatory subunit of 3′,5′-cAMP-dependent protein kinase of yeast Saccharomyces cerevisiae

Cited by 17 publications

References 18 publications

IRA1, an inhibitory regulator of the RAS-cyclic AMP pathway in Saccharomyces cerevisiae.

IRA1, an inhibitory regulator of the RAS-cyclic AMP pathway in Saccharomyces cerevisiae.

A meiosis-specific protein kinase homolog required for chromosome synapsis and recombination.

Association of catalytic and regulatory subunits of cyclic AMP-dependent protein kinase requires a negatively charged side group at a conserved threonine.

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