Differential activation of yeast adenylyl cyclase by Ras1 and Ras2 depends on the conserved N terminus.

Hurwitz, Naama; Segal, Marisa; Marbach, Irith; Levitzki, Alexander

doi:10.1073/pnas.92.24.11009

Cited by 15 publications

(16 citation statements)

References 34 publications

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“…Deletion alleles for RAS1, STE20, STE12, and TEC1 were created by replacement of coding sequences by either the HIS3-or the TRP1-selectable marker. pRS314-RAS2⌬C and pRS314-R2N-R1C were obtained by subcloning of a 3-kb SalI-NotI fragment from p413-RAS2⌬ or a 3.5-kb SalI-NotI fragment from p413-r2r1 (Hurwitz et al, 1995) into pRS314 (Sikorski and Hieter, 1989). YCplac22-RAS2 Val19 was obtained by subcloning of a 3-kb RAS2 Val19 EcoRI-HindIII fragment from YCp50-RAS2 Val19 (Michael Wigler, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) into YCplac22.…”

Section: Plasmidsmentioning

confidence: 99%

“…YCplac22-RAS2 Val19 was obtained by subcloning of a 3-kb RAS2 Val19 EcoRI-HindIII fragment from YCp50-RAS2 Val19 (Michael Wigler, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) into YCplac22. YEplac112-RAS1 was obtained by subcloning of a 3-kb BamHI-EcoRI fragment carrying RAS1 from p413-RAS1 (Hurwitz et al, 1995) into YEplac112 (Gietz and Sugino, 1988). pRS314-RAS2⌬C and pRS314-R2N-R1C were obtained by subcloning of a 3-kb SalI-NotI fragment from p413-RAS2⌬ or a 3.5-kb SalI-NotI fragment from p413-r2r1 (Hurwitz et al, 1995) into pRS314 (Sikorski and Hieter, 1989).…”

Section: Plasmidsmentioning

confidence: 99%

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Crosstalk between the Ras2p-controlled Mitogen-activated Protein Kinase and cAMP Pathways during Invasive Growth ofSaccharomyces cerevisiae

Mösch

Kübler

Krappmann

et al. 1999

MBoC

169

149

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The two highly conserved RAS genes of the budding yeast Saccharomyces cerevisiae are redundant for viability. Here we show that haploid invasive growth development depends on RAS2 but not RAS1. Ras1p is not sufficiently expressed to induce invasive growth. Ras2p activates invasive growth using either of two downstream signaling pathways, the filamentation MAPK (Cdc42p/Ste20p/MAPK) cascade or the cAMP-dependent protein kinase (Cyr1p/cAMP/PKA) pathway. This signal branch point can be uncoupled in cells expressing Ras2p mutant proteins that carry amino acid substitutions in the adenylyl cyclase interaction domain and therefore activate invasive growth solely dependent on the MAPK cascade. Both Ras2p-controlled signaling pathways stimulate expression of the filamentation response element-driven reporter gene depending on the transcription factors Ste12p and Tec1p, indicating a crosstalk between the MAPK and the cAMP signaling pathways in haploid cells during invasive growth.

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Section: Plasmidsmentioning

confidence: 99%

Section: Plasmidsmentioning

confidence: 99%

Crosstalk between the Ras2p-controlled Mitogen-activated Protein Kinase and cAMP Pathways during Invasive Growth ofSaccharomyces cerevisiae

Mösch

Kübler

Krappmann

et al. 1999

MBoC

169

149

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show abstract

“…3B). ras2⌬ cells have lower cyclic AMP levels than isogenic ras1⌬ cells, and purified recombinant Ras2 protein is a more effective activator of adenylyl cyclase than Ras1 (13,29). We reasoned therefore that Erf2 is required in ras2⌬ cells because the overall Ras activity in the cell is reduced.…”

Section: Bmentioning

confidence: 99%

Erf2, a Novel Gene Product That Affects the Localization and Palmitoylation of Ras2 in Saccharomyces cerevisiae

Bartels

Mitchell

Dong

et al. 1999

Molecular and Cellular Biology

167

200

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Ras proteins are small membrane-associated GTP binding proteins that cycle between active (GTP-bound) and inactive (GDP-bound) states to regulate cell growth and differentiation. In Saccharomyces cerevisiae, two Ras proteins (Ras1 and Ras2) affect such diverse processes as vegetative growth, sporulation, carbon source utilization, stress response, and pseudohyphal growth (12,25,45,61,64). Ras-dependent growth requires plasma membrane localization, which in turn depends on a series of posttranslational modifications that occur on a carboxyl-terminal CaaX box (C is cysteine, a is any aliphatic residue, X is the carboxy-terminal residue) (15,20,21,57). The first step is the farnesylation of the CaaX box cysteine by a soluble, heterodimer farnesyl protein transferase encoded by the RAM1 and RAM2 genes in yeast (34,39,53). The aaX sequence is removed by one of two endoplasmic reticulum (ER)-associated proteases encoded by RCE1 and AFC1/STE24 (9, 58). The newly exposed cysteinyl ␣-carboxyl is then methyl esterified by the product of STE14, an integral membrane protein which also colocalizes with the ER in yeast and mammalian cells (16,18,22,54,56).The mature form of Ras is localized primarily on the cytoplasmic surface of the plasma membrane. Mutations in either the CaaX box or the genes encoding the posttranslational modification enzymes cause a reduction in Ras plasma membrane localization and a corresponding reduction in the ability of Ras to support growth (15, 57). However, the mechanism by which prenylation and subsequent posttranslational modifications direct Ras proteins to the plasma membrane is not known. It is clear that prenylation alone is not sufficient for efficient plasma membrane targeting of Ras. Yeast mutants that fail to carry out the palmitoylation step have reduced amounts of Ras protein at the plasma membrane and increased resistance to heat shock in the presence of activated Ras2(V19) alleles (6). Palmitoylation, unlike the CaaX processing steps, is reversible, making it a likely regulatory step. Interestingly, oncogenic forms of Ras are also rendered nontransforming by mutating the palmitoylation sites (69). Unfortunately, palmitoylation is the least well understood step in the Ras posttranslational modification pathway. A major obstacle has been the failure to identify a protein palmitoyltransferase, although reports of partial purification of palmitoyltransferase activities have appeared (3,19,37). The matter is further complicated by the fact that protein palmitoylation can occur nonenzymatically; however, the reaction rate is considerably lower than that observed in vivo (1,23,66). The issue is unlikely to be resolved until there is a better understanding of the requirements for and biological consequences of palmitoylation in vivo.The multistep nature of Ras modification suggests that subcellular targeting may be an ordered process involving distinct intracellular membrane compartments. In support of this idea is the recent demonstration that the -aaX proteases (Afc1 and Rce1) and methylt...

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“…The Ras pathway also promotes mating via the MAPK pathway (1). Although Ras plays a prominent role in regulating adenylyl cyclase and cAMP signaling in Saccharomyces cerevisiae (22,33,39), in both C. neoformans and Ustilago maydis Ras lacks the domain involved in cyclase binding and no longer functions in cAMP signaling (1,28,29).The cAMP-protein kinase A (PKA)-dependent signaling pathway is of particular interest based on its ability to control both virulence factors (melanin and capsule production) and morphological differentiation (mating and filamentous growth) of C. neoformans. Elements of this pathway include the G␣ protein Gpa1, adenylyl cyclase Cac1, and PKA consisting of the catalytic subunits Pka1/Pka2 and the regulatory subunit Pkr1 (2,3,14,21).…”

mentioning

confidence: 99%

mentioning

confidence: 99%

Adenylyl Cyclase-Associated Protein Aca1 Regulates Virulence and Differentiation of Cryptococcus neoformans via the Cyclic AMP-Protein Kinase A Cascade

Bahn¹,

Hicks²,

Giles³

et al. 2004

Eukaryot Cell

109

119

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The evolutionarily conserved cyclic AMP (cAMP) signaling pathway controls cell functions in response to environmental cues in organisms as diverse as yeast and mammals. In the basidiomycetous human pathogenic fungus Cryptococcus neoformans, the cAMP pathway governs virulence and morphological differentiation. Here we identified and characterized adenylyl cyclase-associated protein, Aca1, which functions in parallel with the G␣ subunit Gpa1 to control the adenylyl cyclase (Cac1). Aca1 interacted with the C terminus of Cac1 in the yeast two-hybrid system. By molecular and genetic approaches, Aca1 was shown to play a critical role in mating by regulating cell fusion and filamentous growth in a cAMP-dependent manner. Aca1 also regulates melanin and capsule production via the Cac1-cAMP-protein kinase A pathway. Genetic epistasis studies support models in which Aca1 and Gpa1 are necessary and sufficient components that cooperate to activate adenylyl cyclase. Taken together, these studies further define the cAMP signaling cascade controlling virulence of this ubiquitous human fungal pathogen.Cryptococcus neoformans is a heterothallic, basidiomycetous, pathogenic fungus that causes serious infections of the central nervous system in individuals immunocompromised by AIDS or undergoing organ transplantation, cytotoxic chemotherapy, or corticosteroid therapy (6, 31). The virulence of C. neoformans is influenced by several factors, including the production of an antiphagocytic polysaccharide capsule (7,20,27), the use of melanin as an antioxidant (26, 51), growth at host physiological temperature (37 to 39°C) (27, 29), and prototrophy (41). Although not directly involved in the virulence of C. neoformans, mating and filamentous growth may play a survival role in the environment and also promote dissemination of the pathogen into the host. Signaling cascades regulating virulence and differentiation of C. neoformans have been extensively studied, including a mitogen-activated protein kinase (MAPK) pathway, a G-protein-regulated cyclic AMP (cAMP) pathway, a Ras-specific pathway, and the calcineurin pathway (for reviews, see references 29 and 48).The MAPK cascade regulates mating processes involving morphological differentiation, such as the dikaryotic mycelia, basidia, and basidiospores, which are produced in response to peptide pheromones secreted by opposite mating-type cells (24,32,43). The MAPK pathway is composed of mating-typespecific (Ste3␣/a, Ste20␣/a, Ste11␣/a, and Ste12␣/a) and nonspecific (Gpb1, Ste7, and Cpk1) elements (12, 49). Gene disruption experiments revealed that the MAPK pathway is required for mating and cell type-specific differentiation but not for virulence (12). However, mating type has been associated with the virulence of serotype D (variety neoformans) strains by Kwon-Chung et al., who showed that the ␣-mating type is more virulent than the a-mating type (25). Furthermore, Del Poeta et al. demonstrated that the MF␣1 pheromone gene is induced during the late stages of central nervous system infection...

show abstract

Differential activation of yeast adenylyl cyclase by Ras1 and Ras2 depends on the conserved N terminus.

Cited by 15 publications

References 34 publications

Crosstalk between the Ras2p-controlled Mitogen-activated Protein Kinase and cAMP Pathways during Invasive Growth ofSaccharomyces cerevisiae

Crosstalk between the Ras2p-controlled Mitogen-activated Protein Kinase and cAMP Pathways during Invasive Growth ofSaccharomyces cerevisiae

Erf2, a Novel Gene Product That Affects the Localization and Palmitoylation of Ras2 in Saccharomyces cerevisiae

Adenylyl Cyclase-Associated Protein Aca1 Regulates Virulence and Differentiation of Cryptococcus neoformans via the Cyclic AMP-Protein Kinase A Cascade

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