Regulation of the cell cycle by protein phosphatase 2A in Saccharomyces cerevisiae.

Protein phosphatase 2A (PP2A) has long been implicated in cell cycle regulation in many different organisms. In the yeast Saccharomyces cerevisiae, PP2A controls cell cycle progression mainly through modulation of cyclin-dependent kinase (CDK) at the G(2)/M transition. However, CDK does not appear to be a direct target of PP2A. PP2A affects CDK activity through its roles in checkpoint controls. Inactivation of PP2A downregulates CDK by activating the morphogenesis checkpoint and, consequently, delays mitotic entry. Defects in PP2A also compromise the spindle checkpoint and predispose the cell to an error-prone mitotic exit. In addition, PP2A is involved in controlling the G(1)/S transition and cytokinesis. These findings suggest that PP2A functions in many stages of the cell cycle and its effect on cell cycle progression is pleiotropic.     

ADP regulates SNF1, the Saccharomyces cerevisiae homolog of AMP-activated protein kinase

The SNF1 protein kinase complex plays an essential role in regulating gene expression in response to the level of extracellular glucose in budding yeast. SNF1 shares structural and functional similarities with mammalian AMP-activated protein kinase. Both kinases are activated by phosphorylation on a threonine residue within the activation loop segment of the catalytic subunit. Here we show that ADP is the long-sought metabolite that activates SNF1 in response to glucose limitation by protecting the enzyme against dephosphorylation by Glc7, its physiologically relevant protein phosphatase. We also show that the regulatory subunit of SNF1 has two ADP binding sites. The tighter site binds AMP, ADP, and ATP competitively with NADH, whereas the weaker site does not bind NADH, but is responsible for mediating the protective effect of ADP on dephosphorylation. Mutagenesis experiments suggest that the general mechanism by which ADP protects against dephosphorylation is strongly conserved between SNF1 and AMPK.     

The Dcr2p phosphatase destabilizes Sic1p in Saccharomyces cerevisiae

Initiation of cell division is controlled by an irreversible switch. In Saccharomyces cerevisiae degradation of the Sic1p protein, an inhibitor of mitotic cyclin/cyclin-dependent kinase complexes, takes place before initiation of DNA replication, at a point called START. Sic1p is phosphorylated by multiple kinases, which can differentially affect the stability of Sic1p. How phosphorylations that stabilize Sic1p are reversed is unknown. Here we show that the Dcr2p phosphatase functionally and physically interacts with Sic1p. Over-expression of Dcr2p destabilizes Sic1p and leads to phenotypes associated with destabilized Sic1p, such as genome instability. Our results identify a novel factor that affects the stability of Sic1p, possibly contributing to mechanisms that trigger initiation of cell division.     

Regulation of protein kinase cascades by protein phosphatase 2A.

Many protein kinases themselves are regulated by reversible phosphorylation. Upon cell stimulation, specific kinases are transiently phosphorylated and activated. Several of these protein kinases are substrates for protein phosphatase 2A (PP2A), and PP2A appears to be the major kinase phosphatase in eukaryotic cells that downregulates activated protein kinases. This idea is substantiated by the observation that some viral proteins and naturally occurring toxins target PP2A and modulate its activity. There is increasing evidence that PP2A activity is regulated by extracellular signals and during the cell cycle. Thus, PP2A is likely to play an important role in determining the activation kinetics of protein kinase cascades.     

Molecular genetic analysis of Rts1p, a B' regulatory subunit of Saccharomyces cerevisiae protein phosphatase 2A.

The Saccharomyces cerevisiae gene RTS1 encodes a protein homologous to a variable B-type regulatory subunit of the mammalian heterotrimeric serine/threonine protein phosphatase 2A (PP2A). We present evidence showing that Rts1p assembles into similar heterotrimeric complexes in yeast. Strains in which RTS1 has been disrupted are temperature sensitive (ts) for growth, are hypersensitive to ethanol, are unable to grow with glycerol as their only carbon source, and accumulate at nonpermissive temperatures predominantly as large-budded cells with a 2N DNA content and a nondivided nucleus. This cell cycle arrest can be overcome and partial suppression of the ts phenotype of rts1-null cells occurs if the gene CLB2, encoding a Cdc28 kinase-associated B-type cyclin, is expressed on a high-copy-number plasmid. However, CLB2 overexpression has no suppressive effects on other aspects of the rts1-null phenotype. Expression of truncated forms of Rts1p can also partially suppress the ts phenotype and can fully suppress the inability of cells to grow on glycerol and the hypersensitivity of cells to ethanol. By contrast, the truncated forms do not suppress the accumulation of large-budded cells at high temperatures. Coexpression of truncated Rts1p and high levels of Clb2p fully suppresses the ts phenotype, indicating that the inhibition of growth of rts1-null cells at high temperatures is due to both stress-related and cell cycle-related defects. Genetic analyses show that the role played by Rts1p in PP2A regulation is distinctly different from that played by the other known variable B regulatory subunit, Cdc55p, a protein recently implicated in checkpoint control regulation.     

The Saccharomyces cerevisiae checkpoint gene BUB1 encodes a novel protein kinase.

Normal cell multiplication requires that the events of mitosis occur in a carefully ordered fashion. Cells employ checkpoints to prevent cycle progression until some prerequisite step has been completed. To explore the mechanisms of checkpoint enforcement, we previously screened for mutants of Saccharomyces cerevisiae which are unable to recover from a transient treatment with a benzimidazole-related microtubule inhibitor because they fail to inhibit subsequent cell cycle steps. Two of the identified genes, BUB2 and BUB3, have been cloned and described (M. A. Hoyt, L. Totis, and B. T. Roberts, Cell 66:507-517, 1991). Here we present the characterization of the BUB1 gene and its product. Genetic evidence was obtained suggesting that Bub1 and Bub3 are mutually dependent for function, and immunoprecipitation experiments demonstrated a physical association between the two. Sequence analysis of BUB1 revealed a domain with similarity to protein kinases. In vitro experiments confirmed that Bub1 possesses kinase activity; Bub1 was able to autophosphorylate and to catalyze phosphorylation of Bub3. In addition, overproduced Bub1 was found to localize to the cell nucleus.     

Protein phosphatase 2Bw and protein phosphatase Z are Saccharomyces cerevisiae enzymes.

cDNAs encoding three protein phosphatases, termed PP2Bw (Da Cruz e Silva, E.F. and Cohen, P.T.W. (1989) Biochim. Biophys. Acta 1009, 293-296), PPZ1 and PPZ2 that have been isolated from a Clontech 'rabbit brain' library are shown to be Saccharomyces cerevisiae clones. PPZ1 and PPZ2 are two novel yeast phosphatases showing 93% amino acid sequence identity to one another. PPZ1 shows approx. 60% sequence identity to S. cerevisiae or mammalian PP1 and approx. 40% identity to S. cerevisiae or mammalian PP2A. These and other observations suggest that the two isoforms of PPZ have functions distinct from those of PP1.     

Saccharomyces cerevisiae GTPase complex: Gtr1p-Gtr2p regulates cell-proliferation through Saccharomyces cerevisiae Ran-binding protein, Yrb2p

A Gtr1p GTPase, the GDP mutant of which suppresses both temperature-sensitive mutants of Saccharomyces cerevisiae RanGEF/Prp20p and RanGAP/Rna1p, was presently found to interact with Yrb2p, the S. cerevisiae homologue of mammalian Ran-binding protein 3. Gtr1p bound the Ran-binding domain of Yrb2p. In contrast, Gtr2p, a partner of Gtr1p, did not bind Yrb2p, although it bound Gtr1p. A triple mutant: yrb2delta gtr1delta gtr2delta was lethal, while a double mutant: gtr1delta gtr2delta survived well, indicating that Yrb2p protected cells from the killing effect of gtr1delta gtr2delta. Recombinant Gtr1p and Gtr2p were purified as a complex from Escherichia coli. The resulting Gtr1p-Gtr2p complex was comprised of an equal amount of Gtr1p and Gtr2p, which inhibited the Rna1p/Yrb2 dependent RanGAP activity. Thus, the Gtr1p-Gtr2p cycle was suggested to regulate the Ran cycle through Yrb2p.     

Hog1p mitogen-activated protein kinase determines acetic acid resistance in Saccharomyces cerevisiae

When glucose-repressed, Saccharomyces cerevisiae cannot use acetic acid as a carbon source and is inhibited in growth by high levels of this compound, especially at low pH. Cultures exposed to a 100 mM acetate stress activate both the Hog1p and Slt2p stress-activated MAP kinases. Nevertheless, only active Hog1p, not Slt2p, is needed for the acquisition of acetate resistance. Hog1p undergoes more rapid activation by acetate in pH 4.5, than in pH 6.8 cultures, an indication that the acid may have to enter the cells in order to generate the Hog1p activatory signal. Acetate activation of Hog1p is absent in the ssk1Delta and pbs2Delta mutants, but is present in sho1Delta and ste11Delta, showing that it involves the Sln1p branch of the high-osmolarity glycerol (HOG) pathway signaling to Pbs2p. In low-pH (pH 4.5) cultures, the acetate-activated Hog1p, although conferring acetate resistance, does not generate the GPD1 gene or intracellular glycerol inductions that are hallmarks of activation of the HOG pathway by hyperosmotic stress.     

Regulation of phospholipid biosynthesis in Saccharomyces cerevisiae by cyclic AMP-dependent protein kinase.

The addition of cyclic AMP (cAMP) to Saccharomyces cerevisiae cyr1 mutant cells resulted in an increase in the rate of phosphatidylinositol synthesis at the expense of phosphatidylserine synthesis. The decrease in phosphatidylserine synthesis correlated with the down regulation of phosphatidylserine synthase activity by cAMP-dependent protein kinase phosphorylation. The increase in phosphatidylinositol synthesis was not due to the regulation of phosphatidylinositol synthase by cAMP-dependent protein kinase.     

Pag1p, a novel protein associated with protein kinase Cbk1p, is required for cell morphogenesis and proliferation in Saccharomyces cerevisiae

Protein kinases in the Cot-1/Orb6/Ndr/Warts family are important regulators of cell morphogenesis and proliferation. Cbk1p, a member of this family in Saccharomyces cerevisiae, has previously been shown to be required for normal morphogenesis in vegetatively growing cells and in haploid cells responding to mating pheromone. A mutant of PAG1, a novel gene in S. cerevisiae, displayed defects similar to those of cbk1 mutants. pag1 and cbk1 mutants share a common set of suppressors, including the disruption of SSD1, a gene encoding an RNA binding protein, and the overexpression of Sim1p, an extracellular protein. These genetic results suggest that PAG1 and CBK1 act in the same pathway. Furthermore, we found that Pag1p and Cbk1p localize to the same polarized peripheral sites and that they coimmunoprecipitate with each other. Pag1p is a conserved protein. The homologs of Pag1p in other organisms are likely to form complexes with the Cbk1p-related kinases and function with those kinases in the same biological processes.     

Elm1p is one of three upstream kinases for the Saccharomyces cerevisiae SNF1 complex

BACKGROUND: The yeast SNF1 protein kinase and the mammalian AMP-activated protein kinase are highly conserved heterotrimeric complexes that are "metabolic master switches" involved in the switch from fermentative/anaerobic to oxidative metabolism. They are activated by cellular stresses that deplete cellular ATP, and SNF1 is essential in the response to glucose starvation. In both cases, activation requires phosphorylation at a conserved threonine residue within the activation loop of the kinase domain, but identifying the upstream kinase(s) responsible for this has been a challenging, unsolved problem. RESULTS: Using a library of strains that express 119 yeast protein kinases as GST fusions, we identified Elm1p as the sole kinase that could activate the kinase domain of AMP-activated protein kinase in vitro. Elm1p also activated the purified SNF1 complex, and this correlated with phosphorylation of Thr210 in the activation loop. Removal of the C-terminal domain increased the Elm1p kinase activity, indicating that it is auto-inhibitory. Expression of activated, truncated Elm1p from its own promoter gave a constitutive pseudohyphal growth phenotype that was rescued by deletion of SNF1, showing that Snf1p was acting downstream of Elm1p. Deletion of ELM1 does not give an snf- phenotype. However, Elm1p is closely related to Pak1p and Tos3p, and a pak1Delta tos3Delta elm1Delta triple mutant had an snf1- phenotype, i.e., it would not grow on raffinose and did not display hyperphosphorylation of the SNF1 target, Mig1p, in response to glucose starvation. CONCLUSIONS: Elm1p, Pak1p, and Tos3p are upstream kinases for the SNF1 complex that have partially redundant functions.     

The Saccharomyces cerevisiae type 2A protein phosphatase Pph22p is biochemically different from mammalian PP2A.

The Saccharomyces cerevisiae type 2A protein phosphatase (PP2A) Pph22p differs from the catalytic subunits of PP2A (PP2Ac) present in mammals, plants and Schizosaccharomyces pombe by a unique N-terminal extension of approximately 70 amino acids. We have overexpressed S. cerevisiae Pph22p and its N-terminal deletion mutant Delta N-Pph22p in the GS115 strain of Pichia pastoris and purified these enzymes to apparent homogeneity. Similar to other heterologous systems used to overexpress PP2Ac, a low yield of an active enzyme was obtained. The recombinant enzymes designed with an 8 x His-tag at their N-terminus were purified by ion-exchange chromatography on DEAE-Sephacel and affinity chromatography on Ni2+-nitrilotriacetic acid agarose. Comparison of biochemical properties of purified Pph22p and Delta N-Pph22p with purified human 8 x His PP2Ac identified similarities and differences between these two enzymes. Both enzymes displayed similar specific activities with 32P-labelled phosphorylase a as substrate. Furthermore, selected inhibitors and metal ions affected their activities to the same extend. In contrast to the mammalian catalytic subunit PP2Ac, but similar to the dimeric form of mammalian PP2A, Pph22p, but not Delta N-Pph22p, interacted strongly with protamine. Also with regard to the effects of protamine and polylysine on phosphatase activity Pph22p, but not Delta N-Pph22p, behaved similarly to the PP2Ac-PR65 dimer, indicating a regulatory role for the N-terminal extension of Pph22p. The N-terminal extension appears also responsible for interactions with phospholipids. Additionally Pph22p has different redox properties than PP2Ac; in contrast to human PP2Ac it cannot be reactivated by reducing agents. These properties make the S. cerevisiae Pph22p phosphatase a unique enzyme among all type 2A protein phosphatases studied so far.     

Regulation of phosphatidylinositol kinase activity in Saccharomyces cerevisiae.

The effects of growth phase and carbon source on membrane-associated phosphatidylinositol kinase in cell extracts of Saccharomyces cerevisiae were examined. Phosphatidylinositol kinase activity increased 2- and 2.5-fold in glucose- and glycerol-grown cells, respectively, in the stationary phase as compared with the exponential phase of growth. The increase in phosphatidylinositol kinase activity in the stationary phase of growth correlated with an increase in the relative amounts of phosphatidylinositol 4-phosphate, the product of the reaction. The increase in phosphatidylinositol kinase activity was not due to the presence of water-soluble effector molecules in cell extracts as indicated by mixing experiments. Phosphatidylinositol kinase activity decreased in cell extracts of exponential-phase cells preincubated under phosphorylation conditions which favor cyclic AMP-dependent protein kinase activity. Phosphatidylinositol kinase activity was not affected in cell extracts of stationary-phase cells preincubated under phosphorylation conditions.     

Identification of a glycogen synthase phosphatase from yeast Saccharomyces cerevisiae as protein phosphatase 2A.

A glycogen synthase phosphatase was purified from the yeast Saccharomyces cerevisiae. The purified yeast phosphatase displayed one major protein band which coincided with phosphatase activity on nondenaturing polyacrylamide gel electrophoresis. This phosphatase had a molecular mass of about 160,000 Da determined by gel filtration and was comprised of three subunits, termed A, B, and C. The subunit molecular weights estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 60,000 (A), 53,000 (B), and 37,000 (C), indicating that this yeast glycogen synthase phosphatase is a heterotrimer. On ethanol treatment, the enzyme was dissociated to an active species with a molecular weight of 37,000 estimated by gel filtration. The yeast phosphatase dephosphorylated yeast glycogen synthase, rabbit muscle glycogen phosphorylase, casein, and the alpha subunit of rabbit muscle phosphorylase kinase, was not sensitive to heat-stable protein phosphatase inhibitor 2, and was inhibited 90% by 1 nM okadaic acid. Dephosphorylation of glycogen synthase, phosphorylase, and phosphorylase kinase by this yeast enzyme could be stimulated by histone H1 and polylysines. Divalent cations (Mg2+ and Ca2+) and chelators (EDTA and EGTA) had no effect on dephosphorylation of glycogen synthase or phosphorylase while Mn2+ stimulated enzyme activity by approximately 50%. The specific activity and kinetics for phosphorylase resembled those of mammalian phosphatase 2A. An antibody against a synthetic peptide corresponding to the carboxyl terminus of the catalytic subunit of rabbit skeletal muscle protein phosphatase 2A reacted with subunit C of purified yeast phosphatase on immunoblots, whereas the analogous peptide antibody against phosphatase 1 did not. These data show that this yeast glycogen synthase phosphatase has structural and catalytic similarity to protein phosphatase 2A found in mammalian tissues.