John F. Cannon

Inhibitor-1 Interaction Domain That Mediates the Inhibition of Protein Phosphatase-1

Inhibitor-1 (I-1), a cyclic AMP-regulated phosphoprotein, inhibits protein phosphatase-1 (PP1) activity in response to hormones. The molecular mechanism for PP1 inhibition by I-1 remains unknown. Mutation of nine acidic residues lining a proposed I-1-binding channel in rabbit PP1α yielded one mutant (E256A) slightly impaired in its inhibition by I-1, with the IC50 increased by 3-fold, and one mutant (E275R) located in the β12–β13 loop that showed 4-fold enhanced inhibition by I-1. Substituting Tyr-272, a proposed binding site for the toxins okadaic acid and microcystin-LR, in the β12–β13 loop with Trp, Phe, Asp, Arg, or Ala impaired PP1α inhibition by I-1 by 8–10-fold. Chemical mutagenesis of the Saccharomyces cerevisiae PP1 gene (GLC7) yielded 20 point mutations in the PP1 coding region. Two-hybrid analyses and biochemical assays of these yeast enzymes identified four additional residues in the β12–β13 loop that were required for PP1 binding and inhibition by I-1. Ten-fold higher concentrations of I-1 were required to inhibit these mutants. Finally, deletion of the β12–β13 loop from PP1α maintained full enzyme activity, but attenuated inhibition by I-1 by >100-fold. These data identified the β12–β13 loop in the PP1 catalytic subunit as a domain that mediates binding and enzyme inhibition by I-1.

Glc8 is a glucose-repressible activator of Glc7 protein phosphatase-1

Regulation of Glc7 type 1 protein phosphatase stability and activity was studied in budding yeast. We found that the Glc7 protein has a half-life of over 180min, which is sufficient for several generations. Glc7 protein stability was constant during the cell cycle and in batch culture growth. Furthermore, deletion of regulatory subunit Gac1, Reg1, Reg2, Sds22, or Glc8 had no influence on Glc7 protein half-life. The activity of Glc7 assayed as okadaic acid-resistant phosphorylase phosphatase activity was constant during the cell cycle. Deletion of the aforementioned regulatory subunits revealed that only Glc8 deletion had a significant effect in reducing Glc7 activity. Glc7 activity was induced during stationary phase in a Glc8-dependent manner. In addition, extracellular glucose repressed the induction of Glc7 activity. These results are consistent with glucose repression of Glc8 expression and favor the role of Glc8 as a major Glc7 activator.

Roles of trehalose phosphate synthase in yeast glycogen metabolism and sporulation

Trehalose is a major storage carbohydrate in budding yeast, Saccharomyces cerevisiae. Alterations in trehalose synthesis affect carbon source‐dependent growth, accumulation of glycogen and sporulation. Trehalose is synthesized by trehalose phosphate synthase (TPS), which is a complex of at least four proteins. In this work, we show that the Tps1p subunit protein catalyses trehalose phosphate synthesis in the absence of other TPS components. The tps1–H223Y allele (glc6–1) that causes a semidominant decrease in glycogen accumulation exhibits greater enzyme activity than wild‐type TPS1 because, unlike the wild‐type enzyme, TPS activity in tps1–H223Y cells is not inhibited by phosphate. Poor sporulation in tps1 null diploids is caused by reduced expression of meiotic inducers encoded by IME1, IME2 and MCK1. Furthermore, high‐copy MCK1 or heterozygous hxk2 mutations can suppress the tps1 sporulation trait. These results suggest that the trehalose‐6‐phosphate inhibition of hexokinase activity is required for full induction of MCK1 in sporulating yeast cells.

Function of protein phosphatase-1, Glc7, in Saccharomyces cerevisiae.

Budding yeast, Saccharomyces cerevisiae, and its close relatives are unique among eukaryotes in having a single gene, GLC7, encoding protein phosphatase-1 (PP1). This enzyme with a highly conserved amino acid sequence controls many processes in all eukaryotic cells. Therefore, the study of Glc7 function offers a unique opportunity to gain a comprehensive understanding of this critical regulatory enzyme. This review summarizes our current knowledge of how Glc7 function modulates processes in the cytoplasm and nucleus. Additionally, global Glc7 regulation is described.

Characterization of genes that are synthetically lethal with ade3 or leu2 in Saccharomyces cerevisiae

Combinations of two non‐lethal mutations that result in cell death are synthetically lethal. Such a genetic relationship suggests a functional interaction between the corresponding gene products. Frequently, an ade2 ade3 colony‐sectoring assay is used to screen for synthetic lethal mutants. In these screens, mutants are sought that fail to lose a plasmid that bears a gene of interest. However, a subset of mutants is often found that is dependent on plasmid components other than the target gene. To understand the mechanism of this dependence, we characterized those mutants that, although prevalent in most mutant hunts, are usually discarded. Using a LEU2–ADE3 plasmid, plasmid‐dependent mutations were found in the SHM2, PTR3, BAP2 and SSY1 genes. Double shm2 ade3 mutants are non‐viable because the two pathways for tetrahydrofolate synthesis are blocked. Mutations in PTR3, BAP2 and SSY1 disrupt sensing and transport of extracellular leucine. Therefore, ptr3, bap2 or ssy1 mutants must be leucine prototrophs to grow on rich media. In light of these findings, we propose modifications that should improve the efficiency of synthetic lethal screening procedures. Copyright

How phosphorylation activates the protein phosphatase-1 • inhibitor-2 complex

Phosphorylation regulates activity of many proteins; however, atomic level details are known for very few examples. Inhibitor-2 (I2) squelches the ubiquitous protein phosphatase-1 (PP1) enzyme activity by blocking access to the metal-containing active site. I2 Thr74 phosphorylation results in PP1 activation without I2 dissociation from the PP1-I2 complex. The dynamic disordered structure of the 73-residue segment of I2 containing Thr74, prevented visualization by X-ray crystallography of PP1-I2. In this work, I generated structures of this segment using simulated annealing to NMR restraints, fused them to the crystallographic PP1-I2 coordinates, and used molecular dynamics to study the impact of Thr74 phosphorylation on structural alterations leading to PP1 activation. Frequencies of I2 Tyr149 displacement from the PP1 active site, rotation of the phenolic Tyr149 side chain to prevent its reinsertion, and repositioning the I2 inhibitory helix to expose the PP1 active site to solvent and substrates significantly increased upon I2 Thr74 phosphorylation. After these steps, a second metal bound to produce PP1-Mn(2)-I2, which held the phosphorylated form of I2 to its active site less tightly than it held dephosphorylated I2. I2 Thr74 lies on the edge of variable dynamic communities of residues where it forms various allosteric pathways that induce motions at the PP1 active site 20Å away. These molecular dynamics simulations show how an unstructured region of I2 can harness enhanced rapid movements around phosphorylated Thr74 to pry I2 residues away from the PP1 active site in early steps of PP1-I2 activation.

AMBER force-field parameters for guanosine triphosphate and its imido and methylene analogs

Parameters were derived for guanosine triphosphate (GTP) and GTP analogs suitable for the AMBER force field. Electrostatically derived net atomic charges and force parameters were extracted from MNDO semiempirical calculations. The later parameters came from fitting MNDO and AMBER atom–atom forces in a manner that is extensible to other compounds that lack sufficient vibrational spectral data. The geometric parameters for these compounds were obtained from model compounds in the Cambridge crystallographic data base. Dynamic simulations of Na4 GTP and Na2 Mg GTP of 140 and 100 ps, respectively, indicated a strong preference for a syn C2′ exo conformation in solution.