A protein inhibitor of rabbit liver phosphorylase phosphatase

Brandt, Howard; Lee, Ernest Y.C.; Killilea, S.Derek

doi:10.1016/0006-291x(75)90661-0

Cited by 126 publications

(27 citation statements)

References 5 publications

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“…I-2 was first identified as one of two heat-stable proteins from rabbits that can inhibit the activity of PP1 C (12,41). I-2-like activity has since been found in diverse organisms, ranging from Drosophila melanogaster to mammals (32,34,42,50,59,63,68,77).…”

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confidence: 99%

Regulation of Chromosome Segregation by Glc8P, a Structural Homolog of Mammalian Inhibitor 2 That Functions as both an Activator and an Inhibitor of Yeast Protein Phosphatase 1

Tung¹,

Wang²,

Chan³

1995

Molecular and Cellular Biology

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The Ipl1 protein kinase is essential for proper chromosome segregation and cell viability in the budding yeast Saccharomyces cerevisiae. We have previously shown that the temperature-sensitive growth phenotype of conditional ipl1-1 ts mutants can be suppressed by a partial loss-of-function mutation in the GLC7 gene, which encodes the catalytic subunit (PP1 C ) of protein phosphatase 1, thus suggesting that this enzyme acts in opposition to the Ipl1 protein kinase in regulating yeast chromosome segregation. We report here that the Glc8 protein, which is related in primary sequence to mammalian inhibitor 2, also participates in this regulation. Like inhibitor 2, the Glc8 protein is heat stable, exhibits anomalous electrophoretic mobility, and functions in vitro as an inhibitor of yeast as well as rabbit skeletal muscle PP1 C . Interestingly, overexpression as well as deletion of the GLC8 gene results in a partial suppression of the temperature-sensitive growth phenotype of ipl1 ts mutants and also moderately reduces the amount of protein phosphatase 1 activity which is assayable in crude yeast lysates. In addition, the chromosome missegregation phenotype caused by an increase in the dosage of GLC7 is totally suppressed by the glc8-⌬101::LEU2 deletion mutation. These findings together suggest that the Glc8 protein is involved in vivo in the activation of PP1 C and that when the Glc8 protein is overproduced, it may also inhibit PP1 C function. Furthermore, site-directed mutagenesis studies of GLC8 suggest that Thr-118 of the Glc8 protein, which is equivalent to Thr-72 of inhibitor 2, may play a central role in the ability of this protein to activate and/or inhibit PP1 C in vivo.The reversible phosphorylation of proteins is a major mechanism for the control of protein functions in eukaryotes. Phosphorylation and dephosphorylation of proteins on serine, threonine, and tyrosine residues has been shown to be involved in the control of many essential cellular processes, including chromosome segregation, metabolism, transcription, translation, and cell division. The phosphorylation state of a given protein is determined by a dynamic equilibrium between the activities of the protein kinase(s) and protein phosphatase(s) that recognize it as a substrate. Protein phosphatases that are specific for phosphoserine and phosphothreonine can be divided biochemically into at least four major classes: protein phosphatases 1, 2A, 2B, and 2C (PP1, PP2A, PP2B, and PP2C, respectively). These four classes of enzymes differ in their substrate specificity, cation requirements, and sensitivity to certain inhibitors (reviewed in references 20 and 54). Protein phosphatase activities characteristic of all four classes are found in diverse organisms, ranging from Saccharomyces cerevisiae to mammals (22,45,59).PP1 proteins purified from different sources all contain a catalytic subunit (PP1 C ) of ϳ37 kDa which is often associated with other proteins. These associated proteins include the glycogen-and sarcoplasmic reticulum-binding G subunit from...

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confidence: 99%

Regulation of Chromosome Segregation by Glc8P, a Structural Homolog of Mammalian Inhibitor 2 That Functions as both an Activator and an Inhibitor of Yeast Protein Phosphatase 1

Tung¹,

Wang²,

Chan³

1995

Molecular and Cellular Biology

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“…The enzyme can be partially purified by procedures similar to those described for the preparation of the corresponding enzymes from animal tissues (2)(3)(4)(5)(6)(7)(8). Ethanol precipitation at room temperature apparently is an important step in the purification.…”

Section: Discussionmentioning

confidence: 99%

Phosphoprotein Phosphatase of Soybean Hypocotyls

Lin

Mori²,

Key³

1980

Plant Physiol.

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A soybean histone-type protein kinase was used to prepare 32P-labeled histone Hi as substrate for purification and characterization of a phosphoprotein phosphatase (EC 3.13.16) phorylation and dephosphorylation has been suggested to be an important mechanism in the control of metabolic processes in higher plants (12,25). Randall and his colleagues (21) have shown that phosphorylation of the pyruvate dehydrogenase complex from broccoli mitochondria results in inactivation of the enzyme. Additionally, the nuclear (17), ribosomal (20, 24) and membrane proteins (1) of higher plants are also subject to modification by phosphorylation, although the biological significance of the phosphorylation is unknown. The phosphorylation of these proteins apparently is catalyzed by several protein kinases, including histone-type and casein (or phosvitin)-type enzymes (9,12,25). Although much work has been carried out on phosphorylation of proteins and demonstration of the activities of protein kinases, little attention has been paid to phosphoprotein phosphatase (EC 3.1.3.16) which catalyzes the protein dephosphorylation reaction. There apparently has been no characterization of the phosphatase from higher plants, except a brief mention of the presence of phosphohistone phosphatase activity in pea seedlings (16). Here, we describe the purification and properties of a phosphoprotein phosphatase from soybean hypocotyls. The enzyme is distinct from the phosphatases which catalyze the hydrolysis ofnonprotein low mol wt phosphoesters. Additionally, several lines of evidence indicate that the soybean enzyme is a nonspecific phosphoprotein phosphatase acting on phosphorylated histones, protamine, and, possibly, phosphorylated casein and phosvitin. MATERIALS AND METHODSUnless otherwise stated, the chemicals and biochemical compounds used in this study were obtained from the sources as indicated in the previous paper (12). [y-32PJATP was prepared according to the method of Schendel and Wells (22). All other chemicals were of reagent grade. Soybean seeds (Glycine max var. Wayne) were germinated in the dark and the mature hypocotyls were harvested as described (12).Preparation of 32P-labeled and Nonradioactive Phosphohistone Proteins. Lysine-rich histone HI and other histone species (H2A, H2B, H3, and H4) were purified from the total calfthymus histone mixture (Sigma II or IIA) by column chromatographies on BioGel P-60 and P-10 (12, 14). A histone-type protein kinase with high specificity for histone HI was partially purified from soybean hypocotyls as described (12); the kinase was used to phosphorylate histone H1. For the preparation of 32P-labeled histone species other than histone H1, a cAMP-dependent histone kinase from rat liver cytosol (4) was used. The liver kinase was partially purified by ammonium sulfate fractionation and DEAE-Sephadex A-25 column chromatography.To prepare a large quantity of 32P-labeled histone H1, the reaction mixture in a total volume of 10 ml contained 20 mM Tris-HCI (pH 8.

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“…However, the optimum reaction conditions of GPb and Dg GBE are pH 6.9 to 7.4/38 • C (Brandt, Cafwlong, & Lee, 1975) and pH 8.0/34 • C, respectively. By varying the pH value we noticed that by an increase of the pH value, starting with the lowest value of 6, the degree of branching increases and reaches the highest value of around 13% for a pH of 6.7 and afterwards starts to decrease continuously until the highest analyzed value of pH 9.…”

Section: Effects Of Ph On the Degree Of Branchingmentioning

confidence: 99%