Tautomycetin, Protein Phosphatase 1 Specific Inhibitor, opened the Door for understanding the Role of PP1 in Minkowski Space

Maintenance of cation homoeostasis is a key process for any living organism. Specific mutations in Glc7, the essential catalytic subunit of yeast protein phosphatase 1, result in salt and alkaline pH sensitivity, suggesting a role for this protein in cation homoeostasis. We screened a collection of Glc7 regulatory subunit mutants for altered tolerance to diverse cations (sodium, lithium and calcium) and alkaline pH. Among 18 candidates, only deletion of REF2 (RNA end formation 2) yielded increased sensitivity to these conditions, as well as to diverse organic toxic cations. The Ref2F374A mutation, which renders it unable to bind Glc7, did not rescue the salt-related phenotypes of the ref2 strain, suggesting that Ref2 function in cation homoeostasis is mediated by Glc7. The ref2 deletion mutant displays a marked decrease in lithium efflux, which can be explained by the inability of these cells to fully induce the Na+-ATPase ENA1 gene. The effect of lack of Ref2 is additive to that of blockage of the calcineurin pathway and might disrupt multiple mechanisms controlling ENA1 expression. ref2 cells display a striking defect in vacuolar morphogenesis, which probably accounts for the increased calcium levels observed under standard growth conditions and the strong calcium sensitivity of this mutant. Remarkably, the evidence collected indicates that the role of Ref2 in cation homoeostasis may be unrelated to its previously identified function in the formation of mRNA via the APT (for associated with Pta1) complex.

Download Full-text

N-Acetyl-β-D-glucopyranosylamine 6-phosphate is a specific inhibitor of glycogen-bound protein phosphatase 1

Biochemical Journal ◽

10.1042/bj3280695 ◽

1997 ◽

Vol 328 (2) ◽

pp. 695-700 ◽

Cited By ~ 3

Author(s):

Mary BOARD

Keyword(s):

Phosphatase Activity ◽

Protein Phosphatase ◽

Glycogen Phosphorylase ◽

Glycogen Synthase ◽

Specific Inhibitor ◽

Competitive Inhibitor ◽

Protein Phosphatase 1 ◽

P Concentration ◽

Glucose Analogue ◽

Stimulation Of

Previous work has shown that the C-1-substituted glucose-analogue N-acetyl-β-D-glucopyranosylamine (1-GlcNAc) is a competitive inhibitor of glycogen phosphorylase (GP) and stimulates the inactivation of this enzyme by GP phosphatase. In addition to its effects on GP, 1-GlcNAc also prevents the glucose-led activation of glycogen synthase (GS) in whole hepatocytes. Such an effect on GS was thought to be due to the formation of 1-GlcNAc-6-P by the action of glucokinase within the hepatocyte [Board, Bollen, Stalmans, Kim, Fleet and Johnson (1995) Biochem. J. 311, 845-852]. To investigate this possibility further, a pure preparation of 1-GlcNAc-6-P was synthesized. The effects of the phosphorylated glucose analogue on the activity of protein phosphatase 1 (PP1), the enzyme responsible for dephosphorylation and activation of GS, are reported. During the present study, 1-GlcNAc-6-P inhibited the activity of the glycogen-bound form of PP1, affecting both the GSb phosphatase and GPa phosphatase activities. A level of 50% inhibition of GSb phosphatase activity was achieved with 85 μM 1-GlcNAc-6-P in the absence of Glc-6-P and with 135 μM in the presence of 10 mM Glc-6-P. At either Glc-6-P concentration, 500 μM 1-GlcNAc-6-P completely inhibited activity. The Glc-6-P stimulation of the GPa phosphatase activity of PP1 was negated by 1-GlcNAc-6-P but there was no inhibition of the basal rate in the absence of Glc-6-P. 1-GlcNAc-6-P inhibition was specific for the glycogen-bound form of PP1 and did not inhibit the GSb phosphatase activity of the cytosolic form of the enzyme. The present work explains our previous observations on the inactivating effects on GS of incubating whole hepatocytes with 1-GlcNAc. These observations have their basis in the inhibition of glycogen-bound PP1 by 1-GlcNAc-6-P. A novel inhibitor of PP1, specific for the glycogen-bound form of the enzyme, is presented.

Download Full-text

Inhibition of protein phosphatase 1 reverses alcohol-induced ciliary dysfunction

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00336.2014 ◽

2015 ◽

Vol 308 (6) ◽

pp. L577-L585 ◽

Cited By ~ 15

Author(s):

Michael E. Price ◽

Jacqueline A. Pavlik ◽

Joseph H. Sisson ◽

Todd A. Wyatt

Keyword(s):

Protein Phosphatase ◽

Mucociliary Clearance ◽

Specific Inhibitor ◽

Alcohol Exposure ◽

Inhibitory Effect ◽

Regulatory Proteins ◽

Protein Phosphatase 1 ◽

Tissue Cell ◽

Inhaled Particles ◽

Phosphatase Activation

Airway mucociliary clearance is a first-line defense of the lung against inhaled particles and debris. Among individuals with alcohol use disorders, there is an increase in lung diseases. We previously identified that prolonged alcohol exposure impairs mucociliary clearance, known as alcohol-induced ciliary dysfunction (AICD). Cilia-localized enzymes, known as the ciliary metabolon, are key to the pathogenesis of AICD. In AICD, cyclic nucleotide-dependent ciliary kinases, which modulate phosphorylation to regulate cilia beat, are desensitized. We hypothesized that alcohol activates cilia-associated protein phosphatase 1 (PP1) activity, driving phosphorylation changes of cilia motility regulatory proteins. To test this hypothesis we identified the effects of prolonged alcohol exposure on phosphatase activity, cilia beat, and kinase responsiveness and cilia-associated phosphorylation targets when stimulated by β-agonist or cAMP. Prolonged alcohol activated PP1 and blocked cAMP-dependent cilia beat and protein kinase A (PKA) responsiveness and phosphorylation of a 29-kDa substrate of PKA. Importantly, prolonged alcohol-induced phosphatase activation was inhibited by the PP1 specific inhibitor, inhibitor-2 (I-2), restoring cAMP-stimulated cilia beat and PKA responsiveness and phosphorylation of the 29-kDa substrate. The I-2 inhibitory effect persisted in tissue, cell, and isolated cilia-organelle models, highlighting the association of ciliary metabolon-localized enzymes to AICD. Prolonged alcohol exposure drives ciliary metabolon-localized PP1 activation. PP1 activation modifies phosphorylation of a 29-kDa protein related to PKA activity. These data reinforce our previous findings that alcohol is acting at the level of the ciliary metabolon to cause ciliary dysfunction and identifies PP1 as a therapeutic target to prevent or reverse AICD.

Download Full-text

Xenopus Cdc7 executes its essential function early in S phase and is counteracted by checkpoint-regulated protein phosphatase 1

Open Biology ◽

10.1098/rsob.130138 ◽

2014 ◽

Vol 4 (1) ◽

pp. 130138 ◽

Cited By ~ 18

Author(s):

Wei Theng Poh ◽

Gaganmeet Singh Chadha ◽

Peter J. Gillespie ◽

Philipp Kaldis ◽

J. Julian Blow

Keyword(s):

Protein Phosphatase ◽

Replication Timing ◽

S Phase ◽

Replication Initiation ◽

Protein Phosphatase 1 ◽

Checkpoint Kinases ◽

Anti Cancer ◽

Egg Extracts ◽

Mcm Proteins

The initiation of DNA replication requires two protein kinases: cyclin-dependent kinase (Cdk) and Cdc7. Although S phase Cdk activity has been intensively studied, relatively little is known about how Cdc7 regulates progression through S phase. We have used a Cdc7 inhibitor, PHA-767491, to dissect the role of Cdc7 in Xenopus egg extracts. We show that hyperphosphorylation of mini-chromosome maintenance (MCM) proteins by Cdc7 is required for the initiation, but not for the elongation, of replication forks. Unlike Cdks, we demonstrate that Cdc7 executes its essential functions by phosphorylating MCM proteins at virtually all replication origins early in S phase and is not limiting for progression through the Xenopus replication timing programme. We demonstrate that protein phosphatase 1 (PP1) is recruited to chromatin and rapidly reverses Cdc7-mediated MCM hyperphosphorylation. Checkpoint kinases induced by DNA damage or replication inhibition promote the association of PP1 with chromatin and increase the rate of MCM dephosphorylation, thereby counteracting the previously completed Cdc7 functions and inhibiting replication initiation. This novel mechanism for regulating Cdc7 function provides an explanation for previous contradictory results concerning the control of Cdc7 by checkpoint kinases and has implications for the use of Cdc7 inhibitors as anti-cancer agents.

Download Full-text

Expression of Human Protein Phosphatase-1 inSaccharomyces cerevisiaeHighlights the Role of Phosphatase Isoforms in Regulating Eukaryotic Functions

Journal of Biological Chemistry ◽

10.1074/jbc.m701272200 ◽

2007 ◽

Vol 282 (30) ◽

pp. 21838-21847 ◽

Cited By ~ 23

Author(s):

Jennifer A. Gibbons ◽

Lukasz Kozubowski ◽

Kelly Tatchell ◽

Shirish Shenolikar

Keyword(s):

Protein Phosphatase ◽

Human Protein ◽

Protein Phosphatase 1

Download Full-text

The role of protein phosphatase-1 in the modulation of synaptic and structural plasticity

FEBS Letters ◽

10.1016/j.febslet.2004.03.121 ◽

2004 ◽

Vol 567 (1) ◽

pp. 121-128 ◽

Cited By ~ 62

Author(s):

Richard P. Munton ◽

Sándor Vizi ◽

Isabelle M. Mansuy

Keyword(s):

Protein Phosphatase ◽

Structural Plasticity ◽

Protein Phosphatase 1

Download Full-text

The role of autophosphorylation of cAMP-dependent protein kinase II in the inhibition of protein phosphatase-1

International Journal of Biochemistry ◽

10.1016/0020-711x(89)90056-6 ◽

1989 ◽

Vol 21 (10) ◽

pp. 1137-1141 ◽

Cited By ~ 7

Author(s):

György Vereb ◽

Pal Gergely

Keyword(s):

Protein Kinase ◽

Protein Phosphatase ◽

Protein Phosphatase 1 ◽

Dependent Protein Kinase ◽

Camp Dependent Protein Kinase ◽

Protein Kinase Ii ◽

Dependent Protein

Download Full-text

The Role of Rif1 in telomere length regulation is separable from its role in origin firing

10.1101/2020.04.24.060491 ◽

2020 ◽

Author(s):

Calla B. Shubin ◽

Carol W. Greider

Keyword(s):

Telomere Length ◽

Protein Phosphatase ◽

Protein Phosphatase 1 ◽

Origin Firing ◽

Replication Complex ◽

Telomere Elongation ◽

Length Regulation ◽

Telomere Length Regulation ◽

Telomere Lengthening

AbstractTo examine the established link between DNA replication and telomere length, we tested whether firing of telomeric origins would cause telomere lengthening. We found that RIF1 mutants that block Protein Phosphatase 1 (PP1) binding activated telomeric origins but did not elongate telomeres. In a second approach, we found overexpression of ΔN-Dbf4 and Cdc7 increased DDK activity and activated telomeric origins, yet telomere length was unchanged. We tested a third mechanism to activate origins using the sld3-A mcm5-bob1 mutant that deregulates the pre-replication complex, and again saw no change in telomere length. Finally, we tested whether mutations in RIF1 that cause telomere elongation would affect origin firing. We found that neither rif1-Δ1322 nor rif1HOOK affected firing of telomeric origins. We conclude that telomeric origin firing does not cause telomere elongation, and the role of Rif1 in regulating origin firing is separable from its role in regulating telomere length.

Download Full-text