The mutant type 1 protein phosphatase encoded by glc7-1 from Saccharomyces cerevisiae fails to interact productively with the GAC1-encoded regulatory subunit

1994 ◽  
Vol 14 (2) ◽  
pp. 896-905
Author(s):  
J S Stuart ◽  
D L Frederick ◽  
C M Varner ◽  
K Tatchell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Phosphatase ◽  
Glycogen Synthase ◽  
Regulatory Subunit ◽  
Loss Of Function ◽  
Mutant Type ◽  
Glycogen Accumulation

Loss-of-function gac1 mutants of Saccharomyces cerevisiae fail to accumulate normal levels of glycogen because of low glycogen synthase activity. Increased dosage of GAC1 results in increased activity of glycogen synthase and a corresponding hyperaccumulation of glycogen. The glycogen accumulation phenotype of gac1 is similar to that of glc7-1, a type 1 protein phosphatase mutant. We have partially characterized the GAC1 gene product (Gac1p) and show that levels of Gac1p increase during growth with the same kinetics as glycogen accumulation. Gac1p is phosphorylated in vivo and is hyperphosphorylated in a glc7-1 mutant. Gac1p and the type 1 protein phosphatase directly interact in vitro, as assayed by coimmunoprecipitation, and in vivo, as determined by the dihybrid assay described elsewhere (S. Fields and O.-k. Song, Nature [London] 340:245-246, 1989). The interaction between Gac1p and the glc7-1-encoded form of the type 1 protein phosphatase is defective, as assayed by either immunoprecipitation or the dihybrid assay. Increased dosage of GAC1 partially suppresses the glycogen defect of glc7-1. Collectively, our data support the hypotheses that GAC1 encodes a regulatory subunit of type 1 protein phosphatase and that the glycogen accumulation defect of glc7-1 is due at least in part to the inability of the mutant phosphatase to interact with its regulatory subunit.

scholarly journals The mutant type 1 protein phosphatase encoded by glc7-1 from Saccharomyces cerevisiae fails to interact productively with the GAC1-encoded regulatory subunit.

1994 ◽  
Vol 14 (2) ◽  
pp. 896-905 ◽  
Author(s):  
J S Stuart ◽  
D L Frederick ◽  
C M Varner ◽  
K Tatchell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Phosphatase ◽  
Glycogen Synthase ◽  
Regulatory Subunit ◽  
Loss Of Function ◽  
Mutant Type ◽  
Glycogen Accumulation

Loss-of-function gac1 mutants of Saccharomyces cerevisiae fail to accumulate normal levels of glycogen because of low glycogen synthase activity. Increased dosage of GAC1 results in increased activity of glycogen synthase and a corresponding hyperaccumulation of glycogen. The glycogen accumulation phenotype of gac1 is similar to that of glc7-1, a type 1 protein phosphatase mutant. We have partially characterized the GAC1 gene product (Gac1p) and show that levels of Gac1p increase during growth with the same kinetics as glycogen accumulation. Gac1p is phosphorylated in vivo and is hyperphosphorylated in a glc7-1 mutant. Gac1p and the type 1 protein phosphatase directly interact in vitro, as assayed by coimmunoprecipitation, and in vivo, as determined by the dihybrid assay described elsewhere (S. Fields and O.-k. Song, Nature [London] 340:245-246, 1989). The interaction between Gac1p and the glc7-1-encoded form of the type 1 protein phosphatase is defective, as assayed by either immunoprecipitation or the dihybrid assay. Increased dosage of GAC1 partially suppresses the glycogen defect of glc7-1. Collectively, our data support the hypotheses that GAC1 encodes a regulatory subunit of type 1 protein phosphatase and that the glycogen accumulation defect of glc7-1 is due at least in part to the inability of the mutant phosphatase to interact with its regulatory subunit.

scholarly journals The REG2 gene of Saccharomyces cerevisiae encodes a type 1 protein phosphatase-binding protein that functions with Reg1p and the Snf1 protein kinase to regulate growth.

1996 ◽  
Vol 16 (6) ◽  
pp. 2922-2931 ◽  
Author(s):  
D L Frederick ◽  
K Tatchell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Protein Phosphatase ◽  
Slow Growth ◽  
Glucose Repression ◽  
Growth Defect ◽  
Loss Of Function ◽  
Cell Extracts

The GLC7 gene of Saccharomyces cerevisiae encodes the catalytic subunit of type 1 protein phosphatase (PP1) and is essential for cell growth. We have isolated a previously uncharacterized gene, REG2, on the basis of its ability to interact with Glc7p in the two-hybrid system. Reg2p interacts with Glc7p in vivo, and epitope-tagged derivatives of Reg2p and Glc7p coimmunoprecipitate from cell extracts. The predicted protein product of the REG2 gene is similar to Reg1p, a protein believed to direct PP1 activity in the glucose repression pathway. Mutants with a deletion of reg1 display a mild slow-growth defect, while reg2 mutants exhibit a wild-type phenotype. However, mutants with deletions of both reg1 and reg2 exhibit a severe growth defect. Overexpression of REG2 complements the slow-growth defect of a reg1 mutant but does not complement defects in glycogen accumulation or glucose repression, two traits also associated with a reg1 deletion. These results indicate that REG1 has a unique role in the glucose repression pathway but acts together with REG2 to regulate some as yet uncharacterized function important for growth. The growth defect of a reg1 reg2 double mutant is alleviated by a loss-of-function mutation in the SNF1-encoded protein kinase. The snf1 mutation also suppresses the glucose repression defects of reg1. Together, our data are consistent with a model in which Reg1p and Reg2p control the activity of PP1 toward substrates that are phosphorylated by the Snf1p kinase.

scholarly journals Propofol attenuates lung ischemia/reperfusion injury though the involvement of the MALAT1/microRNA-144/GSK3β axis

2021 ◽  
Vol 27 (1) ◽  
Author(s):  
Jian-Ping Zhang ◽  
Wei-Jing Zhang ◽  
Miao Yang ◽  
Hua Fang
Keyword(s):  
Cell Apoptosis ◽  
Ischemia Reperfusion Injury ◽  
Glycogen Synthase ◽  
Ischemia Reperfusion ◽  
Inflammatory Factors ◽  
Therapeutic Effects ◽  
Protective Effects ◽  
Loss Of Function

Abstract Background Propofol, an intravenous anesthetic, was proven to protect against lung ischemia/reperfusion (I/R) injury. However, the detailed mechanism of Propofol in lung I/R injury is still elusive. This study was designed to explore the therapeutic effects of Propofol, both in vivo and in vitro, on lung I/R injury and the underlying mechanisms related to metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)/microRNA-144 (miR-144)/glycogen synthase kinase-3β (GSK3β). Methods C57BL/6 mice were used to establish a lung I/R injury model while pulmonary microvascular endothelial cells (PMVECs) were constructed as hypoxia/reperfusion (H/R) cellular model, both of which were performed with Propofol treatment. Gain- or loss-of-function approaches were subsequently employed, followed by observation of cell apoptosis in lung tissues and evaluation of proliferative and apoptotic capabilities in H/R cells. Meanwhile, the inflammatory factors, autophagosomes, and autophagy-related proteins were measured. Results Our experimental data revealed that Propofol treatment could decrease the elevated expression of MALAT1 following I/R injury or H/R induction, indicating its protection against lung I/R injury. Additionally, overexpressing MALAT1 or GSK3β promoted the activation of autophagosomes, proinflammatory factor release, and cell apoptosis, suggesting that overexpressing MALAT1 or GSK3β may reverse the protective effects of Propofol against lung I/R injury. MALAT1 was identified to negatively regulate miR-144 to upregulate the GSK3β expression. Conclusion Overall, our study demonstrated that Propofol played a protective role in lung I/R injury by suppressing autophagy and decreasing release of inflammatory factors, with the possible involvement of the MALAT1/miR-144/GSK3β axis.

scholarly journals The GLC7 type 1 protein phosphatase is required for glucose repression in Saccharomyces cerevisiae.

1994 ◽  
Vol 14 (10) ◽  
pp. 6789-6796 ◽  
Author(s):  
J Tu ◽  
M Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Genetic Analysis ◽  
Protein Phosphatase ◽  
Glucose Repression ◽  
Regulatory Mechanisms ◽  
Genetic Studies ◽  
Glycogen Accumulation ◽  
Regulatory Response

We cloned the GLC7/DIS2S1 gene by complementation of the cid1-226 mutation, which relieves glucose repression in Saccharomyces cerevisiae. GLC7 encodes the catalytic subunit of type 1 protein phosphatase (PP1). Genetic analysis and sequencing showed that cid1-226 is an allele of GLC7, now designated glc7-T152K, which alters threonine 152 to lysine. We also show that the glc7-1 and glc7-T152K alleles cause distinct phenotypes: glc7-1 causes a severe defect in glycogen accumulation but does not relieve glucose repression, whereas glc7-T152K does not prevent glycogen accumulation. These findings are discussed in light of evidence that interaction with different regulatory or targeting subunits directs the participation of PP1 in diverse cellular regulatory mechanisms. Finally, genetic studies suggest that PP1 functions antagonistically to the SNF1 protein kinase in the regulatory response to glucose.

scholarly journals Loss of a Protein Phosphatase 2A Regulatory Subunit (Cdc55p) Elicits Improper Regulation of Swe1p Degradation

2000 ◽  
Vol 20 (21) ◽  
pp. 8143-8156 ◽  
Author(s):  
Haifeng Yang ◽  
Wei Jiang ◽  
Matthew Gentry ◽  
Richard L. Hallberg
Keyword(s):  
Phosphatase Activity ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Cell Types ◽  
Regulatory Subunit ◽  
Cyclin Dependent Kinase ◽  
Wild Type ◽  
Phosphatase 2A

ABSTRACT CDC55 encodes a Saccharomyces cerevisiaeprotein phosphatase 2A (PP2A) regulatory subunit.cdc55-null cells growing at low temperature exhibit a failure of cytokinesis and produce abnormally elongated buds, butcdc55-null cells producing the cyclin-dependent kinase Cdc28-Y19F, which is unable to be inhibited by Y19 phosphorylation, show a loss of the abnormal morphology. Furthermore,cdc55-null cells exhibit a hyperphosphorylation of Y19. For these reasons, we have examined in wild-type and cdc55-null cells the levels and activities of the kinase (Swe1p) and phosphatase (Mih1p) that normally regulate the extent of Cdc28 Y19 phosphorylation. We find that Mih1p levels are comparable in the two strains, and an estimate of the in vivo and in vitro phosphatase activity of this enzyme in the two cell types indicates no marked differences. By contrast, while Swe1p levels are similar in unsynchronized and S-phase-arrested wild-type and cdc55-null cells, Swe1 kinase is found at elevated levels in mitosis-arrestedcdc55-null cells. This excess Swe1p incdc55-null cells is the result of ectopic stabilization of this protein during G2 and M, thereby accounting for the accumulation of Swe1p in mitosis-arrested cells. We also present evidence indicating that, in cdc55-null cells, misregulated PP2A phosphatase activity is the cause of both the ectopic stabilization of Swe1p and the production of the morphologically abnormal phenotype.

scholarly journals Mutations in the Gal83 Glycogen-Binding Domain Activate the Snf1/Gal83 Kinase Pathway by a Glycogen-Independent Mechanism

2004 ◽  
Vol 24 (1) ◽  
pp. 352-361 ◽  
Author(s):  
Heather A. Wiatrowski ◽  
Bryce J. W. van Denderen ◽  
Cristin D. Berkey ◽  
Bruce E. Kemp ◽  
David Stapleton ◽  
...  
Keyword(s):  
Glycogen Synthase ◽  
Metabolic Stress ◽  
Binding Domain ◽  
Glycogen Accumulation ◽  
Snf1 Kinase ◽  
Transcriptional Regulatory ◽  
Amp Activated Protein Kinase

ABSTRACT The yeast Snf1 kinase and its mammalian ortholog, AMP-activated protein kinase (AMPK), regulate responses to metabolic stress. Previous studies identified a glycogen-binding domain in the AMPK β1 subunit, and the sequence is conserved in the Snf1 kinase β subunits Gal83 and Sip2. Here we use genetic analysis to assess the role of this domain in vivo. Alteration of Gal83 at residues that are important for glycogen binding of AMPK β1 abolished glycogen binding in vitro and caused diverse phenotypes in vivo. Various Snf1/Gal83-dependent processes were upregulated, including glycogen accumulation, expression of RNAs encoding glycogen synthase, haploid invasive growth, the transcriptional activator function of Sip4, and activation of the carbon source-responsive promoter element. Moreover, the glycogen-binding domain mutations conferred transcriptional regulatory phenotypes even in the absence of glycogen, as determined by analysis of a mutant strain lacking glycogen synthase. Thus, mutation of the glycogen-binding domain of Gal83 positively affects Snf1/Gal83 kinase function by a mechanism that is independent of glycogen binding.

scholarly journals TORC1 signaling modulates Cdk8-dependent GAL gene expression in Saccharomyces cerevisiae

Genetics ◽  
2021 ◽  
Author(s):  
Riley Horvath ◽  
Nicole Hawe ◽  
Cindy Lam ◽  
Konstantin Mestnikov ◽  
Mariam Eji-Lasisi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Nuclear Localization ◽  
Protein Phosphatase ◽  
Regulatory Subunit ◽  
Gal Genes ◽  
Dependent Site ◽  
Insight Into

Abstract Cdk8 of the RNA Polymerase II mediator kinase complex regulates gene expression by phosphorylating sequence-specific transcription factors. This function is conserved amongst eukaryotes, but the signals and mechanisms regulating Cdk8 activity and phosphorylation of its substrates are unknown. Full induction of the GAL genes in yeast requires phosphorylation of the transcriptional activator Gal4 by Cdk8. We used a screen to identify regulators of the Cdk8-dependent phosphorylation on Gal4, from which we identified multiple mutants with defects in TORC1 signaling. One mutant, designated gal four throttle 1 (gft1) was identified as a recessive allele of hom3, encoding aspartokinase, and mutations in hom3 caused effects typical of inhibition of TORC1, including rapamycin sensitivity and enhanced nuclear localization of the TORC1-responsive transcription factor Gat1. Mutations in hom3 also inhibit phosphorylation of Gal4 in vivo at the Cdk8-dependent site on Gal4, as did mutations of tor1, but these mutations did not affect activity of Cdk8 assayed in vitro. Disruption of cdc55, encoding a regulatory subunit of the TORC1-regulated protein phosphatase PP2A, suppressed the effect of hom3 and tor1 mutations on GAL expression, and also restored phosphorylation of Gal4 at the Cdk8-dependent site in vivo. These observations demonstrate that TORC1 signaling regulates GAL induction through the activity of PP2A/Cdc55, and suggest that Cdk8-dependent phosphorylation of Gal4 is opposed by PP2A/Cdc55 dephosphorylation. These results provide insight into how induction of transcription by a specific inducer can be modulated by global nutritional signals through regulation of Cdk8-dependent phosphorylation.

The GLC7 type 1 protein phosphatase is required for glucose repression in Saccharomyces cerevisiae

1994 ◽  
Vol 14 (10) ◽  
pp. 6789-6796
Author(s):  
J Tu ◽  
M Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Genetic Analysis ◽  
Protein Phosphatase ◽  
Glucose Repression ◽  
Regulatory Mechanisms ◽  
Genetic Studies ◽  
Glycogen Accumulation ◽  
Regulatory Response

We cloned the GLC7/DIS2S1 gene by complementation of the cid1-226 mutation, which relieves glucose repression in Saccharomyces cerevisiae. GLC7 encodes the catalytic subunit of type 1 protein phosphatase (PP1). Genetic analysis and sequencing showed that cid1-226 is an allele of GLC7, now designated glc7-T152K, which alters threonine 152 to lysine. We also show that the glc7-1 and glc7-T152K alleles cause distinct phenotypes: glc7-1 causes a severe defect in glycogen accumulation but does not relieve glucose repression, whereas glc7-T152K does not prevent glycogen accumulation. These findings are discussed in light of evidence that interaction with different regulatory or targeting subunits directs the participation of PP1 in diverse cellular regulatory mechanisms. Finally, genetic studies suggest that PP1 functions antagonistically to the SNF1 protein kinase in the regulatory response to glucose.

scholarly journals Cyclin Partners Determine Pho85 Protein Kinase Substrate Specificity In Vitro and In Vivo: Control of Glycogen Biosynthesis by Pcl8 and Pcl10

1998 ◽  
Vol 18 (6) ◽  
pp. 3289-3299 ◽  
Author(s):  
Dongqing Huang ◽  
Jason Moffat ◽  
Wayne A. Wilson ◽  
Lynda Moore ◽  
Christine Cheng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acid Phosphatase ◽  
Protein Kinase ◽  
Substrate Specificity ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Glycogen Accumulation ◽  
Acid Phosphatase Gene

ABSTRACT In Saccharomyces cerevisiae, PHO85 encodes a cyclin-dependent protein kinase (Cdk) with multiple roles in cell cycle and metabolic controls. In association with the cyclin Pho80, Pho85 controls acid phosphatase gene expression through phosphorylation of the transcription factor Pho4. Pho85 has also been implicated as a kinase that phosphorylates and negatively regulates glycogen synthase (Gsy2), and deletion of PHO85 causes glycogen overaccumulation. We report that the Pcl8/Pcl10 subgroup of cyclins directs Pho85 to phosphorylate glycogen synthase both in vivo and in vitro. Disruption of PCL8 and PCL10 caused hyperaccumulation of glycogen, activation of glycogen synthase, and a reduction in glycogen synthase kinase activity in vivo. However, unlikepho85 mutants, pcl8 pcl10 cells had normal morphologies, grew on glycerol, and showed proper regulation of acid phosphatase gene expression. In vitro, Pho80-Pho85 complexes effectively phosphorylated Pho4 but had much lower activity toward Gsy2. In contrast, Pcl10-Pho85 complexes phosphorylated Gsy2 at Ser-654 and Thr-667, two physiologically relevant sites, but only poorly phosphorylated Pho4. Thus, both the in vitro and in vivo substrate specificity of Pho85 is determined by the cyclin partner. Mutation ofPHO85 suppressed the glycogen storage deficiency ofsnf1 or glc7-1 mutants in which glycogen synthase is locked in an inactive state. Deletion of PCL8and PCL10 corrected the deficit in glycogen synthase activity in both the snf1 and glc7-1 mutants, but glycogen synthesis was restored only in the glc7-1mutant strain. This genetic result suggests an additional role for Pho85 in the negative regulation of glycogen accumulation that is independent of Pcl8 and Pcl10.

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