scholarly journals Interaction of SNF1 Protein Kinase with Its Activating Kinase Sak1

2011 ◽  
Vol 10 (3) ◽  
pp. 313-319 ◽  
Author(s):  
Yang Liu ◽  
Xinjing Xu ◽  
Marian Carlson
Keyword(s):  
Protein Kinase ◽  
Kinase Domain ◽  
Catalytic Subunit ◽  
Glucose Limitation ◽  
Content Type ◽  
Snf1 Kinase ◽  
Phosphorylation State ◽  
Glucose Depletion ◽  
C Terminus ◽  
Amp Activated Protein Kinase

ABSTRACT The Saccharomyces cerevisiae SNF1 protein kinase, a member of the SNF1/AMP-activated protein kinase (AMPK) family, is activated by three kinases, Sak1, Tos3, and Elm1, which phosphorylate the Snf1 catalytic subunit on Thr-210 in response to glucose limitation and other stresses. Sak1 is the primary Snf1-activating kinase and is associated with Snf1 in a complex. Here we examine the interaction of Sak1 with SNF1. We report that Sak1 coimmunopurifies with the Snf1 catalytic subunit from extracts of both glucose-replete and glucose-limited cultures and that interaction occurs independently of the phosphorylation state of Snf1 Thr-210, Snf1 catalytic activity, and other SNF1 subunits. Sak1 interacts with the Snf1 kinase domain, and nonconserved sequences C terminal to the Sak1 kinase domain mediate interaction with Snf1 and augment the phosphorylation and activation of Snf1. The Sak1 C terminus is modified in response to glucose depletion, dependent on SNF1 activity. Replacement of the C terminus of Elm1 (or Tos3) with that of Sak1 enhanced the ability of the Elm1 kinase domain to interact with and phosphorylate Snf1. These findings indicate that the C terminus of Sak1 confers its function as the primary Snf1-activating kinase and suggest that the physical association of Sak1 with SNF1 facilitates responses to environmental change.

scholarly journals Differential Roles of the Glycogen-Binding Domains of β Subunits in Regulation of the Snf1 Kinase Complex

2009 ◽  
Vol 9 (1) ◽  
pp. 173-183 ◽  
Author(s):  
Simmanjeet Mangat ◽  
Dakshayini Chandrashekarappa ◽  
Rhonda R. McCartney ◽  
Karin Elbing ◽  
Martin C. Schmidt
Keyword(s):  
Protein Kinase ◽  
Phosphorylation Sites ◽  
Constitutive Activity ◽  
Glucose Limitation ◽  
Α Subunit ◽  
Snf1 Kinase ◽  
Binding Domains ◽  
Conserved Domain ◽  
Amp Activated Protein Kinase

ABSTRACT Members of the AMP-activated protein kinase family, including the Snf1 kinase of Saccharomyces cerevisiae, are activated under conditions of nutrient stress. AMP-activated protein kinases are heterotrimeric complexes composed of a catalytic α subunit and regulatory β and γ subunits. In this study, the role of the β subunits in the regulation of Snf1 activity was examined. Yeasts express three isoforms of the AMP-activated protein kinase consisting of Snf1 (α), Snf4 (γ), and one of three alternative β subunits, either Sip1, Sip2, or Gal83. The Gal83 isoform of the Snf1 complex is the most abundant and was analyzed in the greatest detail. All three β subunits contain a conserved domain referred to as the glycogen-binding domain. The deletion of this domain from Gal83 results in a deregulation of the Snf1 kinase, as judged by a constitutive activity independent of glucose availability. In contrast, the deletion of this homologous domain from the Sip1 and Sip2 subunits had little effect on Snf1 kinase regulation. Therefore, the different Snf1 kinase isoforms are regulated through distinct mechanisms, which may contribute to their specialized roles in different stress response pathways. In addition, the β subunits are subjected to phosphorylation. The responsible kinases were identified as being Snf1 and casein kinase II. The significance of the phosphorylation is unclear since the deletion of the region containing the phosphorylation sites in Gal83 had little effect on the regulation of Snf1 in response to glucose limitation.

scholarly journals Dosage-dependent modulation of glucose repression by MSN3 (STD1) in Saccharomyces cerevisiae.

1994 ◽  
Vol 14 (3) ◽  
pp. 1972-1978 ◽  
Author(s):  
E J Hubbard ◽  
R Jiang ◽  
M Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Glucose Repression ◽  
Binding Studies ◽  
Glucose Limitation ◽  
Snf1 Kinase ◽  
Multicopy Suppressor ◽  
C Terminus ◽  
In The Wild

The SNF1 protein kinase of Saccharomyces cerevisiae is required to relieve glucose repression of transcription. To identify components of the SNF1 pathway, we isolated multicopy suppressors of defects caused by loss of SNF4, an activator of the SNF1 kinase. Increased dosage of the MSN3 gene restored invertase expression in snf4 mutants and also relieved glucose repression in the wild type. Deletion of MSN3 caused no substantial phenotype, and we identified a homolog, MTH1, encoding a protein 61% identical to MSN3. Both are also homologous to chicken fimbrin, human plastin, and yeast SAC6 over a 43-residue region. Deletion of MSN3 and MTH1 together impaired derepression of invertase in response to glucose limitation. Finally, MSN3 physically interacts with the SNF1 protein kinase, as assayed by a two-hybrid system and by in vitro binding studies. MSN3 is the same gene as STD1, a multicopy suppressor of defects caused by overexpression of the C terminus of TATA-binding protein (R. W. Ganster, W. Shen, and M. C. Schmidt, Mol. Cell. Biol. 13:3650-3659, 1993). Taken together, these data suggest that MSN3 modulates the regulatory response to glucose and may couple the SNF1 pathway to transcription.

scholarly journals Springing into Action: Reg2 Negatively Regulates Snf1 Protein Kinase and Facilitates Recovery from Prolonged Glucose Starvation in Saccharomyces cerevisiae

2016 ◽  
Vol 82 (13) ◽  
pp. 3875-3885 ◽  
Author(s):  
Marcin Maziarz ◽  
Aishwarya Shevade ◽  
LaKisha Barrett ◽  
Sergei Kuchin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Carbon Source ◽  
Glucose Deprivation ◽  
Regulatory Subunit ◽  
Glucose Starvation ◽  
Wild Type ◽  
Glucose Limitation ◽  
Content Type ◽  
Glucose Depletion

ABSTRACTGlucose is the preferred carbon source for the yeastSaccharomyces cerevisiae. Glucose limitation activates Snf1 protein kinase, a key regulator of energy homeostasis that promotes utilization of alternative carbon sources and enforces energy conservation. Snf1 activation requires phosphorylation of its T-loop threonine (Thr210) by upstream kinases. When glucose is abundant, Snf1 is inhibited by Thr210 dephosphorylation. This involves the function of the type 1 protein phosphatase Glc7, which is targeted to Snf1 by a regulatory subunit, Reg1. Thereg1mutation causes increased Snf1 activity and mimics various aspects of glucose limitation, including slower growth. Reg2 is another Glc7 regulatory subunit encoded by a paralogous gene,REG2. Previous evidence indicated that thereg2mutation exacerbates the Snf1-dependent slow-growth phenotype caused byreg1, suggesting a link between Reg2 and Snf1. Here, we explore this link in more detail and present evidence that Reg2 contributes to Snf1 Thr210 dephosphorylation. Consistent with this role, Reg2 interacts with wild-type Snf1 but not with nonphosphorylatable Snf1-T210A. Reg2 accumulation increases in a Snf1-dependent manner during prolonged glucose deprivation, and glucose-starved cells lacking Reg2 exhibit delayed Snf1 Thr210 dephosphorylation and slower growth recovery upon glucose replenishment. Accordingly, cells lacking Reg2 are outcompeted by wild-type cells in the course of several glucose starvation/replenishment cycles. Collectively, our results support a model in which Reg2-Glc7 contributes to the negative control of Snf1 in response to glucose refeeding after prolonged starvation. The competitive growth advantage provided by Reg2 underscores the evolutionary significance of this paralog forS. cerevisiae.IMPORTANCEThe ability of microorganisms to respond to stress is essential for their survival. However, rapid recovery from stress could be equally crucial in competitive environments. Therefore, a wise stress response program should prepare cells for quick recovery upon reexposure to favorable conditions. Glucose is the preferred carbon source for the yeastS. cerevisiae. Glucose depletion activates the stress response protein kinase Snf1, which functions to limit energy-consuming processes, such as growth. We show that prolonged glucose deprivation also leads to Snf1-dependent accumulation of Reg2 and that this protein helps to inhibit Snf1 and to accelerate growth recovery upon glucose replenishment. Cells lacking Reg2 are readily outcompeted by wild-type cells during glucose depletion/replenishment cycles. Thus, while prolonged glucose deprivation might seem to put yeast cells “on their knees,” concomitant accumulation of Reg2 helps configure the cells into a “sprinter's crouch start position” to spring into action once glucose becomes available.

scholarly journals An inhibited conformation for the protein kinase domain of theSaccharomyces cerevisiaeAMPK homolog Snf1

2010 ◽  
Vol 66 (9) ◽  
pp. 999-1002 ◽  
Author(s):  
Michael J. Rudolph ◽  
Gabriele A. Amodeo ◽  
Liang Tong
Keyword(s):  
Protein Kinase ◽  
Active Site ◽  
Energy Homeostasis ◽  
Kinase Domain ◽  
Α Subunit ◽  
Protein Kinase Domain ◽  
Cellular Energy ◽  
Glucose Depletion ◽  
Inhibitory Domain ◽  
Amp Activated Protein Kinase

AMP-activated protein kinase (AMPK) is a master metabolic regulator for controlling cellular energy homeostasis. Its homolog in yeast, SNF1, is activated in response to glucose depletion and other stresses. The catalytic (α) subunit of AMPK/SNF1 in yeast (Snf1) contains a protein Ser/Thr kinase domain (KD), an auto-inhibitory domain (AID) and a region that mediates interactions with the two regulatory (β and γ) subunits. Here, the crystal structure of residues 41–440 of Snf1, which include the KD and AID, is reported at 2.4 Å resolution. The AID is completely disordered in the crystal. A new inhibited conformation of the KD is observed in a DFG-out conformation and with the glycine-rich loop adopting a structure that blocks ATP binding to the active site.

Dosage-dependent modulation of glucose repression by MSN3 (STD1) in Saccharomyces cerevisiae

1994 ◽  
Vol 14 (3) ◽  
pp. 1972-1978
Author(s):  
E J Hubbard ◽  
R Jiang ◽  
M Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Glucose Repression ◽  
Binding Studies ◽  
Glucose Limitation ◽  
Snf1 Kinase ◽  
Multicopy Suppressor ◽  
C Terminus ◽  
In The Wild

The SNF1 protein kinase of Saccharomyces cerevisiae is required to relieve glucose repression of transcription. To identify components of the SNF1 pathway, we isolated multicopy suppressors of defects caused by loss of SNF4, an activator of the SNF1 kinase. Increased dosage of the MSN3 gene restored invertase expression in snf4 mutants and also relieved glucose repression in the wild type. Deletion of MSN3 caused no substantial phenotype, and we identified a homolog, MTH1, encoding a protein 61% identical to MSN3. Both are also homologous to chicken fimbrin, human plastin, and yeast SAC6 over a 43-residue region. Deletion of MSN3 and MTH1 together impaired derepression of invertase in response to glucose limitation. Finally, MSN3 physically interacts with the SNF1 protein kinase, as assayed by a two-hybrid system and by in vitro binding studies. MSN3 is the same gene as STD1, a multicopy suppressor of defects caused by overexpression of the C terminus of TATA-binding protein (R. W. Ganster, W. Shen, and M. C. Schmidt, Mol. Cell. Biol. 13:3650-3659, 1993). Taken together, these data suggest that MSN3 modulates the regulatory response to glucose and may couple the SNF1 pathway to transcription.

Mutational analysis of the Saccharomyces cerevisiae SNF1 protein kinase and evidence for functional interaction with the SNF4 protein

1989 ◽  
Vol 9 (11) ◽  
pp. 5034-5044
Author(s):  
J L Celenza ◽  
M Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Mutational Analysis ◽  
Gene Dosage ◽  
Regulatory System ◽  
Kinase Domain ◽  
Regulatory Function ◽  
Protein Kinase Activity ◽  
Glucose Limitation

The SNF1 gene of Saccharomyces cerevisiae encodes a protein-serine/threonine kinase that is required for derepression of gene expression in response to glucose limitation. We present evidence that the protein kinase activity is essential for SNF1 function: substitution of Arg for Lys in the putative ATP-binding site results in a mutant phenotype. A polyhistidine tract near the N terminus was found to be dispensable. Deletion of the large region C terminal to the kinase domain only partially impaired SNF1 function, causing expression of invertase to be somewhat reduced but still glucose repressible. The function of the SNF4 gene, another component of the regulatory system, was required for maximal in vitro activity of the SNF1 protein kinase. Increased SNF1 gene dosage partially alleviated the requirement for SNF4. C-terminal deletions of SNF1 also reduced dependence on SNF4. Our findings suggest that SNF4 acts as a positive effector of the kinase but does not serve a regulatory function in signaling glucose availability.

scholarly journals Conformational heterogeneity of the allosteric drug and metabolite (ADaM) site in AMP-activated protein kinase (AMPK)

2018 ◽  
Vol 293 (44) ◽  
pp. 16994-17007 ◽  
Author(s):  
Xin Gu ◽  
Michael D. Bridges ◽  
Yan Yan ◽  
Parker W. de Waal ◽  
X. Edward Zhou ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Energy Homeostasis ◽  
Metabolic Diseases ◽  
Kinase Domain ◽  
Carbohydrate Binding ◽  
Conformational Heterogeneity ◽  
Α Subunit ◽  
Distance Distributions ◽  
Double Electron Electron Resonance ◽  
Amp Activated Protein Kinase

AMP-activated protein kinase (AMPK) is a master regulator of energy homeostasis and a promising drug target for managing metabolic diseases such as type 2 diabetes. Many pharmacological AMPK activators, and possibly unidentified physiological metabolites, bind to the allosteric drug and metabolite (ADaM) site at the interface between the kinase domain (KD) in the α-subunit and the carbohydrate-binding module (CBM) in the β-subunit. Here, using double electron–electron resonance (DEER) spectroscopy, we demonstrate that the CBM–KD interaction is partially dissociated and the interface highly disordered in the absence of pharmacological ADaM site activators as inferred from a low depth of modulation and broad DEER distance distributions. ADaM site ligands such as 991, and to a lesser degree phosphorylation, stabilize the KD–CBM association and strikingly reduce conformational heterogeneity in the ADaM site. Our findings that the ADaM site, formed by the KD–CBM interaction, can be modulated by diverse ligands and by phosphorylation suggest that it may function as a hub for integrating regulatory signals.

scholarly journals PRKAA1 Promotes Proliferation and Inhibits Apoptosis of Gastric Cancer Cells Through Activating JNK1 and Akt Pathways

2020 ◽  
Vol 28 (3) ◽  
pp. 213-223 ◽  
Author(s):  
Yangmei Zhang ◽  
Xichang Zhou ◽  
Long Cheng ◽  
Xiang Wang ◽  
Qinglin Zhang ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Protein Kinase ◽  
Cancer Cells ◽  
Akt Signaling ◽  
Catalytic Subunit ◽  
Gastric Cancer Cells ◽  
In Vivo Experiments ◽  
Compound C ◽  
Amp Activated Protein Kinase

PRKAA1 (protein kinase AMP-activated catalytic subunit α 1) is a catalytic subunit of AMP-activated protein kinase (AMPK), which plays a key role in regulating cellular energy metabolism through phosphorylation, and genetic variations in the PRKAA1 have been found to be associated with gastric cancer risk. However, the effect and underlying molecular mechanism of PRKAA1 on gastric cancer tumorigenesis, especially the proliferation and apoptosis, are not fully understood. Our data showed that PRKAA1 is highly expressed in BGC-823 and MKN45 cells and is expressed low in SGC-7901 and MGC-803 cells in comparison with the other gastric cancer cells. PRKAA1 downregulation by shRNA or treatment of AMPK inhibitor compound C significantly inhibited proliferation as well as promoted cell cycle arrest and apoptosis of BGC-823 and MKN45 cells. Moreover, the expression of PCNA and Bcl-2 and the activity of JNK1 and Akt signaling were also reduced in BGC-823 and MKN45 cells after PRKAA1 downregulation. In vivo experiments demonstrated that tumor growth in nude mice was significantly inhibited after PRKAA1 silencing. Importantly, inactivation of JNK1 or Akt signaling pathway significantly inhibited PRKAA1 overexpression-induced increased cell proliferation and decreased cell apoptosis in MGC-803 cells. In conclusion, our findings suggest that PRKAA1 increases proliferation and restrains apoptosis of gastric cancer cells through activating JNK1 and Akt pathways.

scholarly journals Pak1 Protein Kinase Regulates Activation and Nuclear Localization of Snf1-Gal83 Protein Kinase

2004 ◽  
Vol 24 (18) ◽  
pp. 8255-8263 ◽  
Author(s):  
Kristina Hedbacker ◽  
Seung-Pyo Hong ◽  
Marian Carlson
Keyword(s):  
Protein Kinase ◽  
Nuclear Localization ◽  
Kinase Activity ◽  
Fluorescent Protein ◽  
Protein Kinase Activity ◽  
Activation Loop ◽  
Glucose Limitation ◽  
Subunit Isoforms ◽  
Green Fluorescent ◽  
Amp Activated Protein Kinase

ABSTRACT Three kinases, Pak1, Tos3, and Elm1, activate Snf1 protein kinase in Saccharomyces cerevisiae. This cascade is conserved in mammals, where LKB1 activates AMP-activated protein kinase. We address the specificity of the activating kinases for the three forms of Snf1 protein kinase containing the β-subunit isoforms Gal83, Sip1, and Sip2. Pak1 is the most important kinase for activating Snf1-Gal83 in response to glucose limitation, but Elm1 also has a significant role; moreover, both Pak1 and Elm1 affect Snf1-Sip2. These findings exclude the possibility of a one-to-one correspondence between the activating kinases and the Snf1 complexes. We further identify a second, unexpected role for Pak1 in regulating Snf1-Gal83: the catalytic activity of Pak1 is required for the nuclear enrichment of Snf1-Gal83 in response to carbon stress. The nuclear enrichment of Snf1 fused to green fluorescent protein (GFP) depends on both Gal83 and Pak1 and is abolished by a mutation of the activation loop threonine; in contrast, the nuclear enrichment of Gal83-GFP occurs in a snf1Δ mutant and depends on Pak1 only when Snf1 is present. Snf1-Gal83 is the only form of the kinase that localizes to the nucleus. These findings, that Pak1 both activates Snf1-Gal83 and controls its nuclear localization, implicate Pak1 in regulating nuclear Snf1 protein kinase activity.

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