Nutrient sensing systems for rapid activation of the protein kinase A pathway in yeast

2005 ◽  
Vol 33 (1) ◽  
pp. 253-256 ◽  
Author(s):  
J.M. Thevelein ◽  
R. Geladé ◽  
I. Holsbeeks ◽  
O. Lagatie ◽  
Y. Popova ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Point Mutations ◽  
Nutrient Sensing ◽  
Amino Acid Permease ◽  
Yeast Saccharomyces Cerevisiae ◽  
Receptor System ◽  
Pka Pathway ◽  
Rapid Activation ◽  
Kinase A

The cAMP-protein kinase A (PKA) pathway in the yeast Saccharomyces cerevisiae controls a variety of properties that depend on the nutrient composition of the medium. High activity of the pathway occurs in the presence of rapidly fermented sugars like glucose or sucrose, but only as long as growth is maintained. Growth arrest of fermenting cells or growth on a respirative carbon source, like glycerol or ethanol, is associated with low activity of the PKA pathway. We have studied how different nutrients trigger rapid activation of the pathway. Glucose and sucrose activate cAMP synthesis through a G-protein-coupled receptor system, consisting of the GPCR Gpr1, the Gα protein Gpa2 and its RGS protein Rgs2. Glucose is also sensed intracellularly through its phosphorylation. Specific mutations in Gpr1 abolish glucose but not sucrose signalling. Activation of the PKA pathway by addition of a nitrogen source or phosphate to nitrogen- or phosphate-starved cells, respectively, is not mediated by an increase in cAMP. Activation by amino acids is triggered by the general amino acid permease Gap1, which functions as a transporter/receptor. Short truncation of the C-terminus results in constitutively activating alleles. Activation by ammonium uses the ammonium permeases Mep1 and Mep2 as receptor. Specific point mutations in Mep2 uncouple signalling from transport. Activation by phosphate is triggered a.o. by the Pho84 phosphate permease. Several mutations in Pho84 separating transport and signalling or triggering constitutive activation have been obtained.

scholarly journals Cooperative Activation of Lipolysis by Protein Kinase A and Protein Kinase C Pathways in 3T3-L1 Adipocytes

Endocrinology ◽  
2004 ◽  
Vol 145 (11) ◽  
pp. 4940-4947 ◽  
Author(s):  
Katrin Fricke ◽  
Aleksandra Heitland ◽  
Erik Maronde
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Signaling Pathways ◽  
Hormone Sensitive Lipase ◽  
Glycerol Release ◽  
Kinase C ◽  
Lipolytic Effect ◽  
Pka Pathway ◽  
Kinase A

Abstract In the present study, we investigate the coherence of signaling pathways leading to lipolysis in 3T3-L1 adipocytes. We observe two linear signaling pathways: one well known, acting via cAMP and protein kinase A (PKA) activation, and a second one induced by phorbol 12-myristate 13-acetate treatment involving protein kinase C (PKC) and MAPK. We demonstrate that both the PKA regulatory subunits RIα and RIIβ are expressed in 3T3-L1 adipocytes and are responsible for the lipolytic effect mediated via the cAMP/PKA pathway. Inhibition of the PKA pathway by the selective PKA inhibitor Rp-8-CPT-cAMPS does not impair lipolysis induced by PKC activation, and neither PD98059 nor U0126, as known MAPK kinase inhibitors, changes the level of glycerol release caused by PKA activation, indicating no cross-talk between these two pathways when only one is activated. However, when both are activated, they act synergistically on glycerol release. Additional experiments focusing on this synergy show no involvement of MAPK phosphorylation and cAMP formation. Phosphorylation of hormone-sensitive lipase is similar upon stimulation of either pathway, but we demonstrate a difference in the ability of both PKA and the PKC pathway activation to phosphorylate perilipin, which in turn may be an explanation for the different maximal lipolytic effect of both pathways.

scholarly journals The activation of the chymotrypsin-like activity of the proteasome is regulated by soluble adenyl cyclase/cAMP/protein kinase A pathway and required for human sperm capacitation

2019 ◽  
Vol 25 (10) ◽  
pp. 587-600 ◽  
Author(s):  
Héctor Zapata-Carmona ◽  
Lina Barón ◽  
Lidia M Zuñiga ◽  
Emilce Silvina Díaz ◽  
Milene Kong ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Current Knowledge ◽  
Human Sperm ◽  
Adenyl Cyclase ◽  
Sperm Capacitation ◽  
Time Treatment ◽  
Proteasome Subunits ◽  
Pka Pathway ◽  
Kinase A

Abstract One of the first events of mammalian sperm capacitation is the activation of the soluble adenyl cyclase/cAMP/protein kinase A (SACY/cAMP/PKA) pathway. Here, we evaluated whether the increase in PKA activity at the onset of human sperm capacitation is responsible for the activation of the sperm proteasome and whether this activation is required for capacitation progress. Viable human sperm were incubated with inhibitors of the SACY/cAMP/PKA pathway. The chymotrypsin-like activity of the sperm proteasome was evaluated using a fluorogenic substrate. Sperm capacitation status was evaluated using the chlortetracycline assay and tyrosine phosphorylation. To determine whether proteasomal subunits were phosphorylated by PKA, the proteasome was immunoprecipitated and tested on a western blot using an antibody against phosphorylated PKA substrates. Immunofluorescence microscopy analysis and co-immunoprecipitation (IPP) were used to investigate an association between the catalytic subunit alpha of PKA (PKA-Cα) and the proteasome. The chymotrypsin-like activity of the sperm proteasome significantly increased after 5 min of capacitation (P < 0.001) and remained high for the remaining incubation time. Treatment with H89, KT5720 or KH7 significantly decreased the chymotrypsin-like activity of the proteasome (P < 0.001). IPP experiments indicated that PKA inhibition significantly modified phosphorylation of proteasome subunits. In addition, PKA-Cα colocalized with the proteasome in the equatorial segment and in the connecting piece, and co-immunoprecipitated with the proteasome. This is the first demonstration of sperm proteasome activity being directly regulated by SACY/PKA-Cα. This novel discovery extends our current knowledge of sperm physiology and may be used to manage sperm capacitation during assisted reproductive technology procedures.

scholarly journals Genetic Analysis of the Kinetochore DASH Complex Reveals an Antagonistic Relationship with the Ras/Protein Kinase A Pathway and a Novel Subunit Required for Ask1 Association

2005 ◽  
Vol 25 (2) ◽  
pp. 767-778 ◽  
Author(s):  
Ju-mei Li ◽  
Yumei Li ◽  
Stephen J. Elledge
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Chromosome Segregation ◽  
Biochemical Analysis ◽  
Genetic Interaction ◽  
Negative Regulator ◽  
Ras Protein ◽  
Sister Chromatids ◽  
Pka Pathway ◽  
Kinase A

ABSTRACT DASH is a microtubule- and kinetochore-associated complex required for proper chromosome segregation and bipolar attachment of sister chromatids on the mitotic spindle. We have undertaken a genetic and biochemical analysis of the DASH complex and uncovered a strong genetic interaction of DASH with the Ras/protein kinase A (PKA) pathway. Overexpression of PDE2 or deletion of RAS2 rescued the temperature sensitivity of ask1-3 mutants. Ras2 negatively regulates DASH through the PKA pathway. Constitutive PKA activity caused by mutation of the negative regulator BCY1 is toxic to DASH mutants such as ask1 and dam1. In addition, we have discovered two novel subunits of DASH, Hsk2 and Hsk3 (helper of Ask1), which are microproteins of fewer than 75 amino acids, as dosage suppressors of ask1 mutants. These are essential genes that colocalize with DASH components on spindles and kinetochores and are present in the DASH complex. Mutants in hsk3 arrest cells in mitosis with short spindles and broken spindle structures characteristic of other DASH mutants. Hsk3 is critical for the integrity of the DASH complex because in hsk3 mutants the association of Dam1, Duo1, Spc34, and Spc19 with Ask1 is greatly diminished. We propose that Hsk3 acts to incorporate Ask1 into the DASH complex.

scholarly journals Functional Heterogeneity of Protein Kinase A Activation in Multipotent Stromal Cells

2020 ◽  
Vol 21 (12) ◽  
pp. 4442
Author(s):  
Pyotr A. Tyurin-Kuzmin ◽  
Maxim N. Karagyaur ◽  
Konstantin Yu. Kulebyakin ◽  
Daniyar T. Dyikanov ◽  
Vadim I. Chechekhin ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Stromal Cells ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Multipotent Stromal Cells ◽  
Adenylyl Cyclases ◽  
Functional Heterogeneity ◽  
Pka Pathway ◽  
Kinase A

Multipotent stromal cells (MSC) demonstrate remarkable functional heterogeneity; however, its molecular mechanisms remain largely obscure. In this study, we explored MSC response to hormones, which activate Gs-protein / cyclic AMP (cAMP) / protein kinase A (PKA) dependent signaling, at the single cell level using genetically encoded biosensor PKA-Spark. For the first time, we demonstrated that about half of cultured MSCs are not able to activate the cAMP/PKA pathway, possibly due to the limited availability of adenylyl cyclases. Using this approach, we showed that MSC subpopulations responding to various hormones largely overlapped, and the share of responding cells did not exceed 40%. Using clonal analysis, we showed that signaling heterogeneity of MSC could be formed de novo within 2 weeks.

scholarly journals The role of the protein kinase A pathway in the response to alkaline pH stress in yeast

2011 ◽  
Vol 438 (3) ◽  
pp. 523-533 ◽  
Author(s):  
Carlos Casado ◽  
Asier González ◽  
Maria Platara ◽  
Amparo Ruiz ◽  
Joaquín Ariño
Keyword(s):  
Stress Response ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Substantial Part ◽  
Alkaline Stress ◽  
Alkaline Ph ◽  
Phenotypic Analysis ◽  
Ph Tolerance ◽  
Pka Pathway ◽  
Kinase A

Exposure of Saccharomyces cerevisiae to alkaline pH provokes a stress condition that generates a compensatory reaction. In the present study we examined a possible role for the PKA (protein kinase A) pathway in this response. Phenotypic analysis revealed that mutations that activate the PKA pathway (ira1 ira2, bcy1) tend to cause sensitivity to alkaline pH, whereas its deactivation enhances tolerance to this stress. We observed that alkalinization causes a transient decrease in cAMP, the main regulator of the pathway. Alkaline pH causes rapid nuclear localization of the PKA-regulated Msn2 transcription factor which, together with Msn4, mediates a general stress response by binding with STRE (stress response element) sequences in many promoters. Consequently, a synthetic STRE–LacZ reporter shows a rapid induction in response to alkaline stress. A msn2 msn4 mutant is sensitive to alkaline pH, and transcriptomic analysis reveals that after 10 min of alkaline stress, the expression of many induced genes (47%) depends, at least in part, on the presence of Msn2 and Msn4. Taken together, these results demonstrate that inhibition of the PKA pathway by alkaline pH represents a substantial part of the adaptive response to this kind of stress and that this response involves Msn2/Msn4-mediated genome expression remodelling. However, the relevance of attenuation of PKA in high pH tolerance is probably not restricted to regulation of Msn2 function.

scholarly journals Role for YakA, cAMP, and Protein Kinase A in Regulation of Stress Responses ofDictyostelium discoideumCells

2002 ◽  
Vol 13 (7) ◽  
pp. 2266-2275 ◽  
Author(s):  
Alexandre Taminato ◽  
Raquel Bagattini ◽  
Renata Gorjão ◽  
Guokai Chen ◽  
Adam Kuspa ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Stress Responses ◽  
Site Mutation ◽  
Camp Synthesis ◽  
Different Types ◽  
Arrest Growth ◽  
Pka Pathway ◽  
Kinase A

The Dictyostelium protein kinase YakA is required for the growth-to-development transition. During growth YakA controls the cell cycle, regulating the intervals between cell divisions. When starved for nutrients Dictyosteliumcells arrest growth and undergo changes in gene expression, decreasing vegetative mRNAs and inducing the expression of pkaC. YakA is an effector of these changes, being necessary for the decrease of vegetative mRNA expression and the increase of protein kinase A (PKA) activity that will ultimately regulate expression of adenylyl cyclase, cAMP synthesis, and the induction of development. We report a role for this kinase in the response to nitrosoative or oxidative stress of Dictyostelium cells. Hydrogen peroxide and sodium nitroprusside arrest the growth of cells and trigger cAMP synthesis and activation of PKA in a manner similar to the well-established response to nutrient starvation. We have found thatyakA null cells are hypersensitive to nitrosoative/oxidative stress and that a second-site mutation inpkaC suppresses this sensitivity. The response to different stresses has been investigated and YakA, cAMP, and PKA have been identified as components of the pathway that regulate the growth arrest that follows treatment with compounds that generate reactive oxygen species. The effect of different types of stress was evaluated in Dictyostelium and the YakA/PKA pathway was also implicated in the response to heat stress.

scholarly journals Protein Kinase A and Sch9 Cooperatively Regulate Induction of Autophagy in Saccharomyces cerevisiae

2007 ◽  
Vol 18 (10) ◽  
pp. 4180-4189 ◽  
Author(s):  
Tomohiro Yorimitsu ◽  
Shadia Zaman ◽  
James R. Broach ◽  
Daniel J. Klionsky
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factors ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Signaling Pathways ◽  
Nutrient Sensing ◽  
Regulatory Mechanisms ◽  
Eukaryotic Cells ◽  
Tor Kinase ◽  
Kinase A

Autophagy is a highly conserved, degradative process in eukaryotic cells. The rapamycin-sensitive Tor kinase complex 1 (TORC1) has a major role in regulating induction of autophagy; however, the regulatory mechanisms are not fully understood. Here, we find that the protein kinase A (PKA) and Sch9 signaling pathways regulate autophagy cooperatively in yeast. Autophagy is induced in cells when PKA and Sch9 are simultaneously inactivated. Mutant alleles of these kinases bearing a mutation that confers sensitivity to the ATP-analogue inhibitor C3-1′-naphthyl-methyl PP1 revealed that autophagy was induced independently of effects on Tor kinase. The PKA–Sch9-mediated autophagy depends on the autophagy-related 1 kinase complex, which is also essential for TORC1-regulated autophagy, the transcription factors Msn2/4, and the Rim15 kinase. The present results suggest that autophagy is controlled by the signals from at least three partly separate nutrient-sensing pathways that include PKA, Sch9, and TORC1.

scholarly journals Protein Kinase A, TOR, and Glucose Transport Control the Response to Nutrient Repletion in Saccharomyces cerevisiae

2007 ◽  
Vol 7 (2) ◽  
pp. 358-367 ◽  
Author(s):  
Matthew G. Slattery ◽  
Dritan Liko ◽  
Warren Heideman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Glucose Transport ◽  
Transcriptional Response ◽  
Nutrient Sensing ◽  
Fresh Medium ◽  
Transcriptional Responses ◽  
Nutrient Regulation ◽  
Kinase A

ABSTRACT Nutrient repletion leads to substantial restructuring of the transcriptome in Saccharomyces cerevisiae. The expression levels of approximately one-third of all S. cerevisiae genes are altered at least twofold when a nutrient-depleted culture is transferred to fresh medium. Several nutrient-sensing pathways are known to play a role in this process, but the relative contribution that each pathway makes to the total response has not been determined. To better understand this, we used a chemical-genetic approach to block the protein kinase A (PKA), TOR (target of rapamycin), and glucose transport pathways, alone and in combination. Of the three pathways, we found that loss of PKA produced the largest effect on the transcriptional response; however, many genes required both PKA and TOR for proper nutrient regulation. Those genes that did not require PKA or TOR for nutrient regulation were dependent on glucose transport for either nutrient induction or repression. Therefore, loss of these three pathways is sufficient to prevent virtually the entire transcriptional response to fresh medium. In the absence of fresh medium, activation of the cyclic AMP/PKA pathway does not induce cellular growth; nevertheless, PKA activation induced a substantial fraction of the PKA-dependent genes. In contrast, the absence of fresh medium strongly limited gene repression by PKA. These results account for the signals needed to generate the transcriptional responses to glucose, including induction of growth genes required for protein synthesis and repression of stress genes, as well as the classical glucose repression and hexose transporter responses.

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