The crystal structure of yeast regulatory subunit reveals key evolutionary insights into Protein Kinase A oligomerization.

Protein Kinase A (PKA) is a widespread enzyme that plays a key role in many signaling pathways from lower eukaryotes to metazoans. In mammals, the regulatory (R) subunits sequester and target the catalytic (C) subunits to proper subcellular locations. This targeting is accomplished by the dimerization and docking (D/D) domain of the R subunits. The activation of the holoenzyme depends on the binding of the second messenger cAMP. The only available structures of the D/D domain proceed from mammalian sources. Unlike dimeric mammalian counterparts, the R subunit from Saccharomyces cerevisiae (Bcy1) forms tetramers in solution. Here we describe the first high-resolution structure of a non-mammalian D/D domain. The tetramer in the crystals of the Bcy1 D/D domain is a dimer of dimers that retain the classical D/D domain fold. By using phylogenetic and structural analyses combined with site-directed mutagenesis, we found that fungal R subunits present an insertion of a single amino acid at the D/D domain that shifts the position of a downstream, conserved arginine. This residue participates in intra-dimer interactions in mammalian D/D domains, while due to this insertion it is involved in inter-dimer contacts in Bcy1, which are crucial for the stability of the tetramer. This surprising finding challenges well-established concepts regarding the oligomeric state within the PKAR protein family and provides important insights into the yet unexplored structural diversity of the D/D domains and the molecular determinants of R subunit oligomerization.

cAMP-PKA signal transduction specificity in Saccharomyces cerevisiae.

Living cells have developed a set of complex signaling responses, which allow them to withstand different environmental challenges. Signaling pathways enable the cell to monitor external and internal states and to articulate the appropriate physiological responses. Cellular signal transmission requires the dynamic formation of spatiotemporal controlled molecular interactions. One of the most important signaling circuits in Saccharomyces cerevisiae is the one controlled by cAMP-Protein Kinase A (PKA). In budding yeast, extracellular glucose and a plethora of signals related with growth and stress conditions regulate the intracellular cAMP levels that modulate PKA activity which in turn regulates a broad range of cellular processes. The cAMP-PKA signaling output requires a controlled specificity of the PKA responses. In this review we discuss the molecular mechanisms that are involved in the establishment of the specificity in the cAMP-PKA signaling pathway in S.cerevisiae.

Overexpression of the regulatory subunit of protein kinase A increases heterologous protein expression in Pichia pastoris.

OBJECTIVE: Translational regulation plays an important role in protein synthesis. Our goal was to screen translation-related factors to improve heterologous protein expression in Pichia pastoris. RESULTS: Twenty-eight translation-related factors were overexpressed in P. pastoris GS115 expressing enhanced green fluorescent protein (eGFP). The results showed that overexpression of Bcy1, the regulatory subunit of protein kinase A (PKA), significantly increased both eGFP expression and cell biomass by 20% under methanol induction for 120 h. Additionally, overexpression of Bcy1 elevated the growth rate by 18% and increased production of the industrial enzyme Phytase (Phy) by 26%. Transcriptome analysis indicated that the overall expression of ribosomal protein genes was significantly downregulated and that postdiauxic shift genes and stress response element genes were upregulated. CONCLUSIONS: Bcy1 regulates ribosome protein genes, postdiauxic shift genes and stress response element genes, leading to improved cell growth and heterologous protein expression. This study provides a convenient and universal factor for heterologous protein production.

Transcriptional regulation of the protein kinase a subunits in Saccharomyces cerevisiae during fermentative growth.

Yeast cells can adapt their growth in response to the nutritional environment. Glucose is the favourite carbon source of Saccharomyces cerevisiae, which prefers a fermentative metabolism despite the presence of oxygen. When glucose is consumed, the cell switches to the aerobic metabolism of ethanol, during the so-called diauxic shift. The difference between fermentative and aerobic growth is in part mediated by a regulatory mechanism called glucose repression. During glucose derepression a profound gene transcriptional reprogramming occurs and genes involved in the utilization of alternative carbon sources are expressed. Protein kinase A (PKA) controls different physiological responses following the increment of cAMP as a consequence of a particular stimulus. cAMP-PKA is one of the major pathways involved in the transduction of glucose signalling. In this work the regulation of the promoters of the PKA subunits during respiratory and fermentative metabolism are studied. It is demonstrated that all these promoters are upregulated in the presence of glycerol as carbon source through the Snf1/Cat8 pathway. However, in the presence of glucose as carbon source, the regulation of each PKA promoter subunits is different and only TPK1 is repressed by the complex Hxk2/Mig1 in the presence of active Snf1. Copyright © 2017 John Wiley & Sons, Ltd.

The PKA regulatory subunit from yeast forms a homotetramer: Low-resolution structure of the N-terminal oligomerization domain.

The cAMP dependent protein kinase (PKA) is a key enzyme involved in many cellular processes in eukaryotes. In mammals, the regulatory (R) subunit localises the catalytic (C) subunit to specific subcellular sites through the interaction of its N-terminal homodimeric docking and dimerization (D/D) domain with specific scaffold proteins. The structure of the D/D domain has been extensively studied in mammals, but there is little information from non-mammalian species. In this work, we present the structural analysis of the D/D domain of Bcy1, the R subunit of PKA from Saccharomyces cerevisiae. Using chemical crosslinking experiments and static light scattering measurements we found that this R subunit forms a tetramer in solution, unlike its dimeric mammalian counterparts. We determined that the D/D domain is responsible for this unusual oligomeric state. Using biophysical techniques including size-exclusion chromatography, sucrose gradient sedimentation, small angle X-ray scattering (SAXS), and circular dichroism, we performed a detailed structural characterization of the tetrameric D/D domain of Bcy1. We used homology modelling in combination with computer-aided docking methods and ab initio SAXS modelling methods to develop structural models for the D/D domain tetramer. The models consist of two homodimers with a canonical D/D domain fold that generate a dimer of dimers with novel putative interaction surfaces. These findings indicate that the oligomerization states of PKA R subunits is more diverse than previously thought, and suggest that this might allow some forms of PKA to interact with a wide range of intracellular partners.

Identification of novel transcriptional regulators of PKA subunits in Saccharomyces cerevisiae by quantitative promoter-reporter screening.

The cAMP-dependent protein kinase (PKA) signaling is a broad pathway that plays important roles in the transduction of environmental signals triggering precise physiological responses. However, how PKA achieves the cAMP-signal transduction specificity is still in study. The regulation of expression of subunits of PKA should contribute to the signal specificity. Saccharomyces cerevisiae PKA holoenzyme contains two catalytic subunits encoded by TPK1, TPK2 and TPK3 genes, and two regulatory subunits encoded by BCY1 gene. We studied the activity of these gene promoters using a fluorescent reporter synthetic genetic array screen, with the goal of systematically identifying novel regulators of expression of PKA subunits. Gene ontology analysis of the identified modulators showed enrichment not only in the category of transcriptional regulators, but also in less expected categories such as lipid and phosphate metabolism. Inositol, choline and phosphate were identified as novel upstream signals that regulate transcription of PKA subunit genes. The results support the role of transcription regulation of PKA subunits in cAMP specificity signaling. Interestingly, known targets of PKA phosphorylation are associated with the identified pathways opening the possibility of a reciprocal regulation. PKA would be coordinating different metabolic pathways and these processes would in turn regulate expression of the kinase subunits.

Transcriptional regulation of the protein kinase A subunits in Saccharomyces cerevisiae: autoregulatory role of the kinase A activity.

Protein kinase A (PKA) is a broad specificity protein kinase that controls a physiological response following the increment of cAMP as a consequence of a particular stimulus. The specificity of cAMP-signal transduction is maintained by several levels of control acting all together. Herein we present the study of the regulation of the expression of each PKA subunit, analyzing the activity of their promoters. The promoter of each isoform of TPK and of BCY1 is differentially activated during the growth phase. A negative mechanism of isoform-dependent autoregulation directs TPKs and BCY1 gene expressions. TPK1 promoter activity is positively regulated during heat shock and saline stress. The kinase Rim15, but not the kinase Yak1, positively regulates TPK1 promoter. Msn2/4, Gis1, and Sok2 are transcription factors involved in the regulation of TPK1 expression during stress. TPK2, TPK3, and BCY1 promoters, unlike TPK1, are not activated under stress conditions, although all the promoters are activated under low or null protein kinase A activity. These results indicate that subunits share an inhibitory autoregulatory mechanism but have different mechanisms involved in response to heat shock or saline stress.

Construction of industrial baker's yeast with high level of cAMP.

Cyclic adenosine monophosphate (cAMP) plays an important role in modulating the activity of microbe cell. In this study, PKA (protein kinase A) activity was weakened through truncation of TPK2 promoter (-150 bp and -300 bp) and gene deletion of BCY1 (encodes the regulatory subunit of PKA), TPK1 and TPK3, generating strains BY9a-T2-150 and BY9a-T2-300, respectively. High-performance liquid chromatography analysis showed cAMP levels in BY9a-T2-150 and BY9a-T2-300 were increased by 5- and 18-fold, respectively, compared with that of parent strain, BY9a. The expression levels of TPK2 gene in two engineered strains were decreased by 95% and 97% compared with that of BY9a, respectively. The PKA activity reflected by heat resistance of engineered strains enhanced compared with parent strain BY9a. This study show a new method to increase the intracellular cAMP concentration in industrial yeast by fine-tuning of PKA activity, without influence in growth and fermentation properties. PRACTICAL APPLICATIONS: cAMP as the "second messenger," is essential for plant, animal, and microorganisms and human life. But its synthesis is still limited by expensive cost and time-consuming method. We constructed the industrial baker's yeast with high level of cAMP and desired to be used to produce functional food for relaxing smooth muscle, expanding blood vessels, improving liver function, and promoting nerve regeneration and as a food additive for treating hyperthyreosis and hepatopathy. The methods of two step homologous recombination and backcross operated in this study eliminate the exogenous gene in engineered strains, made it safety to be used in food production. Fine-tuning of PKA activity in engineered strains ensure produce high level of cAMP and exhibit normal growth performance in engineering strains. Therefore, this work is significant in functional foods product and has the potential to be used in practical application.

The Regulatory Subunit of Protein Kinase A (Bcy1) in Candida albicans Plays Critical Roles in Filamentation and White-Opaque Switching but Is Not Essential for Cell Growth.

The conserved cAMP-dependent protein kinase (PKA) is composed of the regulatory and catalytic subunits and acts as the central component of the cAMP signaling pathway. In the human fungal pathogen Candida albicans, the PKA regulatory subunit Bcy1 plays a critical role in the regulation of cell differentiation and death. It has long been considered that Bcy1 is essential for cell viability in C. albicans. In the current study, surprisingly, we found that Bcy1 is not required for cell growth, and we successfully generated a bcy1/bcy1 null mutant in C. albicans. Deletion of BCY1 leads to multiple cellular morphologies and promotes the development of filaments. Filamentous and smooth colonies are two typical morphological types of the bcy1/bcy1 mutant, which can undergo spontaneous switching between the two types. Cells of filamentous colonies grow better on a number of different culture media and have a higher survival rate than cells of smooth colonies. In addition, deletion of BCY1 significantly increased the frequency of white-to-opaque switching on N-acetylglucosamine (GlcNAc)-containing medium. The bcy1/bcy1 null mutant generated herein provides the field a new resource to study the biological functions of the cAMP signaling pathway in C. albicans.

First international consensus guidelines for breast cancer in young women (BCY1).

The 1st International Consensus Conference for Breast Cancer in Young Women (BCY1) took place in November 2012, in Dublin, Ireland organized by the European School of Oncology (ESO). Consensus recommendations for management of breast cancer in young women were developed and areas of research priorities were identified. This manuscript summarizes these international consensus recommendations, which are also endorsed by the European Society of Breast Specialists (EUSOMA).

Interacting proteins of protein kinase A regulatory subunit in Saccharomyces cerevisiae.

cAMP-dependent protein kinase mediates many extracellular signals in eukaryotes. The compartmentalization of PKA is an important level of control of the specificity of signal transduction mediated by cAMP. Unlike mammalian PKA for which proof insights in the mechanism that controls its localization through anchoring proteins (AKAPs) has been obtained, in the case of Saccharomyces cerevisiae PKA there was little information available. In this work, we present results that demonstrate the isolation and identification of yeast PKA regulatory subunit (Bcy1) associated proteins using a MS-based proteomic analysis and a bioinformatic approach. The verification of some of these interactions was assessed by immunoprecipitation, pull down and co-localization by subcellular fractionation. The key role of positively charged residues present in the interaction domain of the identified proteins was demonstrated. The defined interaction domain has therefore different molecular characteristics than conventional AKAP domains. Finally we assess initial experiments to visualize the physiological relevance of the interaction of both Ira2 and Hsp60 with Bcy1. Bcy1 interacts with Ira2 tethering PKA to the Ras complex and Hsp60 chaperone localizes PKA to mitochondria and has a role in the kinase stability. BIOLOGICAL SIGNIFICANCE: Our work has an important impact in the field of signal transduction especially of protein kinase A. Components of the cAMP signaling cascade are localized in the cell via scaffold proteins named AKAPs that contribute to the high level specific regulation of the cAMP-PKA-signaling pathway. In the unicellular eukaryote Saccharomyces cerevisiae PKA has a pleiotropic role in the cell and the compartmentalization therefore is key to achieve the specificity in the response. At present all AKAPs have been described in mammals and it is unknown whether functional homologs of mammalian AKAPs exist in yeast. Therefore, it is unknown which molecular features of the mammalian anchoring proteins are general and which are distinctive. We have identified and characterized interacting proteins of protein kinase A regulatory subunit in Saccharomyces cerevisiae, through a proteomic and bioinformatic approach. Bcy1 tethering proteins have a domain in which charged positives residues are key for the interaction with regulatory subunit of PKA and Bcy1 N-terminus is important in the interaction. In mammalian AKAPs a hydrophobic amino acid face of an amphipathic α-helix is essential for the high affinity of the binding interaction. The results obtained in this work seem to indicate that the domains identified in the interacting Bcy1 proteins have a structural nature of the interaction different than those defined for mammalian AKAPs-R interaction. Not only positive charged residues are involved as distinctive molecular determinants but also the hydrophobic face of the helix in which they are included was not relevant in the interaction with Bcy1. Even though generally the use of very well characterized models is essential to answer questions, as would be in this case AKAPs from mammals, the study of other alternative models contributes to the building of more universal concepts.

Suppression mechanism of the calcium sensitivity in Saccharomyces cerevisiae ptp2Δmsg5Δ double disruptant involves a novel HOG-independent function of Ssk2, transcription factor Msn2 and the protein kinase A component Bcy1.

In Saccharomyces cerevisiae, disruption of both protein phosphatase genes, PTP2 and MSG5, causes calcium sensitivity while additional disruption of protein kinase genes BCK1, MKK1, SLT2, MCK1, YAK1 and SSK2 confers calcium tolerance. Although the roles of BCK1, MKK1 and SLT2 have been characterized recently, the mechanism of suppression of the calcium sensitivity by SSK2 disruption is poorly understood. In this study, genetic analysis revealed a novel, high osmolarity glycerol (HOG)-independent suppressor function of Ssk2 in relation to the Ptp2 and Msg5-mediated calcium signaling. Through microarray analysis, we identified 19 genes with distinct pattern of expression that is likely involved in the calcium sensitive phenotype of the ptp2Δmsg5Δ double disruptant. Furthermore, we found msn2Δ and bcy1Δ as suppressors of the calcium sensitive phenotype. Our results suggest the interrelationship of a HOG-independent function of Ssk2, transcription factor Msn2, protein kinase A-related protein Bcy1 and 19 rise and fall genes as responsible for the suppression mechanism of the ptp2Δmsg5Δ double disruptant by ssk2Δ disruption.