Cross regulation between Candida albicans catalytic and regulatory subunits of protein kinase A (original) (raw)
Related papers
Yeast, 1990
Candida albicans cAMP-dependent protein kinase (PKA) is coded by two catalytic subunits (TPK1 and TPK2 ) and one regulatory subunit (BCY1 ). In this organism the cAMP/PKA signalling pathway mediates basic cellular processes, such as the yeastto-hyphae transition and cell cycle regulation. In the present study, we investigated the role of C. albicans PKA in response to saline, heat and oxidative stresses as well as in glycogen storage. To fine-tune the analysis, we performed the studies on several C. albicans PKA mutants having heterozygous or homozygous deletions of TPK1 and/or TPK2 in a different BCY1 genetic background. We observed that tpk1 /tpk1 strains developed a lower tolerance to saline exposure, heat shock and oxidative stress, while wild-type and tpk2 /tpk2 mutants were resistant to these stresses, indicating that both isoforms play different roles in the stress response pathway. We also found that regardless of the TPK background, heterozygous and homozygous BCY1 mutants were highly sensitive to heat treatment. Surprisingly, we observed that those strains devoid of one or both TPK1 alleles were defective in glycogen storage, while strains lacking Tpk2 accumulated higher levels of the polysaccharide, indicating that Tpk1 and Tpk2 have opposite roles in carbohydrate metabolism.
cAMP-PKA pathway to induce morphogenesis in Candida albicans
2000
Tel: +32 16 321512 Fax: +32 16 321979 Email: patrick.vandijck@bio.kuleuven.ac.be key-words: GPCR/signal transduction/yeast-to-hyphae/nutrient sensing/methionine Running title: GPCR-induced morphogenesis in C. albicans 2 Abstract We investigated the role in cell morphogenesis and pathogenicity of the Candida albicans GPR1 gene, encoding the G protein coupled receptor Gpr1. Deletion of C. albicans GPR1 has only minor effects in liquid hypha-inducing media but results in strong defects in the yeast-to-hypha transition on solid hypha-inducing media. Addition of cAMP, expression of a constitutively active allele of the Gα protein Gpa2 or of the catalytic protein kinase A subunit TPK1 restores the wild type phenotype of the CaGPR1-deleted strain. Overexpression of HST7, encoding a component of the Mitogen-Activated Protein Kinase (MAPK) pathway, does not suppress the defect in filamentation. These results indicate that CaGpr1 functions upstream in the cAMP-
2001
In the yeast Saccharomyces cerevisiae, three genes TPKl, TPK2, and TPK3 encode catalytic subunits of CAMP-dependent protein kinase. We have purified and characterized the catalytic subunit, C1, encoded by the TPKl gene. In order to purify CI completely free of Cp and CS, a strain was constructed that contained only the TPKl gene and genetic disruptions of the other two TPK genes. The cellular level of C1 was increased by expressing the genes for C1 (TPKl) and yeast regulatory subunit (BCYl) on multiple copy plasmids within this strain. Purification was accomplished by a two-column procedure in which holoenzyme was chromatographed on Sephacryl-290, then bound to an antiregulatory subunit immunoaffinity column. Pure C1 was released from the antibody column by addition of CAMP. The protein migrated on a sodium dodecyl sulfate-polyacrylamide gel with an M, of 52,000. Kinetic analysis showed that the apparent K,,, for ATP and Leu-Arg-Arg-Ala-Ser-Leu-Gly was 33 and 101 PM, respectively. T...
Journal of Biological Chemistry, 1988
In the yeast Saccharomyces cerevisiae, three genes TPKl, TPK2, and TPK3 encode catalytic subunits of CAMP-dependent protein kinase. We have purified and characterized the catalytic subunit, C1, encoded by the TPKl gene. In order to purify CI completely free of Cp and CS, a strain was constructed that contained only the TPKl gene and genetic disruptions of the other two TPK genes. The cellular level of C1 was increased by expressing the genes for C1 (T P K l) and yeast regulatory subunit (BCYl) on multiple copy plasmids within this strain. Purification was accomplished by a two-column procedure in which holoenzyme was chromatographed on Sephacryl-290, then bound to an antiregulatory subunit immunoaffinity column. Pure C1 was released from the antibody column by addition of CAMP. The protein migrated on a sodium dodecyl sulfate-polyacrylamide gel with an M, of 52,000. Kinetic analysis showed that the apparent K,,, for ATP and Leu-Arg-Arg-Ala-Ser-Leu-Gly was 33 and 101 PM, respectively. The kcat was determined to be 640 min-l. The protein weakly autophosphorylated, incorporating less than 0.1 mol of phosphate/mol of catalytic subunit. NH2-terminal sequencing revealed that the protein was blocked. CAMP-dependent protein kinase has been shown to play an important role in the regulation of cellular processes (reviewed in Refs. 1-3). It is ubiquitous in eukaryotes from Dictyosteliurn discoideum and Saccharomyces cerevisiae to mammals. This enzyme serves as the link between hormonal stimulation of adenylate cyclase and a biological response. CAMP-dependent protein kinase functions to phosphorylate protein substrates thereby changing their biological properties. The best studied systems have been in mammalian liver, skeletal muscle, and adipocytes, in which CAMP-dependent protein kinase is involved in the regulation of the enzymes of glycogen and fatty acid metabolism. Recently, expression of several genes has been shown to be controlled by cAMP (4). These findings suggest that regulators of gene transcription may also be substrates of CAMP-dependent protein kinase (5). The inactive holoenzyme of CAMP-dependent protein ki-* This work was supported in part by National Institutes of Health Grant GM33986 (to M. J. Z.
The Yak1 Kinase Is Involved in the Initiation and Maintenance of Hyphal Growth in Candida albicans
Molecular Biology of the Cell, 2008
Members of the dual-specificity tyrosine-phosphorylated and regulated kinase (DYRK) family perform a variety of functions in eukaryotes. We used gene disruption, targeted pharmacologic inhibition, and genome-wide transcriptional profiling to dissect the function of the Yak1 DYRK in the human fungal pathogen Candida albicans. C. albicans strains with mutant yak1 alleles showed defects in the yeast-to-hypha transition and in maintaining hyphal growth. They also could not form biofilms. Despite their in vitro filamentation defect, C. albicans yak1⌬/yak1⌬ mutants remained virulent in animal models of systemic and oropharyngeal candidiasis. Transcriptional profiling showed that Yak1 was necessary for the up-regulation of only a subset of hypha-induced genes. Although downstream targets of the Tec1 and Bcr1 transcription factors were down-regulated in the yak1⌬/yak1⌬ mutant, TEC1 and BCR1 were not. Furthermore, 63% of Yak1-dependent, hypha-specific genes have been reported to be negatively regulated by the transcriptional repressor Tup1 and inactivation of TUP1 in the yak1⌬/yak1⌬ mutant restored filamentation, suggesting that Yak1 may function upstream of Tup1 in governing hyphal emergence and maintenance. † Present address: Evolva SA, Hagmattstrasse 6, 4123 Allschwil, Switzerland.
2021
ABSTRACTMicrobial pathogens grow in a wide range of different morphologies that provide distinct advantages for virulence. In the fungal pathogen Candida albicans, adenylyl cyclase (Cyr1) is thought to be a master regulator of the switch to invasive hyphal morphogenesis and biofilm formation. However, faster growing cyr1Δ/Δ pseudorevertant (PR) mutants were identified that form hyphae in the absence of cAMP. Isolation of additional PR mutants revealed that their improved growth was due to loss of one copy of BCY1, the negative regulatory subunit of protein kinase (A) from the left arm of chromosome 2. Furthermore, hyphal morphogenesis was improved in some of PR mutants by multigenic haploinsufficiency resulting from loss of large regions of the left arm of chromosome 2, including global transcriptional regulators. Interestingly, hyphal-associated genes were also induced in a manner that was independent of cAMP. This indicates that basal protein kinase A activity is an important prer...
The Journal of biological chemistry, 1991
The regulatory subunit of cAMP-dependent protein kinase in yeast, encoded by the BCY1 gene, is known to be required under certain conditions such as growth on nonfermentable carbon sources and entry into stationary phase. We have identified novel isoforms of Bcy1 in cells under these conditions. The isoforms are distinguishable by their migration on one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 2-dimensional nonequilibrium pH gradient gel electrophoresis. The isoforms observed by one-dimensional SDS-PAGE bind cAMP, as determined by [32P]8-azido-cAMP labeling (diagnostic of Bcy1 protein). Proteins isolated from cells grown to stationary phase in rich medium exhibit five antibody-reactive bands, by one-dimensional SDS-PAGE immunoblot analysis, with apparent molecular masses of 50, 52, 55, 59 and 61 kDa. Total Bcy1 protein increases at least 8-fold between exponential and stationary phase. Analysis of proteins from a variety of yeast mutants i...
Microbiology, 2001
BCY1-encoded protein kinase A (PKA) wild-type and mutant regulatory (R) subunits from Saccharomyces cerevisiae were inducibly overexpressed in their corresponding background strains containing the same mutation in the bcy1 gene. The aim of this approach was to shift the catalytic activity of PKA within the cell to the undissociated holoenzyme form(s) in order to evaluate whether the wild-type or the mutant forms of the holoenzyme could display catalytic activity. Two mutants of R subunits were used : bcy1-16, with a complete deletion of cAMP-binding domain B ; and bcy1-14, with a small deletion in the carboxy terminus of cAMP-binding domain A. Their overexpression caused an increase in the level of R subunits in the range 40-90-fold, as detected by cAMP-binding activity, Coomasie-stained SDS-PAGE and Western blot analysis. The change in PKA activity attained by overexpression of R was assessed in three ways : (i) through the analysis of PKA-dependent phenotypes, and (ii, iii) by measurement of PKA activity N/M cAMP using the specific substrate kemptide in crude extracts (ii) and permeabilized cells (iii). Upon overexpression of the R subunits, PKAdependent phenotypes were less severe when compared with their own background. However, a gradient in the degree of severity of phenotypes bcy1-14 Sbcy1-16 S wild-type was observed in the background strains and was maintained in the strains overexpressing the R subunits. cAMP levels measured in background and in R-overexpressing strains showed an increase of around two orders accompanying the overexpression of the R subunits. Three main conclusions could be drawn from the PKA activity measurements N/M cAMP in crude extracts : (i) catalytic activity was not increased in compensation for the increase in R subunits in any of the three cases (wild-type, bcy1-16 or bcy1-14 overexpression) ; (ii) PKA activity assayed in the absence of cAMP was lower in the case of extracts from strains overexpressing wild-type or bcy1-16 R subunits when compared with the corresponding extracts without overexpression ; and (iii) in these two cases, the great excess of R subunits in the crude extracts displayed additional inhibitory capacity towards exogenously added catalytic (C) subunits. To provide an estimate of the in vivo activation of PKA, permeabilized cells from control strains and strains transformed with either wild-type, bcy1-16 or bcy1-14 R subunits were used to measure PKA activity in the presence of variable concentrations of cAMP. There were two main observations from the results : (i) the activity of PKA detected in the absence of exogenous cAMP was decreased in the strains overexpressing