scholarly journals Integrative multi-omics profiling reveals cAMP-independent mechanisms regulating hyphal morphogenesis in Candida albicans

2021 ◽  
Vol 17 (8) ◽  
pp. e1009861
Author(s):  
Kyunghun Min ◽  
Thomas F. Jannace ◽  
Haoyu Si ◽  
Krishna R. Veeramah ◽  
John D. Haley ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Adenylyl Cyclase ◽  
Regulatory Subunit ◽  
Chromosome 2 ◽  
Casein Kinase 1 ◽  
Hyphal Morphogenesis ◽  
Wide Range ◽  
Kinase A

Microbial 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 (PKA) 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 prerequisite to induce hyphae, but activation of adenylyl cyclase is not needed. Instead, phosphoproteomic analysis indicated that the Cdc28 cyclin-dependent kinase and the casein kinase 1 family member Yck2 play key roles in promoting polarized growth. In addition, integrating transcriptomic and proteomic data reveals hyphal stimuli induce increased production of key transcription factors that contribute to polarized morphogenesis.

scholarly journals Integrative multi-omics profiling reveals cAMP-independent mechanisms regulating hyphal morphogenesis in Candida albicans

2021 ◽  
Author(s):  
Kyunghun Min ◽  
Thomas F. Jannace ◽  
Haoyu Si ◽  
Krishna R. Veeramah ◽  
John D. Haley ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Microbial Pathogens ◽  
Regulatory Subunit ◽  
Chromosome 2 ◽  
Proteomic Data ◽  
Hyphal Morphogenesis ◽  
Wide Range ◽  
Kinase A

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) has been 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 prerequisite to induce hyphae, but activation of the cAMP pathway is not needed. Instead, phosphoproteomic analysis indicated that the Cdc28 cyclin-dependent kinase and the casein kinase Yck2 play key roles in promoting polarized growth. In addition, integrating transcriptomic and proteomic data reveals hyphal stimuli induce increased production of key transcription factors that contribute to polarized morphogenesis.

scholarly journals Candida albicans Lacking the Gene Encoding the Regulatory Subunit of Protein Kinase A Displays a Defect in Hyphal Formation and an Altered Localization of the Catalytic Subunit

2004 ◽  
Vol 3 (1) ◽  
pp. 190-199 ◽  
Author(s):  
Alejandro Cassola ◽  
Marc Parrot ◽  
Susana Silberstein ◽  
Beatrice B. Magee ◽  
Susana Passeron ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Protein Kinase ◽  
Fusion Protein ◽  
Protein Kinase A ◽  
Double Mutant ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Wild Type ◽  
Gfp Fusion Protein ◽  
Kinase A

ABSTRACT The fungal pathogen Candida albicans switches from a yeast-like to a filamentous mode of growth in response to a variety of environmental conditions. We examined the morphogenetic behavior of C. albicans yeast cells lacking the BCY1 gene, which encodes the regulatory subunit of protein kinase A. We cloned the BCY1 gene and generated a bcy1 tpk2 double mutant strain because a homozygous bcy1 mutant in a wild-type genetic background could not be obtained. In the bcy1 tpk2 mutant, protein kinase A activity (due to the presence of the TPK1 gene) was cyclic AMP independent, indicating that the cells harbored an unregulated phosphotransferase activity. This mutant has constitutive protein kinase A activity and displayed a defective germinative phenotype in N-acetylglucosamine and in serum-containing medium. The subcellular localization of a Tpk1-green fluorescent protein (GFP) fusion protein was examined in wild-type, tpk2 null, and bcy1 tpk2 double mutant strains. The fusion protein was observed to be predominantly nuclear in wild-type and tpk2 strains. This was not the case in the bcy1 tpk2 double mutant, where it appeared dispersed throughout the cell. Coimmunoprecipitation of Bcy1p with the Tpk1-GFP fusion protein demonstrated the interaction of these proteins inside the cell. These results suggest that one of the roles of Bcy1p is to tether the protein kinase A catalytic subunit to the nucleus.

scholarly journals Noncanonical protein kinase A activation by oligomerization of regulatory subunits as revealed by inherited Carney complex mutations

2021 ◽  
Vol 118 (21) ◽  
pp. e2024716118
Author(s):  
Naeimeh Jafari ◽  
Jason Del Rio ◽  
Madoka Akimoto ◽  
Jung Ah Byun ◽  
Stephen Boulton ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Molecular Mechanisms ◽  
Carney Complex ◽  
Regulatory Subunit ◽  
Regulatory Subunits ◽  
Inhibitory Function ◽  
Wide Range ◽  
Liver Cancers ◽  
Kinase A

Familial mutations of the protein kinase A (PKA) R1α regulatory subunit lead to a generalized predisposition for a wide range of tumors, from pituitary adenomas to pancreatic and liver cancers, commonly referred to as Carney complex (CNC). CNC mutations are known to cause overactivation of PKA, but the molecular mechanisms underlying such kinase overactivity are not fully understood in the context of the canonical cAMP-dependent activation of PKA. Here, we show that oligomerization-induced sequestration of R1α from the catalytic subunit of PKA (C) is a viable mechanism of PKA activation that can explain the CNC phenotype. Our investigations focus on comparative analyses at the level of structure, unfolding, aggregation, and kinase inhibition profiles of wild-type (wt) PKA R1α, the A211D and G287W CNC mutants, as well as the cognate acrodysostosis type 1 (ACRDYS1) mutations A211T and G287E. The latter exhibit a phenotype opposite to CNC with suboptimal PKA activation compared with wt. Overall, our results show that CNC mutations not only perturb the classical cAMP-dependent allosteric activation pathway of PKA, but also amplify significantly more than the cognate ACRDYS1 mutations nonclassical and previously unappreciated activation pathways, such as oligomerization-induced losses of the PKA R1α inhibitory function.

scholarly journals Targeting the Regulatory Subunit R2Alpha of Protein Kinase A in Human Glioblastoma through shRNA-Expressing Lentiviral Vectors

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1361
Author(s):  
Maira Zorzan ◽  
Claudia Del Vecchio ◽  
Stefania Vogiatzis ◽  
Elisa Saccon ◽  
Cristina Parolin ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Lentiviral Vectors ◽  
Brain Parenchyma ◽  
Regulatory Subunit ◽  
Glioblastoma Cells ◽  
Cellular Models ◽  
Therapeutic Setting ◽  
Promising Target ◽  
Kinase A

Glioblastoma is the most malignant and most common form of brain tumor, still today associated with a poor 14-months median survival from diagnosis. Protein kinase A, particularly its regulatory subunit R2Alpha, presents a typical intracellular distribution in glioblastoma cells compared to the healthy brain parenchyma and this peculiarity might be exploited in a therapeutic setting. In the present study, a third-generation lentiviral system for delivery of shRNA targeting the regulatory subunit R2Alpha of protein kinase A was developed. Generated lentiviral vectors are able to induce an efficient and stable downregulation of R2Alpha in different cellular models, including non-stem and stem-like glioblastoma cells. In addition, our data suggest a potential correlation between silencing of the regulatory subunit of protein kinase A and reduced viability of tumor cells, apparently due to a reduction in replication rate. Thus, our findings support the role of protein kinase A as a promising target for novel anti-glioma therapies.

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

2021 ◽  
pp. 107732
Author(s):  
Nicolás González Bardeci ◽  
Enzo Tofolón ◽  
Felipe Trajtenberg ◽  
Julio Caramelo ◽  
Nicole Larrieux ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Regulatory Subunit ◽  
Kinase A

scholarly journals The Role of Uncoupling Protein 1 in the Metabolism and Adiposity of RIIβ-Protein Kinase A-Deficient Mice

2004 ◽  
Vol 18 (9) ◽  
pp. 2302-2311 ◽  
Author(s):  
Michael A. Nolan ◽  
Maria A. Sikorski ◽  
G. Stanley McKnight
Keyword(s):  
Adipose Tissue ◽  
Oxygen Consumption ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Uncoupling Protein ◽  
Mrna Level ◽  
Regulatory Subunit ◽  
Uncoupling Protein 1 ◽  
Brown Adipose ◽  
Kinase A

Abstract Mice lacking the RIIβ regulatory subunit of protein kinase A exhibit a 50% reduction in white adipose tissue stores compared with wild-type littermates and are resistant to diet-induced obesity. RIIβ−/− mice also have an increase in resting oxygen consumption along with a 4-fold increase in the brown adipose-specific mitochondrial uncoupling protein 1 (UCP1). In this study, we examined the basis for UCP1 induction and tested the hypothesis that the induced levels of UCP1 in RIIβ null mice are essential for the lean phenotype. The induction of UCP1 occurred at the protein but not the mRNA level and correlated with an increase in mitochondria in brown adipose tissue. Mice lacking both RIIβ and UCP1 (RIIβ−/−/Ucp1−/−) were created, and the key parameters of metabolism and body composition were studied. We discovered that RIIβ−/− mice exhibit nocturnal hyperactivity in addition to the increased oxygen consumption at rest. Disruption of UCP1 in RIIβ−/− mice reduced basal oxygen consumption but did not prevent the nocturnal hyperactivity. The double knockout animals also retained the lean phenotype of the RIIβ null mice, demonstrating that induction of UCP1 and increased resting oxygen consumption is not the cause of leanness in the RIIβ mutant mice.

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