Analysis of the Protein Kinase A-Regulated Proteome ofCryptococcus neoformansIdentifies a Role for the Ubiquitin-Proteasome Pathway in Capsule Formation

ABSTRACTThe opportunistic fungal pathogenCryptococcus neoformanscauses life-threatening meningitis in immunocompromised individuals. The expression of virulence factors, including capsule and melanin, is in part regulated by the cyclic-AMP/protein kinase A (cAMP/PKA) signal transduction pathway. In this study, we investigated the influence of PKA on the composition of the intracellular proteome to obtain a comprehensive understanding of the regulation that underpins virulence. Through quantitative proteomics, enrichment and bioinformatic analyses, and an interactome study, we uncovered a pattern of PKA regulation for proteins associated with translation, the proteasome, metabolism, amino acid biosynthesis, and virulence-related functions. PKA regulation of the ubiquitin-proteasome pathway inC. neoformansshowed a striking parallel with connections between PKA and protein degradation in chronic neurodegenerative disorders and other human diseases. Further investigation of proteasome function with the inhibitor bortezomib revealed an impact on capsule production as well as hypersusceptibility for strains with altered expression or activity of PKA. Parallel studies with tunicamycin also linked endoplasmic reticulum stress with capsule production and PKA. Taken together, the data suggest a model whereby expression of PKA regulatory and catalytic subunits and the activation of PKA influence proteostasis and the function of the endoplasmic reticulum to control the elaboration of the polysaccharide capsule. Overall, this study revealed both broad and conserved influences of the cAMP/PKA pathway on the proteome and identified proteostasis as a potential therapeutic target for the treatment of cryptococcosis.IMPORTANCEFungi cause life-threatening diseases, but very few drugs are available to effectively treat fungal infections. The pathogenic fungusCryptococcus neoformanscauses a substantial global burden of life-threatening meningitis in patients suffering from HIV/AIDS. An understanding of the mechanisms by which fungi deploy virulence factors to cause disease is critical for developing new therapeutic approaches. We employed a quantitative proteomic approach to define the changes in the protein complement that occur upon modulating the cAMP signaling pathway that regulates virulence inC. neoformans. This approach identified a conserved role for cAMP signaling in the regulation of the ubiquitin-proteasome pathway and revealed a link between this pathway and elaboration of a major virulence determinant, the polysaccharide capsule. Targeting the ubiquitin-proteasome pathway opens new therapeutic options for the treatment of cryptococcosis.

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cAMP signaling through protein kinase A and Epac2 induces substance P release in the rat spinal cord

Neuropharmacology ◽

10.1016/j.neuropharm.2021.108533 ◽

2021 ◽

pp. 108533

Author(s):

Wenling Chen ◽

James A. McRoberts ◽

Helena S. Ennes ◽

Juan Carlos Marvizon

Keyword(s):

Spinal Cord ◽

Protein Kinase ◽

Substance P ◽

Protein Kinase A ◽

Camp Signaling ◽

Rat Spinal Cord ◽

P Release ◽

Substance P Release ◽

Kinase A

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Protein kinase A inhibitor proteins (PKIs) divert GPCR‐Gαs‐cAMP signaling toward EPAC and ERK activation and are involved in tumor growth

The FASEB Journal ◽

10.1096/fj.202001515r ◽

2020 ◽

Vol 34 (10) ◽

pp. 13900-13917 ◽

Cited By ~ 1

Author(s):

James J. Hoy ◽

Natalia Salinas Parra ◽

Jeannie Park ◽

Skyler Kuhn ◽

Ramiro Iglesias‐Bartolome

Keyword(s):

Protein Kinase ◽

Tumor Growth ◽

Protein Kinase A ◽

Camp Signaling ◽

Erk Activation ◽

Kinase A

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cAMP-dependent protein kinase A in grass carp Ctenopharyngodon idella: Molecular characterization, gene structure, tissue distribution and mRNA expression in endoplasmic reticulum stress-induced adipocyte lipolysis

Comparative Biochemistry and Physiology Part B Biochemistry and Molecular Biology ◽

10.1016/j.cbpb.2020.110479 ◽

2020 ◽

Vol 250 ◽

pp. 110479

Author(s):

Shanghong Ji ◽

Jian Sun ◽

Chenchen Bian ◽

Xiaocheng Huang ◽

Zhiguang Chang ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Protein Kinase ◽

Endoplasmic Reticulum Stress ◽

Protein Kinase A ◽

Gene Structure ◽

Ctenopharyngodon Idella ◽

Dependent Protein Kinase ◽

Grass Carp Ctenopharyngodon Idella ◽

Dependent Protein ◽

Kinase A

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Impairment of the Ubiquitin-Proteasome Pathway Is a Downstream Endoplasmic Reticulum Stress Response Induced by Extracellular Human Islet Amyloid Polypeptide and Contributes to Pancreatic -Cell Apoptosis

Diabetes ◽

10.2337/db07-0178 ◽

2007 ◽

Vol 56 (9) ◽

pp. 2284-2294 ◽

Cited By ~ 86

Author(s):

S. Casas ◽

R. Gomis ◽

F. M. Gribble ◽

J. Altirriba ◽

S. Knuutila ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Stress Response ◽

Islet Amyloid Polypeptide ◽

Human Islet ◽

Endoplasmic Reticulum Stress Response ◽

Islet Amyloid ◽

Pancreatic Cell ◽

Ubiquitin Proteasome Pathway ◽

Ubiquitin Proteasome ◽

Proteasome Pathway

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Potential Role for Protein Kinases in Regulation of Bidirectional Endoplasmic Reticulum-to-Golgi Transport Revealed by Protein Kinase Inhibitor H89

Molecular Biology of the Cell ◽

10.1091/mbc.11.8.2577 ◽

2000 ◽

Vol 11 (8) ◽

pp. 2577-2590 ◽

Cited By ~ 58

Author(s):

Tina H. Lee ◽

Adam D. Linstedt

Keyword(s):

Endoplasmic Reticulum ◽

Protein Kinase ◽

Protein Kinase A ◽

Protein Kinases ◽

Potential Role ◽

Kinase Inhibitor ◽

Transport Processes ◽

Protein Kinase Inhibitors ◽

Er Export ◽

Kinase A

Recent evidence suggests a regulatory connection between cell volume, endoplasmic reticulum (ER) export, and stimulated Golgi-to-ER transport. To investigate the potential role of protein kinases we tested a panel of protein kinase inhibitors for their effect on these steps. One inhibitor, H89, an isoquinolinesulfonamide that is commonly used as a selective protein kinase A inhibitor, blocked both ER export and hypo-osmotic-, brefeldin A-, or nocodazole-induced Golgi-to-ER transport. In contrast, H89 did not block the constitutive ER Golgi-intermediate compartment (ERGIC)-to-ER and Golgi-to-ER traffic that underlies redistribution of ERGIC and Golgi proteins into the ER after ER export arrest. Surprisingly, other protein kinase A inhibitors, KT5720 and H8, as well as a set of protein kinase C inhibitors, had no effect on these transport processes. To test whether H89 might act at the level of either the coatomer protein (COP)I or the COPII coat protein complex we examined the localization of βCOP and Sec13 in H89-treated cells. H89 treatment led to a rapid loss of Sec13-labeled ER export sites but βCOP localization to the Golgi was unaffected. To further investigate the effect of H89 on COPII we developed a COPII recruitment assay with permeabilized cells and found that H89 potently inhibited binding of exogenous Sec13 to ER export sites. This block occurred in the presence of guanosine-5′-O-(3-thio)triphosphate, suggesting that Sec13 recruitment is inhibited at a step independent of the activation of the GTPase Sar1. These results identify a requirement for an H89-sensitive factor(s), potentially a novel protein kinase, in recruitment of COPII to ER export sites, as well as in stimulated but not constitutive Golgi-to-ER transport.

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