A Novel Phosphoregulatory Switch Controls the Activity and Function of the Major Catalytic Subunit of Protein Kinase A in <italic toggle="yes">Aspergillus fumigatus</italic>

ABSTRACT Invasive aspergillosis (IA), caused by the filamentous fungal pathogen Aspergillus fumigatus, is a major cause of death among immunocompromised patients. The cyclic AMP/protein kinase A (PKA) signaling pathway is essential for hyphal growth and virulence of A. fumigatus, but the mechanism o...

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Autores principales:	E. Keats Shwab, Praveen R. Juvvadi, Greg Waitt, Erik J. Soderblom, M. Arthur Moseley, Nathan I. Nicely, Yohannes G. Asfaw, William J. Steinbach
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/140506a1f19b4a60a82b864e85bf4afe
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spelling	oai:doaj.org-article:140506a1f19b4a60a82b864e85bf4afe2021-11-15T15:51:07ZA Novel Phosphoregulatory Switch Controls the Activity and Function of the Major Catalytic Subunit of Protein Kinase A in <italic toggle="yes">Aspergillus fumigatus</italic>10.1128/mBio.02319-162150-7511https://doaj.org/article/140506a1f19b4a60a82b864e85bf4afe2017-03-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02319-16https://doaj.org/toc/2150-7511ABSTRACT Invasive aspergillosis (IA), caused by the filamentous fungal pathogen Aspergillus fumigatus, is a major cause of death among immunocompromised patients. The cyclic AMP/protein kinase A (PKA) signaling pathway is essential for hyphal growth and virulence of A. fumigatus, but the mechanism of regulation of PKA remains largely unknown. Here, we discovered a novel mechanism for the regulation of PKA activity in A. fumigatus via phosphorylation of key residues within the major catalytic subunit, PkaC1. Phosphopeptide enrichment and tandem mass spectrometry revealed the phosphorylation of PkaC1 at four sites (S175, T331, T333, and T337) with implications for important and diverse roles in the regulation of A. fumigatus PKA. While the phosphorylation at one of the residues (T333) is conserved in other species, the identification of three other residues represents previously unknown PKA phosphoregulation in A. fumigatus. Site-directed mutagenesis of the phosphorylated residues to mimic or prevent phosphorylation revealed dramatic effects on kinase activity, growth, conidiation, cell wall stress response, and virulence in both invertebrate and murine infection models. Three-dimensional structural modeling of A. fumigatus PkaC1 substantiated the positive or negative regulatory roles for specific residues. Suppression of PKA activity also led to downregulation of PkaC1 protein levels in an apparent novel negative-feedback mechanism. Taken together, we propose a model in which PkaC1 phosphorylation both positively and negatively modulates its activity. These findings pave the way for future discovery of fungus-specific aspects of this key signaling network. IMPORTANCE Our understanding of signal transduction networks in pathogenic fungi is limited, despite the increase in invasive fungal infections and rising mortality rates in the immunosuppressed patient population. Because PKA is known to be essential for hyphal growth and virulence of A. fumigatus, we sought to identify fungus-specific regulatory mechanisms governing PKA activity. In this study, we identify, for the first time, a novel mechanism for the regulation of PKA signaling in which differential phosphorylation of the PkaC1 catalytic subunit can lead to either positive or negative regulation of activity. Furthermore, we show that inactivation of PKA signaling leads to downregulation of catalytic subunit protein levels in a negative-feedback mechanism distinct from expression patterns previously reported in the yeasts. Our findings represent a divergence in the regulation of PKA signaling in A. fumigatus, which could potentially be exploited as a target and also open the avenue for discovery of fungus-specific downstream effectors of PKA.E. Keats ShwabPraveen R. JuvvadiGreg WaittErik J. SoderblomM. Arthur MoseleyNathan I. NicelyYohannes G. AsfawWilliam J. SteinbachAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 8, Iss 1 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 E. Keats Shwab Praveen R. Juvvadi Greg Waitt Erik J. Soderblom M. Arthur Moseley Nathan I. Nicely Yohannes G. Asfaw William J. Steinbach A Novel Phosphoregulatory Switch Controls the Activity and Function of the Major Catalytic Subunit of Protein Kinase A in <italic toggle="yes">Aspergillus fumigatus</italic>
description	ABSTRACT Invasive aspergillosis (IA), caused by the filamentous fungal pathogen Aspergillus fumigatus, is a major cause of death among immunocompromised patients. The cyclic AMP/protein kinase A (PKA) signaling pathway is essential for hyphal growth and virulence of A. fumigatus, but the mechanism of regulation of PKA remains largely unknown. Here, we discovered a novel mechanism for the regulation of PKA activity in A. fumigatus via phosphorylation of key residues within the major catalytic subunit, PkaC1. Phosphopeptide enrichment and tandem mass spectrometry revealed the phosphorylation of PkaC1 at four sites (S175, T331, T333, and T337) with implications for important and diverse roles in the regulation of A. fumigatus PKA. While the phosphorylation at one of the residues (T333) is conserved in other species, the identification of three other residues represents previously unknown PKA phosphoregulation in A. fumigatus. Site-directed mutagenesis of the phosphorylated residues to mimic or prevent phosphorylation revealed dramatic effects on kinase activity, growth, conidiation, cell wall stress response, and virulence in both invertebrate and murine infection models. Three-dimensional structural modeling of A. fumigatus PkaC1 substantiated the positive or negative regulatory roles for specific residues. Suppression of PKA activity also led to downregulation of PkaC1 protein levels in an apparent novel negative-feedback mechanism. Taken together, we propose a model in which PkaC1 phosphorylation both positively and negatively modulates its activity. These findings pave the way for future discovery of fungus-specific aspects of this key signaling network. IMPORTANCE Our understanding of signal transduction networks in pathogenic fungi is limited, despite the increase in invasive fungal infections and rising mortality rates in the immunosuppressed patient population. Because PKA is known to be essential for hyphal growth and virulence of A. fumigatus, we sought to identify fungus-specific regulatory mechanisms governing PKA activity. In this study, we identify, for the first time, a novel mechanism for the regulation of PKA signaling in which differential phosphorylation of the PkaC1 catalytic subunit can lead to either positive or negative regulation of activity. Furthermore, we show that inactivation of PKA signaling leads to downregulation of catalytic subunit protein levels in a negative-feedback mechanism distinct from expression patterns previously reported in the yeasts. Our findings represent a divergence in the regulation of PKA signaling in A. fumigatus, which could potentially be exploited as a target and also open the avenue for discovery of fungus-specific downstream effectors of PKA.
format	article
author	E. Keats Shwab Praveen R. Juvvadi Greg Waitt Erik J. Soderblom M. Arthur Moseley Nathan I. Nicely Yohannes G. Asfaw William J. Steinbach
author_facet	E. Keats Shwab Praveen R. Juvvadi Greg Waitt Erik J. Soderblom M. Arthur Moseley Nathan I. Nicely Yohannes G. Asfaw William J. Steinbach
author_sort	E. Keats Shwab
title	A Novel Phosphoregulatory Switch Controls the Activity and Function of the Major Catalytic Subunit of Protein Kinase A in <italic toggle="yes">Aspergillus fumigatus</italic>
title_short	A Novel Phosphoregulatory Switch Controls the Activity and Function of the Major Catalytic Subunit of Protein Kinase A in <italic toggle="yes">Aspergillus fumigatus</italic>
title_full	A Novel Phosphoregulatory Switch Controls the Activity and Function of the Major Catalytic Subunit of Protein Kinase A in <italic toggle="yes">Aspergillus fumigatus</italic>
title_fullStr	A Novel Phosphoregulatory Switch Controls the Activity and Function of the Major Catalytic Subunit of Protein Kinase A in <italic toggle="yes">Aspergillus fumigatus</italic>
title_full_unstemmed	A Novel Phosphoregulatory Switch Controls the Activity and Function of the Major Catalytic Subunit of Protein Kinase A in <italic toggle="yes">Aspergillus fumigatus</italic>
title_sort	novel phosphoregulatory switch controls the activity and function of the major catalytic subunit of protein kinase a in <italic toggle="yes">aspergillus fumigatus</italic>
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/140506a1f19b4a60a82b864e85bf4afe
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A Novel Phosphoregulatory Switch Controls the Activity and Function of the Major Catalytic Subunit of Protein Kinase A in <italic toggle="yes">Aspergillus fumigatus</italic>

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