Comprehensive Analysis of Aspergillus nidulans PKA Phosphorylome Identifies a Novel Mode of CreA Regulation

Abstract

In filamentous fungi, an important kinase responsible for adaptation to changes in available nutrients is cyclic AMP (cAMP)-dependent protein kinase (protein kinase A [PKA]). This kinase has been well characterized at a molecular level, but its systemic action and direct/indirect targets are generally not well understood in filamentous fungi. In this work, we used a pkaA deletion strain (ΔpkaA) to identify Aspergillus nidulans proteins for which phosphorylation is dependent (either directly or indirectly) on PKA. A combination of phosphoproteomic and transcriptomic analyses revealed both direct and indirect targets of PKA and provided a global perspective on its function. One of these targets was the transcription factor CreA, the main repressor responsible for carbon catabolite repression (CCR). In the ΔpkaA strain, we identified a previously unreported phosphosite in CreA, S319, which (based on motif analysis) appears to be a direct target of Stk22 kinase (AN5728). Upon replacement of CreA S319 with an alanine (i.e., phosphonull mutant), the dynamics of CreA import to the nucleus are affected. Collectively, this work provides a global overview of PKA function while also providing novel insight regarding significance of a specific PKA-mediated phosphorylation event. IMPORTANCE The cyclic AMP (cAMP)-dependent protein kinase A (PKA) signaling pathway is well conserved across eukaryotes, and previous work has shown that it plays an important role in regulating development, growth, and virulence in a number of fungi. PKA is activated in response to extracellular nutrients and acts to regulate metabolism and growth. While a number of components in the PKA pathway have been defined in filamentous fungi, current understanding does not provide a global perspective on PKA function. Thus, this work is significant in that it comprehensively identifies proteins and functional pathways regulated by PKA in a model filamentous fungus. This information enhances our understanding of PKA action and may provide information on how to manipulate it for specific purposes.

Precision Engineering of the Transcription Factor Cre1 in Hypocrea jecorina (Trichoderma reesei) for Efficient Cellulase Production in the Presence of Glucose

“…In Aspergillus fumigatus and Neurospora crassa, PKA is activated in the presence of glucose, thereby inhibiting alternative carbon source usage due to CCR (Brown et al, 2014;Huberman et al, 2016). In accordance with a previous study (Ribeiro et al, 2019), the pkaA deletion strain showed a significant improvement in cellulase activity in Aspergillus nidulans.…”

The High Osmolarity Glycerol Mitogen-Activated Protein Kinase regulates glucose catabolite repression in filamentous fungi

“…Based on the aforementioned results, our MS analysis and previous studies [7,11,15], we propose a model for the interaction between the different signaling pathways that ultimately control CCR (Fig 8). In the presence of the alternative carbon source xylan, formation of transient protein complexes is observed that change upon the addition of glucose.…”

“…Several studies have supported the role of phosphorylation in the post-translational regulation of CreA [8][9][10][11]. In Saccharomyces cerevisiae and A. nidulans, the AMP-activated protein kinase Snf1p/SnfA regulates cellular localization of Mig1p (CreA homologue)/CreA.…”

“…In T. reesei, phosphorylation of CRE1 (CreA homologue) at serine 241, is catalyzed by casein kinase and is required for stabilizing CRE1 in the nucleus [10]. In A. nidulans, CreA was shown to be phosphorylated directly at serine 262 by casein kinase A (CkiA) and indirectly at serine 319 by PkaA in the presence of glucose, suggesting that CreA phosphorylation is required for repression in this fungus [9,11].…”

The Aspergillus fumigatus Phosphoproteome Reveals Roles of High-Osmolarity Glycerol Mitogen-Activated Protein Kinases in Promoting Cell Wall Damage and Caspofungin Tolerance

“…Phosphoproteomics provides information about which protein or pathway might be activated, since generally there is a correlation between a change in protein phosphorylation and protein activity. Phosphoproteomics has been used for understanding mitotic exit and cell wall integrity pathways in Saccharomyces cerevisiae (49,50); hyphal morphogenesis in Candida albicans (51); asexual development and multiple stress responses in Beauveria bassiana (52); cellular processes involved in morphology, secretion, protein kinase A regulation, and catabolite repression in A. nidulans (53,54); and the aflatoxin production in Aspergillus flavus (55). Here, we evaluate the global A. fumigatus phosphoproteome exposed to cell wall-damaging agents, identifying 485 proteins putatively involved in the cell wall stress response.…”