Abstract
The utilization of different carbon sources in filamentous fungi underlies a complex regulatory network governed by signaling events of different protein kinase pathways, including the high osmolarity glycerol (HOG) and protein kinase A (PKA) pathways. This work unraveled cross-talk events between these pathways in governing the utilization of preferred (glucose) and non-preferred (xylan, xylose) carbon sources in the reference fungus Aspergillus nidulans. An initial screening of a library of 103 non-essential protein kinase (NPK) deletion strains identified several mitogen-activated protein kinases (MAPKs) to be important for carbon catabolite repression (CCR). We selected the MAPKs Ste7, MpkB, and PbsA for further characterization and show that they are pivotal for HOG pathway activation, PKA activity, CCR via regulation of CreA cellular localization and protein accumulation, as well as for hydrolytic enzyme secretion. Protein-protein interaction studies show that Ste7, MpkB, and PbsA are part of the same protein complex that regulates CreA cellular localization in the presence of xylan and that this complex dissociates upon the addition of glucose, thus allowing CCR to proceed. Glycogen synthase kinase (GSK) A was also identified as part of this protein complex and shown to potentially phosphorylate two serine residues of the HOG MAPKK PbsA. This work shows that carbon source utilization is subject to cross-talk regulation by protein kinases of different signaling pathways. Furthermore, this study provides a model where the correct integration of PKA, HOG, and GSK signaling events are required for the utilization of different carbon sources.
Highlights
Protein phosphorylation, which is catalysed by protein kinases, is crucial for target protein function and/or cellular localization, resulting in the regulation of a variety of cellular processes and signalling pathways
In order to determine which protein kinases are important for carbon catabolite repression (CCR), an A. nidulans library, containing 103 non-essential protein kinase (NPK) deletion strains [30], was screened for growth in the presence of 2-deoxyglucose (2DG) and allyl alcohol (AA). 2DG and AA are indicators for defects in CCR [31,32], with 2DG being a glucose analogue that cannot be metabolized, and AA being converted to the highly toxic compound acrolein by alcohol dehydrogenase (ADH) [33,34,35]
The addition of glucose caused an accumulation of CreA-GFP in the nuclei of the WT and ΔmpkB strains (~100%), whereas CreA-GFP nuclear localization was significantly reduced in the Δste7 (~69%) and ΔpbsA (~35%) strains (Fig 3B). These results indicate that the protein kinases Ste7 and PbsA, but not MpkB, are important for CreA cellular localization under CCR condition
Summary
Protein phosphorylation, which is catalysed by protein kinases, is crucial for target protein function and/or cellular localization, resulting in the regulation of a variety of cellular processes and signalling pathways. In the filamentous fungus A. nidulans, a role for protein kinases in the regulation of carbon utilisation pathways, such as carbon catabolite repression (CCR), has been shown [1]. In the presence of glucose, CreA localizes to the nucleus, where it represses genes required for the utilisation of non-glycolytic carbon sources, whereas the absence of glucose causes translocation of CreA to the cytoplasm [7]. Mig1p re-localizes to the cytoplasm, relieving gene repression and allowing induction of genes encoding enzymes required for alternative carbon source utilisation [12,13,14]. 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]
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