Functional Characterization of <em>Aspergillus nidulans ypkA,</em> a Homologue of the Mammalian Kinase SGK

<div><p>The serum- and glucocorticoid-regulated protein kinase (SGK) is an AGC kinase involved in signal cascades regulated by glucocorticoid hormones and serum in mammals. The <i>Saccharomyces cerevisiae ypk1</i> and <i>ypk2</i> genes were identified as SGK homologues and Ypk1 was shown to regulate the balance of sphingolipids between the inner and outer plasma membrane. This investigation characterized the <i>Aspergillus nidulans YPK1</i> homologue, YpkA, representing the first filamentous fungal <i>YPK1</i> homologue. Two conditional mutant strains were constructed by replacing the endogenous <i>ypk1</i> promoter with two different regulatable promoters, <i>alcA</i> (from the alcohol dehydrogenase gene) and <i>niiA</i> (from the nitrate reductase gene). Both constructs confirmed that <i>ypkA</i> was an essential gene in <i>A. nidulans</i>. Repression of <i>ypkA</i> caused decreased radial growth, a delay in conidial germination, deficient polar axis establishment, intense branching during late stages of growth, a lack of asexual spores, and a terminal phenotype. Membrane lipid polarization, endocytosis, eisosomes and vacuolar distribution were also affected by <i>ypkA</i> repression, suggesting that YpkA plays a role in hyphal morphogenesis via coordinating the delivery of cell membrane and wall constituents to the hyphal apex. The <i>A. nidulans</i> Pkh1 homologue <i>pkhA</i> was also shown to be an essential gene, and preliminary genetic analysis suggested that the ypkA gene is not directly downstream of <i>pkhA</i> or epistatic to <i>pkhA</i>, rather, <i>ypkA</i> and <i>pkhA</i> are genetically independent or in parallel. <i>BarA</i> is a homologue of the yeast <i>Lag1</i> acyl-CoA-dependent ceramide synthase, which catalyzes the condensation of phytosphingosine with a fatty acyl-CoA to form phytoceramide. When <i>barA</i> was absent, <i>ypkA</i> repression was lethal to the cell. Therefore, there appears to be a genetic interaction between <i>ypkA</i>, <i>barA</i>, and the sphingolipid synthesis. Transcriptional profiling of <i>ypkA</i> overexpression and down-regulation revealed several putative YpkA targets associated with the observed phenotypes.</p> </div

The <i>A. nidulans</i> YpkA interacts with BarA.

<p>The radial growth of the wild-type, <i>niiA::ypkA</i>, <i>barA1</i>, and <i>niiA::ypkA barA1</i> mutant strains were grown for 72 hours at 37°C on MM+sodium nitrate 10 mM or MM+ammonium tartrate 50 mM (Wt = Wild-type).</p

polarized delivery of membrane lipids and cell wall deposition was not confined to the hyphal apex in the <i>niiA::ypkA</i> mutant upon repression.

<p>In all experiments, germlings were grown for 16 hours at 37°C on inducing (sodium nitrate) and repressing conditions (ammonium tartrate). Stains utilized: (A) Hoescht, (B) Filipin, (C) FITC-conjugated wheat germ, and (D) CFW. Bars, 5 and 10 µm.</p

<i>A. nidulans</i> YpkA is involved in polarized growth.

<p>The percentage of wild-type and <i>niiA::ypkA</i> mutant germlings that exhibited polar growth (defined here as the emergence of the germ tube) (A) and the number of nuclear per germling (B). Conidia were grown for 2 to 8 hours at 37°C. Averages (± standard deviation) represent 100 germlings from three independent experiments (Wt = Wild-type). (C) The germination pattern of the wild-type and <i>niiA::ypkA</i> conidiospores. Conidia were allowed to germinate on MM media for 8 to 16 hours. Conidia possessing germ tubes were classified as displaying (left to right) unipolar (1), bipolar (2), unipolar plus lateral branches (3) or bipolar plus lateral branches (4).</p

YpkA expression affects eisosomes distribution in <i>A. nidulans</i>.

<p>The <i>niiA::ypkA pilA::gfp</i> (A) and <i>niiA::ypkA pilB::gfp</i> (B) strains were grown for 16 hours at 37°C in MM+10 mM sodium nitrate or 50 mM ammonium tartrate. Bars, 5 µm.</p

Evaluation of the effect of YpkA depletion and overexpression on the growth of <i>A. nidulans</i> under different experimental conditions.

<p>Five µl of a ten-fold dilution series starting at a concentration of 2×10⁷ for the wild-type (A-D), <i>alcA::ypkA</i> (A-B), and <i>niiA::ypkA</i> (C–D) strains were spotted on different growth media and grown for 72 hours at 37°C, except for experiments where the temperature was evaluated (A and C). CFW = calcofluor white; CR = congo red; MR = myriocin; PHS = phytosphingosine; and SDS = sodium dodecyl sulphate.</p

The <i>pkhA</i> gene is essential to <i>A. nidulans</i> and interacts with <i>ypkA</i>.

<p>(A) The wild-type and <i>niiA::pkhA</i> mutant strains were grown for 72 hours at 37°C on MM+sodium nitrate 10 mM or MM+ammonium tartrate 50 mM (Wt = Wild-type). (B) The <i>alcA::ypkA</i>, <i>niiA::pkhA</i>, and <i>alcA::ypkA niiA::pkhA</i> strains were grown for 72 hours at 37°C on different combinations of MM+glucose 2% or glycerol 2% plus threonine 100 mM plus sodium nitrate 10 mM or ammonium tartrate 50 mM (Wt = Wild-type).</p

The <i>ypkA</i> gene is to essential <i>A. nidulans</i>.

<p>(A) Wild-type and primary <i>ΔypkA</i> transformant were grown (left and center panel) or streaked (right panel) on YAG medium for 96 hours at 37°C. (B) The <i>alcA::ypkA</i> strain was grown for 6 hours in MM+4% glucose or MM+2% glycerol +threonine 100 mM (C) The <i>niiA::ypkA</i> strain was grown for 6 hours in MM+sodium nitrate 10 mM or MM+ammonium tartrate 50 mM. The relative quantitation of <i>ypkA</i> and tubulin gene expression was determined by a standard curve (i.e., C_T –values plotted against a logarithm of the DNA copy number). The presented results are the means (± standard deviation) of four biological replicates. The growth phenotypes of the <i>alcA::ypkA</i> (D) and <i>niiA::ypkA</i> (E) mutant strains. The <i>A. nidulans</i> wild-type, <i>alcA::ypkA</i>, and <i>niiA::ypkA</i> mutant strains were grown for 72 hours at 37°C either on MM+4% glucose or MM+2% glycerol and MM+sodium nitrate 10 mM or MM+ammonium tartrate 50 mM.</p

Protein Kinase C Overexpression Suppresses Calcineurin-Associated Defects in <i>Aspergillus nidulans</i> and Is Involved in Mitochondrial Function

<div><p>In filamentous fungi, intracellular signaling pathways which are mediated by changing calcium levels and/or by activated protein kinase C (Pkc), control fungal adaptation to external stimuli. A rise in intracellular Ca²⁺ levels activates calcineurin subunit A (CnaA), which regulates cellular calcium homeostasis among other processes. Pkc is primarily involved in maintaining cell wall integrity (CWI) in response to different environmental stresses. Cross-talk between the Ca²⁺ and Pkc-mediated pathways has mainly been described in <i>Saccharomyces cerevisiae</i> and in a few other filamentous fungi. The presented study describes a genetic interaction between CnaA and PkcA in the filamentous fungus <i>Aspergillus nidulans</i>. Overexpression of <i>pkcA</i> partially rescues the phenotypes caused by a <i>cnaA</i> deletion. Furthermore, CnaA appears to affect the regulation of a mitogen-activated kinase, MpkA, involved in the CWI pathway. Reversely, PkcA is involved in controlling intracellular calcium homeostasis, as was confirmed by microarray analysis. Furthermore, overexpression of <i>pkcA</i> in a <i>cnaA</i> deletion background restores mitochondrial number and function. In conclusion, PkcA and CnaA-mediated signaling appear to share common targets, one of which appears to be MpkA of the CWI pathway. Both pathways also regulate components involved in mitochondrial biogenesis and function. This study describes targets for PkcA and CnaA-signaling pathways in an <i>A. nidulans</i> and identifies a novel interaction of both pathways in the regulation of cellular respiration.</p></div

Number of genes and the associated functions of the proteins they encode that had restored or elevated expression in the <i>alcA::pkcA ΔcnaA</i> strain when compared to the Δ<i>cnaA</i> strain.

<p>sP is number of genes in each category.</p