Abstract
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 Saccharomyces cerevisiae ypk1 and ypk2 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 Aspergillus nidulans YPK1 homologue, YpkA, representing the first filamentous fungal YPK1 homologue. Two conditional mutant strains were constructed by replacing the endogenous ypk1 promoter with two different regulatable promoters, alcA (from the alcohol dehydrogenase gene) and niiA (from the nitrate reductase gene). Both constructs confirmed that ypkA was an essential gene in A. nidulans. Repression of ypkA 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 ypkA 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 A. nidulans Pkh1 homologue pkhA was also shown to be an essential gene, and preliminary genetic analysis suggested that the ypkA gene is not directly downstream of pkhA or epistatic to pkhA, rather, ypkA and pkhA are genetically independent or in parallel. BarA is a homologue of the yeast Lag1 acyl-CoA-dependent ceramide synthase, which catalyzes the condensation of phytosphingosine with a fatty acyl-CoA to form phytoceramide. When barA was absent, ypkA repression was lethal to the cell. Therefore, there appears to be a genetic interaction between ypkA, barA, and the sphingolipid synthesis. Transcriptional profiling of ypkA overexpression and down-regulation revealed several putative YpkA targets associated with the observed phenotypes.
Highlights
Protein kinases are key regulators of cellular function that act via phosphorylating target proteins directing their activity, location and function
The mammalian Serum- and glucocorticoid-regulated protein kinases (SGK) is activated in response to the follicle-stimulating hormone that is produced during brain damage [2] and the transfection of mammary epithelial cells with the p53 transcription factor [3]
The serum- and glucocorticoid-inducible kinase (SGK) was initially identified in a screen of a cDNA library generated from mammary tumor cells of rats treated with glucocorticoids [1]
Summary
Protein kinases are key regulators of cellular function that act via phosphorylating target proteins directing their activity, location and function. Serum- and glucocorticoid-regulated protein kinases (SGK) are members of the AGC kinase subfamily (PKA, PKC, PKC) and are involved in signal cascades which are regulated in mammals by serum and glucocorticoid hormones [1]. The threonine residue (Thr256), located on the SGK activation loop of the catalytic domain, is phosphorylated by the 3-phosphoinositide-dependent protein kinase-1 (PDK1), which serves as a central integrator of signaling cascades. Complementation of the Dypk S. cerevisiae strain with the mammalian SGK restored viability. The S. cerevisiae pkh protein kinase possesses a catalytic domain that resembles the mammalian PDK1 catalytic domain. Complementation of the Dpkh with PDK1 restored viability, while Ypk was demonstrated to be phosphorylated by Pkh on the Thr504 residue, indicating that PDK1 and pkh are functional homologues [6]
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