duces the phosphorylation of certain protein kinases in vitro. In S. cerevisiae, sphingolipids induce Pkh1 auto-phosphorylation, which in turn activates several other proteins kinases, including Ypk1. Mutations that block the early stages of endocytosis show a reduction in the assimilation of the fatty acids, indicating that Ypk1 affects the uptake of fatty acids by regulating endocytosis. In addition to signaling events and vesicular transport, the control 22392765 of cell growth involves the synthesis of a new cell wall skeleton and the reorganization of actin. The actin cytoskeleton directs the delivery of components to the hyphal apex and it is essential for maintaining polarized growth. In fungi, the cell wall determines the shape of, and confers rigidity to, the cell. Furthermore, the cell wall is a dynamic structure that functions as a sensor of turgor and is continuously remodeling in response to environmental stimuli and growth. 
The S. cerevisiae protein kinase C activates the cell wall maintenance MAP kinase signaling cascade in response to osmotic stress and restrictive temperatures, mediating the signal from TORC2, to regulate the actin cytoskeleton. Pkh2 and MedChemExpress KPT-9274 possibly Pkh1 phosphorylate and activate Pkc1. In addition, ypk mutants demonstrate a random distribution of the actin cytoskeleton and are deficient in the activation of MAP cascade. This study presents the first YPK1 homologue, ypkA, to be characterized in a filamentous fungus. In A. nidulans, ypkA was shown to be an essential gene that is involved in polar axis establishment and germling growth. Membrane lipid polarization was deficient when the ypkA conditional mutant was grown in repressing conditions, suggesting that YpkA plays an important role in hyphal morphogenesis that also affects endocytosis and vacuolar distribution in A. nidulans. Results The A. nidulans ypkA Gene is Essential A BLASTp search of the A. nidulans genome database using the S. cerevisiae Ypk1p and Ypk2p as queries revealed a single open reading frame with significant similarity. The potential homologue, AN5973, is predicted to be a 640-amino acid protein kinase with high identity to Ypk1p and Ypk2 mainly within the C-terminal region. YpkA has two defined domains, a protein kinase domain from residues 293 to 550 and an AGC-kinase C-terminal domain from residues 551 to 622. An in vivo S. cerevisiae fusion-based approach was used in an attempt to generate an A. nidulans ypkA null mutant. Several primary transformants were isolated. However, these transformants displayed a pronounced phenotypic defect on MM, i.e. very small non-sporulating colonies. These colonies were unable to grow or produce a significant amount of mycelium in liquid medium, suggesting a terminal phenotype for these strains. Heterokaryon rescue confirmed that ypkA is an essential gene. Subsequently, two conditional mutants for ypkA were constructed by replacing the endogenous ypkA promoter with the either the alcA or niiA promoters. The alcA promoter 22440900 is repressed by glucose, derepressed by glycerol and induced to high levels by ethanol or L-threonine. Transformants that accumulated approximately 3-fold higher ypkA mRNA when transferred to glycerol 2%+threonine than when transferred to glucose 4% were selected. The second promoter from niiA is induced by sodium nitrate and repressed by ammonium tartrate. Transformants that showed an approximate four-fold decrease in ypkA mRNA accumulation when transferred to ammonium tartrate than when
