Right here we report on a lipid-signalling pathway in plants that is downstream of phosphatidic acid and involves the protein kinase AGC2-1 regulated by the 3′-phosphoinositide-dependent kinase-1 (AtPDK1). the AtPDK1-regulated AGC2-1 kinase indicative of a role in growth and cell division. Cellular localisation of GFP-AGC2-1 fusion protein is highly dynamic in root hairs and at some stages confined to root hair tips and to nuclei. The knockout mutation results in a reduction of root hair length suggesting a role for AGC2-1 in root hair growth and development. (Deak and rice PDK1 kinases lack two conserved amino-acid residues that are required for high-affinity binding to PI(3 4 Rabbit Polyclonal to Mouse IgG (H/L). 5 In addition the PH domain of PDK1 was shown to bind a wide spectrum of lipids (Deak AGC kinase family (Turck AGC kinase family which we call AGC1-1 and AGC2-1. Here we show that AtPDK1 directly regulates AGC2-1 and that phosphatidic acid is the lipid activator of this AtPDK1 signalling pathway. Results AtPDK1 interacts WZ4002 with members of the AGC kinase family To search for potential components of the AtPDK1 signalling pathway a yeast two-hybrid screen was carried out using full-length AtPDK1 as bait and a pACT2 prey cDNA library prepared from cultured cells (Nemeth by 39 members. In a recent WZ4002 review we follow a nomenclature for the AGC kinase family in that provides working names for all members but incorporate and keep published names wherever available (http://www.arabidopsis.org/info/genefamily/AGC.html; Bogre AtPDK1-1 and its analogue AtPDK1-2 interacted with AGC1-1 and AGC2-1 in yeast two-hybrid assays (Figure 1A). AGC2-1 appeared to recognise the AtPDK1 kinase domain as no interaction could be detected with the PH domain of AtPDK1 (Figure 1A). PDK1 binding to selected AGC kinases in animals such as to S6K PKB aPKC or to SGK has been shown to require a C-terminal hydrophobic FXXF motif called PDK1 interacting fragment (PIF) (Biondi and Nebreda 2003 We generated two modified kinase constructs by exchanging the two conserved phenylalanines to alanine within the FXXF motif (M1) or replacing the final six C-terminal proteins with an unrelated series RGSGC (M2) (Shape 1B). Both mutated variations of AGC1-1 and AGC2-1 were not able to connect to WZ4002 PDK1 (Shape 1A). These data WZ4002 support the discovering that both phenylalanines in the PDK1 interacting fragment (PIF) site are necessary for interaction. Shape 1 Discussion of AtPDK1 with AGC1-1 and AGC2-1. (A) Candida two-hybrid discussion assay with wild-type AtPDK1-1 or AtPDK1-2 and with the pleckstrin homology (PH) site of AtPDK1-1 fused towards the Gal4 DNA-binding site as well as the wild-type AGC2-1 or AGC1-1 … AGC1-1 and AGC2-1 talk about substrate specificities with proteins kinase A To look for the enzymatic activity and substrate specificities of AGC1-1 and AGC2-1 kinases we performed kinase assays with different artificial peptides regarded as phosphorylated by pet AGC kinases (Obata AGC kinases had been labelled with N-terminal haemagglutinin (HA) epitope tags indicated in transfected cells and immunopurified for kinase assays one day after transfection using immobilised monoclonal anti-HA IgG. The kemptide peptide which can be easily phosphorylated WZ4002 by pet PKA (Kemp and exactly how this interaction impacts the enzyme activity of AGC2-1. Because of this we cotransfected a Myc-tagged AtPDK1 kinase with GST-tagged AGC2-1 into protoplasts ready from cultured cells and examined for the current presence of Myc-AtPDK1 in GST pull-down tests when compared with the input quantities in crude components. Proteins kinase activity of AGC2-1 was established using the kemptide peptide. These studies confirmed that Myc-AtPDK1 can be recruited by GST-AGC2-1 (Shape 1D). To answer fully the question if the activity of AGC2-1 kinase is necessary for discussion with AtPDK1 we performed identical GST pull-down assays utilizing a customized kinase GST-AGC2-1 (K45R). This inactivated kinase when a important lysine residue necessary for phosphotransfer was transformed to arginine demonstrated no modification in its capability to connect to AtPDK1 (Shape 1D). To check if the conserved AtPDK1 phosphorylation site on AGC2-1 at S235 inside the activation loop is crucial for AGC2-1 activity or its discussion with AtPDK1 we exchanged this serine residue to alanine (S235A). As the activity of the mutated AGC2-1 was decreased to background amounts the phosphorylation site mutation got no influence on the AGC2-1 discussion with AtPDK1. These data.