Design, Synthesis, and Characterization of a Highly Effective Hog1 Inhibitor: A Powerful Tool for Analyzing MAP Kinase Signaling in Yeast

The Saccharomyces cerevisiae High-Osmolarity Glycerol (HOG) pathway is a conserved mitogen-activated protein kinase (MAPK) signal transduction system that often serves as a model to analyze systems level properties of MAPK signaling. Hog1, the MAPK of the HOG-pathway, can be activated by various environmental cues and it controls transcription, translation, transport, and cell cycle adaptations in response to stress conditions. A powerful means to study signaling in living cells is to use kinase inhibitors; however, no inhibitor targeting wild-type Hog1 exists to date. Herein, we describe the design, synthesis, and biological application of small molecule inhibitors that are cell-permeable, fast-acting, and highly efficient against wild-type Hog1. These compounds are potent inhibitors of Hog1 kinase activity both in vitro and in vivo. Next, we use these novel inhibitors to pinpoint the time of Hog1 action during recovery from G1 checkpoint arrest, providing further evidence for a specific role of Hog1 in regulating cell cycle resumption during arsenite stress. Hence, we describe a novel tool for chemical genetic analysis of MAPK signaling and provide novel insights into Hog1 action

Application of a peptide-based assay to characterize inhibitors targeting protein kinases from yeast

Chemical molecules that inhibit protein kinase activity are important tools to assess the functions of protein kinases in living cells. To develop, test and characterize novel inhibitors, a convenient and reproducible kinase assay is of importance. Here, we applied a biotinylated peptide-based method to assess adenosine triphosphate-competitive inhibitors that target the yeast kinases Hog1, Elm1 and Elm1-as. The peptide substrates contained 13 amino acids, encompassing the consensus sequence surrounding the phosphorylation site. To test whether the lack of distal sites affects inhibitor efficacy, we compared the peptide-based assay with an assay using full-length protein as substrate. Similar inhibitor efficiencies were obtained irrespective of whether peptide or full-length protein was used as kinase substrates. Thus, we demonstrate that the peptide substrates used previously (Dinér et al. in PLoS One 6(5):e20012, 2011) give accurate results compared with protein substrates. We also show that the peptide-based method is suitable for selectivity assays and for inhibitor screening. The use of biotinylated peptide substrates provides a simple and reliable assay for protein kinase inhibitor characterization. The utility of this approach is discussed. © 2014 Springer-Verlag Berlin Heidelberg

Positional Scanning Peptide Libraries for Kinase Substrate Specificity Determinations: Straightforward and Reproducible Synthesis Using Pentafluorophenyl Esters.

An efficient method to synthesize positional scanning synthetic combinatorial libraries (PS-SCLs) for studying the specificity of protein kinases is presented. Isokinetic ratios for pentafluorophenyl esters were determined iteratively using a new approach incorporating high performance liquid chromatography (HPLC) quantification and statistical experimental design. In the development process a large amount of work was put in to find efficient ways of screening for new isokinetic mixtures and to optimize the process of PS-SCL synthesis. The newly developed methods for the screening of isokinetic mixtures could be used for the screening of other interesting mixtures, but more importantly, the isokinetic ratios determined for the preactivated pentafluorophenyl esters were incorporated into a new efficient protocol. This straightforward protocol allows for a convenient synthesis of high quality PS-SCLs regardless of previous experience in solid phase synthesis

Determination of primary sequence specificity of Arabidopsis MAPKs MPK3 and MPK6 leads to identification of new substrates

MAPKs (mitogen-activated protein kinases) are signalling components highly conserved among eukaryotes. Their diverse biological functions include cellular differentiation and responses to different extracellular stress stimuli. Although some substrates of MAPKs have been identified in plants, no information is available about whether amino acids in the primary sequence other than proline-directed phosphorylation (pS-P) contribute to kinase specificity towards substrates. in the present study, we used a random positional peptide library to search for consensus phosphorylation sequences for Arabidopsis MAPKs MPK3 and MPK6. These experiments indicated a preference towards the sequence L/P-P/X-S-P-R/K for both kinases. After bioinformatic processing, a number of novel candidate MAPK substrates were predicted and subsequently confirmed by in vitro kinase assays using bacterially expressed native Arabidopsis proteins as substrates. MPK3 and MPK6 phosphorylated all proteins tested more efficiently than did another MAPK, MPK4. These results indicate that the amino acid residues in the primary sequence surrounding the phosphorylation site of Arabidopsis MAPK substrates can contribute to MAPK specificity. Further characterization of one of these new substrates confirmed that AtIg80180.1 was phosphorylated in planta in a MAPK-dependent manner. Phenotypic analyses of Arabidopsis expressing phosphorylation site mutant forms of AtIg80180.1 showed clustered stomata and higher stomatal index in cotyledons expressing the phosphomimetic form of AtIg80180.1, providing a link between this new MAPK substrate and the defined role for MPK3 and MPK6 in stomatal patterning

Elucidating the response of Kluyveromyces lactis to arsenite and peroxide stress and the role of the transcription factor KlYap8.

All organisms need to sense and respond to a range of stress conditions. In this study, we used transcriptional profiling to identify genes and cellular processes that are responsive during arsenite and tert-butyl hydroperoxide exposure in Kluyveromyces lactis. Many arsenite-responsive genes encode proteins involved in redox processes, protein folding and stabilization, and transmembrane transport. The majority of peroxide-responsive genes encode functions related to transcription, translation, redox processes, metabolism and transport. A substantial number of these stress-regulated genes contain binding motifs for the AP-1 like transcription factors KlYap1 and KlYap8. We demonstrate that KlYap8 binds to and regulates gene expression through a 13 base-pair promoter motif, and that KlYap8 provides protection against arsenite, antimonite, cadmium and peroxide toxicity. Direct transport assays show that Klyap8Δ cells accumulate more arsenic and cadmium than wild type cells and that the Klyap8Δ mutant is defective in arsenic and cadmium export. KlYap8 regulates gene expression in response to both arsenite and peroxide, and might cooperate with KlYap1 in regulation of specific gene targets. Comparison of KlYap8 with its Saccharomyces cerevisiae orthologue ScYap8 indicates that KlYap8 senses and responds to multiple stress signals whereas ScYap8 is only involved in the response to arsenite and antimonite. Thus, our data suggest that functional specialization of ScYap8 has occurred after the whole genome duplication event. This is the first genome-wide stress response analysis in K. lactis and the first demonstration of KlYap8 function