Analysis of mitogen-activated protein kinase activity in yeast.
Mitogen-activated protein (MAP) kinases play central roles in transmitting extracellular and intracellular information in a wide variety of situations in eukaryotic cells. Their activities are perturbed in a large number of diseases, and their activating kinases are currently therapeutic targets in cancer. MAPKs are highly conserved among all eukaryotes. MAPKs were first cloned from the yeast Saccharomyces cerevisiae. Yeast has five MAPKs and one MAPK-like kinase. The mating MAPK Fus3 is the best characterized yeast MAPK. Members of all subfamilies of human MAPKs can functionally substitute S. cerevisiae MAPKs, providing systems to use genetic approaches to study the functions of either yeast or human MAPKs and to identify functionally relevant amino acid residues that enhance or reduce the effects of therapeutically relevant inhibitors and regulatory proteins. Here, we describe an assay to measure Fus3 activity in immune complexes prepared from S. cerevisiae extracts. The assay conditions are applicable to other MAPKs, as well.
Elion
Elaine A
EA
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA. elaine_elion@hms.harvard.edu
Sahoo
Rupam
R
D002457:Cell Extracts; D049109:Cell Proliferation; D057075:Enzyme Assays / Q000379:methods*; D047468:Immunoprecipitation; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
D002457:Cell Extracts; D029701:Saccharomyces cerevisiae Proteins; C075580:FUS3 protein, S cerevisiae; C541984:KSS1 protein, S cerevisiae; D020928:Mitogen-Activated Protein Kinases