Isolation of hyperactive mutants of the MAPK p38/Hog1 that are independent of MAPK kinase activation.
Mitogen-activated protein kinases (MAPKs) play pivotal roles in growth, development, differentiation, and apoptosis. The exact role of a given MAPK in these processes is not fully understood. This question could be addressed using active forms of these enzymes that are independent of external stimulation and upstream regulation. Yet, such molecules are not available. MAPK activation requires dual phosphorylation, on neighboring Tyr and Thr residues, catalyzed by MAPK kinases (MAPKKs). It is not known how to force MAPK activation independent of MAPKK phosphorylation. Here we describe a series of nine hyperactive (catalytically and biologically), MAPKK-independent variants of the MAPK Hog1. Each of the active molecules contains just a single point mutation. Six mutations are in the conserved L16 domain of the protein. The active Hog1 mutants were obtained through a novel genetic screen that could be applied for isolation of active MAPKs of other families. Equivalent mutations, introduced to the human p38alpha, rendered the enzyme active even when produced in Escherichia coli, showing that the mutations increased the intrinsic catalytic activity of p38. It implies that the activating mutations could be directly used for production of active forms of MAPKs from yeasts to humans and could open the way to revealing their biological functions.
Bell
M
M
Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
Capone
R
R
Pashtan
I
I
Levitzki
A
A
Engelberg
D
D
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D004789:Enzyme Activation; D006801:Humans; D048369:MAP Kinase Kinase 1; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017354:Point Mutation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D013912:Threonine / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases
D029701:Saccharomyces cerevisiae Proteins; D013912:Threonine; D014443:Tyrosine; D017346:Protein Serine-Threonine Kinases; C088589:HOG1 protein, S cerevisiae; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; C482127:Map2k1 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases; C089356:PBS2 protein, S cerevisiae