Isolation of intrinsically active mutants of MAP kinases via genetic screens in yeast.
Intrinsically active variants of a protein are powerful tools for deciphering the specific functions of that protein. Since the catalytic activity of such variants is spontaneously active in vivo, they can disclose very accurately biochemical and biological functions of the parental protein. It is particularly important to obtain intrinsically active variants of individual MAP kinases. This is because in response to extracellular signals, more than one MAPK is typically concomitantly activated making it difficult to reveal their individual functions and downstream targets. Until very recently intrinsically active variants were not available for MAP kinases because of their unusual mechanism of activation that requires dual phosphorylation on neighboring Thr and Tyr residues. It is not known how to mimic the phospho-Thr-Xaa-phospho-Tyr motif by mutagenesis. We describe here a genetic screen in yeast that we successfully used to isolate bona fide intrinsically active variants of the yeast MAP kinase Hog1 and all isoforms of the human p38 family. We further established a screen for isolation of intrinsically active ERKs. The rationale of our screening approach is to search for MAPK molecules that are active in the absence of their activators. The method could be applied to the discovery of intrinsically active variants of any MAP kinase of any organism. We describe in detail the rationale, the steps that should be taken for establishment of such a screen and a step-by-step protocol for carrying out the screen.
Engelberg
David
D
The Department of Biological Chemistry, The Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel. Engelber@cc.huji.ac.il
Livnah
Oded
O
D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005821:Genetic Techniques; D020930:MAP Kinase Kinase Kinases / Q000235:genetics / Q000378:metabolism; D020935:MAP Kinase Signaling System; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D015003:Yeasts / Q000235:genetics*
D005656:Fungal Proteins; D020928:Mitogen-Activated Protein Kinases; D020930:MAP Kinase Kinase Kinases