Identification of p21-activated kinase specificity determinants in budding yeast: a single amino acid substitution imparts Ste20 specificity to Cla4.
Two closely related p21-activated kinases from Saccharomyces cerevisiae, Ste20 and Cla4, interact with and are regulated by Cdc42, a small Rho-like GTPase. These kinases are argued to perform a common essential function, based on the observation that the single mutants are viable whereas the double mutant is inviable. Despite having a common upstream regulator and at least one common function, these molecules also have many distinct cellular signaling roles. Ste20 signals upstream of several mitogen-activated protein kinase cascades (e.g., pheromone response, filamentous growth, and high osmolarity), and Cla4 signals during budding and cytokinesis. In order to investigate how these kinases are directed to distinct functions, we sought to identify specificity determinants within Ste20 and Cla4. To this end, we constructed both chimeric fusions and point mutants and tested their ability to perform unique and shared cellular roles. Specificity determinants for both kinases were mapped to the C-terminal kinase domains. Remarkably, the substitution of a single amino acid, threonine 818, from Ste20 into an otherwise wild-type Cla4, Cla4D772T, conferred the ability to perform many Ste20-specific functions.
Keniry
Megan E
ME
Department of Biology and Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229, USA.
Sprague
George F
GF
D015153:Blotting, Western; D005982:Glutathione Transferase / Q000378:metabolism; D006238:Haploidy; D047908:Intracellular Signaling Peptides and Proteins; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D004718:Saccharomycetales / Q000201:enzymology* / Q000378:metabolism; D015398:Signal Transduction; D013912:Threonine / Q000737:chemistry; D001616:beta-Galactosidase / Q000378:metabolism; D054462:p21-Activated Kinases
D047908:Intracellular Signaling Peptides and Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D013912:Threonine; D005982:Glutathione Transferase; C095212:CLA4 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D020930:MAP Kinase Kinase Kinases; C078754:STE20 protein, S cerevisiae; C067018:Ste11 protein, S cerevisiae; D001616:beta-Galactosidase