THE BYR2 KINASE TRANSLOCATES TO THE PLASMA-MEMBRANE IN A RAS1-DEPENDENT MANNER

The activation of mitogen-activated protein kinase cascades by the Ras GTPase is an evolutionarily conserved signal transduction mechanism. To better understand the interaction between Ras and its target kinase , we study the yeast Schizosaccharomyces pombe where the Ras1 GTPase a ctivates the Byr2 kinase. Cell fractionation and immunofluorescence sh owed that Ras1 was localized to the plasma membrane and that Byr2 was in the cytoplasm. When Ras1 was overexpressed, Byr2 was translocated t o the plasma membrane. Byr2 translocation was dependent on binding to Ras1 since Ras1-V12, an activated mutant of Ras1, caused more Byr2 tra nslocation than Ras1, since Ras1-D38E, an effector domain mutant, did not cause Byr2 translocation, and since the Ras1-binding domain of Byr 2 was necessary and sufficient to cause Byr2 translocation. The Byr2 p rotein was usually not uniform around the plasma membrane, but was fre quently enriched at the cell ends and at the region of septal depositi on. This uneven membrane localization depended upon regions of the Byr 2 regulatory domain, in addition to those required for Ras1 binding, s uggesting that these Byr2 domains participate in protein-protein inter actions. (C) 1998 Academic Press.