ABSTRACT
Rck2p is a Ser/Thr kinase that binds to, and is activated by, Hog1p. Expression of the MAP kinase kinase Pbs2pDD from a GAL1-driven plasmid hyperactivates the HOG MAP kinase pathway, and leads to cessation of growth. This toxic effect is reduced by deletion of RCK2. We studied the structural and functional basis for the role of Rck2p in mediating the growth arrest phenotype associated with overexpression of Pbs2pDD. Rck2p kinase activity is required for the effect, because Rck2p(Delta487-610), as well as full-length Rck2p, is toxic with Pbs2pDD, but kinase-defective versions of either protein with a K201R mutation are not. Thus, the C-terminal portion of Rck2p is not required provided the protein is activated by removal of the autoinhibitory domain. Relief of inhibition in Rck2p normally requires phosphorylation by Hog1p, and Rck2p contains a putative MAP kinase docking site (TILQR589R590KKVQ) in its C-terminal segment. The Rck2p double mutant R589A/R590A expressed from a centromeric plasmid did not detectably bind Hog1p-GFP and was functionally inactive in mediating the toxic effect of Pbs2pDD, equivalent to an RCK2 deletion. However, overexpression of Rck2p R589A/R590A from a multicopy plasmid restored function. In contrast, RCK2-K201R acted as a multicopy suppressor of PBS2DD, markedly reducing its toxicity. This suppressor activity required the K201R mutation, and the effect was largely lost when the docking site was mutated, suggesting suppression by inhibition of Hog1p functions. We also studied the effect of replacing the predicted T379 and established S520 phosphorylation sites in Rck2p by glutamic acid. Surprisingly, the T379E mutant markedly reduced Pbs2pDD toxicity, and toxicity was only partially rescued by S520E. Rck2 T379E was sufficiently inactive in an rck2Delta strain to allow some cells to survive PBS2DD toxicity even when overexpressed. The significance of these findings for our understanding of Rck2p function is discussed.
