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Molecular Genetics and Genomics

, Volume 271, Issue 2, pp 208–219 | Cite as

Analyses of the effects of Rck2p mutants on Pbs2pDD-induced toxicity in Saccharomyces cervisiae identify a MAP kinase docking motif, and unexpected functional inactivation due to acidic substitution of T379

  • L. Jiang
  • S. Niu
  • K. L. Clines
  • D. J. Burke
  • T. W. SturgillEmail author
Original Paper

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(Δ487–610), 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 PBS2 DD, 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 rck2Δ strain to allow some cells to survive PBS2 DD toxicity even when overexpressed. The significance of these findings for our understanding of Rck2p function is discussed.

Keywords

Yeast  RCK2  PBS2  HOG1 MAP kinases 

Notes

Acknowledgements

T.W.S. is indebted to Tobias Schmelzle and other members of Michael Hall’s laboratory (Biozentrum, Basel). Colleagues Jeremy Thorner (University of California at Berkeley) and Per Sunnerhagen (University of Göteborg) were very generous in providing their reagents, as were Gustav Ammerer (University of Vienna, Biocenter, Vienna), Haru Saito and Pam Silver (Dana-Farber, Boston). This project was supported by National Institutes of Health Grant GM62890 (to T. W. S.)

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Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • L. Jiang
    • 1
  • S. Niu
    • 1
  • K. L. Clines
    • 1
  • D. J. Burke
    • 2
  • T. W. Sturgill
    • 1
    Email author
  1. 1.Department of PharmacologyUniversity of Virginia Health Sciences CenterCharlottesvilleUSA
  2. 2.Department of Biochemistry and Molecular GeneticsUniversity of Virginia Health Sciences CenterCharlottesvilleUSA

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