Rck2 Kinase Is a Substrate for the Osmotic Stress-Activated Mitogen-Activated Protein Kinase Hog1

Abstract

Exposure of yeast cells to increases in extracellular osmolarity activates the Hog1 mitogen-activated protein kinase (MAPK). Activation of Hog1 MAPK results in induction of a set of osmoadaptive responses, which allow cells to survive in high-osmolarity environments. Little is known about how the MAPK activation results in induction of these responses, mainly because no direct substrates for Hog1 have been reported. We conducted a two-hybrid screening using Hog1 as a bait to identify substrates for the MAPK, and the Rck2 protein kinase was identified as an interactor for Hog1. Both two-hybrid analyses and coprecipitation assays demonstrated that Hog1 binds strongly to the C-terminal region of Rck2. Upon osmotic stress, Rck2 was phosphorylated in vivo in a Hog1-dependent manner. Furthermore, purified Hog1 was able to phosphorylate Rck2 when activated both in vivo and in vitro. Rck2 phosphorylation occurred specifically at Ser519, a residue located within the C-terminal putative autoinhibitory domain. Interestingly, phosphorylation at Ser519 by Hog1 resulted in an increase of Rck2 kinase activity. Overexpression of Rck2 partially suppressed the osmosensitive phenotype of hog1Δ and pbs2Δ cells, suggesting that Rck2 is acting downstream of Hog1. Consistently, growth arrest caused by hyperactivation of the Hog1 MAPK was abolished by deletion of the RCK2 gene. Furthermore, overexpression of a catalytically impaired (presumably dominant inhibitory) Rck2 kinase resulted in a decrease of osmotolerance in wild-type cells but not in hog1Δ cells. Taken together, our data suggest that Rck2 acts downstream of Hog1, controlling a subset of the responses induced by the MAPK upon osmotic stress.

ACKNOWLEDGMENTS

We thank Despina Alexandraki and Jean-Claude Jauniaux for valuable advice regarding two-hybrid technology and Mireia Zaguirre and Anna Vilalta for their technical assistance.

This work was supported by grants from the Swedish Cancer Fund (2163-B97-08XAC) and the Swedish Radiation Protection Institute (1092.98) and by grants from AstraZeneca to P.S., grants PB95-0663 and PB98-0565-C4-02 from the Dirección General de Investigación Científica y Técnica (Ministry of Education, Spain) to J.A., grant GM50909 from the National Institutes of Health to H.S., and grant PM99-0028 from the Dirección General de Investigación Científica y Técnica (Ministry of Education, Spain) to F.P. F.P. was the recipient of a postdoctoral research contract from the MEC, Spain.