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Molecular and Cellular Biology, September 2008, p. 5172-5183, Vol. 28, No. 17
0270-7306/08/$08.00+0     doi:10.1128/MCB.00589-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Phosphorylated Ssk1 Prevents Unphosphorylated Ssk1 from Activating the Ssk2 Mitogen-Activated Protein Kinase Kinase Kinase in the Yeast High-Osmolarity Glycerol Osmoregulatory Pathway{triangledown}

Tetsuro Horie,{dagger} Kazuo Tatebayashi,{dagger} Rika Yamada, and Haruo Saito*

Division of Molecular Cell Signaling, Institute of Medical Sciences, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan

Received 11 April 2008/ Returned for modification 12 May 2008/ Accepted 16 June 2008

In Saccharomyces cerevisiae, external high osmolarity activates the Hog1 mitogen-activated protein kinase (MAPK), which controls various aspects of osmoadaptation. Ssk1 is a homolog of bacterial two-component response regulators and activates the Ssk2 MAPK kinase kinase upstream of Hog1. It has been proposed that unphosphorylated Ssk1 (Ssk1-OH) is the active form and that Ssk1 phosphorylated (Ssk1~P) at Asp554 by the Sln1-Ypd1-Ssk1 multistep phosphorelay mechanism is the inactive form. In this study, we show that constitutive activation of Ssk2 occurs when Ssk1 phosphorylation is blocked by either an Ssk1 mutation at the phosphorylation site or an Ssk1 mutation that inhibits its interaction with Ypd1, the donor of phosphate to Ssk1. Thus, Ssk1-OH is indeed necessary for Ssk2 activation. However, overexpression of wild-type Ssk1 or of an Ssk1 mutant that cannot bind Ssk2 prevents constitutively active Ssk1 mutants from activating Ssk2. Therefore, Ssk1 has a dual function as both an activator of Ssk2 and an inhibitor of Ssk1 itself. We also found that Ssk1 exists mostly as a dimer within cells. From mutant phenotypes, we deduce that only the Ssk1-OH/Ssk1-OH dimer can activate Ssk2 efficiently. Hence, because Ssk1~P binds to and inhibits Ssk1-OH, moderate fluctuation of the level of Ssk1-OH does not lead to nonphysiological and detrimental activation of Hog1.

* Corresponding author. Mailing address: Institute of Medical Sciences, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Phone: 81 3 5449 5505. Fax: 81 3 5449 5701. E-mail: h-saito{at}ims.u-tokyo.ac.jp

{triangledown} Published ahead of print on 23 June 2008.

{dagger} T.H. and K.T. contributed equally to this work.

Molecular and Cellular Biology, September 2008, p. 5172-5183, Vol. 28, No. 17
0270-7306/08/$08.00+0     doi:10.1128/MCB.00589-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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