Requirement of STE50 for Osmostress-Induced Activation of the STE11 Mitogen-Activated Protein Kinase Kinase Kinase in the High-Osmolarity Glycerol Response Pathway

Abstract

Exposure of yeast cells to increases in extracellular osmolarity activates the HOG1 mitogen-activated protein (MAP) kinase cascade, which is composed of three tiers of protein kinases: (i) the SSK2, SSK22, and STE11 MAP kinase kinase kinases (MAPKKKs), (ii) the PBS2 MAPKK, and (iii) the HOG1 MAP kinase. Activation of the MAP kinase cascade is mediated by two upstream mechanisms. The SLN1-YPD1-SSK1 two-component osmosensor activates the SSK2 and SSK22 MAPKKKs by direct interaction of the SSK1 response regulator with these MAPKKKs. The second mechanism of HOG1 MAP kinase activation is independent of the twocomponent osmosensor and involves the SHO1 transmembrane protein and the STE11 MAPKKK. Only PBS2 and HOG1 are common to the two mechanisms. We conducted an exhaustive mutant screening to identify additional elements required for activation of STE11 by osmotic stress. We found that strains with mutations in the STE50 gene, in combination with ssk2⌬ ssk22⌬ mutations, were unable to induce HOG1 phosphorylation after osmotic stress. Both two-hybrid analyses and coprecipitation assays demonstrated that the N-terminal domain of STE50 binds strongly to the N-terminal domain of STE11. The binding of STE50 to STE11 is constitutive and is not affected by osmotic stress. Furthermore, the two proteins relocalize similarly after osmotic shock. It was concluded that STE50 fulfills an essential role in the activation of the high-osmolarity glycerol response pathway by acting as an integral subunit of the STE11 MAPKKK.Mitogen-activated protein (MAP) kinase cascades are common signaling modules found in both higher and lower eukaryotic cells. A typical MAP kinase cascade is composed of three tiers of protein kinases, a MAP kinase (MAPK), a MAPK kinase (MAPKK), and a MAPKK kinase (MAPKKK) (27). Yeast cells have several distinct MAP kinase cascades that transduce distinct extracellular stimuli (e.g., mating pheromone, high osmolarity, low osmolarity, and nitrogen starvation) (10, 19). Budding yeast (Saccharomyces cerevisiae) responds to increases in osmolarity in the extracellular environment by activating the HOG1 MAP kinase cascade. This cascade is essential for the survival of yeast in high-osmolarity environments (4, 5). Because a major outcome of the activation of this MAPK pathway is the elevated synthesis of glycerol, this pathway is referred to as the HOG (high-osmolarity glycerol response) pathway (2, 5). Extracellular hyperosmolarity is detected by either of two transmembrane proteins, SLN1 and SHO1 (Fig. 1).SLN1 is a part of a complex regulatory system with homology to prokaryotic two-component signal transducers. The yeast two-component osmosensor is composed of SLN1, a transmembrane protein that contains an extracellular sensor domain and cytoplasmic histidine kinase and receiver domains, the intermediary protein YPD1, and the response regulator SSK1 (14,15,20). The SLN1-YPD1-SSK1 two-component osmosensor works by a multistep phosphorelay mechanism (20). The unphosphorylated form of SSK1 activates SSK2 and SSK22 MAPKK...

Binding the Atypical RA Domain of Ste50p to the Unfolded Opy2p Cytoplasmic Tail Is Essential for the High-Osmolarity Glycerol Pathway

“…In addition to the HOG pathway, Ste50p also plays a critical role in modulating at least two other of the five distinct MAP kinase pathways in S. cerevisiae: the pheromone response and pseudohyphal growth control pathways (Gustin et al, 1998;Posas et al, 1998a;Wu et al, 1999;Jansen et al, 2001;O'Rourke et al, 2002). The main function of Ste50p in the activation of each of these MAP kinase pathways is in plasma membrane localization of the Ste11p MAPKKK (Posas et al, 1998b;Wu et al, 1999Wu et al, , 2006Jansen et al, 2001). The Ste50p adaptor achieves this membrane association through the interaction of its RA (Ras association) domain with Opy2p (Wu et al, 2006) or with other membrane-anchored proteins, such as the small GTPase Cdc42p (Tatebayashi et al, 2006;Truckses et al, 2006).…”

“…The yeast Saccharomyces cerevisiae adapts to extracellular hyperosmolarity stress by activating its high-osmolarity glycerol (HOG) response pathway to direct the accumulation of intracellular glycerol as a compatible osmolyte (Brewster et al, 1993;Hohmann, 2002). In the HOG pathway, two upstream osmosensing branches, containing either the Sln1p or the Sho1p membrane protein, share a redundant function in the activation of a common downstream mitogen-activated protein kinase (MAPK) cascade consisting of the Pbs2p MAPKK and the Hog1p MAPK (Posas and Saito, 1997;O'Rourke and Herskowitz, 1998;Posas et al, 1998b;Wu et al, 1999). The activation of the HOG pathway by the SLN1 branch requires the action of two functionally redundant MAPKKKs, Ssk2p and Ssk22p, and their activator Ssk1p; the SHO1 branch uses the Ste11p MAPKKK to activate the pathway.…”

In vivo measurement of signaling cascade dynamics

“…Two input branches, the SLN1, 16,17 and SHO1 [18][19][20] branch, converge on the core MAP kinase kinase Pbs2. These two branches are redundant for cell survival under conditions of moderate osmotic stress.…”

Scaffold Protein Ahk1, Which Associates with Hkr1, Sho1, Ste11, and Pbs2, Inhibits Cross Talk Signaling from the Hkr1 Osmosensor to the Kss1 Mitogen-Activated Protein Kinase

“…Our recent finding that osmostress induces Ste50-Sho1 binding might explain this activation step (22). Because Ste50 is constitutively bound to Ste11 (27,28), and Sho1 is constitutively bound to Pbs2 (19), an enhanced Ste50-Sho1 interaction will inevitably lead to an increased Ste11-Pbs2 interaction. However, it is unlikely that this is the only activation step that is regulated by osmostress in the SHO1 branch (29).…”

“…Opy2 is a single-path transmembrane protein whose cytoplasmic tail binds to the Ste50 adaptor protein (36,37). Because Ste50 also binds to Ste11 (27,28), Opy2 indirectly recruits Ste11 to the plasma membrane.…”