Special Viewpoints

When the Stress of Your Environment Makes You Go HOG Wild

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Science  26 Nov 2004:
Vol. 306, Issue 5701, pp. 1511-1512
DOI: 10.1126/science.1104879


When exposed to increased dissolved solute in their environment (hyperosmotic stress), all eukaryotic cells respond by rapidly activating a conserved mitogen-activated protein kinase cascade, known in budding yeast Saccharomyces cerevisiae as the high osmolarity glycerol (HOG) pathway. Intensive genetic and biochemical analysis in this organism has revealed the presumptive osmosensors, downstream signaling components, and metabolic and transcriptional changes that allow cells to cope with this stressful condition. These findings have had direct application to understanding stress sensing and control of transcription by stress-activated mitogen-activated protein kinases in mammalian cells.

Cells must constantly adapt their physiology to respond to changing environmental conditions and stimuli. These adjustments are frequently achieved by signal transduction pathways that result in the activation of protein kinases of the conserved mitogen-activated protein kinase (MAPK) family (1). After initial response and propagation of the signal and once cells are adapted to the altered condition, the signaling machinery is deactivated. One environmental stress is exposure to a milieu containing a dissolved solute concentration that is sufficiently high to compromise cell turgor pressure (2). To adapt to hyperosmotic conditions, cells can produce various small molecules that increase the total intracellular solute concentration, thereby providing osmotic stabilization. The budding yeast Saccharomyces cerevisiae, for example, produces the solute, glycerol, and thus the signal transduction network necessary to achieve this end is called the high osmolarity glycerol (HOG) pathway. It includes a MAPK cascade that leads to phosphorylation and activation of the MAPK, Hog1 (its mammalian homolog is the stress-activated MAPK, p38) (3) (Fig. 1). Response to hyperosmotic stress has revealed multiple levels of complex regulation.

Fig. 1.

The requirement for the Hog1 MAPK for cell survival under hyperosmotic conditions is illustrated by plating serial 10-fold dilutions of wild-type (wt) cells and an otherwise isogenic derivative lacking the HOG1 gene (hog1Δ) on normal rich medium (YEPD) or on the same medium containing 1 M sorbitol, as indicated.

HOG Signaling

As our STKE Connections Map (4) indicates, there are two discrete branches of the HOG pathway that can lead to the phosphorylation and activation of the MAPK kinase (MAPKK) Pbs2 and its cognate MAPK, Hog1 (5). In the first branch, two plasma membrane proteins provide the input. Sho1 (an integral membrane protein with four transmembrane segments) may be a turgor and heat sensor (6), and Msb2 (a mucin-like protein with a single transmembrane segment) may serve as a gauge of plasma membrane–cell wall connectivity (7). Sho1 and Msb2 physically interact. Maximal stimulation through this branch in response to hyperosmotic conditions requires both Sho1 and Msb2 and leads to activation of the membrane-associated Rho family guanine triphosphatase (GTPase) Cdc42 through GTP binding. Cdc42, in turn, binds to and stimulates the protein kinase, Ste20 (8). How Sho1 and Msb2 stimulate production of GTP-bound Cdc42 is not known, but the C-terminal cytosolic tail of Msb2 itself binds activated Cdc42 (7). Cdc42 is tethered to the plasma membrane by a C-terminal geranylgeranyl substituent. Thus, GTP-bound Cdc42 also recruits Ste20 to the plasma membrane. The target of Ste20 is the MAPK kinase kinase (MAPKKK), Ste11. Ste11 is positioned at the plasma membrane by direct association with the cytosolic tail of Sho1 (9); by direct association with the MAPKK Pbs2, which is itself anchored to Sho1 (10); and, lastly, through interaction with Cdc42 mediated by the adaptor protein Ste50. Once activated, Ste11 phosphorylates and activates the MAPKK Pbs2. This mechanism results in Hog1 activation in response to strongly hyperosmotic conditions (for example, 0.5 to 1.0 M NaCl). The activation pathway for mammalian p38 resembles this Sho1- and Msb2-dependent branch (11).

The second branch leading to phosphorylation and activation of Pbs2 occurs upon less severe hyperosmotic shifts (for example, 0.125 to 0.25 M NaCl) and involves another transmembrane protein, Sln1, which serves as a osmosensor. Sln1 has protein-histidine kinase activity and is situated at the head of a phospho-relay cascade (Sln1 → Ypd1 → Ssk1) akin to the so-called two-component signaling systems first described in prokaryotic cells (12). Under isoosmotic conditions, Sln1 is autophosphorylated and transfers the phosphate group to an intermediate protein, Ypd1, which in turn transfers the phosphate group to an aspartate residue on the response regulator protein, Ssk1 (13). Ssk1 binding to the MAPKKKs Ssk2 and Ssk22 is required for their function. However, only the unphosphorylated form of Ssk1 binds to and activates Ssk2 and Ssk22 (14). Hence, Sln1-dependent phosphotransfer to Ssk1 blocks activation of Ssk2 and Ssk22. Under osmotic stress, Sln1 no longer undergoes autophosphorylation, phosphate is not transferred to Ssk1, and Ssk2 and Ssk22 are stimulated. Although Ssk2 and Ssk22 seem redundant for their ability to initiate HOG signaling by phosphorylating Pbs2, Ssk2 has an additional role in the recovery of the actin cytoskeleton after osmotic shock. All three MAPKKKs (Ste11, Ssk2, and Ssk22) phosphorylate Pbs2, and activated Pbs2 phosphorylates Hog1 on both a Thr and a Tyr residue.

Dual phosphorylation of Hog1 occurs within minutes of exposing cells to hyperosmotic conditions. Initial exposure to high salt causes nonspecific dissociation of many proteins from chromatin. Activated Hog1 first provides short-term protection for the transcriptional capacity of the cell by phosphorylating both a Na+-H+ antiporter (Nha1) and a potassium channel (Tok1) in the plasma membrane, thereby stimulating Na+ efflux (15). Hog1-stimulated Na+ ejection allows proteins to reassociate with DNA. Hog1 action also affects translation and cell cycle progression. In addition, phosphorylation of Hog1 promotes its nuclear import, apparently mediated by the karyopherin Nmd5 (16). Once inside the nucleus, activated Hog1 leads to increased expression of ∼600 genes (17).

Nuclear Hog1 interacts with various transcriptional activators and repressors. A transcriptional repressor complex (Sko1/Tup1/Ssn6) is bound to the promoters of many of the genes that undergo increased transcription in response to hyperosmotic stress. Activated Hog1 binds to and phosphorylates Sko1, modifying its association with Tup1-Ssn6 and allowing recruitment of the SAGA and Swi/Snf chromatin-modifying and remodeling complexes, thereby inducing gene expression (18). Under conditions of severe salt stress (1 M NaCl), Sko1 gets exported from the nucleus; curiously this translocation is not Hog1-dependent but depends instead on the action of cyclic adenosine monophosphate–dependent protein kinase. Various stressful conditions, including hyperosmotic stress, induce the expression of genes controlled by two highly related, zinc finger–containing transcriptional activators, Msn2 and Msn4. Nuclear Hog1 binds to Msn2-Msn4 at the promoters of certain stress-responsive genes, which in turn recruits another transcriptional activator, Hot1, to the same complexes. At other loci, like the promoter of the GPD1 gene, Hot1 is bound constitutively and independently of Msn2-Msn4 interactions. In this case, nuclear Hog1 binds to and phosphorylates Hot1, promotes recruitment of yet another transcriptional activator, Msn1, and associates with components of the general transcription machinery. All of these actions are necessary for optimal transcription of this gene (19). GPD1 encodes the glycerol 3-phosphate dehydrogenase essential for glycerol production, which converts triose phosphate (namely, dihydroxyacetone phosphate) and NADH to glycerol-3-phosphate and nicotinamide adenine dinucleotide (NAD+). Lastly, there is evidence that Hog1 also promotes expression of the genes under its control by recruiting the Rpd3-Sin3 histone deacetylase complex and thereby remodeling in some unique way the chromatin structure around the promoters to which Hog1 is targeted (20).

By about 20 to 30 min after osmotic shock, concomitant with onset of glycerol production and restoration of osmotic balance, Hog1 is dephosphorylated and exported from the nucleus via the karyopherin, Xpo1 (16). Multiple phosphoprotein phosphatases, some phospho-Tyr-specific (Ptp2 and Ptp3) and some phospho-Thr-specific (Ptc1, Ptc2, and Ptc3), maintain low basal levels of Hog1 activity and down-regulate Hog1 after its activation. The Ssk2 (and Ssk22) activator Ssk1 is degraded in a ubiquitin- and proteasome-dependent manner during the adaptation process (21). However, further increase of the external solute concentration elicits rephosphorylation and nuclear reentry of Hog1 with kinetics nearly identical to those seen in the initial response, suggesting that the Sho1-dependent branch may be the primary input in this situation.

Intriguing mechanistic questions remain about operation of the HOG pathway. How changes in external solute concentration are perceived by the osmosensors remains undetermined, although the fact that Sho1 is activated by heat suggests that it may undergo a conformational change. Three different MAPK modules in S. cerevisiae—pheromone response, filamentatous growth, and one branch of the HOG pathway—all activate the same MAPKKK, Ste11. How specificity is maintained in propagation of the signals from Ste11 remains unclear. Indeed, in the absence of either Pbs2 or Hog1, osmotic stress does lead to activation of Ste7 and Fus3, the MAPKK and MAPK of the pheromone response pathway, and induction of genes normally only expressed in response to mating pheromone (22). Understanding signaling specificity of this sort may shed light on how cells adapt to chronic stress conditions and how to lessen the impact of diseases that arise from adventitious activation of one pathway by another.

References and Notes

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