Francesc Posas

Yeast HOG1 MAP Kinase Cascade Is Regulated by a Multistep Phosphorelay Mechanism in the SLN1–YPD1–SSK1 “Two-Component” Osmosensor

An osmosensing mechanism in the budding yeast (Saccharomyces cerevisiae) involves both a two-component signal transducer (Sln1p, Ypd1p and Ssk1p) and a MAP kinase cascade (Ssk2p/Ssk22p, Pbs2p, and Hog1p). The transmembrane protein Sln1p contains an extracellular sensor domain and cytoplasmic histidine kinase and receiver domains, whereas the cytoplasmic protein Ssk1p contains a receiver domain. Ypd1p binds to both Sln1p and Ssk1p and mediates the multistep phosphotransfer reaction (phosphorelay). This phosphorelay system is initiated by the autophosphorylation of Sln1p at His576. This phosphate is then sequentially transferred to Sln1p-Asp-1144, then to Ypd1p-His64, and finally to Ssk1p-Asp554. We propose that the multistep phosphorelay mechanism is a universal signal transduction apparatus utilized both in prokaryotes and eukaryotes.

The transcriptional response of yeast to saline stress.

Adaptation to changes in extracellular salinity is a critical event for cell survival. Genome-wide DNA chip analysis has been used to analyze the transcriptional response of yeast cells to saline stress. About 7% of the genes encoded in the yeast genome are induced more than 5-fold after a mild and brief saline shock (0.4m NaCl, 10 min). Interestingly, most responsive genes showed a very transient expression pattern, as mRNA levels dramatically declined after 20 min in the presence of stress. A quite similar set of genes increased expression in cells subjected to higher saline concentrations (0.8 m NaCl), although in this case the response was delayed. Therefore, our data show that cells respond to saline stress by inducing the expression of a very large number of genes and suggest that stress adaptation requires regulation of many cellular aspects. The transcriptional induction of most genes that are strongly responsive to salt stress was highly or fully dependent on the presence of the stress-activated mitogen-activated protein kinase Hog1, indicating that the Hog1-mediated signaling pathway plays a key role in global gene regulation under saline stress conditions.

REGULATED NUCLEO/CYTOPLASMIC EXCHANGE OF HOG1 MAPK REQUIRES THE IMPORTIN BETA HOMOLOGS NMD5 AND XPO1

MAP kinase signaling modules serve to transduce extracellular signals to the nucleus of eukaryotic cells, but little is known about how signals cross the nuclear envelope. Exposure of yeast cells to increases in extracellular osmolarity activates the HOG1 MAP kinase cascade, which is composed of three tiers of protein kinases, namely the SSK2, SSK22 and STE11 MAPKKKs, the PBS2 MAPKK, and the HOG1 MAPK. Using green fluorescent protein (GFP) fusions of these kinases, we found that HOG1, PBS2 and STE11 localize to the cytoplasm of unstressed cells. Following osmotic stress, HOG1, but neither PBS2 nor STE11, translocates into the nucleus. HOG1 translocation occurs very rapidly, is transient, and correlates with the phosphorylation and activation of the MAP kinase by its MAPKK. HOG1 phosphorylation is necessary and sufficient for nuclear translocation, because a catalytically inactive kinase when phosphorylated is translocated to the nucleus as efficiently as the wild‐type. Nuclear import of the MAPK under stress conditions requires the activity of the small GTP binding protein Ran–GSP1, but not the NLS‐binding importin α/β heterodimer. Rather, HOG1 import requires the activity of a gene, NMD5, that encodes a novel importin β homolog. Similarly, export of dephosphorylated HOG1 from the nucleus requires the activity of the NES receptor XPO1/CRM1. Our findings define the requirements for the regulated nuclear transport of a stress‐activated MAP kinase.

Controlling gene expression in response to stress

Acute stress puts cells at risk, and rapid adaptation is crucial for maximizing cell survival. Cellular adaptation mechanisms include modification of certain aspects of cell physiology, such as the induction of efficient changes in the gene expression programmes by intracellular signalling networks. Recent studies using genome-wide approaches as well as single-cell transcription measurements, in combination with classical genetics, have shown that rapid and specific activation of gene expression can be accomplished by several different strategies. This article discusses how organisms can achieve generic and specific responses to different stresses by regulating gene expression at multiple stages of mRNA biogenesis from chromatin structure to transcription, mRNA stability and translation.

The MAPK Hog1 recruits Rpd3 histone deacetylase to activate osmoresponsive genes

Regulation of gene expression by mitogen-activated protein kinases (MAPKs) is essential for proper cell adaptation to extracellular stimuli. Exposure of yeast cells to high osmolarity results in rapid activation of the MAPK Hog1, which coordinates the transcriptional programme required for cell survival on osmostress. The mechanisms by which Hog1 and MAPKs in general regulate gene expression are not completely understood, although Hog1 can modify some transcription factors. Here we propose that Hog1 induces gene expression by a mechanism that involves recruiting a specific histone deacetylase complex to the promoters of genes regulated by osmostress. Cells lacking the Rpd3–Sin3 histone deacetylase complex are sensitive to high osmolarity and show compromised expression of osmostress genes. Hog1 interacts physically with Rpd3 in vivo and in vitro and, on stress, targets the deacetylase to specific osmostress-responsive genes. Binding of the Rpd3–Sin3 complex to specific promoters leads to histone deacetylation, entry of RNA polymerase II and induction of gene expression. Together, our data indicate that targeting of the Rpd3 histone deacetylase to osmoresponsive promoters by the MAPK Hog1 is required to induce gene expression on stress.

Activation of the yeast SSK2 MAP kinase kinase kinase by the SSK1 two-component response regulator.

Exposure of yeast cells to increased extracellular osmolarity induces the HOG1 mitogen‐activated protein kinase (MAPK) cascade, which is composed of SSK2, SSK22 and STE11 MAPKKKs, PBS2 MAPKK and HOG1 MAPK. The SSK2/SSK22 MAPKKKs are activated by a ‘two‐component’ osmosensor composed of SLN1, YPD1 and SSK1. The SSK1 C‐terminal receiver domain interacts with an N‐terminal segment of SSK2. Upon hyperosmotic treatment, SSK2 is autophosphorylated rapidly, and this reaction requires the interaction of SSK1 with SSK2. Autophosphorylation of SSK2 is an intramolecular reaction, suggesting similarity to the mammalian MEKK1 kinase. Dephosphorylation of SSK2 renders the kinase inactive, but it can be re‐activated by addition of SSK1 in vitro. A conserved threonine residue (Thr1460) in the activation loop of SSK2 is important for kinase activity. Based on these observations, we propose the following two‐step activation mechanism of SSK2 MAPKKK. In the first step, the binding of SSK1 to the SSK1‐binding site in the N‐terminal domain of SSK2 causes a conformational change in SSK2 and induces its latent kinase activity. In the second step, autophosphorylation of SSK2 renders its activity independent of the presence of SSK1. A similar mechanism might be applicable to other MAPKKKs from both yeast and higher eukaryotes.

Response to Hyperosmotic Stress

An appropriate response and adaptation to hyperosmolarity, i.e., an external osmolarity that is higher than the physiological range, can be a matter of life or death for all cells. It is especially important for free-living organisms such as the yeast Saccharomyces cerevisiae. When exposed to hyperosmotic stress, the yeast initiates a complex adaptive program that includes temporary arrest of cell-cycle progression, adjustment of transcription and translation patterns, and the synthesis and retention of the compatible osmolyte glycerol. These adaptive responses are mostly governed by the high osmolarity glycerol (HOG) pathway, which is composed of membrane-associated osmosensors, an intracellular signaling pathway whose core is the Hog1 MAP kinase (MAPK) cascade, and cytoplasmic and nuclear effector functions. The entire pathway is conserved in diverse fungal species, while the Hog1 MAPK cascade is conserved even in higher eukaryotes including humans. This conservation is illustrated by the fact that the mammalian stress-responsive p38 MAPK can rescue the osmosensitivity of hog1Δ mutations in response to hyperosmotic challenge. As the HOG pathway is one of the best-understood eukaryotic signal transduction pathways, it is useful not only as a model for analysis of osmostress responses, but also as a model for mathematical analysis of signal transduction pathways. In this review, we have summarized the current understanding of both the upstream signaling mechanism and the downstream adaptive responses to hyperosmotic stress in yeast.

Yeast Cdc42 GTPase and Ste20 PAK-like kinase regulate Sho1-dependent activation of the Hog1 MAPK pathway

The adaptive response to hyperosmotic stress in yeast, termed the high osmolarity glycerol (HOG) response, is mediated by two independent upstream pathways that converge on the Pbs2 MAP kinase kinase (MAPKK), leading to the activation of the Hog1 MAP kinase. One branch is dependent on the Sho1 transmembrane protein, whose primary role was found to be the binding and translocation of the Pbs2 MAPKK to the plasma membrane, and specifically to sites of polarized growth. The yeast PAK homolog Ste20 is essential for the Sho1‐dependent activation of the Hog1 MAP kinase in response to severe osmotic stress. This function of Ste20 in the HOG pathway requires binding of the small GTPase Cdc42. Overexpression of Cdc42 partially complements the osmosensitivity of ste20Δ mutants, perhaps by activating another PAK‐like kinase, while a dominant‐negative Cdc42 mutant inhibited signaling through the SHO1 branch of the HOG pathway. Since activated Cdc42 translocates Ste20 to sites of polarized growth, the upstream and downstream elements of the HOG pathway are brought together through the membrane targeting function of Sho1 and Cdc42.

Distributed biological computation with multicellular engineered networks

Ongoing efforts within synthetic and systems biology have been directed towards the building of artificial computational devices using engineered biological units as basic building blocks. Such efforts, inspired in the standard design of electronic circuits, are limited by the difficulties arising from wiring the basic computational units (logic gates) through the appropriate connections, each one to be implemented by a different molecule. Here, we show that there is a logically different form of implementing complex Boolean logic computations that reduces wiring constraints thanks to a redundant distribution of the desired output among engineered cells. A practical implementation is presented using a library of engineered yeast cells, which can be combined in multiple ways. Each construct defines a logic function and combining cells and their connections allow building more complex synthetic devices. As a proof of principle, we have implemented many logic functions by using just a few engineered cells. Of note, small modifications and combination of those cells allowed for implementing more complex circuits such as a multiplexer or a 1-bit adder with carry, showing the great potential for re-utilization of small parts of the circuit. Our results support the approach of using cellular consortia as an efficient way of engineering complex tasks not easily solvable using single-cell implementations.

Dealing with osmostress through MAP kinase activation

In response to changes in the extracellular environment, cells coordinate intracellular activities to maximize their probability of survival and proliferation. Eukaryotic cells, from yeast to mammals, transduce diverse extracellular stimuli through the cell by multiple mitogen‐activated protein kinase (MAPK) cascades. Exposure of cells to increases in extracellular osmolarity results in rapid activation of a highly conserved family of MAPKs, known as stress‐activated MAPKs (SAPKs). Activation of SAPKs is essential for the induction of adaptive responses required for cell survival upon osmostress. Recent studies have begun to shed light on the broad effects of SAPK activation in the modulation of several aspects of cell physiology, ranging from the control of gene expression to the regulation of cell division.