Negative feedback-loop mechanisms regulating HOG- and pheromone-MAPK signaling in yeast.

The pheromone response and the high osmolarity glycerol (HOG) pathways are considered the prototypical MAPK signaling systems. They are the best-understood pathways in eukaryotic cells, yet they continue to provide insights in how cells relate with the environment. These systems are subjected to tight regulatory circuits to prevent hyperactivation in length and intensity. Failure to do this may be a matter of life or death specially for unicellular organisms such as Saccharomyces cerevisiae. The signaling pathways are fine-tuned by positive and negative feedback loops exerted by pivotal control elements that allow precise responses to specific stimuli, despite the fact that some elements of the systems are common to different signaling pathways. Here we describe the experimentally proven negative feedback loops that modulate the pheromone response and the HOG pathways. As described in this review, MAP kinases are central mechanistic components of these feedback loops. They have the capacity to modulate basal signaling activity, a fast extranuclear response, and a longer-lasting transcriptional process.

Activation of the Hog1 MAPK by the Ssk2/Ssk22 MAP3Ks, in the absence of the osmosensors, is not sufficient to trigger osmostress adaptation in Saccharomyces cerevisiae.

Yeast cells respond to hyperosmotic stress by activating the high-osmolarity glycerol (HOG) pathway, which consists of two branches, Hkr1/Msb2-Sho1 and Sln1, which trigger phosphorylation and nuclear internalization of the Hog1 mitogen-activated protein kinase. In the nucleus, Hog1 regulates gene transcription and cell cycle progression, which allows the cell to respond and adapt to hyperosmotic conditions. This study demonstrates that the uncoupling of the known sensors of both branches of the pathway at the level of Ssk1 and Ste11 impairs cell growth in hyperosmotic medium. However, under these conditions, Hog1 was still phosphorylated and internalized into the nucleus, suggesting the existence of an alternative Hog1 activation mechanism. In the ssk1ste11 mutant, phosphorylated Hog1 failed to associate with chromatin and to activate transcription of canonical hyperosmolarity-responsive genes. Accordingly, Hog1 also failed to induce glycerol production at the levels of a wild-type strain. Inactivation of the Ptp2 phosphatase moderately rescued growth impairment of the ssk1ste11 mutant under hyperosmotic conditions, indicating that downregulation of the HOG pathway only partially explains the phenotypes displayed by the ssk1ste11 mutant. Cell cycle defects were also observed in response to stress when Hog1 was phosphorylated in the ssk1ste11 mutant. Taken together, these observations indicate that Hog1 phosphorylation by noncanonical upstream mechanisms is not sufficient to trigger a protective response to hyperosmotic stress.

Role of the Sln1-phosphorelay pathway in the response to hyperosmotic stress in the yeast Kluyveromyces lactis.

The Kluyveromyces lactis SLN1 phosphorelay system includes the osmosensor histidine kinase Sln1, the phosphotransfer protein Ypd1 and the response regulator Ssk1. Here we show that K. lactis has a functional phosphorelay system. In vitro assays, using a heterologous histidine kinase, show that the phosphate group is accepted by KlYpd1 and transferred to KlSsk1. Upon hyperosmotic stress the phosphorelay is inactivated, KlYpd1 is dephosphorylated in a KlSln1 dependent manner, and only the version of KlSsk1 that lacks the phosphate group interacts with the MAPKKK KlSsk2. Interestingly, inactivation of the KlPtp2 phosphatase in a ΔKlsln1 mutant did not lead to KlHog1 constitutive phosphorylation. KlHog1 can replace ScHog1p and activate the hyperosmotic response in Saccharomyces cerevisiae, and when ScSln1 is inactivated, KlHog1 becomes phosphorylated and induces cell lethality. All these observations indicate that the phosphorelay negatively regulates KlHog1. Nevertheless, in the absence of KlSln1 or KlYpd1, no constitutive phosphorylation is detected and cells are viable, suggesting that a strong negative feedback that is independent of KlPtp2 operates in K. lactis. Compared with S. cerevisiae, K. lactis has only a moderate accumulation of glycerol and fails to produce trehalose under hyperosmotic stress, indicating that regulation of osmolyte production is different in K. lactis.