The Fission Yeast Mitotic Regulator win1+ Encodes an MAP Kinase Kinase Kinase That Phosphorylates and Activates Wis1 MAP Kinase Kinase in Response to High Osmolarity

The Schizosaccharomyces pombe win1-1 mutant has a defect in the G2-M transition of the cell cycle. Although the defect is suppressed by wis1+ and wis4+, which are components of a stress-activated MAP kinase pathway that links stress response and cell cycle control, the molecular identity of Win1 has not been known. We show here that win1+ encodes a polypeptide of 1436 residues with an apparent molecular size of 180 kDa and demonstrate that Win1 is a MAP kinase kinase kinase that phosphorylates and activates Wis1. Despite extensive similarities between Win1 and Wis4, the two MAP kinase kinase kinases have distinct functions. Wis4 is able to compensate for loss of Win1 only under unstressed conditions to maintain basal Wis1 activity, but it fails to suppress the osmosignaling defect conferred by win1 mutations. The win1-1 mutation is a spontaneous duplication of 16 nucleotides, which leads to a frameshift and production of a truncated protein lacking the kinase domain. We discuss the cell cycle phenotype of the win1-1 cdc25-22 wee1-50 mutant and its suppression by wis genes.

Rck2, a member of the calmodulin-protein kinase family, links protein synthesis to high osmolarity MAP kinase signaling in budding yeast

Rck2, a yeast Ser/Thr protein kinase homologous to mammalian calmodulin kinases, requires phosphorylation for activation. We provide evidence that in budding yeast, this step can be executed by the osmostress-activated mitogen-activated protein kinase Hog1. Rck2 phosphorylation was transiently increased during osmostress or in mutants with a hyperactive high osmolarity glycerol (HOG) pathway. This modification depended on catalytically active Hog1 kinase and two putative mitogen-activated protein kinase phosphorylation sites in Rck2. Immunokinase assays showed that Hog1 can directly phosphorylate Rck2 to stimulate its enzymatic activity toward translation elongation factor 2. We demonstrate that Hog1 and Rck2 are necessary for attenuation of protein synthesis in response to osmotic challenge and show that modification of elongation factor 2 induced by osmostress depends on Rck2 and Hog1 in vivo. Therefore, we propose that the transient down-regulation of protein synthesis after osmotic shock is a response not to damage but to an extracellular signal mediated by Hog1 and Rck2.

Blood pressure reduction and diabetes insipidus in transgenic rats deficient in brain angiotensinogen

Angiotensin produced systemically or locally in tissues such as the brain plays an important role in the regulation of blood pressure and in the development of hypertension. We have established transgenic rats [TGR(ASrAOGEN)] expressing an antisense RNA against angiotensinogen mRNA specifically in the brain. In these animals, the brain angiotensinogen level is reduced by more than 90% and the drinking response to intracerebroventricular renin infusions is decreased markedly compared with control rats. Blood pressure of transgenic rats is lowered by 8 mmHg (1 mmHg = 133 Pa) compared with control rats. Crossbreeding of TGR(ASrAOGEN) with a hypertensive transgenic rat strain exhibiting elevated angiotensin II levels in tissues results in a marked attenuation of the hypertensive phenotype. Moreover, TGR(ASrAOGEN) exhibit a diabetes insipidus-like syndrome producing an increased amount of urine with decreased osmolarity. The observed reduction in plasma vasopressin by 35% may mediate these phenotypes of TGR(ASrAOGEN). This new animal model presenting long-term and tissue-specific down-regulation of angiotensinogen corroborates the functional significance of local angiotensin production in the brain for the central regulation of blood pressure and for the pathogenesis of hypertension.

COS1, a two-component histidine kinase that is involved in hyphal development in the opportunistic pathogen Candida albicans

Two-component histidine kinases recently have been found in eukaryotic organisms including fungi, slime molds, and plants. We describe the identification of a gene, COS1, from the opportunistic pathogen Candida albicans by using a PCR-based screening strategy. The sequence of COS1 indicates that it encodes a homolog of the histidine kinase Nik-1 from the filamentous fungus Neurospora crassa. COS1 is also identical to a gene called CaNIK1 identified in C. albicans by low stringency hybridization using CaSLN1 as a probe [Nagahashi, S., Mio, T., Yamada-Okabe, T., Arisawa, M., Bussey, H. & Yamada-Okabe, H. (1998) Microbiol. 44, 425–432]. We assess the function of COS1/CaNIK1 by constructing a diploid deletion mutant. Mutants lacking both copies of COS1 appear normal when grown as yeast cells; however, they exhibit defective hyphal formation when placed on solid agar media, either in response to nutrient deprivation or serum. In constrast to the Δnik-1 mutant, the Δcos1/Δcos1 mutant does not demonstrate deleterious effects when grown in media of high osmolarity; however both Δnik-1 and Δcos1/Δcos1 mutants show defective hyphal formation. Thus, as predicted for Nik-1, Cos1p may be involved in some aspect of hyphal morphogenesis and may play a role in virulence properties of the organism.

Kinase Activity-dependent Nuclear Export Opposes Stress-induced Nuclear Accumulation and Retention of Hog1 Mitogen-activated Protein Kinase in the Budding Yeast Saccharomyces cerevisiae

Budding yeast adjusts to increases in external osmolarity via a specific mitogen-activated protein kinase signal pathway, the high-osmolarity glycerol response (HOG) pathway. Studies with a functional Hog1–green fluorescent protein (GFP) fusion reveal that even under nonstress conditions the mitogen-activated protein kinase Hog1 cycles between cytoplasmic and nuclear compartments. The basal distribution of the protein seems independent of its activator, Pbs2, and independent of its phosphorylation status. Upon osmotic challenge, the Hog1–GFP fusion becomes rapidly concentrated in the nucleus from which it is reexported after return to an iso-osmotic environment or after adaptation to high osmolarity. The preconditions and kinetics of increased nuclear localization correlate with those found for the dual phosphorylation of Hog1–GFP. The duration of Hog1 nuclear residence is modulated by the presence of the general stress activators Msn2 and Msn4. Reexport of Hog1 to the cytoplasm does not require de novo protein synthesis but depends on Hog1 kinase activity. Thus, at least three different mechanisms contribute to the intracellular distribution pattern of Hog1: phosphorylation-dependent nuclear accumulation, retention by nuclear targets, and a kinase-induced export.

Ssp1 Promotes Actin Depolymerization and Is Involved in Stress Response and New End Take-Off Control in Fission Yeast

The ssp1 gene encodes a protein kinase involved in alteration of cell polarity in Schizosaccharomyces pombe. ssp1 deletion causes stress sensitivity, reminiscent of defects in the stress-activated MAP kinase, Spc1; however, the two protein kinases do not act through the same pathway. Ssp1 is localized mainly in the cytoplasm, but after a rise in external osmolarity it is rapidly recruited to the plasma membrane, preferentially to active growth zones and septa. Loss of Ssp1 function inhibits actin relocalization during osmotic stress, in cdc3 and cdc8 mutant backgrounds, and in the presence of latrunculin A, implicating Ssp1 in promotion of actin depolymerization. We propose a model in which Ssp1 can be activated independently of Spc1 and can partially compensate for its loss. The ssp1 deletion mutant exhibited monopolar actin distribution, but new end take-off (NETO) could be induced in these cells by exposure to KCl or to latrunculin A pulse treatment. This treatment induced NETO in cdc10 cells arrested in G1 but not in tea1 cells. This suggests that cells that contain intact cell end markers are competent to undergo NETO throughout interphase, and Ssp1 is involved in generating the NETO stimulus by enlarging the actin monomer pool.

Mitochondrial Inheritance Is Delayed in Saccharomyces cerevisiae Cells Lacking the Serine/Threonine Phosphatase PTC1

In wild-type yeast mitochondrial inheritance occurs early in the cell cycle concomitant with bud emergence. Cells lacking the PTC1 gene initially produce buds without a mitochondrial compartment; however, these buds later receive part of the mitochondrial network from the mother cell. Thus, the loss of PTC1 causes a delay, but not a complete block, in mitochondrial transport. PTC1 encodes a serine/threonine phosphatase in the high-osmolarity glycerol response (HOG) pathway. The mitochondrial inheritance delay in the ptc1 mutant is not attributable to changes in intracellular glycerol concentrations or defects in the organization of the actin cytoskeleton. Moreover, epistasis experiments with ptc1Δ and mutations in HOG pathway kinases reveal that PTC1 is not acting through the HOG pathway to control the timing of mitochondrial inheritance. Instead, PTC1 may be acting either directly or through a different signaling pathway to affect the mitochondrial transport machinery in the cell. These studies indicate that the timing of mitochondrial transport in wild-type cells is genetically controlled and provide new evidence that mitochondrial inheritance does not depend on a physical link between the mitochondrial network and the incipient bud site.

Heat Stress Activates Fission Yeast Spc1/StyI MAPK by a MEKK-Independent Mechanism

Fission yeast Spc1/StyI MAPK is activated by many environmental insults including high osmolarity, oxidative stress, and heat shock. Spc1/StyI is activated by Wis1, a MAPK kinase (MEK), which is itself activated by Wik1/Wak1/Wis4, a MEK kinase (MEKK). Spc1/StyI is inactivated by the tyrosine phosphatases Pyp1 and Pyp2. Inhibition of Pyp1 was recently reported to play a crucial role in the oxidative stress and heat shock responses. These conclusions were based on three findings: 1) osmotic, oxidative, and heat stresses activate Spc1/StyI in wis4 cells; 2) oxidative stress and heat shock activate Spc1/StyI in cells that express Wis1AA, in which MEKK consensus phosphorylation sites were replaced with alanine; and 3) Spc1/StyI is maximally activated in Δpyp1 cells. Contrary to these findings, we report: 1) Spc1/StyI activation by osmotic stress is greatly reduced in wis4 cells; 2) wis1-AA and Δwis1 cells have identical phenotypes; and 3) all forms of stress activate Spc1/StyI in Δpyp1 cells. We also report that heat shock, but not osmotic or oxidative stress, activate Spc1 in wis1-DD cells, which express Wis1 protein that has the MEKK consensus phosphorylation sites replaced with aspartic acid. Thus osmotic and oxidative stress activate Spc1/StyI by a MEKK-dependent process, whereas heat shock activates Spc1/StyI by a novel mechanism that does not require MEKK activation or Pyp1 inhibition.

Functional Characterization of the Interaction of Ste50p with Ste11p MAPKKK in Saccharomyces cerevisiae

The Saccharomyces cerevisiae Ste11p protein kinase is a homologue of mammalian MAPK/extracellular signal-regulated protein kinase kinase kinases (MAPKKKs or MEKKs) as well as the Schizosaccharomyces pombe Byr2p kinase. Ste11p functions in several signaling pathways, including those for mating pheromone response and osmotic stress response. The Ste11p kinase has an N-terminal domain that interacts with other signaling molecules to regulate Ste11p function and direct its activity in these pathways. One of the Ste11p regulators is Ste50p, and Ste11p and Ste50p associate through their respective N-terminal domains. This interaction relieves a negative activity of the Ste11p N terminus, and removal of this negative function is required for Ste11p function in the high-osmolarity glycerol (HOG) pathway. The Ste50p/Ste11p interaction is also important (but not essential) for Ste11p function in the mating pathway; in this pathway binding of the Ste11p N terminus with both Ste50p and Ste5p is required, with the Ste5p association playing the major role in Ste11p function. In vitro, Ste50p disrupts an association between the catalytic C terminus and the regulatory N terminus of Ste11p. In addition, Ste50p appears to modulate Ste11p autophosphorylation and is itself a substrate of the Ste11p kinase. Therefore, both in vivo and in vitro data support a role for Ste50p in the regulation of Ste11p activity.

Changes in brain cell shape create residual extracellular space volume and explain tortuosity behavior during osmotic challenge

Diffusion of molecules in brain extracellular space is constrained by two macroscopic parameters, tortuosity factor λ and volume fraction α. Recent studies in brain slices show that when osmolarity is reduced, λ increases while α decreases. In contrast, with increased osmolarity, α increases, but λ attains a plateau. Using homogenization theory and a variety of lattice models, we found that the plateau behavior of λ can be explained if the shape of brain cells changes nonuniformly during the shrinking or swelling induced by osmotic challenge. The nonuniform cellular shrinkage creates residual extracellular space that temporarily traps diffusing molecules, thus impeding the macroscopic diffusion. The paper also discusses the definition of tortuosity and its independence of the measurement frame of reference.

Regulation of Cell Cycle Progression by Swe1p and Hog1p Following Hypertonic Stress

Exposure of yeast cells to an increase in external osmolarity induces a temporary growth arrest. Recovery from this stress is mediated by the accumulation of intracellular glycerol and the transcription of several stress response genes. Increased external osmolarity causes a transient accumulation of 1N and 2N cells and a concomitant depletion of S phase cells. Hypertonic stress triggers a cell cycle delay in G2 phase cells that appears distinct from the morphogenesis checkpoint, which operates in early S phase cells. Hypertonic stress causes a decrease in CLB2 mRNA, phosphorylation of Cdc28p, and inhibition of Clb2p-Cdc28p kinase activity, whereas Clb2 protein levels are unaffected. Like the morphogenesis checkpoint, the osmotic stress-induced G2 delay is dependent upon the kinase Swe1p, but is not tightly correlated with inhibition of Clb2p-Cdc28p kinase activity. Thus, deletion of SWE1 does not prevent the hypertonic stress-induced inhibition of Clb2p-Cdc28p kinase activity. Mutation of the Swe1p phosphorylation site on Cdc28p (Y19) does not fully eliminate the Swe1p-dependent cell cycle delay, suggesting that Swe1p may have functions independent of Cdc28p phosphorylation. Conversely, deletion of the mitogen-activated protein kinase HOG1 does prevent Clb2p-Cdc28p inhibition by hypertonic stress, but does not block Cdc28p phosphorylation or alleviate the cell cycle delay. However, Hog1p does contribute to proper nuclear segregation after hypertonic stress in cells that lack Swe1p. These results suggest a hypertonic stress-induced cell cycle delay in G2 phase that is mediated in a novel way by Swe1p in cooperation with Hog1p.

Remodeling of Yeast Genome Expression in Response to Environmental Changes

We used genome-wide expression analysis to explore how gene expression in Saccharomyces cerevisiae is remodeled in response to various changes in extracellular environment, including changes in temperature, oxidation, nutrients, pH, and osmolarity. The results demonstrate that more than half of the genome is involved in various responses to environmental change and identify the global set of genes induced and repressed by each condition. These data implicate a substantial number of previously uncharacterized genes in these responses and reveal a signature common to environmental responses that involves ∼10% of yeast genes. The results of expression analysis with MSN2/MSN4 mutants support the model that the Msn2/Msn4 activators induce the common response to environmental change. These results provide a global description of the transcriptional response to environmental change and extend our understanding of the role of activators in effecting this response.