Simultaneous inactivation of pypl and pyp2 PTPases in fission yeast leads to aberrant cell morphology and growth arrest. Spontaneous recessive mutations that bypass the requirement for pypl and pyp2 and reside in two complementation groups were isolated, styl and sty2. styl-and sty2-mutant cells are substantially delayed in the timing of mitotic initiation. We have isolated the styl gene, which encodes a MAP kinase that is closely related to a subfamily of MAP kinases regulated by osmotic stress including Saccharomyces cerevisiae HOG1 and human CSBP1. We find that sty2 is allelic to the wisl MAP kinase kinase and that 9 styl and Awisl cells are unable to grow in high osmolarity medium. Osmotic stress induces both tyrosine phosphorylation of Styl and a reduction in cell size at division. Pyp2 associates with and tyrosine dephosphorylates Styl in vitro. We find that wisl-dependent induction of pyp2 mRNA is responsible for tyrosine dephosphorylation of Styl in vivo on prolonged exposure to osmotic stress. We conclude that Pypl and Pyp2 are tyrosine-specific MAP kinase phosphatases that inactivate an osmoregulated MAP kinase, Styl, which acts downstream of the Wisl MAP kinase kinase to control cell size at division in fission yeast.

The atfl ÷ gene of Schizosaccharomyces pombe encodes a bZIP transcription factor with strong homology to the mammalian factor ATF-2. ATF-2 is regulated through phosphorylation in mammalian cells by the stress-activated mitogen-activated protein (MAP) kinases SAPK/JNK and p38. We show here that the fission yeast Atfl factor is also regulated by a stress-activated kinase, Styl. The Styl kinase is stimulated by a variety of different stress conditions including osmotic and oxidative stress and heat shock. Deletion of the atfl + gene results in many, but not all, of the phenotypes associated with loss of Styl, including sensitivity to environmental stress and inability to undergo sexual conjugation. Furthermore, we identify a number of target genes that are induced rapidly in a manner dependent upon both the Styl kinase and the Atfl transcription factor. These genes include gpdl +, which is important for the response of cells to osmotic stress, the catalase gene ~ important for cells to combat oxidative stress, and pyp2 +, which encodes a tyrosine-specific MAP kinase phosphatase. Induction of Pyp2 by Atfl is direct in that it does not require de novo protein synthesis and results in a negative feedback loop that serves to control signaling through the Styl/Wisl pathway. We show that Atfl associates stably and is phosphorylated by the Styl kinase in vitro. Taken together, these results indicate that the interaction between Atfl and Styl is direct. These findings highlight a remarkable level of conservation in transcriptional control by stress-activated MAP kinase pathways between fission yeast and mammalian cells.

The fission yeast Styl MAP kinase is required for cell cycle control, initiation of sexual differentiation, and protection against cellular stress. Like the mammalian JNK/SAPK and p38/CSBP1 MAP kinases, Styl is activated by a range of environmental insults including osmotic stress, hydrogen peroxide, menadione, heat shock, and the protein synthesis inhibitor anisomycin. We have identified an upstream regulator that mediates activation of the Styl MAP kinase by multiple environmental stresses as the product of the mitotic catastrophe suppressor, mcs4. Mcs4 is structurally and functionally homologous to the budding yeast SSK1 response regulator, suggesting that the eukaryotic stress-activated MAP kinase pathway is controlled by a conserved two-component system. Mcs4 acts upstream of Wakl, a homolog of the SSK2 and SSK22 MEK kinases, which transmits the stress signal to the Wisl MEK. We show that the Wisl MEK is controlled by an additional pathway that is independent of both Mcs4 and the Wakl MEK kinase. Furthermore, we demonstrate that Mcs4 is required for the correct timing of mitotic initiation by mechanisms both dependent and independent on Styl, indicating that Mcs4 coordinately controls cell cycle progression with the cellular response to environmental stress.

In an often rapidly changing environment, cells must adapt by monitoring and reacting quickly to extracellular stimuli detected by membrane-bound receptors and proteins. Reversible phosphorylation of intracellular regulatory proteins has emerged as a crucial mechanism effecting the transmission and modulation of such signals and is determined by the relative activities of protein kinases and phosphatases within the cell. These are often arranged into complex signaling networks that may function independently or be subject to cross-regulation. Recently, genetic and biochemical analyses have identified the universally conserved mitogen-activated protein (MAP) kinase cascade as one of the most ubiquitous signal transduction systems. This pathway is activated after a variety of cellular stimuli and regulates numerous physiological processes, particularly the cell division cycle. Progression through the cell cycle is critically dependent on the presence of environmental growth factors and stress stimuli, and failure to correctly integrate such signals into the cell cycle machinery can lead to the accumulation of genetic damage and genomic instability characteristic of cancer cells. Here we focus on the MAP kinase cascade and discuss the molecular mechanisms by which these extensively studied signaling pathways influence cell growth and proliferation.

The fission yeast Sty1/Spc1 mitogen-activated protein (MAP) kinase is a member of the eukaryotic stressactivated MAP kinase (SAPK) family. We have identified a protein, Sin1, that interacts with Sty1/Spc1 which is a member of a new evolutionarily conserved gene family. Cells lacking Sin1 display many, but not all, of the phenotypes of cells lacking the Sty1/Spc1 MAP kinase including sterility, multiple stress sensitivity and a cell-cycle delay. Sin1 is phosphorylated after stress but this is not Sty1/Spc1-dependent. Importantly, Sin1 is not required for activation of Sty1/Spc1 but is required for stress-dependent transcription via its substrate, Atf1. We find that in the absence of Sin1, Sty1/ Spc1 appears to translocate to the nucleus but Atf1 is not fully phosphorylated and becomes unstable in response to environmental stress. Sin1 is also required for effective transcription via the AP-1 factor Pap1 but does not prevent its nuclear translocation. Remarkably chimaeric fusions of sin1 with chicken sin1 sequences rescue loss of sin1 function. We conclude that Sin1 is a novel component of the eukaryotic SAPK pathway.

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The fission yeast Sty1 mitogen-activated protein (MAP) kinase (MAPK) and its activator the Wis1 MAP kinase kinase (MAPKK) are required for cell cycle control, initiation of sexual differentiation, and protection against cellular stress. Like the mammalian JNK/ SAPK and p38/CSBP1 MAPKs, Sty1 is activated by a range of environmental insults including osmotic stress, hydrogen peroxide, UV light, menadione, heat shock, and the protein synthesis inhibitor anisomycin. We have recently identified two upstream regulators of the Wis1 MAPKK, namely the Wak1 MAPKKK and the Mcs4 response regulator. Cells lacking Mcs4 or Wak1, however, are able to proliferate under stressful conditions and undergo sexual differentiation, suggesting that additional pathway(s) control the Wis1 MAPKK. We now show that this additional signal information is provided, at least in part, by the Win1 mitotic regulator. We show that Wak1 and Win1 coordinately control activation of Sty1 in response to multiple environmental stresses, but that Wak1 and Win1 perform distinct roles in the control of Sty1 under poor nutritional conditions. Our results suggest that the stress-activated Sty1 MAPK integrates information from multiple signaling pathways.

Mitogen and stress-activated kinase-1 (MSK1) is a serine/threonine protein kinase that is activated by either p38 or p42ERK MAPKs in response to stress or mitogenic extracellular stimuli. MSK1 belongs to a family of protein kinases that contain two distinct kinase domains in one polypeptide chain. We report the 1.8 A crystal structure of the N-terminal kinase domain of MSK1. The crystal structure reveals a unique inactive conformation with the ATP binding site blocked by the nucleotide binding loop. This inactive conformation is stabilized by the formation of a new three-stranded beta sheet on the N lobe of the kinase domain. The three beta strands come from residues at the N terminus of the kinase domain, what would be the alphaB helix in the active conformation, and the activation loop. The new three-stranded beta sheet occupies a position equivalent to the N terminus of the alphaC helix in active protein kinases.