Protein Phosphatase 2C Acts Independently of Stress-activated Kinase Cascade to Regulate the Stress Response in Fission Yeast

Abstract

Stress-activated signal transduction pathways, which are largely conserved among a broad spectrum of eukaryotic species, have a crucial role in the survival of many forms of stress. It is therefore important to discover how these pathways are both positively and negatively regulated. Recent genetic studies have implicated protein phosphatase 2C (PP2C) as a novel negative regulator of stress response pathways in both budding and fission yeasts. Moreover, it was hypothesized that PP2C dephosphorylates one or more components of protein kinase cascades that are at the core of stress-activated signal transduction pathways. Herein we present genetic and biochemical studies of the fission yeast Schizosaccharomyces pombe that disprove this hypothesis and indicate that PP2C instead negatively regulates a downstream element of the pathway. First, high expression of PP2C produces phenotypes that are inconsistent with negative regulation of the Wik1-Wis1-Spc1 stress-activated kinase cascade. Second, high expression of PP2C leads to sustained activating tyrosine phosphorylation of Spc1. Third, Spc1-dependent phosphorylation of Atf1, a transcription factor substrate of Spc1, is unaffected by high expression of PP2C. Fourth, high expression of PP2C suppresses Atf1-dependent transcription of a stress-response gene. These studies strongly suggest that PP2C acts downstream of Spc1 kinase in the stress-activated signal transduction pathway.Eukaryotic organisms frequently encounter environmental conditions that cause cytotoxic damage; hence, they have developed sophisticated systems of sensing and responding to physiological stress. Protein kinase cascades are at the core of these stress sensor pathways (1). These cascades follow the paradigm established for mitogen-activated protein kinase (MAPK) 1 cascades: a MAPK kinase kinase (MAPKKK) phosphorylates a MAPK kinase (MAPKK), which in turn phosphorylates a MAPK. The MAPKKK and MAPK components are serine-threonine kinases, whereas MAPKKs are dual specificity enzymes, activating MAPK substrates by phosphorylating threonine and tyrosine residues in a conserved motif (2). It is not understood why protein kinase cascades are used to transmit stress signals, although it is likely that the spatial distribution of the cascade elements facilitates rapid signaling from the cell surface to the nucleus where MAPK homologs phosphorylate transcription factor substrates.Recent studies have revealed impressive functional and structural conservation of stress response pathways in yeast, plants, and various metazoan species, including humans (3). The fission yeast Schizosaccharomyces pombe has been focus of some of the most interesting investigations, in part because studies of fission yeast have uncovered a link between stress response pathways and cell cycle control (4, 5). The S. pombe stress-activated kinase cascade consists of Wik1-Wis1-Spc1 kinases (6). The Spc1 MAPK homolog, which is also known as Sty1 and Phh1 (4, 7), is highly similar to mammalian p38 kinases (8) and Hog1p kinase of the bud...

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