Perturbation of the Nucleus: A Novel Hog1p-independent, Pkc1p-dependent Consequence of Hypertonic Shock in Yeast

Abstract

Hypertonic shock of Saccharomyces cerevisiae activates the Hog1p MAP kinase cascade. In contrast, protein kinase C (Pkc1p) and the "cell integrity" MAP kinase cascade are critical for the response to hypotonic shock. We observed that hypertonic shock transiently relocated many, but not all, nuclear and nucleolar proteins to the cytoplasm. We hypothesized that the relocation of nuclear proteins was due to activation of the Hog1p kinase cascade, yet, surprisingly, Hog1p was not required for these effects. In contrast, Pkc1p kinase activity was required, although the Pkc1p MAP kinase cascade and several factors known to lie upstream and downstream of Pkc1p were not. Moreover, sudden induction of a hyperactive form of Pkc1p was sufficient to relocate nuclear proteins. Taken together, these observations show that the scope of involvement of Pkc1p in the organization of the nucleus considerably exceeds what has been characterized previously. The relocation of nuclear proteins is likely to account for the profound inhibition of RNA synthesis that was observed during hypertonic shock. INTRODUCTIONThe cellular effects of changes in tonicity have been investigated extensively (Robbins et al., 1970;Banuett, 1998;Gustin et al., 1998;Lang et al., 1998). In particular, analysis of the yeast Saccharomyces cerevisiae has elucidated major roles for two MAP kinase signaling pathways: the Pkc1p "cell integrity" pathway, which is stimulated by hypotonic shock (Watanabe et al., 1994;Davenport et al., 1995), and the Hog1p pathway, which is stimulated by hypertonic shock (Brewster et al., 1993;Posas and Saito, 1997). Each pathway is required for long-term survival in the corresponding medium. Hypertonic shock profoundly affects the actin cytoskeleton (Chowdhury et al., 1992;Mulholland et al., 1994;Delley and Hall, 1999).The Pkc1p pathway can receive input from plasma membrane glycoproteins of the Wsc family and from the Rho GTPases and their regulatory factors, for example in the context of control of cell polarization and cell wall biosynthesis (Gustin et al., 1998). Interestingly, some signaling events that require Pkc1p appear not to require the corresponding downstream MAP kinase cascade, which is linked to activation of the transcription factors Rlm1p and Swi4p/ Swi6p (Lee and Levin, 1992;Gustin et al., 1998;Delley and Hall, 1999; Li et al., 2000). In contrast, the Hog1p pathway is initiated by a two-component phosphorelay involving the cell surface transmembrane protein Sln1p and the cytosolic protein Ypd1p (Posas et al., 1996). Stimulation of this pathway causes phosphorylation and transient nuclear entry of the terminal kinase, Hog1p, as well as of the transcription factor Msn2p (Ferrigno et al., 1998;Gö rner et al., 1998;Reiser et al., 1999). The downstream substrate(s) of Hog1p kinase is not known.We have observed recently that arrest of the secretory pathway in yeast inhibits nuclear import and causes many nucleolar and nucleoplasmic proteins to relocate to the cytoplasm. These events (the "arrest of secretion response") a...

Pkc1 Acts Through Zds1 and Gic1 to Suppress Growth and Cell Polarity Defects of a Yeast eIF5A Mutant

“…Activation of Mpk1 upregulates transcription of a series of genes involved in cell cycle progression and cell wall synthesis (Gustin et al 1998). While transcription of a great number of genes is mediated by the MAP kinases cascade in response to different cell wall impairments, little is known about other Pkc1 downstream effectors (Ketela et al 1999;Andrews and Stark 2000;Li et al 2000;Nanduri and Tartakoff 2001;Chai et al 2002;Valdivia and Schekman 2003).…”

“…The downstream PKC1-mitogen-activated protein kinase (MAPK) cascade, consisting of Bck1, Mkk1/Mkk2, and Mpk1 kinases, phosphorylates transcription factors that regulate cell wall remodeling and cytoskeleton organization in polarized cell growth (Heinisch et al 1999). Although less well defined, several studies report evidence for other biochemical pathways branching out from Pkc1 (Ketela et al 1999;Andrews and Stark 2000;Li et al 2000;Nanduri and Tartakoff 2001;Chai et al 2002;Valdivia and Schekman 2003;Vilella et al 2005).…”

Hyperosmotic Stress Signaling to the Nucleus Disrupts the Ran Gradient and the Production of RanGTP

“…When yeast experience hyperosmotic stress, the nucleolar protein Fpr3p relocalizes to the cytoplasm; however, over expression of Hog1 in this setting has a protective effect on Fpr3p nuclear localization (Nanduri and Tartakoff, 2001b). We examined the role of p38 MAPK in the nuclear transport factor response to osmotic stress using the p38 MAPK inhibitor, SB203580.…”

“…In addition, hyper-osmotic stress of yeast has been shown to cause delocalization of nuclear proteins. This occurs in a Pkc1-dependent manner and can be inhibited by overexpression of Hog1 (Nanduri and Tartakoff, 2001b).In the course of analyzing the effect of stress signaling on nuclear transport, we found that the Ran protein gradient was disrupted in response to osmotic stress. To characterize the mechanism responsible for disruption of the Ran gradient, a battery of assays were used to define how the localization and activity of the nuclear transport machinery is affected by sorbitol stress signaling.…”

“…In addition, hyper-osmotic stress of yeast has been shown to cause delocalization of nuclear proteins. This occurs in a Pkc1-dependent manner and can be inhibited by overexpression of Hog1 (Nanduri and Tartakoff, 2001b).…”