In the absence of Cbk1 phosphorylation Ssd1-associated mRNAs are redirected from sites of polarized cell growth to stress granules and P-bodies.

Saccharomyces cerevisiae Cbk1 is a LATS/Ndr protein kinase and a downstream component of the regulation of Ace2 and morphogenesis (RAM) signaling network. Cbk1 and the RAM network are required for cellular morphogenesis, cell separation, and maintenance of cell integrity. Here, we examine the phenotypes of conditional cbk1 mutants to determine the essential function of Cbk1. Cbk1 inhibition severely disrupts growth and protein secretion, and triggers the Swe1-dependent morphogenesis checkpoint. Cbk1 inhibition also delays the polarity establishment of the exocytosis regulators Rab-GTPase Sec4 and its exchange factor Sec2, but it does not interfere with actin polarity establishment. Cbk1 binds to and phosphorylates Sec2, suggesting that it regulates Sec4-dependent exocytosis. Intriguingly, Cbk1 inhibition causes a >30% decrease in post-Golgi vesicle accumulation in late secretion mutants, indicating that Cbk1 also functions upstream of Sec2-Sec4, perhaps at the level of the Golgi. In agreement, conditional cbk1 mutants mislocalize the cis-Golgi mannosyltransferase Och1, are hypersensitive to the aminoglycoside hygromycin B, and exhibit diminished invertase and Sim1 glycosylation. Significantly, the conditional lethality and hygromycin B sensitivity of cbk1 mutants are suppressed by moderate overexpression of several Golgi mannosyltransferases. These data suggest that an important function for Cbk1 and the RAM signaling network is to regulate growth and secretion via Golgi and Sec2/Sec4-dependent processes. INTRODUCTIONCell polarity is a common cellular feature among eukaryotic cells that is characterized by asymmetry in cell shape, protein distribution, and cellular function (Nelson, 2003;Pruyne et al., 2004). The establishment and maintenance of polarized growth are critical for the development and function of specialized cells, tissues, and organs. Polarized growth is particularly important for the development and function of neuronal cells, which can reach several meters in length, and for epithelial cells, which regulate homeostasis and maintain physical barriers between biological compartments (Arimura and Kaibuchi, 2005;Dow and Humbert, 2007;Tang, 2008). Dynamic changes in cell polarity are essential for cell migration during cell and tissue development. Polarized growth and morphogenesis are also important for yeast and filamentous fungi development (Harris, 2006).Saccharomyces cerevisiae is an excellent model organism for studying conserved mechanisms of polarized growth and cell division. Saccharomyces cerevisiae cells undergo polarized growth during cell division, mating, and pseudohyphae formation (Pruyne et al., 2004). Many of the evolutionarily conserved elements of cell growth and proliferation were first identified and characterized in yeast (Pruyne et al., 2004). Yeast polarity establishment involves the recruitment and activation of Cdc42 GTPase to a cortical landmark. Cdc42 regulates several polarity processes, including septin assembly and actin cytoskeleton polarization (Park and Bi, 2007). Po...

SUMMARY Mechanisms that control mRNA metabolism are critical for cell function, development and stress response. The S. cerevisiae mRNA-binding protein Ssd1 has been implicated in mRNA processing, aging, stress response and maintenance of cell integrity. Ssd1 is a substrate of the LATS/NDR tumor suppressor orthologue Cbk1 kinase. Previous data indicate that Ssd1 localizes to the cytoplasm, however biochemical interactions suggest that Ssd1 at least transiently localizes to the nucleus. We therefore explored whether nuclear localization is important for Ssd1 cytoplasmic functions. We identified a functional NLS in the N-terminal domain of Ssd1. An Ssd1 derived NLS-GFP fusion protein and several C-terminally truncated Ssd1 proteins, which presumably lack nuclear export sequences, accumulate in the nucleus. Alanine substitution of the Ssd1 NLS prevents Ssd1 nuclear entry, mRNA binding and disrupts Srl1 mRNA localization. Moreover, Ssd1-NLS mutations abolish Ssd1 toxicity in the absence of Cbk1 phosphorylation and cause Ssd1 to localize prominently to cytoplasmic puncta. These data indicate that nuclear shuttling is critical for Ssd1 mRNA binding and Ssd1-mRNA localization in the cytoplasm. Collectively these data support the model that Ssd1 functions analogously to hnRNPs, which bind mRNA co-transcriptionally, are exported to the cytoplasm and target mRNAs to sites of localized translation and P-bodies.

Cbk1 kinase was previously implicated in regulating polarized morphogenesis, gene expression, and cell integrity. This study reveals that Cbk1 regulates heat shock signaling and stress adaptation by modulating Mpk1 activity and MAPK phosphatase localization. A model for Cbk1 and its putative substrate for these functions is presented.