Signaling molecules such as Cdc42 and mitogen-activated protein kinases (MAPKs) can function in multiple pathways in the same cell. Here, we propose one mechanism by which such factors may be directed to function in a particular pathway such that a specific response is elicited. Using genomic approaches, we identify a new component of the Cdc42-and MAPK-dependent signaling pathway that regulates filamentous growth (FG) in yeast. This factor, called Msb2, is a FG-pathway-specific factor that promotes differential activation of the MAPK for the FG pathway, Kss1. Msb2 is localized to polarized sites on the cell surface and interacts with Cdc42 and with the osmosensor for the high osmolarity glycerol response (HOG) pathway, Sho1. Msb2 is glycosylated and is a member of the mucin family, proteins that in mammalian cells promote disease resistance and contribute to metastasis in cancer cells. Remarkably, loss of the mucin domain of Msb2 causes hyperactivity of the FG pathway, demonstrating an inhibitory role for mucin domains in MAPK pathway activation. Taken together, our data suggest that Msb2 is a signaling mucin that interacts with general components, such as Cdc42 and Sho1, to promote their function in the FG pathway.[Keywords: Morphogenesis; cell polarity; signal transduction; pseudohyphal growth; specificity] Supplemental material is available at http://www.genesdev.org.

Haploid yeast invades solid agar in response to nutrient limitation. To decipher the cues that underlie invasion, we have developed a single cell invasive growth assay. Using this assay, as well as the traditional plate-washing assay, we show that invasive growth occurs in response to glucose depletion. In the absence of glucose (or other fermentable sugar), individual cells adopted a nonaxial budding pattern and elongated morphology within the first cell divisions, and invasion into the agar was observed in microcolonies containing as few as 10 cells. In support of this observation, we found that glucose suppressed the hyperinvasive growth morphology of STE11-4, pbs2, hsl7, and RAS2V19 mutations. In addition, removal of glucose from YPD medium caused constitutive invasion in wild-type cells. We tested glucose control proteins for a role in invasion and found that Snf1, a protein required for derepression of glucose-repressed genes, was required for invasive growth. The transcription factor Sip4, which interacts with Snf1 and is induced during the diauxic shift, had an inhibitory role on invasive growth, suggesting that multiple mechanisms are required for glucose depletion-dependent invasion. S everal yeast͞fungal species have the ability to adopt two growth forms, a vegetative (yeast) form and a pseudohyphal or filamentous form. In pathogenic organisms such as Candida albicans, this morphological transition is important for invasion of host tissue and therefore pathogenesis (1). In nonpathogenic organisms, the transition can occur upon nutrient limitation and has therefore been suggested to be a mechanism to permit foraging for nutrients. Insight into the signal transduction mechanisms that are necessary for the morphological change will contribute to an understanding of basic biological phenomena and of pathogenesis.The budding yeast Saccharomyces cerevisiae provides a genetic system to investigate the morphological transition from vegetative to filamentous development. In diploid yeast, loss of environmental fixed nitrogen causes the transition from vegetative growth, in which cells are round and bud in a bipolar fashion, to pseudohyphal growth, in which cells are elongated and bud in a unipolar budding pattern (2). In haploid yeast, nutrient limitation causes a similar developmental switch that allows cells to penetrate the surface of an agar medium in a process called invasive growth (see refs. 3-6 for reviews). For simplicity, we will use the term filamentous form to refer to both pseudohyphal and invasive growth, although there are differences between these two growth responses.At least two signal transduction pathways are required for filamentous growth. One pathway uses a number of components from the pheromone response pathway, notably the p21-activated kinase (PAK) kinase Ste20, the kinases Ste11 and Ste7 from the first two tiers of the mitogen-activated protein (MAP) kinase cascade, and the transcription factor Ste12 (7, 8), but has unique components as well-the MAP kinase Kss1 (9-11) and a second t...

Filamentous growth is a nutrient-regulated growth response that occurs in many fungal species. In pathogens, filamentous growth is critical for host-cell attachment, invasion into tissues, and virulence. The budding yeast Saccharomyces cerevisiae undergoes filamentous growth, which provides a genetically tractable system to study the molecular basis of the response. Filamentous growth is regulated by evolutionarily conserved signaling pathways. One of these pathways is a mitogen activated protein kinase (MAPK) pathway. A remarkable feature of the filamentous growth MAPK pathway is that it is composed of factors that also function in other pathways. An intriguing challenge therefore has been to understand how pathways that share components establish and maintain their identity. Other canonical signaling pathways-rat sarcoma/protein kinase A (RAS/PKA), sucrose nonfermentable (SNF), and target of rapamycin (TOR)-also regulate filamentous growth, which raises the question of how signals from multiple pathways become integrated into a coordinated response. Together, these pathways regulate cell differentiation to the filamentous type, which is characterized by changes in cell adhesion, cell polarity, and cell shape. How these changes are accomplished is also discussed. High-throughput genomics approaches have recently uncovered new connections to filamentous growth regulation. These connections suggest that filamentous growth is a more complex and globally regulated behavior than is currently appreciated, which may help to pave the way for future investigations into this eukaryotic cell differentiation behavior. TABLE OF CONTENTS Abstract 23Introduction 24

Signaling mucins are cell adhesion molecules that activate RAS/RHO guanosine triphosphatases and their effector mitogen-activated protein kinase (MAPK) pathways. We found that the Saccharomyces cerevisiae mucin Msb2p, which functions at the head of the Cdc42p-dependent MAPK pathway that controls filamentous growth, is processed into secreted and cell-associated forms. Cleavage of the extracellular inhibitory domain of Msb2p by the aspartyl protease Yps1p generated the active form of the protein by a mechanism incorporating cellular nutritional status. Activated Msb2p functioned through the tetraspan protein Sho1p to induce MAPK activation as well as cell polarization, which involved the Cdc42p guanine nucleotide exchange factor Cdc24p. We postulate that cleavage-dependent activation is a general feature of signaling mucins, which brings to light a novel regulatory aspect of this class of signaling adhesion molecule.

The Rho-type GTPase, Cdc42, has been implicated in a variety of functions in the yeast life cycle, including septin organization for cytokinesis, pheromone response, and haploid invasive growth. A group of proteins called GTPase-activating proteins (GAPs) catalyze the hydrolysis of GTP to GDP, thereby inactivating Cdc42. At the time this study began, there was one known GAP, Bem3, and one putative GAP, Rga1, for Cdc42. We identified another putative GAP for Cdc42 and named it Rga2 (Rho GTPase-activating protein 2). We confirmed by genetic and biochemical criteria that Rga1, Rga2, and Bem3 act as GAPs for Cdc42. A detailed characterization of Rga1, Rga2, and Bem3 suggested that they regulate different subsets of Cdc42 function. In particular, deletion of the individual GAPs conferred different phenotypes. For example, deletion of RGA1, but not RGA2 or BEM3, caused hyperinvasive growth. Furthermore, overproduction or loss of Rga1 and Rga2, but not Bem3, affected the two-hybrid interaction of Cdc42 with Ste20, a p21-activated kinase (PAK) kinase required for haploid invasive growth. These results suggest Rga1, and possibly Rga2, facilitate the interaction of Cdc42 with Ste20 to mediate signaling in the haploid invasive growth pathway. Deletion of BEM3 resulted in cells with severe morphological defects not observed in rga1⌬ or rga2⌬ strains. These data suggest that Bem3 and, to a lesser extent, Rga1 and Rga2 facilitate the role of Cdc42 in septin organization. Thus, it appears that the GAPs play a role in modulating specific aspects of Cdc42 function. Alternatively, the different phenotypes could reflect quantitative rather than qualitative differences in GAP activity in the mutant strains.In the yeast Saccharomyces cerevisiae, the Cdc42 protein (33) is a member of the Rho subfamily of the Ras superfamily of GTPases, which act as molecular switches and regulate many cellular processes (1, 34). Like all GTPases, Cdc42 can exist in two states, a GTP-bound, active state and a GDP-bound, inactive state. The cycling of Cdc42 between these states is controlled by two sets of proteins: guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). There is one GEF for Cdc42 in yeast, called Cdc24 (62,67,69). At the time this study began, there was one protein with demonstrated biochemical GAP activity for Cdc42, Bem3 (68), and one protein with potential GAP activity based on the presence of a 170-amino-acid GAP domain, Rga1 (14, 63). Although Bem3 and Rga1 are fairly similar in their GAP domains, they are divergent over the remainder of their protein sequences. Rga1 contains two tandem 35,63,66). LIM domains bind zinc ions and are thought to mediate protein-protein interactions (2, 18, 45). In contrast, Bem3 contains a Pleckstrin homology (PH) domain. PH domains are thought to play roles in membrane localization and protein-protein interactions (40).Cdc42 is required for polarization of the actin cytoskeleton, for cytokinesis, and for morphological changes that accompany activation of some signal transd...

A central question in the area of signal transduction is why pathways utilize common components. In the budding yeast Saccharomyces cerevisiae, the HOG and filamentous growth (FG) MAPK pathways require overlapping components but are thought to be induced by different stimuli and specify distinct outputs. To better understand the regulation of the FG pathway, we examined FG in one of yeast's native environments, the grape-producing plant Vitis vinifera. In this setting, different aspects of FG were induced in a temporal manner coupled to the nutrient cycle, which uncovered a multimodal feature of FG pathway signaling. FG pathway activity was modulated by the HOG pathway, which led to the finding that the signaling mucins Msb2p and Hkr1p, which operate at the head of the HOG pathway, differentially regulate the FG pathway. The two mucins exhibited different expression and secretion patterns, and their overproduction induced nonoverlapping sets of target genes. Moreover, Msb2p had a function in cell polarization through the adaptor protein Sho1p that Hkr1p did not. Differential MAPK activation by signaling mucins brings to light a new point of discrimination between MAPK pathways. INTRODUCTIONSignal transduction pathways regulate the cellular response to diverse stimuli. Signaling pathways typically function in highly connected networks where multiple inputs become integrated into a global response (Bhattacharyya et al., 2006). For example, mitogen-activated protein kinase (MAPK) pathways utilize overlapping or shared components to coordinate different aspects of cellular behaviors (Bardwell, 2006). The overlap between pathways can be extensive, and it remains unclear how a particular signal transmitted through an interconnected network elicits a specific response. Given that inappropriate cross talk between MAPK pathways is an underlying cause of cancer and other diseases (Santen et al., 2002), understanding how signaling pathways precisely coordinate cellular behaviors is an important question.In the budding yeast Saccharomyces cerevisiae, MAPK pathways regulate the response to a variety of stimuli (Errede et al., 1995). In response to nutrient limitation, cells undergo filamentous growth (FG; pseudohyphal/invasive growth; Gimeno et al., 1992;Liu et al., 1993;Roberts and Fink, 1994;, a cellular differentiation characteristic of many fungal species including pathogens (Lo et al., 1997;Whiteway and Bachewich, 2007). FG is regulated by a typical MAPK pathway (Roberts and Fink, 1994;Borneman et al., 2007). At the head of the FG pathway, the signaling mucin Msb2p and adaptor protein Sho1p (O'Rourke and Herskowitz, 1998;Cullen et al., 2004) connect to the polarity establishment Rho (Ras homology) GTPase Cdc42p (Peter et al., 1996;Leberer et al., 1997), a global regulator of cell polarity and signaling (Johnson, 1999). In its activated (GTPbound) state, Cdc42p associates with the p21-activated kinase (PAK) Ste20p (Peter et al., 1996;Leberer et al., 1997), which results in the activation of a typical MAPK cascade compos...

In haploid strains of Saccharomyces cerevisiae, glucose depletion causes invasive growth, a foraging response that requires a change in budding pattern from axial to unipolar-distal. To begin to address how glucose influences budding pattern in the haploid cell, we examined the roles of bud-site-selection proteins in invasive growth. We found that proteins required for bipolar budding in diploid cells were required for haploid invasive growth. In particular, the Bud8p protein, which marks and directs bud emergence to the distal pole of diploid cells, was localized to the distal pole of haploid cells. In response to glucose limitation, Bud8p was required for the localization of the incipient bud site marker Bud2p to the distal pole. Three of the four known proteins required for axial budding, Bud3p, Bud4p, and Axl2p, were expressed and localized appropriately in glucose-limiting conditions. However, a fourth axial budding determinant, Axl1p, was absent in filamentous cells, and its abundance was controlled by glucose availability and the protein kinase Snf1p. In thebud8 mutant in glucose-limiting conditions, apical growth and bud site selection were uncoupled processes. Finally, we report that diploid cells starved for glucose also initiate the filamentous growth response.

An important emerging question in the area of signal transduction is how information from different pathways becomes integrated into a highly coordinated response. In budding yeast, multiple pathways regulate filamentous growth, a complex differentiation response that occurs under specific environmental conditions. To identify new aspects of filamentous growth regulation, we used a novel screening approach (called secretion profiling) that measures release of the extracellular domain of Msb2p, the signaling mucin which functions at the head of the filamentous growth (FG) MAPK pathway. Secretion profiling of complementary genomic collections showed that many of the pathways that regulate filamentous growth (RAS, RIM101, OPI1, and RTG) were also required for FG pathway activation. This regulation sensitized the FG pathway to multiple stimuli and synchronized it to the global signaling network. Several of the regulators were required for MSB2 expression, which identifies the MSB2 promoter as a target “hub” where multiple signals converge. Accessibility to the MSB2 promoter was further regulated by the histone deacetylase (HDAC) Rpd3p(L), which positively regulated FG pathway activity and filamentous growth. Our findings provide the first glimpse of a global regulatory hierarchy among the pathways that control filamentous growth. Systems-level integration of signaling circuitry is likely to coordinate other regulatory networks that control complex behaviors.