New Results

Divergent Roles for cAMP–PKA Signaling in the Regulation of Filamentous Growth in Saccharomyces cerevisiae and Saccharomyces bayanus

Ömur Kayikci, Paul M. Magwene
doi: https://doi.org/10.1101/311415

ABSTRACT

The cyclic AMP – Protein Kinase A (cAMP–PKA) pathway is an evolutionarily conserved eukaryotic signaling network that is essential for growth and development. In the fungi, cAMP–PKA signaling plays a critical role in regulating cellular physiology and morphological switches in response to nutrient availability. We undertook a comparative investigation of the role that cAMP-PKA signaling plays in the regulation of filamentous growth in two closely related budding yeast species, Saccharomyces cerevisiae and Saccharomyces bayanus. Using chemical and genetic perturbations of this pathway and its downstream targets we discovered divergent roles for cAMP-PKA signaling in the regulation of filamentous growth. While cAMP-PKA signaling is required for the filamentous growth response in both species, increasing or decreasing the activity of this pathway leads to drastically different phenotypic outcomes. In S. cerevisiae, cAMP-PKA inhibition ameliorates the filamentous growth response while hyper-activation of the pathway leads to increased filamentous growth; the same perturbations in S. bayanus result in the obverse. Divergence in the regulation of filamentous growth between S. cerevisiae and S. bayanus extends to downstream targets of PKA, including several kinases, transcription factors, and effector proteins. Our findings highlight the potential for significant evolutionary divergence in gene network function, even when the constituent parts of such networks are well conserved.

The cyclic AMP-Protein Kinase A (cAMP-PKA) pathway isan evolutionarily conserved signaling network that is important for the regulation of growth, differentiation, and devel-opment in animals, fungi, and amoebae (Toda et al. 1985; Zim-merman et al. 2015; D’Souza and Heitman 2001; Cho-Chung 2004; Das et al. 2007; Rinaldi et al. 2010; Gold et al. 2013; Loomis 2014). The basic principles of eukaryotic cAMP-PKA signal-ing are simple - in response to internal or external stimuli, in-creased adenylate cyclase activity causes a rise in intracellularcAMP levels. cAMP molecules bind to the regulatory domainof the PKA holoenzyme, releasing catalytic PKA subunits thatphosphorylate downstream targets such as other kinases and transcription factors. cAMP production by adenylate cyclase is counter-balanced by cAMP breakdown via phosphodiesterases. Positive and negative feedback loops and temporally and spatially dynamic patterns further help to regulate cAMP-PKA activity (Toda et al. 1985, 1987; Belotti et al. 2012) In the model eukaryote, Saccharomyces cerevisiae (budding yeast), the cAMP-PKA signaling pathway helps to coordinate growth and cell fate decision-making in response to nutrient availability (Za-man et al. 2008; Gancedo 2013).

Filamentous growth is a cAMP-PKA regulated developmental response which is characterized by cell elongation, unipolar budding, physical attachment of mother and daughter cells, and increased adhesion to and invasion of growth substrates (Figure 1A). Nitrogen limitation is the primary trigger for filamentous growth in diploid cells, whereas haploid cells undergo filamentous differentiation in response to glucose limitation. The diploid filamentous growth response is also referred to as pseudohyphal growth, and we use both terms interchangeably in this study. S. cerevisiae filamentous differentiation is positively correlated with the activity of the cAMP-PKA pathway; genetic or biochemical manipulations that increase intracellular cAMP levels or PKA activity result in increased filamentous growth, while manipulations that decrease the net activity of the pathway ameliorate or abolish filamentous growth (Cullenand Sprague 2012; Gimeno and Fink 1994) (Figure 1B). Downstream targets of PKA include several transcription factors that regulate the expression of a cell wall glycoprotein, Flo11, required for filamentous growth in S. cerevisiae (Rupp et al. 1999; Lo and Dranginis 1998; Pan and Heitman 1999). Many of these same transcription factors are regulated in parallel by a MAP-kinase cascade (FG-MAPK). Both cAMP-PKA signaling and the FG-MAPK pathway are regulated by the Ras protein, Ras2.

Figure 1
Figure 1 Filamentous growth in budding yeast.

A) Upon nitrogen depletion, yeasts of the genus Saccharomyces undergo pseudohyphal differentiation in the presence of a fermentable carbon source, such as glucose. B) Flo11, a cell wall adhesin that is required for filamentous growth in S. cerevisiae is regulated in parallel by cAMP-PKA signaling and the filamentous growth MAP kinase pathway.

S. cerevisiae and related yeast within the Saccharomyces sensu stricto clade, provide a powerful comparative framework for understanding the evolution of gene networks (Cliften et al. 2003; Dujon 2010; Replansky et al. 2008; Hittinger 2013; Boynton and Greig 2014). Two additional species, Saccharomyces paradoxus and Saccharomyces bayanus, have received particular attention (Figure S1). S. paradoxus, the closest relative to S. cerevisiae, is primarily isolated from woodland areas and shows little genomic evidence of human facilitated admixture (Sampaio and Gonçalves 2008; Fay and Benavides 2005; Johnson et al. 2004; Naumov et al. 1998; Kowallik et al. 2015). S. bayanus, a lager yeast, is more distantly related to S. cerevisiae and S. paradoxus, and recent studies suggest that the phylogenetic history of the S. bayanus lineage involves a complex history of interspecific hybridization, facilitated by human activity (Sampaioand Gonçalves 2008; Naumov and Naumova 2011; Masneuf-Pomarède et al. 2010; Rodríguez et al. 2014; Pérez-Través et al. 2014). Since the nomenclature for the S. bayanus species complex is in flux (Hittinger 2013), for the purposes of this study we have adopted a conservative approach and refer to all strains belonging to this species complex as S. bayanus. S. cerevisiae, S. paradoxus, and S. bayanus display different physiologies, such as distinct differences in growth and survival strategies (Hittinger 2013; Borneman and Pretorius 2015). Within the Saccharomyces lineage, all of the major components of cAMP-PKA pathway are conserved.

In the present study we marshal phenotypic, biochemical, and genetic data to demonstrate that the regulation of filamentous growth by the cAMP-PKA signaling pathway has diverged significantly between S. bayanus and S. cerevisiae. We find that high levels of cAMP signaling have opposite effects on filamentous growth among these three species, promoting filamentous growth in both S. cerevisiae and S. paradoxus while inhibiting the filamentous response in S. bayanus. Divergent affects on the filamentous growth phenotype extend to downstream targets of PKA as well. In sum, our findings demonstrate that significant rewiring of the cAMP signaling pathway has occurred at multiple points in the cAMP-PKA gene network among the closely related species of the Saccharomyces sensu stricto. Our results, taken together with other recent findings regarding intraspecific variation and the potential for rapid evolution of cAMP-PKA signaling in response to selection, suggest that the cAMP-PKA pathway may be an evolutionary hot-spot for the accumulation of alleles that contribute to adaptation to novel nutrient niches.

Materials and Methods

Strains

Laboratory and environmental isolates of S. cerevisiae, S. paradoxus, and S. bayanus, and their corresponding pseudohyphal growth phenotypes are provided in Tables S1, S2, and S3. Mutants strains used in this study are given in Table S7. For S. bayanus, homozygous null mutants were generated in the NCYC365 background using KanMX4 deletion-cassette (Goldstein and McCusker 1999) with the standard PEG/LiAc protocol modified at the heat shock step, which was performed at 37rC for 45 minutes. The generated mutants, were confirmed with PCR and Sanger sequencing using primers listed in Table S8.

Media and Phenotyping

Strains were grown overnight in YPD to a density of 2 ×107 cells/ml. The cells were then washed twice in sterile water and 106 cells were transferred to agar plates. Pseudohyphal growth was assayed using a modified SLAD medium (SLAD-1%) consisting of 0.17% YNB AA/AS, 1% dextrose, 50 M ammonium sulfate, and 2%Noble agar (Gimeno et al. 1992). For drug treatments, plates were supplemented with the indicated concentrations of cAMP (Enzo), 8-Bromoadenosine 3’,5’-cyclic monophosphate [8-Br-cAMP] (Sigma), 3-isobutyl-1-methylxanthine [IBMX] (Sigma), H-89 (Sigma), MDL 12,330A [MDL] (Sigma), and 2-5-Dideoxyadenosine [ddAdo] (Santa Cruz). For phenotyping, S. cerevisiae and S. paradoxus were incubated at 30řC, and S. bayanus strains were incubated at room temperature (RT). The strains were scored for pseudohyphal growth by the presence or absence of cellular projections at the colony edges, and the response was evaluated qualitatively as increased (+), decreased (−), or no change (Ø) relative to wild-type at 72 hours post plating. Images were collected using a Leica stereo microscope.

Results

Intra- and interspecific variation in pseudohyphal growth

We measured filamentous growth under nitrogen limitation in a genetically diverse panel of S. cerevisiae (36 strains), S. paradoxus(35 strains), and S. bayanus (36 strains) strains (Tables S1, S2, and S3). We adopted a binary classification system, rating each strain as pseudohyphal or non-pseudohyphal after 72 hours of growth on low-nitrogen growth medium (SLAD; see methods). Scoring was done via microscopic observation of the periphery of colonies for the presence of elongated cells, unipolar budding, and characteristic multicellular arrangements of cells into chains and branches. A similar fraction of strains in both S. cerevisiae and S. bayanus exhibited pseudohyphal growth (63.8% and 61.1% respectively). Only 31.4% of S. paradoxus strains showed pseudohyphal after 72 hours of growth on SLAD. For all three species, there was significant variation in the strength of the pseudohyphal response among those strains capable of filamentous growth.

Exogenous cAMP inhibits pseudohyphal growth in S. bayanus

Previous studies have demonstrated that application of exogenous cAMP to the growth medium increases the propensity to form pseudohyphae in S. cerevisiae, and can restore pseudohyphal growth in mutants with reduced cAMP production (Lorenz and Heitman 1997; Kübler et al. 1997). This effect presumably mimics the increased activity of the endogenous adenylate cyclase. To test the generality of this effect across the Saccharomyces sensu stricto clade, we grew pseudohyphal and a non-pseudohyphal strains of S. cerevisiae, S. bayanus, and S. paradoxus under nitrogen-limiting conditions with various concentrations of exogenous cAMP (1 mM, 3 mM, 10 mM) added to the growth media. Most non-pseudohyphal S. cerevisiae and S. paradoxus isolates displayed a strong pseudohyphal phenotype in response to the presence of cAMP, exhibiting numerous filamentous extensions at the colony perimeter as well as increased invasiveness. Similarly, strains of S. cerevisiae and S. paradoxus that already exhibited the ability to undergo pseudohyphal growth showed a qualitative increase in the response upon cAMP treatment. In striking contrast, exogenous cAMP treatment was ineffective in inducing pseudohyphal differentiation in S. bayanus strains. Not only was the cAMP treatment ineffective in inducing the response in non-pseudohyphal S. bayanusisolates but, surprisingly, cAMP treatment suppressed filamentous differentiation in more than half of the normally pseudohyphal S. bayanus strains (Figure 2 and Table S4). We also tested the effect of the cAMP analog 8-Br-cAMP, which is reported to be more membrane permeant and resistant to degredation by phosphodiesterases (Schaap et al. 1993). 8-Br-cAMP at a concentration of 500 M produced a reduction of pseudohyphal growth in S. bayanus and an increase in S. cerevisiae comparable to approximately 3 mM cAMP (Figure S4).

Figure 2
Figure 2 Exogenous cAMP inhibits pseudohyphal growth in S. bayanus.

Pseudohyphal phenotypes are shown for a pseudohyphal (Psh) and a non-pseudohyphal (Non-Psh) strain of each species, grown in the presence of 5 mM cAMP. cAMP treatment promotes pseudohyphal growth in S. cerevisiae and S. paradoxus but inhibits pseudohyphal growth in S. bayanus.

Chemical manipulation of the cAMP-PKA Pathway

In order to further explore the surprising effect that exogenous cAMP had on filamentous growth in S. bayanus, we scored pseudohyphal growth in the presence of four additional chemical agents that have been shown to modify the activity of key enzymes involved in cAMP-PKA signaling. MDL-12,330A and 2-5 Dideoxyadenosine (ddAdo) directly inhibit the activity of adenylate cyclase (Cutuli et al. 2000; Guellaen et al. 1977), and thus should decrease intracellular cAMP levels. H89 is a protein kinase A inhibitor with broad specificity (Murray 2008), but is likely to decrease PKA activity. IBMX is an inhibitor of phosphodiesterases (Van Lookeren Campagne et al. 1990), and thus would tend to favor accumulation of cAMP in cells. Treatment with both MDL and ddAdo lead to a drastic decrease in pseudohyphal growth in S. cerevisiae and a modest decrease in S. paradoxus, but the filamentous response in S. bayanus in the presence of these agents is comparable to the untreated control (Figure S3). A 1 mM IBMX treatment increased the pseudohyphal response in both S. cerevisiae and S. paradoxus, while decreasing the density of pseuodohyphal projections on the margin of colonies in S. bayanus (Figure S4). A higher concentration of IBMX (3 mM), however, led to a dimunution of the response in all three species. The PKA inhibitor H-89 (50 M) had no discernible effects on pseudohyphal growth in S. bayanus, however there was a modest to complete loss of pseudohyphal growth in response to H89 in both S. cerevisiae and S. paradoxus (Figure S4).

Since S. cerevisiae and S. bayanus showed the greatest divergence of filamentous phenotypes in response to nutrient limitation and chemical manipulation, we chose to concentrate further investigations on these two species.

MAPK functions similarly in S. cerevisiae and S. bayanus pseudohyphal response

Both the cAMP-PKA pathway and the FG-MAPK cascade are capable of inducing pseudohyphal growth in S. cerevisiae. To rule out differences in the contribution of the FG-MAPK cascade to filamentous growth in the two species, we carried out gene deletion experiments in S. bayanus to confirm that FG-MAPK mutant phenotypes are similar to those previously reported for S. cerevisiae. Using drug resistance markers, we created deletion mutants of STE7, STE12, TEC1, and DIG1. The mutants of the positively contributing MAPK components, ste7, ste12, and tec1, exhibited smooth colony edges and a lack of invasiveness. The deletion of the negative element, DIG1, led to an increase in the filamentous response (Figure S2). These results are consistent with phenotypes observed for the same mutants in S. cerevisiae (Cook et al. 1996; Madhani and Fink 1997; Oehlen and Cross 1998; Roberts and Fink 1994).

The cAMP-PKA pathway is required for the filamentous response in both S. cerevisiae and S. bayanus

Having ruled out the FG-MAPK pathway as a likely candidate for the differences observed between S. cerevisiae and S. bayanus, we proceeded with systematic genetic manipulation of key genes in the cAMP-PKA pathway. We deleted 11 genes encoding elements of the cAMP pathway in S. bayanus, and compared the resulting filamentous growth phenotypes to those of the same mutants in S. cerevisiae. Unlike FG-MAPK mutants, we found that the effects of gene deletions in the cAMP-PKA pathway often differed in terms of observed phenotypes between S. bayanus and S. cerevisiae. We classified our observations into two categories of effects: 1) mutants with similar phenotypes and 2) mutants with opposite effects (Table S5).

The first category of mutants, exhibiting similar phenotypes in both species, included gpa2, tpk1, tpk2, and tpk3. Deletion of TPK2 ameliorates the FG response in both S. bayanus and S. cerevisiae, indicating that this PKA subunit is required for induction of filamentous growth in both species (Figure 3) (Robertson andFink 1998; Pan and Heitman 1999). tpk1 and tpk3 mutants have the opposite effect relative to tpk2, showing increased pseudohyphal growth in S. bayanus as has been previously reported for S. cerevisiae (Robertson and Fink 1998; Pan and Heitman 1999). This confirms that the distinct roles of the PKA subunits in the regulation of filamentous growth is conserved between the two species. Gpa2 is an activator of the adenylate cyclase Cyr1, and an inhibitor of the kelch repeat proteins Gpb1 and Gpb2. The gpa2 mutants show a loss of pseudohyphal growth in both species (Figure 4). The gpb1 and gpb2 mutants in S. bayanusshow a slight increase in pseudohyphal growth (Figure S6), similar to what has been reported for S. cerevisiae (Harashima and Heitman2002).

Figure 3
Figure 3 Mutations of subunits of the PKA holoenzyme have both similar and opposite effects on filamentous growth in S. cerevisiae and S. bayanus.

The catalytic subunit TPK2 promotes pseudohyphal growth in both species, while TPK1 and TPK3 are negative regulators of pseudohyphal growth. Deletion of the PKA catalytic subunits leads to parallel phenotypes in the two species. By contrast, deletion of the regulatory subunit, BCY1, results in hyper-filamentous growth in S. cerevisiae, but extremely slow growth with no pseudohyphae in S. bayanus (see also supplementary Figure S5). Mutants are on 1278b and NCYC 365 backgrounds for S. cerevisiae and S. bayanus, respectively.

Figure 4
Figure 4 Mutations that affect cAMP levels have primarily opposite effects on filamentous growth in S. cerevisiae and S. bayanus.

With the exception of gpa2, deletion mutations that affect adenylate cyclase activity or cAMP concentration show opposite phenotypic effects in S. cerevisiae and S. bayanus. See text for further discussion.

Mutants with opposite phenotypes in the two species included ras2, pde1, pde2, ira2, and bcy1 (Figures 3 and 4 and summarized in Table S5). The ras2 mutants show a strong decrease of filamentous growth in S. cerevisiae, but no decrease in S. bayanus. The ira2 mutants show an increase of filamentous growth in S. cerevisiae, and a strong decrease in S. bayanus. The pde1 mutants show an increase in filamentous growth in S. cerevisiae, and a strong decrease in S. bayanus, while pde2 mutants show a decrease of filamentous growth in S. cerevisiae and no change or a slight increase in S. bayanus. bcy1 mutants in S. cerevisiae showed abundant pseudohyphae, while the same mutant in S. bayanus is very slow growing and shows insufficient growth after 72 hours to score FG. However, if S. bayanus bcy1 mutants are allowed to grow for 10 days they eventually form a colony, but show no pseudohyphae (Figure S5).

Mutant phenotypes of targets of PKA in S. bayanus

We next examined the phenotypic effects of knockout mutants of four transcription factors – Flo8, Phd1, Sfl1, and Msn2 – that are targets of PKA, and which are known to play key roles in regulating pseudohyphal growth in S. cerevisiae (Figure 5). Flo8 and Phd1 are positive regulators of the pseudohyphal response; while Sfl1 is a repressor. All three are thought to modify pseudohyphal growth primarily through transcriptional regulation of FLO11 (see below). Msn2 is a stress responsive transcription factor that is regulated by both PKA and the TOR pathway.

Figure 5
Figure 5 Downstream targets of cAMP-PKA signaling show a mix of similar and divergent pseudohyphal responses.

Pseudohyphal phenotypes offlo8 mutations differ between S. cerevisiae and S. bayanus, but the response upon deletion of SFL1 and PHD1 is conserved. Deletion of FLO11 eliminates pseudohyphal growth completely in S. cerevisiae; in contrast, deletion of FLO11in S. bayanus causes a delay in the pseudohyphal response. At day three of observation filamentous growth is absent in the flo11 mutants of both S. cerevisiae and S. bayanus, but S. bayanus flo11 mutants start to exhibit pseudohyphal projections by day five.

The phd1 and sfl1 mutant phenotypes are identical between S. cerevisiae and S. bayanus, with PHD1 deletion mutants showing a loss in filamentous growth and SFL1 deletion mutants showing an increase in filamentous growth. Surprisingly, flo8mutants show opposite phenotypes in the two species, with complete abrogation of the pseudohyphal response observed in S. cerevisiae but no change in filamentous growth in S. bayanus. The msn2 mutants show no loss of pseudohyphal growth in S. bayanus, while there is a complete loss of the phenotype in S. cerevisiae. Rim15 is a kinase that is a target of both PKA and TOR signaling, and in turn contributes to the regulation of Msn2. rim15 mutants in S. cerevisiae show a loss of pseudohyphal growth, while S. bayanus mutants show comparable filamentous growth to the wild type background (Figure 5).

FLO11 is not required for filamentous growth in S. bayanus

The cell wall glycoprotein Flo11 is regarded as one of the primary molecular effectors of pseudohyphal growth in S. cerevisiae. flo11 mutants not only show a loss of pseudohyphal growth in S. cerevisiae, but also show an inability to form biofilms and complex colonies (Granek and Magwene2010; Granek et al. 2013; Zara et al. 2009). We compared flo11 mutants in both S. cerevisiae and S. bayanus over the course of five days. S. cerevisiae flo11 mutants show no sign of pseudohyphal growth, even up to five days post-plating. S. bayanus mutants show little filamentous growth at day three post-plating, but begin to show pseudohyphae at the colony margin at day 4, and show substantial pseudohyphae by day 5 (though less than WT) (Figure 5). We conclude that flo11 deletion delays the expression of filamentous growth in S. bayanus, and thus may be a key effector of in both species, but our finding also suggests a Flo11 independent mechanism for producing pseudohyphae in S. bayanus.

Discussion

The findings we describe above, regarding the role of cAMP-PKA signaling in the regulation of pseudohyphal growth in S. bayanus, are surprising in a number of respects. In S. cerevisiae, cAMP-PKA signaling plays an unambiguously positive role in the regulation of filamentous growth. Chemical and genetic manipulations that increase cAMP-PKA signaling lead to increased filamentous growth in S. cerevisiae, while perturbations that decrease cAMP-PKA signaling reduce the strength of the pseudohyphal response. In contrast, we find that in S. bayanus, perturbations that are predicted to increase intracellular levels of cAMP lead to a decrease in the filamentous growth response. These differences between the two species exist despite the fact that the core elements of the cAMP-PKA signaling network are highly conserved at the sequence level throughout the Saccha-romyces sensu stricto species (Table S6).

Chemical and genetic manipulation of cAMP levels produces divergent phenotypes in S. cerevisiae and S. bayanus

The application of exogenous cAMP exaggerates the pseudohyphal response in S. cerevisiae and S. paradoxus, but attenuates the pseudohyphal switch in S. bayanus (Figure 2). Pharmacological agents that modulate cAMP levels also show contrasting effects between S. cerevisiae and S. bayanus (Figures S3 and S4). Consistent with the results by chemical manipulation, genetic perturbation of the feedback mechanisms controlling cAMP levels results in starkly contrasting phenotypes between S. cerevisiae and S. bayanus. For example, knockouts of PDE1and IRA2 increase intracellular cAMP levels and as a consequence pde1 and ira2 mutants exhibit exaggerated pseudohyphal growth in S. cerevisiae (Cullen and Sprague2012). The same mutations in S. bayanus, lead to a striking reduction in pseudohyphal growth. RAS2 mutants, which show a loss of pseudohyphal growth in S. cerevisiae, have wild type pseudohyphal phenotypes in S. bayanus. The one exception to the pattern is the phenotypes observed for gpa2 mutants, where both S. cerevisiae and S. bayanus show a loss of pseudohyphal growth.

PKA mutations and downstream targets produce a mixture of similar and dissimilar phenotypes

In contrast to the generally divergent phenotypes exhibited by S. cerevisiae and S. bayanus upon manipulation of cAMP levels, the results we observed for mutants and chemical agents that affect PKA activity showed a mixture of similar and divergent phenotypes between the two species. Deletions of the PKA regulatory subunit, BCY1, which inhibits PKA activity, shows strong differences between the species. bcy1 mutants show hyper filamentous growth in S. cerevisiae, while the same mutant is slow-growing and non-pseudohyphal in S. bayanus. However, deletions of the PKA catalytic subunits Tpk1, Tpk2, and Tpk3 produced identical phenotypes in both S. cerevisiaeand S. bayanus, with tpk1 and tpk3 mutants both showing increased pseduohyphal growth while tpk2 mutants show decreased pseudohyphal growth.

At the level of downstream targets of PKA, we again see a mix of similar and divergent phenotypes between S. cerevisiaeand S. bayanus among deletion mutants. The transcription factors Phd1 and Sfl1 play similar roles in both species, however deletions of the transcription factors Flo8 and Msn2 produced opposite responses when comparing the species. The ability of S. bayanus to produce pseudohyphae in the absence of Flo8p is especially surprising as this deletion completely abrogates pseudohyphal growth inS. cerevisiae (Liu et al. 1996).

Flo11 is partially dispensable for pseudohyphal growth in S. bayanus

In S. cerevisiae both the cAMP-PKA pathway and the filamentous growth MAPK pathway jointly regulate FLO11, a cell wall adhesin that is thought to be critical for nutrient-induced pseudohyphal growth. Loss-of-function or deletion mutations of FLO11 eliminate nutrient-induced pseudohyphal growth in S. cerevisiae (Cullen and Sprague 2012). As we describe above, S. bayanus flo11 mutants are slow to manifest pseudohyphal growth, but do eventually exhibit pseudohyphae, though the strength of the pseudohyphal response is reduced relative to wild-type. FLO11 independent regulation of filamentous growth is not totally without precedent. For example, Lorenz et al. (Lorenz et al. 2000) reported that FLO11 is dispensable for pseudohyphal growth in the presence of 1% butanol and Halme et al. (Halme et al. 2004) found that ira1 flo11 mutants can undergo FLO10 dependent pseudohyphal growth.

The FG-MAPK cascade is conserved between S. cerevisiae and S. bayanus

In contrast to the numerous differences we documented with respect to the cAMP-PKA pathway, the genetic effects of perturbations to the filamentous growth MAPK cascade appears to be conserved between S. cerevisiae and S. bayanus, with both species showing similar mutant phenotypes for all the genes tested in this pathway. This conservation of genetic effects for FG-MAPK mutants holds even though previous studies have demonstrated significant divergence between S. cerevisiae and S. bayanus in the genes regulated by Ste12 and Tec1, two transcription factors that are targets of the FG-MAPK pathway and which contribute to the regulation of pseudohyphal growth (Borneman et al. 2007; Martin et al. 2012).

Speculative Model and Future Directions

How might we integrate the findings presented above into a model for the role that cAMP-PKA signaling plays in the regulation of pseudohyphal in S. bayanus? Two broad patterns emerge from our chemical and genetic perturbations. The first is that some level of PKA activity is required for pseudohyphal growth in both S. cerevisiae and S. bayanus. The second is that high levels of cAMP are inhibitory of pseudohyphal growth in S. bayanus, while promoting pseudohyphal growth in S. cerevisiae.

Particularly interesting in this regard is the role of Bcy1, the PKA regulatory subunit that directly interacts with cAMP and hence is the critical mediator between intracellular cAMP levels and the downstream effects of PKA activity. High levels of cAMP relieve the inhibitory effects of Bcy1 on the PKA catalytic subunits – Tpk1, Tpk2, and Tpk3. Genetically, Tpk1 and Tpk3 are inhibitors of pseudohyphal growth while Tpk2 is an activator of pseudohyphal growth, as has been previously shown for S. cerevisiae (Robertson and Fink 1998; Pan and Heitman 1999), and as we show here for S. bayanus.

We hypothesize that S. cerevisiae and S. bayanus differ in the relative amount or activity of the PKA catalytic subunits, in response to changes in intracellular cAMP levels. Species specific differences in the relative expression of the different Tpk subunits, or their relative affinity for the PKA regulatory subunit, Bcy1, could favor a shift in the balance between Tpk1/Tpk3 versus Tpk2. We hypothesize that in S. cerevisiae, increased cAMP signaling favors greater activity of Tpk2, while in S. bayanus similar increases in cAMP favor greater Tpk1 and/or Tpk3 activity (Figure 6). This hypothesis can be tested in future studies using a combination of gene deletions and heterologous expression of the various PKA regulatory and catalytic subunits individually and in combination in both S. cerevisiae and S. bayanus.

Figure 6
Figure 6 A proposed model for cAMP-PKA signaling in S. bayanus.

To explain the differences in the regulation of pseudohyphal growth in S. bayanus and S. cerevisiae, we propose a model based on the relative strength of interactions (indicated by line weight) between the regulatory (Bcy1) and catalytic (Tpk1, Tpk2, Tpk3) PKA subunits. We hypothesize that moderate levels of cAMP signaling lead to the preferential release of the catalytic subunit Tpk2, a positive regulator of filamentous growth. At high concentrations of cAMP, the Tpk1 and Tpk3 (repressors of filamentous growth), are also released from the PKA holoenzyme, counteracting the effects of Tpk2 and suppressing pseudohyphal growth.

Our findings also point to differences in the relative importance of downstream effectors of PKA, particularly key transcription factors such as Msn2 and Flo8, for the regulation of pseudohyphal growth. This suggests that rewiring at the level of gene regulation also contributes to the differences between S. cerevisiae and S. bayanus.

More broadly we speculate that the differences we observe in the regulation of pseudohyphal growth by the cAMP-PKA pathway reflects physiological differences between the two species, not only with respect to nitrogen utilization, but other stresses as well (Blein-Nicolas et al. 2013; Masneuf-Pomarède et al. 2010; Serra et al. 2005).

The cAMP-PKA pathway is an evolutionary hot-spot for adaptation in yeast

A number of other recent studies, focusing on variation within S. cerevisiae, highlight how standing genetic variation and de novo mutations in the cAMP-PKA pathway contribute to the genetic architecture of complex traits and adaptation to novel environments. These studies indicate that: 1) among environmental isolates of S. cerevisiae there is substantial genetic variation in the cAMP-PKA pathway and this variation affects a diversity of phenotypic traits (Granek et al. 2013; Taylor et al. 2016; Yadav et al. 2015); and 2) mutations that affect cAMP-PKA signaling are often among the earliest genotypic changes that are favored when yeast populations are subjected to selection in novel nutrient environments (Hong and Gresham2014; Liet al. 2018; Sato et al. 2016; Venkataram et al. 2016). Our findings, taken together with this growing body of work, thus point to the cAMP-PKA pathway as a major driver of evolutionary change in the the Saccharomyces sensu stricto species complex. Given the central role that cAMP-PKA signaling plays in the regulation of morphogenesis across the fungi (Hicks and Heit-man 2007; Klengel et al. 2005; Dürrenberger et al. 1998), we expect that the central importance of this pathway for adaptation and evolution is likely to be recapitulated in many other fungal clades.

Conclusions

This study highlights the evolutionary lability of the cAMP-PKA pathway among the species of the Saccharomyces sensu stricto complex. cAMP-PKA signaling is an key regulator of morphogenetic switches in response to environmental cues for the fungi generally (Boyce and Andrianopoulos 2015; Pérez Martín, José and Di Pietro, Antonio 2012; Turrà et al. 2014) and both inter- and intraspecific variation in cAMP-PKA signaling is likely to be an important genetic determinant of phenotypic variation in many fungal systems. More generally our findings exemplify the potential for conserved eukaryotic signaling pathways to diverge in the regulation of cellular phenotypes even among relatively closely related species.

Author Contributions

Conceived and designed the experiments: OK PM. Performed the experiments: OK. Analyzed the data: OK PMM. Wrote the paper: OK PMM.

Conflicts of Interest

The authors have declared no known conflicts of interest.

Acknowledgments

This research was funded National Science Foundation grant MCB 1330545. We thank John McCusker, Joseph Heitman, and Timothy Galitski for providing strains. We thank Andrew Alspaugh, Fred Nijhout, Amy Schmid, and Rytas Vilgalys for feedback and recommendations during the course of this study. We thank Debra Murray for proofreading and comments on the manuscript.

Footnotes

  • ↵* paul.magwene{at}duke.edu

  • ↵1 paul.magwene{at}duke.edu, Department of Biology, Duke University, Box 90338, Durham, NC 27708

Literature Cited

  1. ↵
    Belotti, F., R. Tisi, C. Paiardi, M. Rigamonti, S. Groppi, et al., 2012 Localization of Ras signaling complex in budding yeast. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1823: 1208–1216.
    OpenUrl
  2. ↵
    Blein-Nicolas, M., W. Albertin, B. Valot, P. Marullo, D. Sicard, et al., 2013 Yeast Proteome Variations Reveal Different Adaptive Responses to Grape Must Fermentation. Mol Biol Evol 30: 1368–1383.
    OpenUrlCrossRefPubMed
  3. ↵
    Borneman, A., P. Chambers, and I. Pretorius, 2007 Yeast systems biology: modelling the winemaker’s art. Trends in Biotechnol 25: 349–355.
    OpenUrl
  4. ↵
    Borneman, A. R. and I. S. Pretorius, 2015 Genomic insights into the Saccharomyces sensu stricto complex. Genetics 199: 281–91.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    Boyce, K. J. and A. Andrianopoulos, 2015 Fungal dimorphism: the switch from hyphae to yeast is a specialized morphogenetic adaptation allowing colonization of a host. FEMS Microbiol Rev 39: 797–811.
    OpenUrlCrossRefPubMed
  6. ↵
    Boynton, P. J. and D. Greig, 2014 The ecology and evolution of non-domesticated Saccharomyces species. Yeast 31: 449–462.
    OpenUrlCrossRefPubMed
  7. ↵
    Cho-Chung, Y. S., 2004 cAMP signaling in cancer genesis and treatment. In Signal Transduction in Cancer, edited by D. A. F. MD, number 115 in Cancer Treatment and Research, pp. 123–143, Springer US.
  8. ↵
    Cliften, P., P. Sudarsanam, A. Desikan, L. Fulton, B. Fulton, et al., 2003 Finding functional features in Saccharomyces genomes by phylogenetic footprinting. Science 301: 71–6.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    Cook, J. G., L. Bardwell, S. J. Kron, and J. Thorner, 1996 Two novel targets of the map kinase kss1 are negative regulators of invasive growth in the yeast Saccharomyces cerevisiae. Genes Dev 10: 2831–48.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    Cullen, P. and G. Sprague, 2012 The regulation of filamentous growth in yeast. Genetics 190: 23–49.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    Cutuli, M., S. Cristiani, J. Lipton, and A. Catania, 2000 Antimicrobial effects of alpha-MSH peptides. J Leukoc Biol 67: 233–9.
    OpenUrlCrossRefPubMedWeb of Science
  12. ↵
    Das, R., V. Esposito, M. Abu-Abed, G. S. Anand, S. S. Taylor, et al., 2007 camp activation of pka defines an ancient signaling mechanism. Proc Natl Acad Sci USA 104: 93–8.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    D’Souza, C. A. and J. Heitman, 2001 Conserved cAMP signaling cascades regulate fungal development and virulence. FEMS Microbiol Rev 25: 349–364.
    OpenUrlCrossRefPubMedWeb of Science
  14. ↵
    Dujon, B., 2010 Yeast evolutionary genomics. Nature Reviews Genetics 11: 512–524.
    OpenUrlCrossRefPubMed
  15. ↵
    Dürrenberger, F., K. Wong, and J. W. Kronstad, 1998 Identification of a cAMP-dependent protein kinase catalytic subunit required for virulence and morphogenesis in Ustilago maydis. Proc Natl Acad Sci USA 95: 5684–9.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    Fay, J. C. and J. A. Benavides, 2005 Evidence for domesticated and wild populations of Saccharomyces cerevisiae. PLoS Genet 1: e5.
    OpenUrlCrossRef
  17. ↵
    Gancedo, J., 2013 Biological roles of cAMP: variations on a theme in the different kingdoms of life. Biol Rev Camb Philos Soc 88: 645–648.
    OpenUrlCrossRef
  18. ↵
    Gimeno, C. and G. Fink, 1994 Induction of pseudohyphal growth by overexpression of PHD1, a Saccharomyces cerevisiae gene related to transcriptional regulators of fungal development. Molecular and Cellular Biology 14: 2100.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    Gimeno, C., P. Ljungdahl, C. Styles, and G. Fink, 1992 Unipolar cell divisions in the yeast S. cerevisiae lead to filamentous growth: regulation by starvation and RAS. Cell 68: 1077–1090.
    OpenUrlCrossRefPubMedWeb of Science
  20. ↵
    Gold, M. G., T. Gonen, and J. D. Scott, 2013 Local cAMP signaling in disease at a glance. J Cell Sci 126: 4537–4543.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Goldstein, A. and J. McCusker, 1999 Three new dominant drug resistance cassettes for gene disruption in Saccharomyces. Yeast 15: 1541–1553.
    OpenUrlCrossRefPubMedWeb of Science
  22. ↵
    Granek, J. and P. Magwene, 2010 Environmental and genetic determinants of colony morphology in yeast. PLoS Genet 6: e1000823.
    OpenUrlCrossRefPubMed
  23. ↵
    Granek, J., D. Murray, Ö. Kayikçi, and P. Magwene, 2013 The genetic architecture of biofilm formation in a clinical isolate of Saccharomyces cerevisiae. Genetics 193: 587–600.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    Guellaen, G., J.-L. Mahu, P. Mavier, P. Berthelot, and J. Hanoune, 1977 RMI 12330 A, an inhibitor of adenylate cyclase in rat liver. Biochimica et Biophysica Acta (BBA) - Enzymology 484: 465–475.
    OpenUrl
  25. ↵
    Halme, A., S. Bumgarner, C. Styles, and G. Fink, 2004 Genetic and epigenetic regulation of the FLO gene family generates cell-surface variation in yeast. Cell 116: 405–415.
    OpenUrlCrossRefPubMedWeb of Science
  26. ↵
    Harashima, T. and J. Heitman, 2002 The Galpha protein Gpa2 controls yeast differentiation by interacting with kelch repeat proteins that mimic Gbeta subunits. Molecular Cell 10: 163–73.
    OpenUrlCrossRefPubMedWeb of Science
  27. ↵
    Hicks, J. K. and J. Heitman, 2007 Divergence of protein kinase a catalytic subunits in Cryptococcus neoformans and Cryptococcus gattii illustrates evolutionary reconfiguration of a signaling cascade. Eukaryot Cell 6: 413–20.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Hittinger, C., 2013 Saccharomyces diversity and evolution: a budding model genus. Trends in Genetics 29: 307–319.
    OpenUrl
  29. ↵
    Hong, J. and D. Gresham, 2014 Molecular specificity, convergence and constraint shape adaptive evolution in nutrient-poor environments. PLoS Genet 10: e1004041.
    OpenUrlCrossRefPubMed
  30. ↵
    Johnson, L., V. Koufopanou, M. Goddard, R. Hetherington, S. Schäfer, et al., 2004 Population genetics of the wild yeast Saccharomyces paradoxus. Genetics 166: 43–52.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    Klengel, T., W.-J. Liang, J. Chaloupka, C. Ruoff, K. Schröppel, et al., 2005 Fungal adenylyl cyclase integrates co2 sensing with camp signaling and virulence. Curr Biol 15: 2021–6.
    OpenUrlCrossRefPubMedWeb of Science
  32. ↵
    Kowallik, V., E. Miller, and D. Greig, 2015 The interaction of Saccharomyces paradoxus with its natural competitors on oak bark. Mol Ecol 24: 1596–1610.
    OpenUrlCrossRef
  33. ↵
    Kübler, E., H. U. Mösch, S. Rupp, and M. P. Lisanti, 1997 Gpa2p, a G-protein alpha-subunit, regulates growth and pseudohyphal development in Saccharomyces cerevisiae via a cAMP-dependent mechanism. J Biol Chem 272: 20321–3.
    OpenUrlAbstract/FREE Full Text
  34. Li, Y., S. Venkataram, A. Agarwala, B. Dunn, D. A. Petrov, et al., 2018 Hidden complexity of yeast adaptation under simple evolutionary conditions. Curr Biol 28: 515–525.e6.
    OpenUrlCrossRef
  35. ↵
    Liu, H., C. Styles, and G. Fink, 1996 Saccharomyces cerevisiaeS288C has a mutation in FLO8, a gene required for filamentous growth. Genetics 144: 967.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    Lo, W. and A. Dranginis, 1998 The cell surface flocculin Flo11 is required for pseudohyphae formation and invasion by Sac-charomyces cerevisiae. Mol.Biol.Cell 9: 161.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    Loomis, W. F., 2014 Cell signaling during development of Dictyostelium. Dev Biol 391: 1–16.
    OpenUrlCrossRefPubMed
  38. ↵
    Lorenz, M., N. Cutler, and J. Heitman, 2000 Characterization of alcohol-induced filamentous growth in Saccharomyces cerevisiae. Mol Biol Cell 11: 183–99.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    Lorenz, M. C. and J. Heitman, 1997 Yeast pseudohyphal growth is regulated by GPA2, a G protein alpha homolog. EMBO J 16: 7008–7018.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    Madhani, H. D. and G. R. Fink, 1997 Combinatorial control required for the specificity of yeast mapk signaling. Science 275: 1314–7.
    OpenUrlAbstract/FREE Full Text
  41. ↵
    Martin, H., J. Roop, J. Schraiber, T. Hsu, and R. Brem, 2012 Evolution of a membrane protein regulon in Saccharomyces. Mol Biol Evol 29: 1747–1756.
    OpenUrlCrossRefPubMedWeb of Science
  42. ↵
    Masneuf-Pomarède, I., M. Bely, P. Marullo, A. Lonvaud-Funel, and D. Dubourdieu, 2010 Reassessment of phenotypic traits for Saccharomyces bayanus var. uvarum wine yeast strains. Int J Food Microbiol 139: 79–86.
    OpenUrlCrossRefPubMed
  43. ↵
    Murray, A., 2008 Pharmacological PKA inhibition: all may not be what it seems. Science Signaling 1: re4.
    OpenUrlAbstract/FREE Full Text
  44. ↵
    Naumov, G. I. and E. S. Naumova, 2011 Genetic identification of African cultured yeasts of the genus Saccharomyces. Microbiology 80: 386–390.
    OpenUrlCrossRef
  45. ↵
    Naumov, G. I., E. S. Naumova, and P. D. Sniegowski, 1998 Saccharomyces paradoxus and Saccharomyces cerevisiae are associated with exudates of North American oaks. Can. J. Microbiol. 44: 1045–1050.
    OpenUrlCrossRefPubMedWeb of Science
  46. ↵
    Oehlen, L. and F. R. Cross, 1998 The mating factor response pathway regulates transcription of tec1, a gene involved in pseudohyphal differentiation of Saccharomyces cerevisiae. FEBS Lett 429: 83–8.
    OpenUrlCrossRefPubMedWeb of Science
  47. ↵
    Pan, X. and J. Heitman, 1999 Cyclic AMP-dependent protein kinase regulates pseudohyphal differentiation in Saccharomyces cerevisiae. Mol.Cell.Biol. 19: 4874–4887.
    OpenUrlAbstract/FREE Full Text
  48. ↵
    1. Pérez Martín, José and
    2. Di Pietro, Antonio
    , editors, 2012 Morphogenesis and Pathogenicity in Fungi, volume 22 of Topics in Current Genetics. Springer.
  49. ↵
    Pérez-Través, L., C. A. Lopes, A. Querol, and E. Barrio, 2014 On the complexity of the saccharomyces bayanus taxon: hybridization and potential hybrid speciation. PLoS One 9: e93729.
    OpenUrlCrossRefPubMed
  50. ↵
    Replansky, T., V. Koufopanou, D. Greig, and G. Bell, 2008 Saccharomyces sensu stricto as a model system for evolution and ecology. Trends Ecol Evol (Amst) 23: 494–501.
    OpenUrl
  51. ↵
    Rinaldi, J., J. Wu, J. Yang, C. Y. Ralston, B. Sankaran, et al., 2010 Structure of yeast regulatory subunit: a glimpse into the evolution of pka signaling. Structure 18: 1471–82.
    OpenUrlCrossRefPubMed
  52. ↵
    Roberts, R. L. and G. R. Fink, 1994 Elements of a single map kinase cascade in Saccharomyces cerevisiae mediate two developmental programs in the same cell type: mating and invasive growth. Genes Dev 8: 2974–85.
    OpenUrlAbstract/FREE Full Text
  53. ↵
    Robertson, L. and G. Fink, 1998 The three yeast A kinases have specific signaling functions in pseudohyphal growth. Proc Natl Acad Sci USA 95: 13783.
    OpenUrlAbstract/FREE Full Text
  54. Rodriguez, M. E., L. Pérez-Través, M. P. Sangorrín, E. Barrio, and C. A. Lopes, 2014 Saccharomyces eubayanus and Saccharomyces uvarum associated with the fermentation of Araucaria araucana seeds in Patagonia. FEMS Yeast Res. 14: 948–965.
    OpenUrlCrossRefPubMed
  55. ↵
    Rupp, S., E. Summers, H. Lo, H. Madhani, and G. Fink, 1999 MAP kinase and cAMP filamentation signaling pathways converge on the unusually large promoter of the yeast FLO11 gene. EMBOJ. 18: 1257–1269.
    OpenUrlAbstract
  56. ↵
    Sampaio, J. and P. Gonçalves, 2008 Natural populations of Sac-charomyces kudriavzevii in Portugal are associated with oak bark and are sympatric with S. cerevisiae and S. paradoxus. Appl Environ Microbiol 74: 2144–2152.
    OpenUrlAbstract/FREE Full Text
  57. ↵
    Sato, T. K., M. Tremaine, L. S. Parreiras, A. S. Hebert, K. S. Myers, et al., 2016 Directed evolution reveals unexpected epistatic interactions that alter metabolic regulation and enable anaerobic xylose use by saccharomyces cerevisiae. PLoS Genet 12: e1006372.
    OpenUrlCrossRef
  58. ↵
    Schaap, P., M. v. Ments-Cohen, R. D. Soede, R. Brandt, R. A. Firtel, et al., 1993 Cell-permeable non-hydrolyzable cAMP derivatives as tools for analysis of signaling pathways controlling gene regulation in Dictyostelium. J. Biol. Chem. 268: 6323–6331.
    OpenUrlAbstract/FREE Full Text
  59. ↵
    Serra, A., P. Strehaiano, and P. Taillandier, 2005 Influence of temperature and pH on Saccharomyces bayanus var. uvarumgrowth; impact of a wine yeast interspecific hybridization on these parameters. Int J Food Microbiol 104: 257–65.
    OpenUrlCrossRefPubMed
  60. ↵
    Taylor, M. B., J. Phan, J. T. Lee, M. McCadden, and I. M. Ehrenreich, 2016 Diverse genetic architectures lead to the same cryptic phenotype in a yeast cross. Nat Commun 7: 11669.
    OpenUrlCrossRef
  61. ↵
    Toda, T., S. Cameron, P. Sass, M. Zoller, and M. Wigler, 1987 Three different genes in S. cerevisiae encode the catalytic subunits of the cAMP-dependent protein kinase. Cell 50: 277.
    OpenUrlCrossRefPubMedWeb of Science
  62. ↵
    Toda, T., I. Uno, T. Ishikawa, S. Powers, T. Kataoka, et al., 1985 In yeast, RAS proteins are controlling elements of adenylate cyclase. Cell 40: 27–36.
    OpenUrlCrossRefPubMedWeb of Science
  63. ↵
    Turrà, D., D. Segorbe, and A. Di Pietro, 2014 Protein kinases in plant-pathogenic fungi: conserved regulators of infection. Annu Rev Phytopathol 52: 267–88.
    OpenUrlCrossRefPubMed
  64. ↵
    Van Lookeren Campagne, M. M., F. V. Diaz, B. Jastorff, E. Winkler, H. G. Genieser, et al., 1990 Characterization of the yeast low Km cAMP-phosphodiesterase with cAMP analogues. Applications in mammalian cells that express the yeast PDE2 gene. J. Biol. Chem. 265: 5847–5854.
    OpenUrlAbstract/FREE Full Text
  65. ↵
    Venkataram, S., B. Dunn, Y. Li, A. Agarwala, J. Chang, et al., 2016 Development of a comprehensive genotype-to-fitness map of adaptation-driving mutations in yeast. Cell 166: 1585–1596.e22.
    OpenUrlCrossRef
  66. ↵
    Yadav, A., A. Radhakrishnan, G. Bhanot, and H. Sinha, 2015 Differential regulation of antagonistic pleiotropy in synthetic and natural populations suggests its role in adaptation. G3 (Bethesda) 5: 699–709.
    OpenUrl
  67. Zaman, S., S. Lippman, X. Zhao, and J. Broach, 2008 How Saccharomyces responds to nutrients. Annu Rev Genet 42: 27–81.
    OpenUrlCrossRefPubMedWeb of Science
  68. ↵
    Zara, G., S. Zara, C. Pinna, and S. Marceddu, 2009 FLO11 gene length and transcriptional level affect biofilm-forming ability of wild flor strains of Saccharomyces cerevisiae. Microbiology 155: 3838–3846.
    OpenUrlCrossRefPubMedWeb of Science
  69. Zimmerman, N. P., I. Roy, A. D. Hauser, J. M. Wilson, C. L. Williams, et al., 2015 Cyclic AMP regulates the migration and invasion potential of human pancreatic cancer cells. Mol Carcinog 54: 203–215.
    OpenUrlCrossRefPubMed
Back to top
Posted April 30, 2018.
Download PDF
Email
Divergent Roles for cAMP–PKA Signaling in the Regulation of Filamentous Growth in Saccharomyces cerevisiae and Saccharomyces bayanus
Ömur Kayikci, Paul M. Magwene
bioRxiv 311415; doi: https://doi.org/10.1101/311415
Reddit logo Twitter logo Facebook logo LinkedIn logo Mendeley logo
Citation Tools

Subject Area