cAMP Signaling in S. cerevisiae
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cAMP Signaling in S. cerevisiae

Yeast Filamentous Growth is regulated by two conserved signal transduction cascades; a G-protein regulated cAMP (Cyclic Adenosine 3,5-monophosphate) signaling and a MAPK (Mitogen-Activated Protein Kinase) cascade. S. cerevisiae (Saccharomyces cerevisiae) expresses at least three members of the G-protein-coupled family of Serpentine receptors among which the GPR1 (G-Protein Coupled Receptor-1) is coupled to Gpa2 (G-Protein-Alpha Subunit), in order to facilitate cAMP Signaling (Ref.1). In response to nitrogen limitation and abundant fermentable carbon source, diploid cells of S. cerevisiae undergo dimorphic transition to a Filamentous Growth form referred to as Pseudohyphal Growth. In the unicellular eukaryote, S. cerevisiae, PKA/cAPK (cAMP-Dependent Protein Kinase) is controlled by a wide variety of effectors. S. cerevisiae is also an excellent model system with great potential to provide insights into signaling in other fungi (Ref.2).

The signaling pathway which functions in parallel with the MAPK pathway to regulate Pseudohyphal Growth is the Nutrient-Sensing cAMP Pathway and involves GPR1, Gpa2, Ras, Adenylyl Cyclase/CDC35, cAMP and PKA. GPR1 acts as a nitrogen sensor, regulating the switch between Yeast-like and Pseudohyphal Growth, by binding PLC1 (Phospholipase-C1) and Gpa2 at its intracellularly oriented carboxyl-terminal domain. Gpa2 regulates cAMP production in response to extracellular Glucose. Glucose triggers a rapid and transient increase in intracellular cAMP levels. Gpa2 stimulates cAMP production by Adenylyl Cyclase/CDC35. Consistent with this, cAMP stimulates Pseudohyphal Growth. Ras activates cAMP via Adenylyl Cyclase/CDC35. The target of cAMP in S. cerevisiae is PKA (Ref.3). In yeast cells, the PKA regulatory subunit, Bcy1, is encoded by a single gene and catalytic subunits of PKA; Tpk1, Tpk2 and Tpk3, are encoded by the three genes. In response to external signals that increase intracellular cAMP levels, cAMP binds to the regulatory subunit and triggers conformational changes that  release the active catalytic subunits (Ref.1).

Catalytic subunits of PKA play redundant roles in Vegetative Growth but specialized roles in Filamentous Growth. The Tpk2 catalytic subunit positively regulates Filamentous Growth by regulating the transcription factor Flo8 (Transcriptional Activator-Flo8), which in turn regulates Flo11 (Flocculin) expression. Flo11 is a GPI (Glycosyl-Phosphatidylinositol)-linked cell surface protein that is required for Pseudohyphal and Haploid Invasive Growth. In particular, Flo11 plays a role in mother-daughter cell adhesion, which is required for the integrity of Pseudohyphal filaments. The unique activating function of the Tpk2 subunit is linked to structural differences in the catalytic region of the kinase and not to differences in gene regulation or the unique amino-terminal region of the protein (Ref.4). Tpk2 enhance Flo8 and Flo11 by regulating a complex set of transcription factors that includes Ste12 and Tec1. The Tpk1 and Tpk3 catalytic subunits play a negative role in regulating filamentous growth, possibly by a feedback loop that inhibits cAMP production. These show an intimate role for the PKA in the regulation of yeast dimorphism (Ref.1).

Rim15 (Serine/Threonine Protein Kinase-Rim15) constitute a downstream effector of PKA, under negative control of PKA. PKA signal transduction pathway transmits the Glucose and Nitrogen signals to Rim15 and inhibits its kinase activity. Rim15 is transcriptionally repressed in the presence of Glucose. This defines a positive regulatory role of Rim15 for entry into stationary phase (Ref.5). The zinc finger protein, Gis1 (Transcriptional Activator/Repressor-Gis1) functions in the Ras-cAMP pathway downstream of Rim15 to control transcription of a set of genes whose products are essential for long-term survival following nutrient limitation. Gis1 acts downstream of Rim15 and stimulate the transcription of growth inhibitory genes via activation of the PDS ((T/A)AGGGAT Post-Diauxic Shift) element. Loss of Gis1 causes a defect in transcriptional derepression following nutrient limitation of various genes, including Ssa3 (Heat Shock Protein-Ssa3), HSP12 (Heat Shock Protein-12) and HSP26 (Heat Shock Protein-26), whose expression is known to be negatively regulated by the cAMP pathway through Rim15. Gis1 activates one or more genes whose products inhibit Cell Proliferation (Ref.6 & 7).

Accordingly, PKA regulate growth directly through modulation of transcriptional activators including MSN2 (Zinc Finger Protein-MSN2) and MSN4 (Zinc Finger Protein-MSN4), as well as by its action on transcriptional repressors such as, for instance, SoK2. The MSN2/4-dependent gene induction through STREs (Stress Response Elements) is negatively controlled by the cAMP pathway (Ref.8). PKA exerts its negative effect on MSN2/4-dependent gene transcription by directly inhibiting MSN2/4 function and/or nuclear localization by phosphorylating MSN2/4. MSN2/4 function is also required for expression of Yak1 (Protein Kinase-Yak1), a gene whose product antagonize PKA-dependent growth. The CCR4-NOT Complex is an essential global regulator of transcription that regulates genes positively and negatively. It often exists as a core 1.2 MDa complex, consisting of CCR4 (Carbon Catabolite Repressor Protein-4), CAF (CCR4-Associated Factor) proteins like CAF1, CAF40 and CAF130; and the five NOT proteins (NOT1 to 5). The CCR4-NOT Complex contributes to the control of MSN2/4-dependent transcription by the cAMP pathway. NOT proteins interact with one of the catalytic subunits of PKA, leading to alteration of post-translational modifications of MSN2/4. In general the CCR4-NOT Complex control transcription by regulating modifying enzymes that acts to control the activity of MSN2/4 (Ref.8 & 9).

PKA antagonizes stress-responsive signaling. Activation of Mpk1/SLT2 (Suppressor of Lyt-2) by heat stress or Ca2+ (Calcium) led to increased phosphorylation of Bcy1, since Bcy1 is a target of the PKC1 (Protein Kinase-C Like-1)-Mpk1/SLT2 pathway. These phosphorylations are mainly dependent on MCK1 (Protein Kinase-MCK1), a known downstream target of Mpk1/SLT2. Phosphorylation of Bcy1 affects its intracellular localization. Bcy1 phosphorylation, however, is not sufficient for cytoplasmic localization but requires also Zds1 and Zds2, two putative yeast AKAPs (A-Kinase Anchor Proteins) that associate with phosphoisoforms of Bcy1 (Ref.1). Association of Bcy1 with Zds facilitates PKA-mediated phosphorylation of components that are involved in cell wall integrity signaling. Alternatively, Zds is a PKA substrate, and phosphorylation leads to activation of its capacity to down-regulate Mpk1/SLT2. Such a negative feedback inhibition exerted by phosphorylation of PKA and Zds help to prevent unbridled activation of Mpk1/SLT2, allowing growth and division as efficiently as possible (Ref.2).

A second signaling pathway which functions in parallel with the cAMP pathway is the Filamentous Growth cascade or MAPK pathway. The components of this MAPK cascade required for Filamentous Growth include the Ste20, Ste11, Ste7 and Kss1 kinases; nuclear proteins like Dig1 and Dig2; and the Ste12 transcription factor. In addition, another transcription factor, Tec1, forms a heterodimer with Ste12 that regulates expression of Tec1 itself and additional targets, such as the cell surface Flo11 required for Agar Invasion and Filamentation (Ref.1 & 2). Kss1 plays both positive and negative regulatory roles during Filamentous Growth, in part by relieving repression of Ste12 by the Dig1 and Dig2 proteins. The pheromones, pheromone receptors and subunits of the pheromone-activated heterotrimeric G-protein are dispensable for Filamentous Growth and are not expressed in diploid cells. During Filamentous Growth, the MAPK pathway is activated by a different mechanism involving the CDC42 (Cell Division Cycle-42) and Ras (Ref.1).

In summary highly conserved G-proteins functions in a Nutrient-Sensing pathway to regulate cAMP production during Pseudohyphal Growth in S. cerevisiae. While the components of this pathway required for activation of PKA are well established, little is known about the mechanisms of activation of the pathway by biological signals (i.e. nutrients), or about the potential biochemical targets of PKA. Accordingly, despite the remarkable progress in understanding of how the components of the cAMP pathway activate PKA in response to nutrient availability, PKA-dependent regulation of cell proliferation remains poorly understood. Elucidation of this mechanism will undoubtedly depend on the identification and characterization of downstream effectors that are specifically involved in Cell Proliferation control (Ref.1).