All organisms require nutrients that provide both building blocks and energy supply to drive their metabolism and synthesize the necessary cellular components. Apart from their essential role in metabolism, certain nutrients are also potent regulators of sophisticated signalling pathways. These nutrient-induced signalling pathways allow for optimal nutrient consumption through adaptations of metabolism and concomitant adjustments of growth properties. Particularly in unicellular organisms, nutrient sensing and signalling events are of utmost importance as these organisms are often faced with dramatic environmental changes where periods of plentiful and abundant nutrient availability alternate with long periods of nutrient shortness. Microorganisms, be it lower eukaryotes or prokaryotes, rapidly resume proliferation when nutritional conditions turn favourable, but prepare themselves to enter into a resting phase (G₀) under condition where essential nutrients start to become limiting 12. In higher multicellular organisms, most cells experience relative environmental homeostasis, while a sub-set of specialised cell types continually monitor the level of key nutrients and produce appropriate metabolic and/or behavioural responses, typically via altered secretion of certain hormones and/or neurotransmitters. A well-known example is the regulation of human blood glucose via the insulin and glucagon hormones produced in specialised pancreatic islet cells 34. Interestingly, recent evidence shows that particular nutrients can initiate cell-signalling events independently of hormonal influences also in higher eukaryotes 56.

Baker's yeast Saccharomyces cerevisiae has proven to be an important model organism for studies related to nutrient-induced signalling events, which is underlined by the fact that many signal transduction mechanisms are highly conserved from yeast to higher eukaryotes 7. For yeast cells, rapidly fermentable sugars are the preferred carbon and energy sources. Hence, when glucose is added to yeast cells grown on a non-fermentable carbon source, they rapidly adapt their metabolism to fermentation during a short lag-phase to ensure optimal and exclusive use of this rich carbon source. This adaptation requires different regulatory pathways such as the glucose-repression pathway and the Ras-cAMP pathway 8. After this initial transition, the cells start to consume and ferment the sugar during the exponential phase where they display a maximal growth rate (Fig. 1). Once glucose becomes limiting, yeast cells enter a second lag-phase, known as diauxic shift during which they reset their metabolic mode from fermentation to respiration. This metabolic switch is followed by a second slow-growing phase during which ethanol, acetate and other products of the initial fermentation process are being used as carbon sources. Finally, when these carbon sources are exhausted, the cells enter a quiescent or stationary phase (G₀). During entry into the G₀ phase, many transcriptional and metabolic rearrangements take place in the yeast cell. Some of these (such as the induction of stress-responsive genes and the accumulation of the reserve carbohydrate trehalose) serve to acquire stress resistance and ensure optimal survival during the starvation period. Other changes such as the repression of genes involved in protein synthesis result in the controlled downregulation of growth. Interestingly, when yeast cells are starved (in the presence of glucose) for essential nutrients such as for instance nitrogen or phosphate, similar physiological adjustments can be observed than the one observed following glucose limitation. This observation indicates that several components involved in the control of typical G₀-traits may be shared between multiple nutrient-induced signalling pathways. Moreover, certain environmental stresses (e.g., heat stress) engender entry of cells into a G₀-like state, indicating that nutritional deprivation can be considered as a specific stress condition 910. Consistent with this idea, nutrient and stress signalling pathways were found to converge on components like Msn2 and Msn4 (Msn2/4), a pair of partially redundant transcription factors that regulate expression of a subset of stress-responsive genes that contain stress-responsive elements (STREs) in their promoters 111213. In this review we focus on the protein kinase Rim15. While originally identified as a regulator of meiosis in diploid cells, recent studies demonstrated that Rim15 is essential in both diploid and haploid cells for the proper entry into G₀. Most interestingly, Rim15 was found to integrate signals derived from several different nutrient-sensory kinases (i.e., PKA, TORC1, Sch9, and Pho85-Pho80) that transmit information on the availability of different nutrients.

Diploid yeast cells undergo meiosis under conditions of nitrogen depletion in the presence of a non-fermentable carbon source such as acetate 1415. At the onset of meiosis,

e

m

Using a genetic screen, several mutations were isolated that caused reduced expression of an ime2-lacZ fusion gene and the corresponding genes were named RIM (

R

Independently of the studies on meiosis, Rim15 was also isolated as a protein that specifically interacted with the Tps1 subunit of the trehalose synthase complex 2021. Detailed phenotypic analyses revealed that loss of Rim15 causes a pleiotropic phenotype in cells grown to stationary phase on rich medium; this phenotype includes defects in trehalose and glycogen accumulation, in transcriptional derepression of HSP12, HSP26, and SSA3, in induction of thermotolerance and starvation resistance, and in proper G₁ arrest. As all of these traits are reminiscent of deficiencies previously ascribed to mutations that cause constitutive activation of PKA, these observations pointed to a role of Riml5 in nutrient signalling at some point in the Ras-cAMP pathway. Tests of epistasis indeed supported the notion that Rim15 may act in this pathway downstream of protein kinase A (PKA). Accordingly, while loss of Rim15 rendered cells independent of PKA activity, overproduction of Rim15 exacerbated the growth defect of strains compromised for PKA activity. Finally, biochemical analyses revealed that PKA-mediated phosphorylation of Rim15 (specifically at five PKA consensus phosphorylation sites; RRXS) strongly inhibited its kinase activity in vitro. Taken together, these results placed Rim15 immediately downstream and under negative control of PKA and defined a positive regulatory role of Rim15 in G₀ entry 21.

Adaptation to nutrient starvation conditions involves critical reprogramming of overall transcription. Interestingly, a dosage-suppressor screen of the defect of rim15Δ mutants to induce transcription of SSA3 following nutrient limitation led to the identification of Gis1, which is a zinc-finger transcription factor that binds the post-diauxic shift (PDS) element in the promoter of genes that become derepressed upon glucose exhaustion at the diauxic shift 22. The core of the PDS-element closely resembles the consensus sequence of the STRE-element, which confers transcriptional induction in response to a variety of stresses including nutrient limitation and oxidative, heat and osmotic stress. Interestingly, the control of STRE-driven gene expression by PKA is mediated by the transcription factors Msn2 and Msn4 111213. Moreover, both Msn2 and Msn4 partially overlap in functions with Gis1 in vivo and appear to regulate transcription of a large set of genes in a cooperative manner 222324. In line with this finding, combination of the gis1Δ mutation with the msn2Δ msn4Δ mutations causes an apparent synthetic growth defect on non-fermentable carbon sources 24. Important to the discussion here is that genome-wide transcriptional profiling indicated the three transcription factors, Msn2, Msn4, and Gis1, to almost entirely account for the Rim15-dependent effects on transcription during glucose limitation at the diauxic shift 23. The mechanisms by which Rim15 controls the functions and activities of Gis1 and Msn2/4, however, are still unknown. Although direct phosphorylation of the transcription factors by Rim15 is conceivable, Rim15 may alternatively regulate the establishment of physiological context-specific interactions between these factors and the general transcription machinery 25 or activate transcription from Msn2/4- and Gis1-dependent promoters through chromatin remodeling 2627.

The data described above established the epistatic relation between PKA, Rim15 and the transcription factors Gis1, Msn2 and Msn4. In addition, with Rim15 being immediately downstream PKA, the data also provided a molecular link between the Ras-cAMP pathway and the regulation of meiosis because activation of PKA inhibits Rim15, thereby preventing the dissociation of Ume6 from Sin3/Rpd3 and ensuring repression of EMGs. This was further confirmed in vivo since Rpd3 is not bound to the EMG promoters in sporulation medium supplemented with 2% glucose in a strain that expresses the constitutively active 5-RRXS/A mutant allele of RIM15 16.

Notably, enhanced synthesis of cAMP is mainly controlled by glucose, but not by any other nutrient 8. Since yeast cells also respond to nitrogen or phosphate starvation by acquiring many of the same G₀-characteristics as glucose-starved cells, additional mechanisms must exist that integrate the information on the availability on these other nutrients 82829. Notably, in this context, entry into meiosis requires integration of two nutritional cues (i.e., the absence of a fermentable carbon source and the limited supply of nitrogen), underlining the proposed existence of additional nutrient integration mechanisms 1415.

Several studies dealing with the regulation of molecular processes associated with the availability of glucose and nitrogen implicated the TOR pathway in the nutrient-responsive signalling network of yeast. The central components of this pathway are the highly conserved protein kinases Tor1 and Tor2, which were both identified based on a screen for mutants that confer resistance to the immunosuppressant rapamycin. Tor1 and Tor2 are present in two functionally distinct multiprotein complexes, i.e. TORC1, which controls ribosome biogenesis, translation, protein turnover and transcription of starvation-specific genes, and TORC2, which controls actin cytoskeleton organization. Only the TORC1 complex is inhibited by the immunosuppressant rapamycin 303132. Cells treated with rapamycin arrest growth and induce a typical G₀ program and therefore phenotypically resemble nutrient-starved cells. Intriguingly, mutants that lack Rim15 fail to properly enter G₀ following rapamycin treatment as exemplified for instance by their corresponding defects both in transcriptional induction of PDS- and STRE-driven genes and in trehalose and glycogen accumulation. The underlying reason for this is that rapamycin treatment and/or inhibition of TORC1 abolishes the cytoplasmic retention and thereby favours the cytoplasmic-to-nuclear translocation of Rim15 33. Mechanistically, inactivation of TORC1 results in dephosphorylation of the pThr¹⁰⁷⁵ in a 14-3-3-binding site of Rim15 thereby causing the release of Rim15 from the cytoplasmic 14-3-3 anchor protein Bmh2 34. Notably, this TORC1 effect on Rim15 appears not to involve the known TORC1-effector Sit4 and it is possibly also independent of the type 2A protein phosphatases Pph21 and Pph22 33.

Another pathway involved in signalling the presence of glucose and nitrogen to yeast cells is the so-called Fermentable Growth Medium (FGM) induced pathway 8. The key component of this pathway is the protein kinase Sch9, an orthologue of mammalian PKB/Akt. Initially, Sch9 was proposed to act as a nutritional regulator that controls the activity of the free catalytic subunits of PKA via a cAMP-independent process 28. Recent genetic data, however, indicated that Sch9 might converge with the Ras-cAMP pathway downstream of PKA on its effector Rim15 2433. Like TORC1, Sch9 is required for cytoplasmic retention of Rim15 during exponential growth on glucose-containing medium 33. While the epistatic relationship between TORC1 and Sch9 remains to be elucidated, it was proposed that Sch9 may be involved in phosphorylation of a second (pThr¹⁰⁷⁵-independent) 14-3-3-binding site in Rim15, which may act in concert with the 14-3-3-binding site flanking Thr1075 to mediate tandem 14-3-3 binding 34. Furthermore, it is worth noting that Rim15 nuclear accumulation can only be visualized using kinase-inactive variant versions of GFP-Rim15 indicating that autophosphorylation of Rim15 stimulates its proper nuclear export 34. Such a mechanism may add an additional layer to the dynamic control of G₀ entry by establishing a corresponding requirement for continued starvation signals to maintain sufficiently high levels of Rim15 in the nucleus.

In addition to its role in glucose- and nitrogen-dependent signalling, Rim15 also controls entry into G₀ in response to a phosphate (Pi) starvation signal 29. Signalling phosphate starvation in yeast is mediated by the PHO pathway, which consists of the cyclin-dependent kinase (CDK) Pho85 and its cyclin partner Pho80, the CDK inhibitor (CKI) Pho81 and the Pho4 transcription factor 35. Under high-Pi conditions, Pho85-Pho80 phosphorylates and thereby inactivates the Pho4 transcription factor. Upon Pi starvation, Pho81 inhibits the Pho85-Pho80 kinase complex, thereby allowing activation of Pho4 and the concomitant transcriptional induction of genes involved in the acquisition, uptake and storage of phosphate 3637. Importantly, Pi starvation induces, in a Rim15-dependent manner, similar G₀- traits as those described above following glucose and nitrogen starvation 2938. In support of these genetic studies, biochemical studies have recently revealed that Rim15 represents a bona fide target of the Pho85-Pho80 kinase complex both in vitro as well as in vivo 34. Surprisingly, a previously defined minimum domain of Pho81, necessary and sufficient to regulate Pho4 action in response to Pi availability 39, is deficient in regulating Rim15 activity in response to Pi starvation 29. Therefore, distinct domains of Pho81 have to be involved in the regulation of the two targets (i.e., Pho4 and Rim15) of the Pho85-Pho80 kinase complex.

Intriguingly, Pho85-Pho80 phosphorylates Thr¹⁰⁷⁵ within the 14-3-3 binding site of Rim15. Thus, Pho85-Pho80 stimulates Bmh2-dependent, cytoplasmic retention of Rim15 via phosphorylation of the 14-3-3 site flanking the residue Thr1075, while TORC1 likely controls the phosphorylation status of the same residue by keeping corresponding pThr¹⁰⁷⁵-targeting phosphatases inactive. Notably, since Pho85-Pho80 is localized in the nucleus and TORC1 in the cytoplasm 4041, both kinases are likely to act on different pools of Rim15. Taken together, based on current knowledge Rim15 integrates signals from at least four nutrient-sensitive protein kinases, namely TORC1, Sch9 and Pho85-Pho80, which control sub-cellular localization, and PKA, which controls the activity of Rim15.

An important question, which remains to be addressed, is whether each of these nutrient-sensitive kinases (i.e., TORC1, Sch9, Pho85-Pho80, and PKA) converges independently on Rim15, or whether they may impinge on each other at different levels. Current data suggest that Pho85-Pho80 acts independently of TORC1 and Sch9 2934. Whether PKA acts independently of TORC1 and/or Sch9, however, is still a matter of debate 24334243. Undoubtedly, understanding of the relationship between these nutrient-signalling kinases will require more detailed knowledge on both the operating feedback mechanisms in the corresponding pathways and the identification of additional critical points of convergence. One such point may be Msn2, whose nuclear exclusion/cytoplasmic retention is apparently controlled independently by both PKA and TORC1 2444454647. Moreover, our current data suggest that Gis1 may actually represent another important point of convergence that integrates signals from both Sch9 and Pho85-Pho80 independently 2429.

Rim15 is a distant member of the NDR family of AGC kinases, which share the presence of an insert between the protein kinase subdomains VII and VIII 182348. In S. cerevisiae, this insert comprises 188 amino acids and contains a binding site for the 14-3-3 protein Bmh2, which anchors Rim15 in the cytoplasm under nutrient-rich conditions 1834. The kinase domain of Rim15 is flanked by large N- and C-terminal extensions containing several functional domains (Fig. 3). These include an evolutionary conserved, amino-terminal PAS (Per-Arnt-Sim) domain, which classifies Rim15 as a new, distinct member of the PAS kinase family 23. PAS domains are a small regulatory modules represented in all kingdoms of life and function as cis-acting regulatory domains that may respond to a wide variety of stimuli, such as changes in light, redox potential, oxygen, small ligands and overall cellular energy levels 4950515253. The identification of a PAS domain in Rim15 opens the perspective that this domain might act as a cis-regulatory switch that regulates Rim15 in response to additional signals besides the nutrient-induced regulation described above. An interesting candidate signal represents oxidative stress. As yeast cells traverse the diauxic shift, the rise in mitochondrial respiratory chain activity produces an increase in reactive oxygen species (ROS) 54. Superoxide, a typical ROS, inactivates the aconitase and other [4Fe-4S] cluster enzymes and may cause loss of mitochondrial function and ultimately cell death 5556. In response to oxidative stress, yeast cells therefore regulate the induction of various protective mechanisms. Rim15 seems to play a crucial role in this process since rim15Δ mutant cells display a decreased chronological life-span when compared to wild-type strains 57. Interestingly, Rim15 was proposed to act downstream of the Ras-cAMP-PKA and Sch9 pathways and to regulate transcriptional induction of the mitochondrial manganese superoxide dismutase-encoding SOD2 gene 235758. Since rim15Δ but not msn2Δ msn4Δ or sod2Δ mutants display a specific short-lived phenotype 5758, Rim15 is likely to regulate longevity via yet additional mechanisms. An intriguing model posits that the PAS domain may sense oxidative stress and/or cellular redox potential and regulate Rim15 activity in response to these signals 23.

Although several regulatory mechanisms controlling Rim15 activity have recently been elucidated, some important questions remain unanswered. These include the nature of the presumed TORC1-controlled protein phosphatase(s) that controls the phosphorylation status of Thr¹⁰⁷⁵. Moreover, the molecular details by which Sch9 controls Rim15 subcellular localisation 33, the role of the Rim15-PAS domain for intra- or inter-molecular regulation of the Rim15 kinase activity 23, or the mechanism by which defined domains in Pho81 regulate Rim15 function 2934, remain key issues that will have to be addressed in the near future.

A major challenge in further understanding Rim15 function lies within the identification of bona fide physiological targets of this kinase. Although Rim15 phosphorylates the Ume6 protein in vitro 19, it is still unclear whether this also occurs in vivo. Additional candidate target proteins may be the Sin3 and/or Rpd3 HDAC proteins 16. Moreover, even though Msn2/4 and Gis1 were genetically identified as downstream targets of Rim15, it is possible that Rim15 may rather indirectly control the activity of these transcription factors 222324. Interestingly, a recent high-throughput in vitro kinase assay has identified several potential candidates of the Rim15 kinase 59. Even though none of the above mentioned putative target proteins were scored in this assay, analysis of the corresponding positive hits may yield most valuable information regarding the cellular function of Rim15.

In conclusion, Rim15 plays a central role in connecting nutrient-induced signalling pathways, thereby regulating crucial processes involved in yeast proliferation, differentiation and aging. As many of these processes are highly conserved among eukaryotes, the studies in yeast may provide important clues on the molecular mechanisms that govern these processes in higher organisms as well.