Systems Biology of AGC Kinases in Fungi

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Science's STKE  12 Sep 2006:
Vol. 2006, Issue 352, pp. re9
DOI: 10.1126/stke.3522006re9


Sch9 appears to be the Saccharomyces cerevisiae homolog of protein kinase B and is involved in the control of numerous nutrient-sensitive processes, including regulation of cell size, cell cycle progression, and stress resistance. Sch9 has also been implicated in the regulation of replicative and chronological life span. Systematic comparison of the phenotypes of sch9 and other AGC kinase mutants in fungal species with their counterparts in model eukaryotic organisms provides insight into the functions of AGC kinases. The availability of data from global studies of protein-protein interactions now makes it possible to predict and validate functional connections between Sch9, its putative substrates, and other proteins. This review highlights several emerging paradigms of AGC kinase signaling that are relevant for growth, development, and aging.

The formation of new cell structures occurs to a large extent by increase in size or replication of what already exists. Every cell emerges by division of an existing cell, so that in theory, all cells can be traced back without interruptions to one single original cell.

— C. Nüsslein-Volhard, Coming To Life: How Genes Drive Development

Introduction to the AGC Family of Kinases

In fungi and multicellular eukaryotes, AGC kinases regulate various signaling events that orchestrate growth and morphogenesis. Their activities contribute to processes that determine how many cells are produced within a tissue, organ, or colony, and the size of each individual cell. The availability of nutrients plays a key role in activating AGC kinases. AGC kinases affect such processes as storage of carbohydrates, synthesis of DNA and protein, ribosomal biogenesis, cell cycle progression, autophagy, and apoptosis (15). In turn, these cellular processes determine the limits of life span for individual cells and for the whole organism. Indeed, recent studies directly implicate Sch9, a member of the Saccharomyces cerevisiae AGC kinase family, in the control of aging (6, 7).

The AGC family of serine-threonine kinases is named for protein kinase A (PKA), protein kinase G (PKG), and protein kinase C (PKC), but also includes phosphoinositide-dependent kinase (PDK), protein kinase B (PKB), and the ribosomal protein S6 kinases. These kinases share sequence similarity, especially within the catalytic kinase domains, and usually also have a common domain organization. Most AGC kinases have (i) one or several N-terminal lipid-binding domain(s) of pleckstrin homology (PH), C1, or C2 type; (ii) a catalytic domain; and (iii) a C-terminal regulatory (H) domain characterized by a consensus hydrophobic sequence (Fig. 1). Typically, AGC kinases form a kinase module, in which the upstream kinase (for example, PDK) phosphorylates the serine (Ser) or threonine (Thr) residue within the conserved regulatory motif of its downstream target that is also an AGC kinase (for example, PKB). AGC kinases affect downstream signaling components through numerous direct and indirect mechanisms, including regulation of nuclear shuttling and activities of transcription factors; phosphorylation-dependent trafficking of other signaling proteins, such as small guanosine triphosphatases (GTPases) (8); and chromatin remodeling to derepress silenced genes (9).

Fig. 1.

Domain organization of AGC family kinases. (A) Generic scheme of typical domain organization: membrane-targeting, lipid-binding domains (C1, C2, or PH), catalytic kinase domain, and regulatory hydrophobic (H) motif. (B) Domain organization of Sch9. On the basis of sequence comparisons, it is expected that the C2 domain is responsible for targeting Sch9 to vacuolar or vesicular membranes. The serine residue within the consensus motif of the H domain might be phosphorylated by the upstream kinase, such as Pkh1. It is presently unknown which motif(s) in Sch9 mediates interactions of the kinase with other proteins (Table 1) and with the activator sphingolipid phytosphingosine (PHS).

Methodologies for Studying AGC Kinases and Aging in Yeast

Peaks and pitfalls in the functional analysis of AGC kinases

Distinguishing among the different aspects of function for a protein kinase within a signaling network can be challenging, because of multiple connections, feedback loops, and secondary effects. Integration of classical reductionism and holistic nonbiased genome- and proteome-wide approaches allows one to predict how genetic perturbations may affect the whole system and how systematic changes may affect the functions of a specific component. Genetic screens, high-throughput gene expression, and protein-protein interaction studies provide a rich source of information for generating new hypotheses—identifying unknown components and connections within the networks of interacting genes, proteins, metabolites, and signaling molecules. These studies are typically followed by experimental validation of selected interactions carried out with traditional lower-throughput methods (validation of DNA microarrays by polymerase chain reaction, and coimmunopurification of protein complexes identified by mass spectrometry or yeast two-hybrid assays). Genome- and proteome-scale data might be prone to a higher error or artifact rate, as compared with studies based on classical reductionism approaches, and thus validation by other methods is a critical part of the analysis. How these different approaches have provided new insight into the functions of Sch9 and other AGC kinases is discussed below.

Interspecies comparisons of AGC kinases are complicated by the presence of multiple high-score homologs with little or no annotation available and by sequences that are, in part, different in domain organization or substrate specificity. Complexities also exist in interpreting the phenotypes of genetic mutants. Many of the genes that encode proteins that regulate growth (for example, the gene encoding mammalian PDK and the gene encoding yeast Pkc1) are essential. In yeast, Dictyostelium, and other organisms, severe growth defects complicate interpretation of the phenotypes in mutants that produce either nonviable or poorly growing progeny. Furthermore, the genetic background in which the AGC kinase mutants occur can also create differences in phenotype or alter the severity of the phenotype. For example, yeast strains with reduced PKA activity in which sch9 has also been deleted exhibit severe growth defects, whereas sch9 deletion in a wild-type background has only a mild, slow growth defect (5, 6). Interpretation of mutant phenotypes is complicated further by the involvement of AGC kinases, such as mammalian PKB, in maintenance of genomic stability. For example, in mammals, some mutations, such as those that inactivate the phosphatase PTEN, result in abnormally high PKB activity, which promotes unstable or heterogeneous clones with variable properties (10). Thus, it can be difficult to determine if the phenotype is a direct result of a change in AGC kinase activity or a secondary effect due to mutations in other genes that arise as a result of genomic instability.

In addition to genetic knockouts, various other methods have been applied to selectively modulate the AGC kinase activity, including the use of pharmacological inhibitors, selective inhibition with RNA interference (RNAi) techniques, and expression-based strategies. Expression-based strategies may use kinase-deficient, dominant-negative or constitutively active mutants, such as those used to study Drosophila S6 kinase (11). Alternatively, a chemical genomics strategy, in which the adenosine 5′-triphosphate (ATP)–binding cassette in a kinase-expressing construct is engineered to be selectively sensitive to chemical inhibitor, may be used. Expression of a selectively sensitive kinase was used to study the functions of Sch9 (12). Because kinase overexpression may, in some cases, be sufficient to cause its unregulated activation, it is especially important to consider the differential copy number or gene dosage effects in (i) homozygotes versus heterozygotes, (ii) haploid versus diploid mutant strains, and (iii) expression constructs driven by heterologous promoters versus native promoters.

When genetic knockouts are used to study AGC kinase family members, several issues need to be considered. Many AGC kinases exist in multiple isoforms with either distinct or partially redundant functions. For example, one may consider the yeast PDK homologs Pkh1 and Pkh2. Single mutations in each of these genes results in a "weak" or "no" growth phenotype that may be explained by compensation due to the activity of the other isoform. To account for this redundancy, organisms with mutations in multiple AGC kinase genes must be analyzed. For example, lethality of the double mutant pkh1/pkh2 in yeast demonstrates the functional redundancy of these two proteins (13). Alternatively, a phenotype may not be apparent due to compensation by increased activity of another signaling pathway. For example, growth defects in sch9 cells are minimized by an increase in the adenosine 3′,5′monophosphate (cAMP)–PKA pathway (14).

Using fungi to study aging

Aging in yeast can be studied in terms of two different outputs: replicative longevity and chronological longevity. Replicative longevity in yeast is defined as the total number of daughter cells generated by a mother cell (15). Classical counting methods rely on recognition of smaller daughter cells (buds) and removal of the daughter cells by micromanipulation on solid agar, which then allows the number of buds generated by each mother cell to be determined. Another method involves isolation of the mother cells from the daughter by labeling the mother cells with biotin, allowing the cells to undergo a number of divisions, and then removing the labeled mother cells with avidin-magnet beads (16). Because these methods are based on counting cell divisions over time, they are not directly applicable to studying aging during filamentous growth.

A method to investigate chronological longevity in yeast was developed by Longo and colleagues (7, 17) and is based on monitoring survival of cells in the minimal glucose-containing medium. Survival is measured every 48 hours by counting the colonies produced by viable yeast cells (colony-forming units, CFUs). Under these conditions, wild-type yeast cells typically have metabolic rates that allow survival for up to 6 to 7 days.

A generally accepted method for investigating aging in filamentous fungi is to represent life span as the time from germination until cessation of growth of individual hyphae, which is considered a senescence stage (18). This type of filamentous growth is typically examined over several weeks in culture, using the classical Race tube method (19). If one assumes that outgrowth is directional along a certain axis, then the increment in length of individual hyphae over time defines the rate of growth. The duration of outgrowth defines the age at which the senescent state is reached. Replicative longevity in filamentous fungi may correlate with the number of new septa and interseptal units formed over time. The combination of microscopy with fluorescent reporter probes is a promising tool for monitoring cell cycle progression, gene expression, and silencing events in individual cells in real time (2022).

AGC Kinases in Fungi

Fungal AGC kinases primarily respond to availability of sugars (glucose) and nitrogen. Similar to mammals and insects, fungal AGC kinases may form signaling modules in which an AGC kinase activates a phosphorylation cascade (Fig. 2). For example, in Schizosaccharomyces pombe, the TOR-PDK-AGC kinase module, which resembles the TOR-PDK-S6K cassette in Drosophila and mammals, has been identified (23) (TOR stands for target of rapamycin). In this yeast, either nutrient (nitrogen) starvation or exposure to stressful conditions (high temperature or osmotic stress) leads to cell cycle arrest in G1 phase. Survival under stressful conditions and the onset of sexual development required the AGC kinase Gad8 (23), which is homologous to S. cerevisiae kinases Ypk1 and 2. gad8 is a high-copy suppressor of sterility and temperature-sensitive growth of tor1-deficient strain. (High-copy suppressors are genes that, when present at high copy number, rescue phenotypes or lethality associated with mutation or deletion of another gene.) Yeast deficient in gad8 cannot grow under stressful conditions, do not arrest in G1 phase of the cell cycle, and fail to mate, which are phenotypes similar to those of tor1 cells. Because overexpression of tor1 failed to suppress the defect of gad8, it was concluded that gad8 is downstream of tor1. Tor1 and the PDK-like kinase Ksg1 activated Gad8 by phosphorylating the regulatory motif and activation loop, respectively (23).

Fig. 2.

Control of aging by Sch9 and other AGC family kinases. The model shows the network of signaling components that control stress responses and aging in S. cerevisiae. Availability of nutrients, sphingolipid metabolites, and exposure to stress factors tune the activity of AGC kinases PDK-like kinases Pkh1 and 2, PKB-like kinase Sch9, SGK-like kinases Ypk1 and 2, and Pkc1. Sch9 is involved in the coordination of several transcriptional responses, including induction of antioxidants, heat shock proteins, ribosomal protein genes, and ribosomal biogenesis, and genes that promote progression through the G1 phase of the cell cycle. These coordinated responses affect metabolic rates and biomass accumulation, and ultimately set the limits of chronological and replicative aging.

In S. cerevisiae, an AGC kinase–initiated phosphorylation cascade has also been identified. Here, the PDK-like kinases Pkh1 and Pkh2 phosphorylate Sch9 (PKB-like), Ypk1, and Ypk2 (SGK-like), and Pkc [a kinase of the mitogen-activated protein kinase (MAPK) cell wall integrity pathway]. These AGC kinases are stimulated by sphingolipid base phytosphingosine (PHS) (24). Ypk1 is a high-copy suppressor gene that allows growth when the synthesis of sphingolipids is inhibited (25).

Recently, the mammalian phosphatidylinositol 3-kinase (PI3K)-PKB pathway was reconstituted in various yeast strains, including the strains deficient (bearing individual deletions or temperature-sensitive mutations) in pkh1, pkh2, tor1, or tor2 (26). In this study, Pkh1 or Pkh2 phosphorylated mammalian PKB on a residue required for PKB kinase activity in a manner similar to that of the mammalian counterpart (PDK1), whereas yeast tor1 and tor2 genes were not required for efficient PKB phosphorylation. These data argue against oversimplification, as far as conservation of the TOR-PKB signaling module is concerned.

At least some downstream components of the insulin-like growth factor (IGF)–PKB pathway may be conserved in fungi. Indeed, on the basis of the sequence information, one may predict other similarities beyond that of the TOR-PDK module. Sequence analysis suggests that fungal genomes contain pten homologs; however, these have not been functionally characterized. Rheb, a guanosine triphosphatase (GTPase), and its activating protein, the TSC1-TSC2 complex, have counterparts in fungal genomes. In S. pombe, tsc1/2 and rhb1 are involved in sensing nitrogen starvation and regulate the expression of permeases, which control the uptake of amino acids (27, 28). Notably, the gene encoding the G1 cyclin pas1 was identified as a high-copy suppressor of the defect in amino acid uptake exhibited by tsc1/2 null cells. This cyclin functions downstream of the TSC1-TSC2 complex to promote G1 arrest, linking amino acid uptake and cell cycle progression (29). The existence of homologs of forkhead transcription factors in fungal genomes suggests that these may be regulated in yeast by AGC kinases in a manner similar to the regulation of FOXO transcription factors by PKB and SGK in mammals. The residues phosphorylated by metazoan PKB are not conserved in any of the four forkhead transcription factors encoded by the S. cerevisiae genome (fkh1, fkh2, fhl1, and hcm1) (26). Nevertheless, FOXO3-like forkhead domains are well conserved among fungi, nematodes, and mammals, including two serine residues that become phosphorylated in mammalian neurons by the mammalian Ste20-like kinases (MST) in response to oxidative stress (30). It is unknown whether these sites are functional phosphorylation sites in fungal species.

Despite the apparent similarities in AGC kinase signaling cascades between yeast and metazoans, there are also fundamental differences in yeast AGC kinase signaling. For example, in yeast, phosphoinositides do not activate Pkh1 and other AGC kinases (13). Those functions that depend on the well-characterized interconnections between p53, PKB, and TOR (31) cannot be conserved in fungi, because p53 and related genes seem to be absent in these organisms.

A Closer Look at the Fungal PKB Homologs

Several fungal AGC kinase genes similar to S. cerevisiae sch9 (14) have been characterized, including sck1 (32) and sck2 (33) in S. pombe and schA in Aspergillus nidulans (34). The sch9 gene was identified as high-copy suppressor of cdc25 temperature-sensitive allele and cAMP-PKA signaling defective mutants: When present at high copy number, it rescued defects of cdc25 and growth defects and lethality in strains lacking the components of the cAMP-PKA signaling pathway (14). Suppressor activity may be explained by the functional overlap in the activities of Sch9, PKA, and TOR. These three kinases together regulate the expression of genes encoding ribosomal proteins and the ribosomal biogenesis regulon, as well as genes involved in the heat shock response and cell cycle progression (1, 12, 35) (Fig. 2).

Sch9 in regulation of cell size and cell number

Strains that are deficient in sch9 form smaller colonies with fewer numbers of cells and cells of smaller size, grow at a slower rate, and show a threefold extension in chronological life span compared to wild-type yeast (12, 36, 37). In contrast, overexpression of sch9 results in a nontoxic large-cell phenotype. Cell-size regulation is dependent on the kinase activity of Sch9. This role of Sch9 in the regulation of cell size and cell number supports the hypothesis that sch9 is the S. cerevesiae homolog of PKB. Indeed, deficiency in PKB isoforms in mice and Drosophila leads to organ- and tissue-specific decreases in cell size and cell number, whereas overexpression of active PKB leads to increases in cell size and number (38, 39). Similarly, Dictyostelium discoideum pkbA cells have a severe defect in the formation of aggregates that is comparable to the formation of small colonies by sch9 cells (40).

The sch9 null strain is characterized by a prolonged G1 phase of the cell cycle, such that the doubling time of sch9 cells is greater than that of wild-type cells (12, 14). The connection between sch9 and cell cycle progression is also supported by the initial observation that sch9 is a suppressor of a temperature-sensitive mutation in cdc25, a guanine nucleotide-exchange factor for Ras1p and Ras2p that has been implicated in cell cycle progression and replicative aging (14). A link between Sch9 and replicative aging was also suggested by the identification of Sch9-regulated genes in a screen for the yeast with extended replicative life span. These genes include rpl31A and rpl6B, which encode components of the large ribosomal subunit (6). Deletion of sch9 leads to a decrease in ribosome biogenesis, which, in turn, correlates with and, in part, accounts for the increase in replicative life-span phenotypes (12).

A role for sch9 and sck1 and sck2 (the homologs of sch9 in S. pombe) in cell cycle progression was predicted from several studies in which DNA microarray analysis was used to identify cell cycle–regulated genes in S. cerevisiae and S. pombe (4, 41, 42). These findings are reminiscent of the cell cycle functions of AGC kinases in other organisms. Mammalian PKB controls cell cycle progression through the number of substrates (43). Systematic genome-wide RNAi analysis of cell cycle defects in cultured Drosophila cells revealed that decreased abundance of nutrient-sensitive AGC kinases PDK (Pk61C) or S6K led to an increase in the proportion of small cells in G1 phase of the cell cycle (3). These similarities between the yeast sch9 and the phenotypes of Drosophila AGC kinase mutants suggest that Sch9 may serve multiple roles that are performed by specific isoforms in higher organisms.

Systematic protein chip analysis of the kinase-substrate specificity of 119 yeast protein kinases, including Sch9, indicated that regulators of the cell cycle, such as the Nim1-related kinase Hsl1 and the serine-threonine kinase Ptk2, may be Sch9 substrates (44). Biochemical evidence implicating Sch9 in control of the cell cycle comes from high-throughput mass spectrometry analysis of protein-protein interactions in yeast (45). Two microtubule- and mitotic spindle–associated proteins, Ats1 and Kip1 (kinesin-related motor protein), associated with Sch9 (45). It is presently unknown whether Sch9 activity requires the PKB consensus motif. Systematic analysis of Sch9 kinase activity toward the candidate substrate proteins will shed light on the functions of this kinase in yeast and related kinases in other organisms. Better understanding of the Sch9 substrates should also provide insight into the mechanisms that link the cell cycle defects to the extended life span in sch9 cells.

Sch9 in the cellular response to stress

Stress resistance is a key feature of many long-lived mutants in various model organisms (46). In yeast, stress resistance is suppressed by activation of Sch9. In contrast, stress resistance is induced by sch9 deletion or inactivating mutations. The most convincing evidence implicating sch9 in stress resistance and longevity is that this gene was identified independently in several nonbiased screens for heat resistance, oxidative stress resistance, or chronological longevity (7, 47).

Sphingolipid metabolites have been implicated in resistance to heat and oxidation. Sphingolipid long-chain bases rapidly accumulate in the endoplasmic reticulum upon exposure of cells to heat stress and may mediate stress response signaling through activation of Sch9 by Pkh1 and Pkh2 (24) (Fig. 2).

The contribution of Sch9 to heat sensitivity is also supported by studies showing a genetic interaction between sch9 and the gene encoding heat shock factor 1 (Hsf1). A yeast strain carrying a deletion in the activation domain of HSF1 exhibits heat sensitivity and low abundance of the heat shock protein 90 (Hsp90) (48). Upon sch9 deletion, there is a suppression of high-temperature growth sensitivity in the strain with the deletion of the activation domain of heat shock factor 1 (HSF1) such that Hsp90 chaperone function was derepressed, increasing tolerance to Hsp90 inhibitors and facilitating Hsp90-dependent signaling pathways (48). A high-throughput proteomics study found that Sch9 formed a complex with Hsp82, which belongs to the Hsp90 family (Table 1) (Fig. 3) (45). A synthetic genetic interaction was also reported between sch9 and the gene encoding the Hsp90 co-chaperone Sse1 (49). Double deletion of sse1 and sch9 genes results in abnormally high PKA activity and also suppresses heat shock resistance at high temperatures (49).

Fig. 3.

Genetic interactions and protein-protein interactions that involve Sch9. The data of Table 1 and Sch9 entry of BIOGRID (35) ( were confirmed by data mining of ResNet Yeast 3 database integrated into PathwayStudio software (Ariadne Genomics Inc.). See;2006/352/re9/DC1 for an interactive version of the figure.

PKA and Sch9 regulate overlapping sets of target genes that are effectors of stress pathways. Therefore, it is not surprising that the activities of these two enzymes would be coordinated. In S. cerevesiae, PKA mediates stress responses by phosphorylating downstream targets (50) and exerting inhibitory effects on the nuclear shuttling and activity of transcription factors, such as Msn2 and Msn4, which are activators of numerous stress-response element (STRE)–regulated genes. The same stress-responsive genes are also implicated in chronological longevity and include those encoding heat shock proteins and manganese superoxide dismutase (Mn-SOD) (1, 7, 17, 50). Given that the heat shock proteins exhibit substantial age-specific decreases in abundance (51), it is tempting to speculate that converging mechanisms link regulation of expression of these genes with aging and stress resistance.

Nutrient-sensitive activity and localization of sch9

The effects of calorie restriction on aging are comparable to the phenotypes of sch9mutants in yeast, as well as PKB deficiency in Caenorhabditis elegans, Drosophila melanogaster, and other organisms. Both paradigms result in reduced signaling through nutrient-sensitive pathways and extension of replicative life span. In S. cerevisiae, the effect of calorie restriction seems to be mediated, at least in part, by decreased kinase activity of Sch9, and calorie restriction cannot further extend replicative life span in sch9 cells (6). Analysis of the localization of Sch9 suggests that it has a role as a nutrient sensor. During the log phase of growth, Sch9 is present throughout the cell, showing a granular pattern (52, 53) ( and enrichment at the vacuolar membrane (12). Notably, Sch9 enrichment at the vacuolar membrane disappeared in response to carbon starvation, pointing to possible connection between membrane localization and the kinase activity (12) (Fig. 1).

Sch9 in control of biosynthetic and catabolic pathways

The analysis of Sch9-associated proteins indicates that this kinase may be involved in regulation of DNA and protein synthesis and cellular metabolism (45) (Table 1) (Fig. 3). For example, identification of the Tfc3 subunit of the transcription initiation complex TFIIIC in a screen for interaction partners for Sch9 suggests that this kinase may be directly involved in transcriptional regulation. Histone Htb1 (H2B in mammals) was also identified in a complex with Sch9 in the proteome-wide screen for protein-protein interactions, and this is consistent with the well-established kinase activity of PKB homologs toward H2B (54). Htb1 is involved in activation of transcription, chromatin assembly, and postreplication DNA repair. Pdc2, a transcription factor that regulates glycolysis by controlling the synthesis of pyruvate decarboxylase, also interacts with Sch9, suggesting a role of Sch9 in storage and utilization of carbohydrates. Finally, protein synthesis may be also under direct control of Sch9. Sch9 forms a complex with, and, presumably, phosphorylates Pab1 and prionlike protein Sup35. Pab1 functions in association between the 5′ cap and 3′ mRNA polyadenylate [poly(A)] tail, and Sup35 functions in the termination of translation. Thus, Sch9 may be part of the mechanism that relays availability of nutrients to utilization of glucose and to the rates of DNA and protein synthesis.

Table 1.

Sch9-associated proteins in S. cerevisiae. The list was assembled from the data of proteome-wide protein-protein interaction studies (45). Genetic interactions that involve Sch9 are summarized at BIOGRID database (35).

Sch9 and protein degradation

One of the interesting outcomes of the proteome-wide analysis of protein-protein interactions in yeast (45) is the finding that Sch9 associates with Shp1, Cdc48, and Ufd1, which form a complex responsible for the recognition and targeting of ubiquitinated proteins to the proteasome for degradation (Table 1) (Fig. 3). What is the result of the interaction of Sch9 with this complex of the protein-degradation machinery? One possibility is that Sch9 itself is ubiquitinated and targeted for degradation by Shp1-Cdc48-Ufd1. An alternative scenario is that Sch9 phosphorylates some of the components of this complex to regulate its activity and connect protein degradation to the availability of nitrogen and amino acids. In this context, Sch9 may be part of the feedback mechanism connecting sensitivity to exogenous nitrogen and amino acids with supply of endogenous amino acids through proteasomal degradation. Such a phosphorylation-dependent mechanism might be able to tune the activity of the Shp1-Cdc48-Ufd1 complex by affecting either substrate recognition or its interaction with the proteasome. Negative feedback would imply relief of starvation by increasing the supply of nutrients through increased proteasomal degradation. Alternatively, a positive-feedback mechanism might serve to maintain higher metabolic rates, so that the rate of degradation is increased to accommodate increased rates of protein synthesis.

Sch9 homologs in other fungi.

Two S. pombe genes, sck1 and sck2, are closely related to sch9 (32, 33). Both genes, when overexpressed, suppress PKA deficiency (slow growth, short cell morphology, and derepression of mating and sporulation). Functional characterization of sck1, sck2, sck1/pka1, and sck2/pka1 cells showed that these gene deletions affected growth and spore germination to various degrees (33). Searches of genomic databases indicate that the homologs of sch9 and sck1/2 are present in other fungal genomes. These genes would be expected to suppress mutations resulting in reduced PKA activity. In contrast to the nonviability of PKA mutants in S. cerevisiae, A. nidulans pkaA strain shows only a mild phenotype characterized by slower kinetics of spore germination. A. nidulans schA deletion does not appear to alter growth (34). Cells that are null for schA have normal colony morphology and normal kinetics of germ tube outgrowth. However, there is an additional delay in germ-tube formation in the schA/pkaA double-mutant compared to the pka strain, indicating that both kinases are involved in germination.

Note added in proof: Despite the differences described above, evidence is emerging for roles for AGC kinases in other yeast species in aging. Rokeach and co-workers demonstrated that AGC kinases Pka1 and Sck2 regulate chronological aging in S. pombe. Pka1/sck2 cells have additional increases in life span and exhibit increased resistance to both oxidative stress and heat shock (55).


Sch9 and other AGC kinases are involved in signaling pathways that sense nutrients and finely tune programs of gene expression, including those that control cell cycle progression, stress resistance, and protein synthesis (5). On the basis of overall similarities in primary sequences, domain organization, epistatic interactions, and functions of Sch9 and PKB kinases in other organisms, it is tempting to speculate that Sch9 should be considered as the yeast PKB ortholog. However, certain differences in domain organization, mode of activation, and functions argue against such a conclusion. Notably, molecular components of Sch9 and PKB pathways are only partially conserved in yeast and metazoans. Whereas human PDK1 cDNA appears to complement growth defects in pkh1/pkh2 null yeast cells (13), so far there is no evidence that sch9 cDNA complements pkb-deficient mutants in other organisms and vice versa, and that metazoan PKB cDNAs complement defects of sch9 cells. Given the selective binding and activation of yeast AGC kinases by sphingolipids versus phospholipids, and protein-localization patterns, it is questionable whether the lipid-binding C2 and PH domains of these proteins are functionally interchangeable between Sch9 and PKB homologs. Comparative genome- and proteome-wide analyses of Sch9 and PKB substrates and their interaction partners will lead to more conclusive evidence for classifying Sch9 and other fungal AGC kinases.


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