A sterol-binding protein integrates endosomal lipid metabolism with TOR signaling and nitrogen sensing.

Kes1, and other oxysterol-binding protein superfamily members, are involved in membrane and lipid trafficking through trans-Golgi network (TGN) and endosomal systems. We demonstrate that Kes1 represents a sterol-regulated antagonist of TGN/endosomal phosphatidylinositol-4-phosphate signaling. This regulation modulates TOR activation by amino acids and dampens gene expression driven by Gcn4, the primary transcriptional activator of the general amino acid control regulon. Kes1-mediated repression of Gcn4 transcription factor activity is characterized by nonproductive Gcn4 binding to its target sequences, involves TGN/endosome-derived sphingolipid signaling, and requires activity of the cyclin-dependent kinase 8 (CDK8) module of the enigmatic "large Mediator" complex. These data describe a pathway by which Kes1 integrates lipid metabolism with TORC1 signaling and nitrogen sensing.

The Tor and cAMP-dependent protein kinase signaling pathways coordinately control autophagy in Saccharomyces cerevisiae.

Macroautophagy (hereafter autophagy) is a conserved membrane trafficking pathway responsible for the turnover of cytosolic protein and organelles during periods of nutrient deprivation. This pathway is also linked to a number of processes important for human health, including tumor suppression, innate immunity and the clearance of protein aggregates. As a result, there is tremendous interest in autophagy as a potential point of therapeutic intervention in a variety of pathological states. To achieve this goal, it is imperative that we develop a thorough understanding of the normal regulation of this process in eukaryotic cells. The Tor protein kinases clearly constitute a key element of this control as Tor activity inhibits this degradative process in all organisms examined, from yeast to man. Here, we discuss recent work indicating that the cAMP-dependent protein kinase (PKA) also plays a critical role in controlling autophagy in the budding yeast, Saccharomyces cerevisiae. A model describing how PKA activity might influence this degradative process, and how this control might be integrated with that of the Tor pathway, is presented.

The Tor and PKA signaling pathways independently target the Atg1/Atg13 protein kinase complex to control autophagy.

Macroautophagy (or autophagy) is a conserved degradative pathway that has been implicated in a number of biological processes, including organismal aging, innate immunity, and the progression of human cancers. This pathway was initially identified as a cellular response to nutrient deprivation and is essential for cell survival during these periods of starvation. Autophagy is highly regulated and is under the control of a number of signaling pathways, including the Tor pathway, that coordinate cell growth with nutrient availability. These pathways appear to target a complex of proteins that contains the Atg1 protein kinase. The data here show that autophagy in Saccharomyces cerevisiae is also controlled by the cAMP-dependent protein kinase (PKA) pathway. Elevated levels of PKA activity inhibited autophagy and inactivation of the PKA pathway was sufficient to induce a robust autophagy response. We show that in addition to Atg1, PKA directly phosphorylates Atg13, a conserved regulator of Atg1 kinase activity. This phosphorylation regulates Atg13 localization to the preautophagosomal structure, the nucleation site from which autophagy pathway transport intermediates are formed. Atg13 is also phosphorylated in a Tor-dependent manner, but these modifications appear to occur at positions distinct from the PKA phosphorylation sites identified here. In all, our data indicate that the PKA and Tor pathways function independently to control autophagy in S. cerevisiae, and that the Atg1/Atg13 kinase complex is a key site of signal integration within this degradative pathway.

Distal recognition sites in substrates are required for efficient phosphorylation by the cAMP-dependent protein kinase.

Protein kinases are important mediators of signal transduction in eukaryotic cells, and identifying the substrates of these enzymes is essential for a complete understanding of most signaling networks. In this report, novel substrate-binding variants of the cAMP-dependent protein kinase (PKA) were used to identify substrate domains required for efficient phosphorylation in vivo. Most wild-type protein kinases, including PKA, interact only transiently with their substrates. The substrate domains identified were distal to the sites of phosphorylation and were found to interact with a C-terminal region of PKA that was itself removed from the active site. Only a small set of PKA alterations resulted in a stable association with substrates, and the identified residues were clustered together within the hydrophobic core of this enzyme. Interestingly, these residues stretched from the active site of the enzyme to the C-terminal substrate-binding domain identified here. This spatial organization is conserved among the entire eukaryotic protein kinase family, and alteration of these residues in a second, unrelated protein kinase also resulted in a stable association with substrates. In all, this study identified distal sites in PKA substrates that are important for recognition by this enzyme and suggests that the interaction of these domains with PKA might influence specific aspects of substrate binding and/or release.

Identifying Atg1 substrates: four means to an end.

Autophagy is essential for normal development and the response to a variety of stress conditions, including nutrient deprivation. The Atg1 serine/threonine-specific protein kinase appears to be a key regulator of many forms of autophagy that occur in eukaryotic cells. Therefore, to fully understand the regulation of autophagy, it is essential that we identify the signaling pathways regulating Atg1 and the physiologically-relevant targets of Atg1 kinase activity. Although some progress has been made on the former question, no Atg1 substrates important for autophagy have yet been identified. In this review, we discuss four different experimental strategies that should facilitate the search for Atg1 substrates.

Coiled coil structures and transcription: an analysis of the S. cerevisiae coilome.

The alpha-helical coiled coil is a simple but widespread motif that is an integral feature of many cellular structures. Coiled coils allow monomeric building blocks to form complex assemblages that can serve as molecular motors and springs. Previous parametrically delimited analyses of the distribution of coiled coils in the genomes of diverse organisms, including Escherichia coli, Saccharomyces cerevisiae, Arabidopsis thaliana, Caenorhabditis elegans and Homo sapiens, have identified conserved biological processes that make use of this versatile motif. Here we present a comprehensive inventory of the set of coiled coil proteins in S. cerevisiae by combining multiple coiled coil prediction algorithms with extensive literature curation. Our analysis of this set of proteins, which we call the coilome, reveals a wider role for this motif in transcription than was anticipated, particularly with respect to the category that includes nucleocytoplasmic shuttling factors involved in transcriptional regulation. We also show that the constitutively nuclear yeast transcription factor Gcr1 is homologous to the mammalian transcription factor MLL3, and that two coiled coil domains conserved between these homologs are important for Gcr1 dimerization and function. These data support the hypothesis that coiled coils are required to assemble structures essential for proper functioning of the transcriptional machinery.

Using substrate-binding variants of the cAMP-dependent protein kinase to identify novel targets and a kinase domain important for substrate interactions in Saccharomyces cerevisiae.

Protein kinases mediate much of the signal transduction in eukaryotic cells and defects in kinase function are associated with a variety of human diseases. To understand and correct these defects, we will need to identify the physiologically relevant substrates of these enzymes. The work presented here describes a novel approach to this identification process for the cAMP-dependent protein kinase (PKA) in Saccharomyces cerevisiae. This approach takes advantage of two catalytically inactive PKA variants, Tpk1K336A/H338A and Tpk1R324A, that exhibit a stable binding to their substrates. Most protein kinases, including the wild-type PKA, associate with substrates with a relatively low affinity. The binding observed here was specific to substrates and was dependent upon PKA residues known to be important for interactions with peptide substrates. The general utility of this approach was demonstrated by the ability to identify both previously described and novel PKA substrates in S. cerevisiae. Interestingly, the positions of the residues altered in these variants implicated a particular region within the PKA kinase domain, corresponding to subdomain XI, in the binding and/or release of protein substrates. Moreover, the high conservation of the residues altered and, in particular, the invariant nature of the R324 position suggest that this approach might be generally applicable to other protein kinases.

Increased phosphoglucomutase activity suppresses the galactose growth defect associated with elevated levels of Ras signaling in S. cerevisiae.

The Ras proteins regulate many aspects of cell growth in the budding yeast, Saccharomyces cerevisiae, via the cAMP-dependent protein kinase (PKA). Here, we show that a RAS2(val19) mutant that exhibits elevated levels of Ras/PKA signaling activity is unable to grow on media with galactose as the sole source of carbon. This growth defect was due, at least in part, to a defect in the expression of genes, like GAL1, that encode enzymes needed for the metabolism of galactose. This growth defect was used as the basis for a genetic screen for dosage suppressors of the RAS2(val19) mutant. This screen identified two genes, PGM1 and PCM1, that encode proteins with phosphoglucomutase activity. This activity is responsible for converting the glucose-1-phosphate produced during the metabolism of galactose to glucose-6-phosphate, a precursor that can be metabolized via the glycolytic pathway. The over-expression of PGM1 was not able to suppress any other RAS2(val19) phenotype or the galactose growth defect associated with a gal1Delta mutant. Overall, these data suggest that the elevated levels of phosphoglucomutase activity allow for the more efficient utilization of the limiting levels of glucose-1-phosphate that are present in the RAS2(val19) mutant.

An evolutionary proteomics approach identifies substrates of the cAMP-dependent protein kinase.

Protein kinases are important mediators of much of the signal transduction that occurs in eukaryotic cells. Unfortunately, the identification of protein kinase substrates has proven to be a difficult task, and we generally know few, if any, of the physiologically relevant targets of any particular kinase. Here, we describe a sequence-based approach that simplified this substrate identification process for the cAMP-dependent protein kinase (PKA) in Saccharomyces cerevisiae. In this method, the evolutionary conservation of all PKA consensus sites in the S. cerevisiae proteome was systematically assessed within a group of related yeasts. The basic premise was that a higher degree of conservation would identify those sites that are functional in vivo. This method identified 44 candidate PKA substrates, 5 of which had been described. A phosphorylation analysis showed that all of the identified candidates were phosphorylated by PKA and that the likelihood of phosphorylation was strongly correlated with the degree of target site conservation. Finally, as proof of principle, the activity of one particular target, Atg1, a key regulator of autophagy, was shown to be controlled by PKA phosphorylation in vivo. These data therefore suggest that this evolutionary proteomics approach identified a number of PKA substrates that had not been uncovered by other methods. Moreover, these data show how this approach could be generally used to identify the physiologically relevant occurrences of any protein motif identified in a eukaryotic proteome.

Reverse recruitment: the Nup84 nuclear pore subcomplex mediates Rap1/Gcr1/Gcr2 transcriptional activation.

The recruitment model for gene activation presumes that DNA is a platform on which the requisite components of the transcriptional machinery are assembled. In contrast to this idea, we show here that Rap1/Gcr1/Gcr2 transcriptional activation in yeast cells occurs through a large anchored protein platform, the Nup84 nuclear pore subcomplex. Surprisingly, Nup84 and associated subcomplex components activate transcription themselves in vivo when fused to a heterologous DNA-binding domain. The Rap1 coactivators Gcr1 and Gcr2 form an important bridge between the yeast nuclear pore complex and the transcriptional machinery. Nucleoporin activation may be a widespread eukaryotic phenomenon, because it was first detected as a consequence of oncogenic rearrangements in acute myeloid leukemia and related syndromes in humans. These chromosomal translocations fuse a homeobox DNA-binding domain to the human homolog (hNup98) of a transcriptionally active component of the yeast Nup84 subcomplex. We conclude that Rap1 target genes are activated by moving to contact compartmentalized nuclear assemblages, rather than through recruitment of the requisite factors to chromatin by means of diffusion. We term this previously undescribed mechanism "reverse recruitment" and discuss the possibility that it is a central feature of eukaryotic gene regulation. Reverse recruitment stipulates that activators work by bringing the DNA to an nuclear pore complex-tethered platform of assembled transcriptional machine components.