Roles of Candida albicans Mig1 and Mig2 in glucose repression, pathogenicity traits, and SNF1 essentiality.

Metabolic adaptation is linked to the ability of the opportunistic pathogen Candida albicans to colonize and cause infection in diverse host tissues. One way that C. albicans controls its metabolism is through the glucose repression pathway, where expression of alternative carbon source utilization genes is repressed in the presence of its preferred carbon source, glucose. Here we carry out genetic and gene expression studies that identify transcription factors Mig1 and Mig2 as mediators of glucose repression in C. albicans. The well-studied Mig1/2 orthologs ScMig1/2 mediate glucose repression in the yeast Saccharomyces cerevisiae; our data argue that C. albicans Mig1/2 function similarly as repressors of alternative carbon source utilization genes. However, Mig1/2 functions have several distinctive features in C. albicans. First, Mig1 and Mig2 have more co-equal roles in gene regulation than their S. cerevisiae orthologs. Second, Mig1 is regulated at the level of protein accumulation, more akin to ScMig2 than ScMig1. Third, Mig1 and Mig2 are together required for a unique aspect of C. albicans biology, the expression of several pathogenicity traits. Such Mig1/2-dependent traits include the abilities to form hyphae and biofilm, tolerance of cell wall inhibitors, and ability to damage macrophage-like cells and human endothelial cells. Finally, Mig1 is required for a puzzling feature of C. albicans biology that is not shared with S. cerevisiae: the essentiality of the Snf1 protein kinase, a central eukaryotic carbon metabolism regulator. Our results integrate Mig1 and Mig2 into the C. albicans glucose repression pathway and illuminate connections among carbon control, pathogenicity, and Snf1 essentiality

Studies of molecular mechanisms integrating carbon metabolism and growth in plants

Plants use light energy, carbon dioxide and water to produce sugars and other carbohydrates, which serve as stored energy reserves and as building blocks for biosynthetic reactions. Supply of light is variable and plants have evolved means to adjust their growth and development accordingly. An increasing body of evidence suggests that the basic mechanisms for sensing and signaling energy availability in eukaryotes are evolutionary conserved and thus shared between plants, animals and fungi. I have used different experimental approaches that take advantage of findings from other eukaryotes in studying carbon and energy metabolism in plants. In the first part, I developed a novel screening procedure in yeast aimed at isolating cDNAs from other organisms encoding proteins with a possible function in sugar sensing or signaling. The feasibility of the method was confirmed by the cloning of a cDNA from Arabidopsis thaliana encoding a new F-box protein named AtGrh1, which is related to the yeast Grr1 protein that is involved in glucose repression. In the second part of the study, plant homologues of key components in the yeast glucose repression pathway were cloned and characterized in the moss Physcomitrella patens, in which gene function can be studied by gene targeting. We first cloned PpHXK1 which was shown to encode a chloroplast localized hexokinase representing a previously overlooked class of plant hexokinases with an N-terminal chloroplast transit peptide. Significantly, PpHxk1 is the major hexokinase in Physcomitrella, accounting for 80% of the glucose phosphorylating activity. A knockout mutant deleted for PpHXK1 exhibits a complex phenotype affecting growth, development and sensitivities to plant hormones. I also cloned and characterized two closely related Physcomitrella genes, PpSNF1a and PpSNF1b, encoding type 1 Snf1-related kinases. A double knockout mutant for these genes was viable even though it lacks detectable Snf1-like kinase activity. The mutant suffers from pleiotropic phenotypes which may reflect a constitutive high energy growth mode. Significantly, the double mutant requires constant high light and is therefore unable to grow in a normal day/night light cycle. These findings are consistent with the proposed role of the Snf1-related kinases as energy gauges which are needed to recognize and respond to low energy conditions

Dissection of GTPase activating proteins reveals functional asymmetry in the COPI coat of budding yeast.

The Arf GTPase controls formation of the COPI vesicle coat. Recent structural models of COPI revealed the positioning of two Arf1 molecules in contrasting molecular environments. Each of these pockets for Arf1 is expected to also accommodate an Arf GTPase-activating protein (ArfGAP). Structural evidence and protein interactions observed between isolated domains indirectly suggests that each niche may preferentially recruit one of the two ArfGAPs known to affect COPI, Gcs1/ArfGAP1 and Glo3/ArfGAP2/3, although only partial structures are available. The functional role of the unique non-catalytic domain of either ArfGAP has not been integrated into the current COPI structural model. Here, we delineate key differences in the consequences of triggering GTP hydrolysis via the activity of one versus the other ArfGAP. We demonstrate that Glo3/ArfGAP2/3 specifically triggers Arf1 GTP hydrolysis impinging on the stability of the COPI coat. We show that the yeast homologue of AMP kinase, Snf1, phosphorylates the region of Glo3 that is critical for this effect and thereby regulates its function in the COPI-vesicle cycle. Our results revise the model of ArfGAP function in the molecular context of COPI

Role of Protein Phosphatase Reg2-Glc7 in the Regulation of the Yeast Stress Response Kinase, Snf1

Kinases of the AMP-activated protein kinase (AMPK) family are conserved in eukaryotes and play central roles in responses to reduced energy availability. AMPK, nicknamed the “fuel gauge” of the cell, monitors cellular energy status via the ratio of AMP to ATP nucleotides. AMPK restores energy homeostasis by reducing energy “spending” and increasing energy “income”. Correspondingly, defects in AMPK signaling have been implicated in diseases including type II diabetes, obesity, and cancer. In yeast, the AMPK homolog is Snf1 protein kinase. Glucose is the preferred carbon/energy source of yeast, and thus limitation for glucose similarly activates Snf1. Snf1 activation requires phosphorylation of its T-loop threonine (Thr210) by upstream kinases. When glucose is abundant, Snf1 is inhibited by Thr210 dephosphorylation. The latter involves the function of type 1 protein phosphatase Glc7, which is targeted to Snf1 by a regulatory subunit, Reg1. The reg1 mutation causes increased Snf1 activity and mimics various aspects of glucose limitation, including slower growth. Reg2 is another Glc7 regulatory subunit encoded by a paralogous gene, REG2. The goal of our study was to determine if Reg2 has a role in Snf1 regulation. Indeed, we have found that Reg2 contributes to Snf1 Thr210 dephosphorylation. Consistent with this role, Reg2 interacts with wild-type Snf1 but not with non-phosphorylatable Snf1-T210A. Additionally, the ability of Reg2 to regulate Snf1 depends on the Reg2-Glc7 interaction. Reg2 accumulation increases in a Snf1-dependent manner during prolonged glucose deprivation, and glucose-starved cells lacking Reg2 exhibit delayed Snf1 Thr210 dephosphorylation and slower growth recovery upon glucose replenishment. Accordingly, cells lacking Reg2 are outcompeted by wild-type cells in the course of several glucose starvation/replenishment cycles. Collectively, our results support a model in which Reg2-Glc7 contributes to the negative control of Snf1 in response to glucose re-feeding after prolonged starvation. The competitive growth advantage provided by Reg2 underscores the evolutionary significance of this paralog for S. cerevisiae

Mitochondrial Voltage-Dependent Anion Channel Protein Por1 Positively Regulates the Nuclear Localization of Saccharomyces cerevisiae AMP-Activated Protein Kinase

ABSTRACT Snf1 protein kinase of the yeast Saccharomyces cerevisiae is a member of the highly conserved eukaryotic AMP-activated protein kinase (AMPK) family, which is involved in regulating responses to energy limitation. Under conditions of carbon/energy stress, such as during glucose depletion, Snf1 is catalytically activated and enriched in the nucleus to regulate transcription. Snf1 catalytic activation requires phosphorylation of its conserved activation loop threonine (Thr210) by upstream kinases. Catalytic activation is also a prerequisite for Snf1’s subsequent nuclear enrichment, a process that is mediated by Gal83, one of three alternate β-subunits of the Snf1 kinase complex. We previously reported that the mitochondrial voltage-dependent anion channel (VDAC) proteins Por1 and Por2 play redundant roles in promoting Snf1 catalytic activation by Thr210 phosphorylation. Here, we show that the por1Δ mutation alone, which by itself does not affect Snf1 Thr210 phosphorylation, causes defects in Snf1 and Gal83 nuclear enrichment and Snf1’s ability to stimulate transcription. We present evidence that Por1 promotes Snf1 nuclear enrichment by promoting the nuclear enrichment of Gal83. Overexpression of Por2, which is not believed to have channel activity, can suppress the localization and transcription activation defects of the por1Δ mutant, suggesting that the regulatory role played by Por1 is separable from its channel function. Thus, our findings expand the positive roles of the yeast VDACs in carbon/energy stress signaling upstream of Snf1. Since AMPK/Snf1 and VDAC proteins are conserved in evolution, our findings in yeast may have implications for AMPK regulation in other eukaryotes, including humans. IMPORTANCE AMP-activated protein kinases (AMPKs) sense energy limitation and regulate transcription and metabolism in eukaryotes from yeast to humans. In mammals, AMPK responds to increased AMP-to-ATP or ADP-to-ATP ratios and is implicated in diabetes, heart disease, and cancer. Mitochondria produce ATP and are generally thought to downregulate AMPK. Indeed, some antidiabetic drugs activate AMPK by affecting mitochondrial respiration. ATP release from mitochondria is mediated by evolutionarily conserved proteins known as voltage-dependent anion channels (VDACs). One would therefore expect VDACs to serve as negative regulators of AMPK. However, our experiments in yeast reveal the existence of an opposite relationship. We previously showed that Saccharomyces cerevisiae VDACs Por1 and Por2 positively regulate AMPK/Snf1 catalytic activation. Here, we show that Por1 also plays an important role in promoting AMPK/Snf1 nuclear localization. Our counterintuitive findings could inform research in areas ranging from diabetes to cancer to fungal pathogenesis

Prolonged Glucose Deprivation Sensitizes Snf1 to Negative Regulation By PKA to Delay Entry into Quiescence

AMPK, the fuel gauge of the cell, and its upstream kinase, LKB1, have been implicated in cancer prevention and stress response associated with energy exhaustion. In the yeast Saccharomyces cerevisiae, Snf1 is the ortholog of mammalian AMPK. In S. cerevisiae, Snf1 is activated by phosphorylation of its T–loop at Thr210, primarily by its upstream kinase Sak1, in absence of the preferred carbon source, glucose, or during some other stress responses. Cyclic AMP–dependent protein kinase A, PKA, is involved in nutrient signaling largely antagonistically to Snf1. Using yeast strains of the Sigma 1278b genetic background, which have a high basal level of cAMP signaling, PKA was previously suggested to downregulate Snf1 by a mechanism that involves phosphorylation of Sak1 on two consensus PKA recognition sites in the non–dashcatalytic C-terminal domain. Sequence analysis suggests that Snf1 or its immediate regulators are also targets for negative regulation by PKA in other genetic lineages of S. cerevisiae and even in other yeast species. Here, we have investigated the possible existence of an antagonistic relationship between PKA and Snf1 in another S. cerevisiae strain lineage, W303, which has a relatively low basal level of cAMP signaling. In addition to short-term glucose limitation, we monitored Snf1 activation under conditions of long–term carbon stress that normally leads to exit from the mitotic cycle and entry into a quiescent state. We observed that W303 ira1 mutant cells with increased PKA signaling have significantly reduced levels of Snf1 activation after long-term carbon stress. The quiescence-associated trait of stress resistance, specifically heat-shock survival, was also evaluated. W303 lacking Snf1 or with significantly higher levels of PKA activity due to the ira1 mutation, did not acquire normal heat-shock resistance, suggesting failure to enter quiescence. This suggests that downregulation of Snf1 represents one mechanism by which PKA inhibits entry into quiescence. Since the ability to enter quiescence offers significant evolutionary advantages, similar relationships between PKA and Snf1 are likely to exist in various fungal species. Moreover, since PKA and Snf1–AMPK are conserved in eukaryotes from yeast to humans, the PKA–AMPK pathway could also regulate quiescence as it pertains to development and tumorigenesis

Regulation of plant energy signaling by components of the abscisic acid pathway

"The capacity to sense and react to fluctuations in nutrient availability is crucial for the survival of all living organisms. In eukaryotes, two highly evolutionarily conserved protein complexes, the Snf1/AMPK/SnRK1 and the TOR protein kinases, play an essential role in the control of energy homeostasis. Low-energy conditions activate the Snf1/AMPK/SnRK1 system, which thereby initiates a transcriptional and metabolic reprogramming to favor catabolic (energy producing) over anabolic (energy consuming) processes and ultimately restore energy homeostasis. One of the main targets of Snf1/AMPK/SnRK1 is the growth- promoting TOR kinase, which is inhibited in conditions of energy deficit that cannot sustain growth. Snf1/AMPK/SnRK1 and TOR constitute the Snf1/AMPK/SnRK1-TOR functional axis, which translates the cellular energy/nutritional status into growth outputs. The capacity to sense the energy status enables the Snf1/AMPK/SnRK1-TOR axis to react to a diversity of stress conditions that impinge on primary energy metabolism. (...)

MELK-a conserved kinase: functions, signaling, cancer, and controversy.

Maternal embryonic leucine zipper kinase (MELK) is a highly conserved serine/threonine kinase initially found to be expressed in a wide range of early embryonic cellular stages, and as a result has been implicated in embryogenesis and cell cycle control. Recent evidence has identified a broader spectrum of tissue expression pattern for this kinase than previously appreciated. MELK is expressed in several human cancers and stem cell populations. Unique spatial and temporal patterns of expression within these tissues suggest that MELK plays a prominent role in cell cycle control, cell proliferation, apoptosis, cell migration, cell renewal, embryogenesis, oncogenesis, and cancer treatment resistance and recurrence. These findings have important implications for our understanding of development, disease, and cancer therapeutics. Furthermore understanding MELK signaling may elucidate an added dimension of stem cell control