Glucose starvation-induced turnover of the yeast glucose transporter Hxt1

Background

The budding yeast Saccharomyces cerevisiae possesses multiple glucose transporters with different affinities for glucose that enable it to respond to a wide range of glucose concentrations. The steady-state levels of glucose transporters are regulated in response to changes in the availability of glucose. This study investigates the glucose regulation of the low affinity, high capacity glucose transporter Hxt1.

Methods and results

Western blotting and confocal microscopy were performed to evaluate glucose regulation of the stability of Hxt1. Our results show that glucose starvation induces endocytosis and degradation of Hxt1 and that this event requires End3, a protein required for endocytosis, and the Doa4 deubiquitination enzyme. Mutational analysis of the lysine residues in the Hxt1 N-terminal domain demonstrates that the two lysine residues, K12 and K39, serve as the putative ubiquitin-acceptor sites by the Rsp5 ubiquitin ligase. We also demonstrate that inactivation of PKA (cAMP-dependent protein kinase A) is needed for Hxt1 turnover, implicating the role of the Ras/cAMP-PKA glucose signaling pathway in the stability of Hxt1.

Conclusion and general significance

Hxt1, most useful when glucose is abundant, is internalized and degraded when glucose becomes depleted. Of note, the stability of Hxt1 is regulated by PKA, known as a positive regulator for glucose induction of HXT1 gene expression, demonstrating a dual role of PKA in regulation of Hxt1.     

Glucose Activates TORC2-Gad8 Protein via Positive Regulation of the cAMP/cAMP-dependent Protein Kinase A (PKA) Pathway and Negative Regulation of the Pmk1 Protein-Mitogen-activated Protein Kinase Pathway

The target of rapamycin (TOR) kinase belongs to the highly conserved eukaryotic family of phosphatidylinositol 3-kinase-related kinases. TOR proteins are found at the core of two evolutionary conserved complexes, known as TORC1 and TORC2. In fission yeast, TORC2 is dispensable for proliferation under optimal growth conditions but is required for starvation and stress responses. TORC2 has been implicated in a wide variety of functions; however, the signals that regulate TORC2 activity have so far remained obscure. TORC2 has one known direct substrate, the AGC kinase Gad8, which is related to AKT in human cells. Gad8 is phosphorylated by TORC2 at Ser-546 (equivalent to AKT Ser-473), leading to its activation. Here, we show that glucose is necessary and sufficient to induce Gad8 Ser-546 phosphorylation in vivo and Gad8 kinase activity in vitro. The glucose signal that activates TORC2-Gad8 is mediated via the cAMP/PKA pathway, a major glucose-sensing pathway. By contrast, Pmk1, similar to human extracellular signal-regulated kinases and a major stress-induced mitogen activated protein kinase (MAPK) in fission yeast, inhibits TORC2-dependent Gad8 phosphorylation and activation. Inhibition of TORC2-Gad8 also occurs in response to ionic or osmotic stress, in a manner dependent on the cAMP/PKA and Pmk1-MAPK signaling pathways. Our findings highlight the significance of glucose availability in regulation of TORC2-Gad8 and indicate a novel link between the cAMP/PKA, Pmk1/MAPK, and TORC2-Gad8 signaling.     

Chemoattractant stimulation of TORC2 is regulated by receptor/G protein–targeted inhibitory mechanisms that function upstream and independently of an essential GEF/Ras activation pathway in Dictyostelium

Global stimulation of Dictyostelium with different chemoattractants elicits multiple transient signaling responses, including synthesis of cAMP and cGMP, actin polymerization, activation of kinases ERK2, TORC2, and phosphatidylinositide 3-kinase, and Ras-GTP accumulation. Mechanisms that down-regulate these responses are poorly understood. Here we examine transient activation of TORC2 in response to chemically distinct chemoattractants, cAMP and folate, and suggest that TORC2 is regulated by adaptive, desensitizing responses to stimulatory ligands that are independent of downstream, feedback, or feedforward circuits. Cells with acquired insensitivity to either folate or cAMP remain fully responsive to TORC2 activation if stimulated with the other ligand. Thus TORC2 responses to cAMP or folate are not cross-inhibitory. Using a series of signaling mutants, we show that folate and cAMP activate TORC2 through an identical GEF/Ras pathway but separate receptors and G protein couplings. Because the common GEF/Ras pathway also remains fully responsive to one chemoattractant after desensitization to the other, GEF/Ras must act downstream and independent of adaptation to persistent ligand stimulation. When initial chemoattractant concentrations are immediately diluted, cells rapidly regain full responsiveness. We suggest that ligand adaptation functions in upstream inhibitory pathways that involve chemoattractant-specific receptor/G protein complexes and regulate multiple response pathways.     

Characterisation of glucose transport in Saccharomyces cerevisiae with plasma membrane vesicles (countertransport) and intact cells (initial uptake) with single Hxt1, Hxt2, Hxt3, Hxt4, Hxt6, Hxt7 or Gal2 transporters

The yeast glucose transporters Hxt1, Hxt2, Hxt3, Hxt4, Hxt6, Hxt7 and Gal2, individually expressed in an hxt1-7 null mutant strain, demonstrate the phenomenon of countertransport. Thus, these transporters, which are the most important glucose transporters in Saccharomyces cerevisiae, are facilitated diffusion transporters. Apparent K(m)-values from high to low affinity, determined from countertransport and initial-uptake experiments, respectively, are: Hxt6 0.9+/-0.2 and 1.4+/-0.1 mM, Hxt7 1.3+/-0.3 and 1.9+/-0.1 mM, Gal2 1.5 and 1.6+/-0.1 mM, Hxt2 2.9+/-0.3 and 4.6+/-0.3 mM, Hxt4 6.2+/-0.5 and 6.2+/-0.3 mM, Hxt3 28.6+/-6.8 and 34.2+/-3.2 mM, and Hxt1 107+/-49 and 129+/-9 mM. From both independent methods, countertransport and initial uptake, the same range of apparent K(m)-values was obtained for each transporter. In contrast to that in human erythrocytes, the facilitated diffusion transport mechanism of glucose in yeast was symmetric. Besides facilitated diffusion there existed in all single glucose transport mutants, except for the HXT1 strain, significant first-order behaviour.     

Localization of Ras signaling complex in budding yeast

In Saccharomyces cerevisiae, cAMP/pKA pathway plays a major role in metabolism, stress resistance and proliferation control. cAMP is produced by adenylate cyclase, which is activated both by Gpr1/Gpa2 system and Ras proteins, regulated by Cdc25/Sdc25 guanine exchange factors and Ira GTPase activator proteins. Recently, both Ras2 and Cdc25 RasGEF were reported to localize not only in plasma membrane but also in internal membranes. Here, the subcellular localization of Ras signaling complex proteins was investigated both by fluorescent tagging and by biochemical cell membrane fractionation on sucrose gradients. Although a consistent minor fraction of Ras signaling complex components was found in plasma membrane during exponential growth on glucose, Cdc25 appears to localize mainly on ER membranes, while Ira2 and Cyr1 are also significantly present on mitochondria. Moreover, PKA Tpk1 catalytic subunit overexpression induces Ira2 protein to move from mitochondria to ER membranes. These data confirm the hypothesis that different branches of Ras signaling pathways could involve different subcellular compartments, and that relocalization of Ras signaling complex components is subject to PKA control.     

Starvation-induced degradation of yeast hexose transporter Hxt7p is dependent on endocytosis, autophagy and the terminal sequences of the permease

The yeast high-affinity glucose transporters Hxt6p and Hxt7p are rapidly degraded during nitrogen starvation in the presence of high concentrations of fermentable carbon sources. Our results suggest that degradation is mainly due to the stimulation of general protein turnover and not caused by a mechanism specifically triggered by glucose. Analysis of Hxt6p/7p stability and cellular distribution in end4, aut2 and apg1 mutants indicates that Hxt7p is internalized by endocytosis, and autophagy is involved in the final delivery of Hxt7p to the vacuole for proteolytic degradation. Internalization and degradation of Hxt7p were blocked after truncation of its N-terminal hydrophilic domain. Nevertheless, this fully functional and stabilized hexose transporter could not maintain fermentation capacity of the yeast cells under starvation conditions, indicating a regulatory constraint on glucose uptake.     

Acetate regulation of spore formation is under the control of the Ras/cyclic AMP/protein kinase A pathway and carbon dioxide in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the Ras/cyclic AMP (cAMP)/protein kinase A (PKA) pathway is a nutrient-sensitive signaling cascade that regulates vegetative growth, carbohydrate metabolism, and entry into meiosis. How this pathway controls later steps of meiotic development is largely unknown. Here, we have analyzed the role of the Ras/cAMP/PKA pathway in spore formation by the meiosis-specific manipulation of Ras and PKA or by the disturbance of cAMP production. We found that the regulation of spore formation by acetate takes place after commitment to meiosis and depends on PKA and appropriate A kinase activation by Ras/Cyr1 adenylyl cyclase but not by activation through the Gpa2/Gpr1 branch. We further discovered that spore formation is regulated by carbon dioxide/bicarbonate, and an analysis of mutants defective in acetate transport (ady2Δ) or carbonic anhydrase (nce103Δ) provided evidence that these metabolites are involved in connecting the nutritional state of the meiotic cell to spore number control. Finally, we observed that the potential PKA target Ady1 is required for the proper localization of the meiotic plaque proteins Mpc70 and Spo74 at spindle pole bodies and for the ability of these proteins to initiate spore formation. Overall, our investigation suggests that the Ras/cAMP/PKA pathway plays a crucial role in the regulation of spore formation by acetate and indicates that the control of meiotic development by this signaling cascade takes places at several steps and is more complex than previously anticipated.     

Ksp1 kinase regulates autophagy via the target of rapamycin complex 1 (TORC1) pathway

Macroautophagy (hereafter autophagy) is a bulk degradation system conserved in all eukaryotes, which engulfs cytoplasmic components within double-membrane vesicles to allow their delivery to, and subsequent degradation within, the vacuole/lysosome. Autophagy activity is tightly regulated in response to the nutritional state of the cell and also to maintain organelle homeostasis. In nutrient-rich conditions, Tor kinase complex 1 (TORC1) is activated to inhibit autophagy, whereas inactivation of this complex in response to stress leads to autophagy induction; however, it is unclear how the activity of TORC1 is controlled to allow precise adjustments in autophagy activity. In this study, we performed genetic analyses in Saccharomyces cerevisiae to identify factors that regulate TORC1 activity. We determined that the Ksp1 kinase functions in part as a negative regulator of autophagy; deletion of KSP1 facilitated dephosphorylation of Atg13, a TORC1 substrate, which correlates with enhanced autophagy. These results suggest that Ksp1 down-regulates autophagy activity via the TORC1 pathway. The suppressive function of Ksp1 is partially activated by the Ras/cAMP-dependent protein kinase A (PKA), which is another negative regulator of autophagy. Our study therefore identifies Ksp1 as a new component that functions as part of the PKA and TORC1 signaling network to control the magnitude of autophagy.     

Ras-mutant Cancer Cells Display B-Raf Binding to Ras That Activates Extracellular Signal-regulated Kinase and Is Inhibited by Protein Kinase A Phosphorylation

The small G protein Ras regulates proliferation through activation of the mitogen-activated protein (MAP) kinase (ERK) cascade. The first step of Ras-dependent activation of ERK signaling is Ras binding to members of the Raf family of MAP kinase kinase kinases, C-Raf and B-Raf. Recently, it has been reported that in melanoma cells harboring oncogenic Ras mutations, B-Raf does not bind to Ras and does not contribute to basal ERK activation. For other types of Ras-mutant tumors, the relative contributions of C-Raf and B-Raf are not known. We examined non-melanoma cancer cell lines containing oncogenic Ras mutations and express both C-Raf and B-Raf isoforms, including the lung cancer cell line H1299 cells. Both B-Raf and C-Raf were constitutively bound to oncogenic Ras and contributed to Ras-dependent ERK activation. Ras binding to B-Raf and C-Raf were both subject to inhibition by the cAMP-dependent protein kinase PKA. cAMP inhibited the growth of H1299 cells and Ras-dependent ERK activation via PKA. PKA inhibited the binding of Ras to both C-Raf and B-Raf through phosphorylations of C-Raf at Ser-259 and B-Raf at Ser-365, respectively. These studies demonstrate that in non-melanocytic Ras-mutant cancer cells, Ras signaling to B-Raf is a significant contributor to ERK activation and that the B-Raf pathway, like that of C-Raf, is a target for inhibition by PKA. We suggest that cAMP and hormones coupled to cAMP may prove useful in dampening the effects of oncogenic Ras in non-melanocytic cancer cells through PKA-dependent actions on B-Raf as well as C-Raf.     

The Ras/cAMP-dependent protein kinase signaling pathway regulates an early step of the autophagy process in Saccharomyces cerevisiae

When faced with nutrient deprivation, Saccharomyces cerevisiae cells enter into a nondividing resting state, known as stationary phase. The Ras/PKA (cAMP-dependent protein kinase) signaling pathway plays an important role in regulating the entry into this resting state and the subsequent survival of stationary phase cells. The survival of these resting cells is also dependent upon autophagy, a membrane trafficking pathway that is induced upon nutrient deprivation. Autophagy is responsible for targeting bulk protein and other cytoplasmic constituents to the vacuolar compartment for their ultimate degradation. The data presented here demonstrate that the Ras/PKA signaling pathway inhibits an early step in autophagy because mutants with elevated levels of Ras/PKA activity fail to accumulate transport intermediates normally associated with this process. Quantitative assays indicate that these increased levels of Ras/PKA signaling activity result in an essentially complete block to autophagy. Interestingly, Ras/PKA activity also inhibited a related process, the cytoplasm to vacuole targeting (Cvt) pathway that is responsible for the delivery of a subset of vacuolar proteins in growing cells. These data therefore indicate that the Ras/PKA signaling pathway is not regulating a switch between the autophagy and Cvt modes of transport. Instead, it is more likely that this signaling pathway is controlling an activity that is required during the early stages of both of these membrane trafficking pathways. Finally, the data suggest that at least a portion of the Ras/PKA effects on stationary phase survival are the result of the regulation of autophagy activity by this signaling pathway.     

Antagonism of rat beta-cell voltage-dependent K+ currents by exendin 4 requires dual activation of the cAMP/protein kinase A and phosphatidylinositol 3-kinase signaling pathways

Antagonism of voltage-dependent K+ (Kv) currents in pancreatic beta-cells may contribute to the ability of glucagon-like peptide-1 (GLP-1) to stimulate insulin secretion. The mechanism and signaling pathway regulating these currents in rat beta-cells were investigated using the GLP-1 receptor agonist exendin 4. Inhibition of Kv currents resulted from a 20-mV leftward shift in the voltage dependence of steady-state inactivation. Blocking cAMP or protein kinase A (PKA) signaling (Rp-cAMP and H-89, respectively) prevented the inhibition of currents by exendin 4. However, direct activation of this pathway alone by intracellular dialysis of cAMP or the PKA catalytic subunit (cPKA) could not inhibit currents, implicating a role for alternative signaling pathways. A number of phosphorylation sites associated with phosphatidylinositol 3 (PI3)-kinase activation were up-regulated in GLP-1-treated MIN6 insulinoma cells, and the PI3 kinase inhibitor wortmannin could prevent antagonism of beta-cell currents by exendin 4. Antagonists of Src family kinases (PP1) and the epidermal growth factor (EGF) receptor (AG1478) also prevented current inhibition by exendin 4, demonstrating a role for Src kinase-mediated trans-activation of the EGF tyrosine kinase receptor. Accordingly, the EGF receptor agonist betacellulin could replicate the effects of exendin 4 in the presence of elevated intracellular cAMP. Downstream, the PKCzeta pseudosubstrate inhibitor could prevent current inhibition by exendin 4. Therefore, antagonism of beta-cell Kv currents by GLP-1 receptor activation requires both cAMP/PKA and PI3 kinase/PKCzeta signaling via trans-activation of the EGF receptor. This represents a novel dual pathway for the control of Kv currents by G protein-coupled receptors.     

The requirement of Ras and Rap1 for the activation of ERKs by cAMP, PACAP, and KCl in cerebellar granule cells

In cerebellar granule cells, the mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase (ERK) cascade mediates multiple functions, including proliferation, differentiation, and survival. In these cells, ERKs are activated by diverse stimuli, including cyclic adenosine monophosphate (cAMP), pituitary adenylate cyclase activating protein (PACAP), depolarization induced by elevated extracellular potassium (KCl), and the neurotrophin brain-derived neurotrophic factor. Extensive studies in neuronal cell lines have implicated the small G proteins Ras and Rap1 in the activation of ERKs by cAMP, PACAP, and KCl. However, the requirement of Ras and Rap1 in these pathways in cerebellar granule cells has not been addressed. In this study, we utilize multiple biochemical assays to determine the mechanisms of action and requirement of Ras and Rap1 in cultured cerebellar granule cells. We show that both Ras and Rap1 can be activated by cAMP or PACAP via protein kinase (PKA)-dependent mechanisms. KCl activation of Ras also required PKA. Using both adenoviral and transgenic approaches, we show that Ras plays a major role in ERK activation by cAMP, PACAP, and KCl, while Rap1 also mediates activation of a selective membrane-associated pool of ERKs. Furthermore, Rap1, but not Ras, activation by PKA appears to require the action of Src family kinases.     

Interaction of the E1A oncoprotein with Yak1p, a novel regulator of yeast pseudohyphal differentiation, and related mammalian kinases

The C-terminal portion of adenovirus E1A suppresses ras-induced metastasis and tumorigenicity in mammalian cells; however, little is known about the mechanisms by which this occurs. In the simple eukaryote Saccharomyces cerevisiae, Ras2p, the homolog of mammalian h-ras, regulates mitogen-activated protein kinase (MAPK) and cyclic AMP-dependent protein kinase A (cAMP/PKA) signaling pathways to control differentiation from the yeast form to the pseudohyphal form. When expressed in yeast, the C-terminal region of E1A induced pseudohyphal differentiation, and this was independent of both the MAPK and cAMP/PKA signaling pathways. Using the yeast two-hybrid system, we identified an interaction between the C-terminal region of E1A and Yak1p, a yeast dual-specificity serine/threonine protein kinase that functions as a negative regulator of growth. E1A also physically interacts with Dyrk1A and Dyrk1B, two mammalian homologs of Yak1p, and stimulates their kinase activity in vitro. We further demonstrate that Yak1p is required in yeast to mediate pseudohyphal differentiation induced by Ras2p-regulated signaling pathways. However, pseudohyphal differentiation induced by the C-terminal region of E1A is largely independent of Yak1p. These data suggest that mammalian Yak1p-related kinases may be targeted by the E1A oncogene to modulate cell growth.