Insulin signaling in the yeast Saccharomyces cerevisiae. 3. Induction of protein phosphorylation by human insulin

A low affinity insulin-binding protein in the plasma membrane of Saccharomyces cerevisiae has been identified recently (Müller, G., Rouveyre, N., Upshon, C., Gross, E., and Bandlow, W., preceding paper in this issue). Since the mammalian insulin receptor functions as a tyrosine kinase with autophosphorylation capacity, kinase studies were performed with the partially purified insulin-binding protein preparation. Incubation with [gamma-32P]ATP in vitro led to phosphorylation of the 53-kDa insulin-binding protein on serine but not on tyrosine residues. In addition, a 70-kDa polypeptide, copurified with the insulin-binding protein preparation, was tyrosine-phosphorylated under the same conditions. Phosphorylation of both proteins was enhanced by human insulin. These results obtained by immunoprecipitation and immunoblotting using specific anti-phosphoserine/threonine/tyrosine antibodies were confirmed by phosphoamino acid analysis of the individual immunoprecipitated and gel-purified 32P-labeled phosphoproteins. During gel filtration, the 53-kDa protein coeluted as a 300-kDa complex together with the 70-kDa phosphotyrosine-containing protein and was coimmunoprecipitated with the latter using an anti-phosphotyrosine antibody, strongly arguing for complex formation between the two proteins. The data presented raise the possibility that stimulation of glycogen synthesis by insulin in yeast is mediated by a 53-kDa insulin-binding protein and a 70-kDa phosphotyrosine-containing protein which are organized in a large plasma membrane-bound signaling complex. Elucidation of the function and molecular mode of interaction of these components in yeast may help to understand metabolic insulin signaling in mammalian cells.     

Insulin signaling in the yeast Saccharomyces cerevisiae. 2. Interaction of human insulin with a putative binding protein

A putative insulin-binding protein (Kd = 0.5 +/- 0.2 microM for human insulin) was partially purified from solubilized plasma membranes of Saccharomyces cerevisiaeby wheat germ agglutinin and insulin affinity chromatographies. The binding affinities of various mutant insulin analogues correlated well with their capacities to activate glycogen synthase and SNF1 kinase in glucose-induced yeast spheroplasts, the ranking of their relative efficacies in yeast and in isolated rat adipocytes being similar. Using a bifunctional cross-linker and two different experimental protocols, a 53-kDa polypeptide contained in the insulin-binding protein preparation was specifically affinity cross-linked to [125I]monoiodo[B26]insulin. The relative rankings of the insulin analogues with respect to inhibition of cross-linking and binding to the partially purified insulin-binding protein were identical. Incubation of intact yeast spheroplasts with [125I]monoiodo[AI4]insulin led to specific and time-dependent association of the radiolabeled insulin with the cell surface followed by its internalization and degradation. These processes were considerably delayed by low temperature and energy depletion of the spheroplasts, suggesting involvement of the ATP-dependent endosomal apparatus. These data provide evidence for the existence of a low-affinity insulin-binding protein in the plasma membrane of Saccharomyces cerevisiae.     

MAP kinase pathways in the yeast Saccharomyces cerevisiae

A cascade of three protein kinases known as a mitogen-activated protein kinase (MAPK) cascade is commonly found as part of the signaling pathways in eukaryotic cells. Almost two decades of genetic and biochemical experimentation plus the recently completed DNA sequence of the Saccharomyces cerevisiae genome have revealed just five functionally distinct MAPK cascades in this yeast. Sexual conjugation, cell growth, and adaptation to stress, for example, all require MAPK-mediated cellular responses. A primary function of these cascades appears to be the regulation of gene expression in response to extracellular signals or as part of specific developmental processes. In addition, the MAPK cascades often appear to regulate the cell cycle and vice versa. Despite the success of the gene hunter era in revealing these pathways, there are still many significant gaps in our knowledge of the molecular mechanisms for activation of these cascades and how the cascades regulate cell function. For example, comparison of different yeast signaling pathways reveals a surprising variety of different types of upstream signaling proteins that function to activate a MAPK cascade, yet how the upstream proteins actually activate the cascade remains unclear. We also know that the yeast MAPK pathways regulate each other and interact with other signaling pathways to produce a coordinated pattern of gene expression, but the molecular mechanisms of this cross talk are poorly understood. This review is therefore an attempt to present the current knowledge of MAPK pathways in yeast and some directions for future research in this area.     

Glutathione depletion in the yeast Saccharomyces cerevisiae

The effect of chosen compounds on the total glutathione (GSH) level in stationary cultures of S. cerevisiae was compared. 1-Chloro-2,4-dinitrobenzene, 1-fluoro-2,4-dinitrobenzene, maleimide, iodacetamide and allyl alcohol (1 mM), and menadione (0.5 mM) caused an almost complete GSH depletion during several minutes. Bromobenzoic acid and chloramine T (I mM), and daunomycin (60 mu M) induced a slower GSH decrease, down to 30-70% after 60 min. Paraquat (1 mM), CuSO(4) (0.5 mM) and cadmium acetate (1 mM) decreased glutathione level down to ca 70%. Diamide (0.5 mM), phenazine methosulphate, phenylhydrazine, acetylphenylhydrazine and H(2)O(2) (1 mM), and t-butyl hydroperoxide (2 mM) did not affect total GSH during 60-min exposure. There was no clear-cut dependence between the ability of various chemicals to deplete cellular GSH and their increased toxicity to a glutathione-poor mutant.     

Stimulation of glucose and amino acid transport and activation of the insulin signaling pathways by insulin lispro in L6 skeletal muscle cells

The monomeric insulin analogue insulin lispro (Lys B28, Pro B29) is a rapid-acting insulin with a shorter duration of activity than human regular insulin. This compound has the advantage of reducing early postprandial hyperglycemia and the accompanying late hypoglycemia, thereby improving overall blood glucose control. To date, all published studies of the functional properties of insulin lispro have been conducted in whole animals. This study aimed to characterize the cellular actions of insulin lispro and the signals it elicits in an insulin-sensitive muscle cell line, L6 cells. Comparing the cellular actions of insulin lispro with those of human regular insulin, a number of observations were made. (1) Insulin lispro stimulated glucose and amino acid transport into L6 myotubes with a dose dependency and time course virtually identical to those of human regular insulin. (2) Insulin lispro was as effective as human regular insulin in stimulating time-dependent phosphorylation of insulin receptor substrate 1 (IRS-1), p70 ribosomal S6 kinase, and two isoforms of mitogen-activated protein kinase (ERK1 and ERK2). (3) Insulin lispro's ability to induce the association of IRS-1 with the p85 subunit of phosphatidylinositol 3-kinase was similar to that of human regular insulin. (4) As with human regular insulin, 100 nmol of the fungal metabolite wortmannin completely inhibited insulin lispro stimulation of glucose uptake. We concluded that the cellular actions of insulin lispro are similar to those of human regular insulin with respect to glucose and amino acid uptake and that the biochemical signals elicited are also comparable.     

The PDE1-encoded low-affinity phosphodiesterase in the yeast Saccharomyces cerevisiae has a specific function in controlling agonist-induced cAMP signaling

The yeast Saccharomyces cerevisiae contains two genes, PDE1 and PDE2, which respectively encode a low-affinity and a high-affinity cAMP phosphodiesterase. The physiological function of the low-affinity enzyme Pde1 is unclear. We show that deletion of PDE1, but not PDE2, results in a much higher cAMP accumulation upon addition of glucose or upon intracellular acidification. Overexpression of PDE1, but not PDE2, abolished the agonist-induced cAMP increases. These results indicate a specific role for Pde1 in controlling glucose and intracellular acidification-induced cAMP signaling. Elimination of a putative protein kinase A (PKA) phosphorylation site by mutagenesis of serine252 into alanine resulted in a Pde1(ala252) allele that apparently had reduced activity in vivo. Its presence in a wild-type strain partially enhanced the agonist-induced cAMP increases compared with pde1Delta. The difference between the Pde1(ala252) allele and wild-type Pde1 was strongly dependent on PKA activity. In a RAS2(val19) pde2Delta background, the Pde1(ala252) allele caused nearly the same hyperaccumulation of cAMP as pde1Delta, while its expression in a PKA-attenuated strain caused the same reduction in cAMP hyperaccumulation as wild-type Pde1. These results suggest that serine252 might be the first target site for feedback inhibition of cAMP accumulation by PKA. We show that Pde1 is rapidly phosphorylated in vivo upon addition of glucose to glycerol-grown cells, and this activation is absent in the Pde1(ala252) mutant. Pde1 belongs to a separate class of phosphodiesterases and is the first member shown to be phosphorylated. However, in vitro the Pde1(ala252) enzyme had the same catalytic activity as wild-type Pde1, both in crude extracts and after extensive purification. This indicates that the effects of the S252A mutation are not caused by simple inactivation of the enzyme. In vitro phosphorylation of Pde1 resulted in a modest and variable increase in activity, but only in crude extracts. This was absent in Pde1(ala252), and phosphate incorporation was strongly reduced. Apparently, phosphorylation of Pde1 does not change its intrinsic activity or affinity for cAMP but appears to be important in vivo for protein-protein interaction or for targeting Pde1 to a specific subcellular location. The PKA recognition site is conserved in the corresponding region of the Schizosaccharomyces pombe and Candida albicans Pde1 homologues, possibly indicating a similar control by phosphorylation.     

Stabilization of microsatellite sequences by variant repeats in the yeast Saccharomyces cerevisiae

We examined the effect of a single variant repeat on the stability of a 51-base pair (bp) microsatellite (poly GT). We found that the insertion stabilizes the microsatellite about fivefold in wild-type strains. The stabilizing effect of the variant base was also observed in strains with mutations in the DNA mismatch repair genes pms1, msh2 and msh3, indicating that this effect does not require a functional DNA mismatch repair system. Most of the microsatellite alterations in the pms1, msh2 and msh3 strains were additions or deletions of single GT repeats, but about half of the alterations in the wild-type and msh6 strains were large (> 8 bp) deletions or additions.     

A novel signal transduction pathway in Saccharomyces cerevisiae defined by Snf3-regulated expression of HXT6

We show that cells deleted for SNF3, HXT1, HXT2, HXT3, HXT4, HXT6, and HXT7 do not take up glucose and cannot grow on media containing glucose as a sole carbon source. The expression of Hxt1, Hxt2, Hxt3, Hxt6, or Gal2 in these cells resulted in glucose transport and allowed growth on glucose media. In contrast, the expression of Snf3 failed to confer glucose uptake or growth on glucose. HXT6 is highly expressed on raffinose, low glucose, or nonfermentable carbon sources but is repressed in the presence of high concentrations of glucose. The maintenance of HXT6 glucose repression is strictly dependent on Snf3 and not on intracellular glucose. In snf3 delta cells expression of HXT6 is constitutive even when the entire repertoire of HXT genes is present and glucose uptake is abundant. In addition, glucose repression of HXT6 does not require glucose uptake by HXT1, HXT2, HXT3 or HXT4. We show that a signal transduction pathway defined by the Snf3-dependent hexose regulation of HXT6 is distinct from but also overlaps with general glucose regulation pathways in Saccharomyces cerevisiae. Finally, glucose repression of ADH2 and SUC2 is intact in snf3 delta hxt1 delta hxt2 delta hxt3 delta hxt4 delta hxt6 delta hxt7 delta gal2 cells, suggesting that the sensing and signaling mechanism for general glucose repression is independent from glucose uptake.     

Cascades of mammalian caspase activation in the yeast Saccharomyces cerevisiae

Caspases (aspartate-specific cysteine proteases) play a critical role in the execution of the mammalian apoptotic program. To address the regulation of human caspase activation, we used the yeast Saccharomyces cerevisiae, which is devoid of endogenous caspases. The apical procaspases, -8beta and -10, were efficiently processed and activated in yeast. Although protease activity, per se, was insufficient to drive cell death, caspase-10 activity had little effect on cell viability, whereas expression of caspase-8beta was cytotoxic. This lethal phenotype was abrogated by co-expression of the pan-caspase inhibitor, baculovirus p35, and by mutation of the active site cysteine of procaspase-8beta. In contrast, autoactivation of the executioner caspase-3 and -6 zymogens was not detected. Procaspase-3 activation required co-expression of procaspase-8 or -10. Surprisingly, activation of procaspase-6 required proteolytic activities other than caspase-8, -10, or -3. Caspase-8beta or -10 activity was insufficient to catalyze the maturation of procaspase-6. Moreover, a constitutively active caspase-3, although cytotoxic in its own right, was unable to induce the processing of wild-type procaspase-6 and vice versa. These results distinguish sequential modes of activation for different caspases in vivo and establish a yeast model system to examine the regulation of caspase cascades. Moreover, the distinct terminal phenotypes induced by various caspases attest to differences in the cellular targets of these apoptotic proteases, which may be defined using this system.     

Regulation of phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae by CDP-diacylglycerol

Regulation of the 45- and 55-kDa forms of Saccharomyces cerevisiae membrane-associated phosphatidylinositol (PI) 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase) by phospholipids was examined using Triton X-100/phospholipid-mixed micelles. CDP-diacylglycerol and phosphatidylglycerol inhibited 45-kDa PI 4-kinase activity in a dose-dependent manner. Kinetic analyses of the 45-kDa PI 4-kinase showed that phosphatidylglycerol was a competitive inhibitor with respect to PI (Ki = 2 mol %), and CDP-diacylglycerol was a mixed type of inhibitor with respect to PI (Ki = 4 mol %) and MgATP (Ki = 5 mol %). 55-kDa PI 4-kinase activity was not significantly affected by phospholipids. The physiological relevance of CDP-diacylglycerol inhibition of 45-kDa PI 4-kinase activity was examined using plasma membranes from inositol auxotrophic (ino1) cells. Immunoblot analysis showed that 45-kDa PI 4-kinase expression in plasma membranes was not affected by inositol starvation of ino1 cells. However, both 45-kDa PI 4-kinase activity and its product PI 4-phosphate were reduced in plasma membranes from inositol-starved ino1 cells. The CDP-diacylglycerol concentration (9.6 mol %) in plasma membranes of inositol-starved ino1 cells was 12-fold higher than its concentration (0.8 mol %) in plasma membranes of inositol-supplemented cells. Plasma membranes of inositol-starved ino1 cells also had increased levels of phosphatidate, phosphatidylserine, phosphatidylethanolamine, and cardiolipin. However, these phospholipids did not affect pure 45-kDa PI 4-kinase activity. The concentration of CDP-diacylglycerol in plasma membranes of inositol-starved ino1 cells was in the range of the inhibitor constants determined for CDP-diacylglycerol by kinetic analyses using pure 45-kDa PI 4-kinase. These results raised the suggestion that 45-kDa PI 4-kinase activity may be regulated in vivo by CDP-diacylglycerol.     

Mammalian 5'-AMP-activated protein kinase non-catalytic subunits are homologs of proteins that interact with yeast Snf1 protein kinase

The 5'-AMP-activated protein kinase is responsible for the regulation of fatty acid synthesis by phosphorylation and inactivation of acetyl-CoA carboxylase. The porcine liver 5'-AMP-activated protein kinase 63-kDa catalytic subunit co-purifies 14,000-fold with a 38- and 40-kDa protein (Mitchelhill, K.I. et al. (1994) J. Biol. Chem. 269, 2361-2364). The 63-kDa subunit is homologous to the Saccharomyces cerevisiae Snf1 protein kinase, which regulates gene expression during glucose derepression. Peptide amino acid and polymerase chain reaction-derived partial cDNA sequences of both the pig and rat liver enzymes show that the 38-kDa protein is homologous to Snf4p (CAT3) and that the 40-kDa protein is homologous to the Sip1p/Spm/GAL83 family of Snf1p interacting proteins. Sucrose density gradient and cross-linking experiments with purified 5'-AMP-activated protein kinase suggest that both the 38- and 40-kDa proteins associate tightly with the 63-kDa catalytic polypeptide in either a heterotrimeric complex or in dimeric complexes. The 40-kDa subunit is autophosphorylated within the 63-kDa subunit complex. The sequence relationships between the mammalian 5'-AMP-activated protein kinase and yeast Snf1p extend to the subunit proteins consistent with conservation of the functional roles of these polypeptides in cellular regulation by this family of metabolite-sensing protein kinases.     

Calcium ion influx during sporulation in the yeast Saccharomyces cerevisiae

The changes in intra- and (or) extra-cellular concentrations of Ca2+, Mg2+, K+, and Na+ during sporulation of a MATa/MAT alpha diploid yeast of Saccharomyces cerevisiae were examined in a nutrition-deprived medium with potassium acetate. Among these, Ca2+ in external medium was preferentially incorporated into cells, and sporulation was induced when the magnitude of free Ca2+ gradient between cytosol [Ca2+]i and external medium [Ca2+]o reached more than 3 x 10(3) ([Ca2+]i/[Ca2+]o = 3.5 x 10(3)). The result indicated that the meiosis and (or) sporulation signal of the yeast S. cerevisiae was generated through elevated Ca2+ influx rather than release from the internal Ca2+ stores.     

Stimulation of IRS-1-associated phosphatidylinositol 3-kinase and Akt/protein kinase B but not glucose transport by beta1-integrin signaling in rat adipocytes

The signal transduction pathway by which insulin stimulates glucose transport is not understood, but a role for complexes of insulin receptor substrate (IRS) proteins and phosphatidylinositol (PI) 3-kinase as well as for Akt/protein kinase B (PKB) has been proposed. Here, we present evidence suggesting that formation of IRS-1/PI 3-kinase complexes and Akt/PKB activation are insufficient to stimulate glucose transport in rat adipocytes. Cross-linking of beta1-integrin on the surface of rat adipocytes by anti-beta1-integrin antibody and fibronectin was found to cause greater IRS-1 tyrosine phosphorylation, IRS-1-associated PI 3-kinase activity, and Akt/PKB activation, detected by anti-serine 473 antibody, than did 1 nM insulin. Clustering of beta1-integrin also significantly potentiated stimulation of insulin receptor and IRS-1 tyrosine phosphorylation, IRS-associated PI 3-kinase activity, and Akt/PKB activation caused by submaximal concentrations of insulin. In contrast, beta1-integrin clustering caused neither a change in deoxyglucose transport nor an effect on the ability of insulin to stimulate deoxyglucose uptake at any concentration along the entire dose-response relationship range. The data suggest that (i) beta1-integrins can engage tyrosine kinase signaling pathways in isolated fat cells, potentially regulating fat cell functions and (ii) either formation of IRS-1/PI 3-kinase complexes and Akt/PKB activation is not necessary for regulation of glucose transport in fat cells or an additional signaling pathway is required.     

Regulation of phosphatidate phosphatase activity from the yeast Saccharomyces cerevisiae by sphingoid bases

The regulation of Saccharomyces cerevisiae membrane-associated phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) activity by sphingoid bases was examined using Triton X-100/lipid-mixed micelles. Sphingosine, phytosphingosine, and sphinganine inhibited purified preparations of the 104- and 45-kDa forms of phosphatidate phosphatase in a dose-dependent manner. The structural requirements for the sphingoid base inhibition of phosphatidate phosphatase activity were a free amino group and a long chain hydrocarbon. A detailed kinetic analysis was performed to determine the mechanism of phosphatidate phosphatase inhibition by sphingoid bases. The phosphatidate phosphatase dependence on phosphatidate was cooperative (Hill numbers of approximately 2) in the absence and presence of sphingoid bases. Sphingosine, phytosphingosine, and sphinganine were parabolic competitive inhibitors of phosphatidate phosphatase activity. This indicated that more than one inhibitor molecule contributed to the exclusion of phosphatidate from the enzyme. The aKi values (inhibitor constants) for sphingosine, phytosphingosine, and sphinganine were 1.5, 0.4, and 0.2 mol %, respectively, and the Km value for phosphatidate was 2.2 mol %. The cellular concentrations of free phytosphingosine and sphinganine were 0.16 and 0.53 mol %, respectively, relative to the total phospholipids in S. cerevisiae. The cellular concentrations of phytosphingosine and sphinganine were in the range of the aKi values for these sphingoid bases. These results raised the suggestion that phosphatidate phosphatase activity may be regulated in vivo by sphingoid bases.     

Adenylosuccinate synthetase of the yeast Saccharomyces cerevisiae: purification and properties

Motor and sensory nerve conduction velocities (MNCV and SNCV) were reduced in the sciatic nerve of rats after 4 weeks of untreated streptozotocin-induced diabetes, and declined further during the following 4 weeks. Treating diabetic rats with the novel peptide HP228 had no effect on the decline of MNCV after the first 4 weeks of diabetes but attenuated the decline in SNCV. HP228 treatment also prevented any further decline in MNCV or SNCV between weeks 4 and 8 of diabetes. Consequently, at the conclusion of the study, the nerve conduction velocities (NCVs) in treated rats were significantly (both P < .001) higher than in untreated diabetic rats. Reduced nerve homogenate Na+,K+-adenosine triphosphatase (ATPase) activity in diabetic rats was significantly (P < .05) increased by HP228 but remained significantly (P < .05) lower than in untreated controls. HP228 treatment also reduced nerve Na+,K+-ATPase activity of control rats compared with untreated controls (P < .05). There was no effect of HP228 on the hyperglycemia, nerve polyol accumulation, myo-inositol depletion, reduced nerve laser Doppler blood flow, thermal hypoalgesia, or reduced mean axonal caliber in diabetic rats or on any of these parameters in control rats. These data demonstrate that a novel peptide may protect against the slowing of nerve conduction in prolonged diabetes and that the mechanism of action is unrelated to aldose reductase inhibition, prevention of nerve ischemia, or axonal atrophy. HP228 may prove a potential therapeutic agent for the treatment of prolonged diabetic neuropathy.