Insulin-like signaling in yeast: modulation of protein phosphatase 2A, protein kinase A, cAMP-specific phosphodiesterase, and glycosyl-phosphatidylinositol-specific phospholipase C activities.
Previously, we have described significant effects of human insulin on glucose metabolism in the yeast Saccharomyces cerevisiae under conditions of growth limitation. These regulations apparently rely on a transmembrane receptor capable of binding human insulin and responding by tyrosine/serine phosphorylation of a specific set of polypeptides [Müller, G., Rouveyre, N., Crecelius, A., and Bandlow, W. (1998) Biochemistry 37, 8683-8695; Müller, G., Rouveyre, N., Upshon, C., Gross, E., and Bandlow, W. (1998) Biochemistry 37, 8696-8704; Müller, G., Rouveyre, N., Upshon, C., and Bandlow, W. (1998) Biochemistry 37, 8705-8713]. To characterize the molecular link between the initial steps in insulin-like signaling in yeast and the changes in the activities of glycogen synthase and glycogen phosphorylase, we examined here the effects of human insulin on a set of key regulatory enzymes of glycogen metabolism, protein phosphatase 2A (PP2A), cAMP-specific phosphodiesterase (cAMP-PDE), and protein kinase A (PKA). PP2A was activated about 2-fold by insulin in spheroplasts and in intact cells, whereas the fraction of active PKA was significantly reduced in a cAMP-independent manner as well as through a subsequent up to 3-fold increase in particulate cAMP-PDE activity accompanied by a 50% decrease in cytosolic cAMP levels. In addition, glycosyl-phosphatidylinositol-specific phospholipase C (GPI-PLC), which in isolated rat adipocytes is activated by insulin, was stimulated to up to 5-fold by glucose and 10-fold by glucose plus insulin in both yeast spheroplasts and intact cells leading to a concentration-dependent leftward shift of the glucose-response curve for activation of the GPI-PLC. GPI-PLC was most pronouncedly stimulated by authentic human insulin compared to various insulin analogues and insulin-like growth factor I. In addition to lipolytic cleavage by GPI-PLC, the GPI anchor of the cAMP-binding ectoprotein, Gce1p, was secondarily processed by a rapid proteolytic event. As the GPI-PLC reaction is rate limiting, the efficiency of the two-step anchor cleavage was significantly increased when insulin was present together with glucose as compared to glucose alone. The insulin concentrations effective in modulating PP2A, PKA, cAMP-PDE, and GPI-PLC activities correlate well with those required for half-saturation of the specific binding sites as well as for stimulation of protein phosphorylation and glycogen accumulation. The data suggest that mammalian insulin-sensitive cells and yeast share (part of) the key regulatory mechanism (consisting of PP2A, PKA, cAMP-PDE, and GPI-PLC) involved in the transduction of the insulin signal from the respective receptor systems to glycogen synthase and phosphorylase.
Müller
G
G
Hoechst Marion Roussel Deutschland GmbH, DG Metabolic Diseases, Building H825, 65926 Frankfurt am Main, Germany. Guenter.Mueller@hmrag.com
Grey
S
S
Jung
C
C
Bandlow
W
W
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D000242:Cyclic AMP / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005656:Fungal Proteins / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology; D006006:Glycogen Synthase / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000494:pharmacology / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D054648:Protein Phosphatase 2; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction / Q000187:drug effects; D013104:Spheroplasts / Q000187:drug effects / Q000201:enzymology; D010738:Type C Phospholipases / Q000378:metabolism*
D005656:Fungal Proteins; C066709:GAS1 protein, S cerevisiae; C102317:GPI1 protein, S cerevisiae; D017261:Glycosylphosphatidylinositols; D007328:Insulin; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D000242:Cyclic AMP; D006006:Glycogen Synthase; D017868:Cyclic AMP-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D010738:Type C Phospholipases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D005947:Glucose