Glucose-induced stimulation of the Ras-cAMP pathway in yeast leads to multiple phosphorylations and activation of 6-phosphofructo-2-kinase.
Yeast cells respond to changes of the environment by complex modifications of the metabolism. An increase of the extracellular glucose concentration activates the Ras-cAMP pathway. Via a production of cAMP this pathway stimulates the cAMP-dependent protein kinase (PKA) which is involved in the posttranslational regulation of the key enzymes of gluconeogenesis and glycolysis. 6-Phosphofructo-2-kinase (PFK2) catalyzes the synthesis of fructose 2,6-bisphosphate, the most potent activator of the glycolytic key enzyme 6-phosphofructo-1-kinase. We investigated the molecular mechanism of the glucose-induced phosphorylation and activation of PFK2 in Saccharomyces cerevisiae. After an incubation of PFK2 with ATP and PKA in vitro, two amino acid residues, Thr157 and Ser644, are phosphorylated and the enzyme is activated. A stimulation of the Ras-cAMP pathway by glucose addition to cultivated yeast cells leads to an in vivo activation of PFK2 which is accompanied by a more complex phosphorylation pattern of the enzyme. The phosphorylation of the protein on Ser644 is the result of PKA stimulation while the protein kinase(s) catalyzing the 5-fold phosphorylation of the peptide fragment T(67)(-)(101) is (are) still unknown. The functional significance of T(67)(-)(101) and its phosphorylation is supported by the finding that PFK2 lacking this peptide is inactive.
Dihazi
Hassan
H
Institute of Biochemistry, Medical Faculty, University of Leipzig, Liebigstrasse 16, Germany.
Kessler
Renate
R
Eschrich
Klaus
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005656:Fungal Proteins; D005947:Glucose / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D025481:Phosphofructokinase-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D017384:Sequence Deletion; D012694:Serine / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013912:Threonine / Q000737:chemistry; D018631:ras Proteins / Q000378:metabolism*
D005656:Fungal Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D013912:Threonine; D012694:Serine; D000242:Cyclic AMP; D025481:Phosphofructokinase-2; D017868:Cyclic AMP-Dependent Protein Kinases; D018631:ras Proteins; D005947:Glucose