14-3-3s regulate fructose-2,6-bisphosphate levels by binding to PKB-phosphorylated cardiac fructose-2,6-bisphosphate kinase/phosphatase.
The cardiac isoform of 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase (PFK-2), regulator of the glycolysis-stimulating fructose-2,6-bisphosphate, was among human HeLa cell proteins that were eluted from a 14-3-3 affinity column using the phosphopeptide ARAApSAPA. Tryptic mass fingerprinting and phospho-specific antibodies showed that Ser466 and Ser483 of 14-3-3-affinity-purified PFK-2 were phosphorylated. 14-3-3 binding was abolished by selectively dephosphorylating Ser483, and 14-3-3 binding was restored when both Ser466 and Ser483 were phosphorylated with PKB, but not when Ser466 alone was phosphorylated by AMPK. Furthermore, the phosphopeptide RNYpS(483)VGS blocked binding of PFK-2 to 14-3-3s. These data indicate that 14-3-3s bind to phosphorylated Ser483. When HeLa cells expressing HA-tagged PFK-2 were co-transfected with active PKB or stimulated with IGF-1, HA-PFK-2 was phosphorylated and bound to 14-3-3s. The response to IGF-1 was abolished by PI 3-kinase inhibitors. In addition, IGF-1 promoted the binding of endogenous PFK-2 to 14-3-3s. When cells were transduced with penetratin-linked AARAApSAPA, we found that this reagent bound specifically to 14-3-3s, blocked the IGF-1-induced binding of HA-PFK-2 to 14-3-3s, and completely inhibited the IGF-1-induced increase in cellular fructose-2,6-bisphosphate. These findings suggest that PKB-dependent binding of 14-3-3s to phospho-Ser483 of cardiac PFK-2 mediates the stimulation of glycolysis by growth factor.
Pozuelo Rubio
Mercedes
M
MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK. m.pozuelorubio@dundee.ac.uk
Peggie
Mark
M
Wong
Barry H C
BH
Morrice
Nick
N
MacKintosh
Carol
C
D048948:14-3-3 Proteins / Q000378:metabolism*; D014408:Biomarkers, Tumor; D002352:Carrier Proteins / Q000378:metabolism; D057846:Cell-Penetrating Peptides; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation; D005092:Exonucleases / Q000378:metabolism*; D005095:Exoribonucleases; D005635:Fructosediphosphates / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006019:Glycolysis; D006367:HeLa Cells; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D009206:Myocardium / Q000201:enzymology*; D009363:Neoplasm Proteins; D010446:Peptide Fragments / Q000378:metabolism; D025481:Phosphofructokinase-2 / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism
D048948:14-3-3 Proteins; D014408:Biomarkers, Tumor; D002352:Carrier Proteins; D057846:Cell-Penetrating Peptides; D005635:Fructosediphosphates; D007527:Isoenzymes; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D012694:Serine; D007334:Insulin-Like Growth Factor I; C027652:fructose 2,6-diphosphate; C414409:penetratin; D005982:Glutathione Transferase; D011494:Protein Kinases; D025481:Phosphofructokinase-2; D017868:Cyclic AMP-Dependent Protein Kinases; D005092:Exonucleases; D005095:Exoribonucleases; C413442:SFN protein, human