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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.

Pubmed ID: 12853467

Authors

  • Pozuelo Rubio M
  • Peggie M
  • Wong BH
  • Morrice N
  • MacKintosh C

Journal

The EMBO journal

Publication Data

July 15, 2003

Associated Grants

None

Mesh Terms

  • 14-3-3 Proteins
  • Carrier Proteins
  • Cyclic AMP-Dependent Protein Kinases
  • Enzyme Activation
  • Exonucleases
  • Exoribonucleases
  • Fructosediphosphates
  • Glutathione Transferase
  • Glycolysis
  • HeLa Cells
  • Humans
  • Insulin-Like Growth Factor I
  • Isoenzymes
  • Myocardium
  • Neoplasm Proteins
  • Peptide Fragments
  • Phosphofructokinase-2
  • Phosphorylation
  • Protein Kinases
  • Recombinant Fusion Proteins
  • Serine
  • Tumor Markers, Biological