14-3-3s regulate fructose-2,6-bisphosphate levels by binding to PKB-phosphorylated cardiac fructose-2,6-bisphosphate kinase/phosphatase

Abstract

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 RNYpS₄₈₃VGS 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.