Pozuelo Rubio Mercedes, Peggie Mark, Wong Barry H C, Morrice Nick, MacKintosh Carol
MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.
EMBO J. 2003 Jul 15;22(14):3514-23. doi: 10.1093/emboj/cdg363.
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.
6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶（PFK-2）的心脏同工型是糖酵解刺激因子果糖-2,6-二磷酸的调节因子，它是从人HeLa细胞蛋白中用磷酸肽ARAApSAPA从14-3-3亲和柱上洗脱下来的。胰蛋白酶质谱指纹图谱和磷酸特异性抗体表明，14-3-3亲和纯化的PFK-2的Ser466和Ser483被磷酸化。通过选择性地使Ser483去磷酸化，14-3-3的结合被消除，当Ser466和Ser483都被蛋白激酶B（PKB）磷酸化时，14-3-3的结合得以恢复，但当Ser466单独被AMPK磷酸化时则不然。此外，磷酸肽RNYpS(483)VGS阻断了PFK-2与14-3-3的结合。这些数据表明14-3-3与磷酸化的Ser483结合。当表达HA标签的PFK-2的HeLa细胞与活性PKB共转染或用胰岛素样生长因子-1（IGF-1）刺激时，HA-PFK-2被磷酸化并与14-3-3结合。PI 3-激酶抑制剂消除了对IGF-1的反应。此外，IGF-1促进内源性PFK-2与14-3-3的结合。当用穿膜肽连接的AARAApSAPA转导细胞时，我们发现该试剂特异性地与14-3-3结合，阻断了IGF-1诱导的HA-PFK-2与14-3-3的结合，并完全抑制了IGF-1诱导的细胞内果糖-2,6-二磷酸的增加。这些发现表明，14-3-3与心脏PFK-2的磷酸化Ser483的PKB依赖性结合介导了生长因子对糖酵解的刺激作用。
