Measurement of binding kinetics between PI3-K and phosphorylated IGF-1R using a surface plasmon resonance biosensor.

Quantitative measurement of biochemical and biophysical parameters of molecular recognition events in signaling pathways is very important for understanding biological function. Binding of insulin-like growth factor-1 receptor (IGF-1R) with IGF-1 activates receptor tyrosine phosphorylation and triggers several signaling pathways including phosphatidylinositol-3 kinase (PI3-K)/AKT and extracellular signal-regulated protein kinase (ERK)/mitogen-activated protein kinase (MAPK) pathways. We have analyzed the interactions between PI3-K and IGF-1R with or without IGF-1 stimulation. The results demonstrate that p85 subunits of PI3-K bind to IGF-1R with IGF-1 stimulation in intact cells. The binding kinetics between PI3-K and IGF-1R with or without IGF-1 stimulation were obtained using surface plasmon resonance biosensor. The affinity constant of the PI3-K to phosphorylated IGF-1R was (2.27 ± 0.12) × 108 M−1, which was about 20 times higher than that of PI3-K to unphosphorylated IGF-1R. Moreover, the kinetic effects of Mg2+, ATP and two kinase inhibitors, genistein and quercetin, on the binding between PI3-K and phosphorylated IGF-1R were studied. The data showed that Mg2+ increased the binding affinity of PI3-K with IGF-1R about 2-fold, while genistein decreased the affinity constant 2.7-fold. On the other hand, ATP and quercetin had no significant effects on the affinity constant, although both k a and k d values were increased or decreased by ATP or quercetin, respectively. This study implicated that the PI3-K binding sites on IGF-IR may be different from its phosphorylation and catalytic sites. [ABSTRACT FROM AUTHOR]