Abstract
The tyrosine kinase activity of insulin-like growth factor I receptor (IGF1R) is under tight control. Ligand binding to the extracellular portion of IGF1R stimulates autophosphorylation at three sites (Tyr1131, Tyr1135, and Tyr1136) in the activation loop within the tyrosine kinase catalytic domain. Autophosphorylation at all three sites is required for maximum enzyme activity, and for IGF1-stimulated cellular activity of the receptor. Previous studies have not clarified the contributions of the individual tyrosines to enzymatic activation. Here, we produced single Tyr-to-Phe mutations at these positions, and compared activities of the purified kinase domains (unphosphorylated and phosphorylated) with wild-type IGF1R. Rates of autophosphorylation of the three mutants were more rapid than for wild-type IGF1R; this was most apparent for the Y1135F mutant. Substrate phosphorylation studies on the unphosphorylated forms of IGF1R confirmed that the value of Vmax for Y1135F was elevated relative to wild-type IGF1R, consistent with a disruption of an autoinhibitory interaction. In contrast, activity measurements on the fully phosphorylated enzymes indicated that kcat/Km values were lowered relative to wild-type IGF1R; this effect was most dramatic for Y1136F. We confirmed these findings using limited proteolysis and tryptophan fluorescence experiments. The results demonstrate that Tyr1135 plays a particularly important role in stabilizing the autoinhibited conformation of the activation loop, while Tyr1136 plays the key role in stabilizing the open, activated conformation of IGF1R.
Highlights
Ligand binding stimulates the intrinsic tyrosine kinase activity of the cytoplasmic domain
The autophosphorylation of IGF1R at three sites within the activation loop is crucial for full catalytic activity and important for the physiological roles of the receptor in normal and transformed cells [13, 19, 20]
Previous studies have shown that there is a preferred sequence of activation loop (A-loop) phosphorylations (Tyr1135, followed by Tyr1131, Tyr1136), and that each phosphorylation increases enzyme activity [16]
Summary
IGF1-induced receptor autophosphorylation, whereas it did The purification of the mutants was accomplished using simnot have an impact on cell proliferation or phosphorylation of ilar procedures as for wild-type IGF1RK [16]. Kinase Assays—The kinetics of IGF1R autophosphorylation and substrate peptide phosphorylation were measured by a continuous spectrophotometric assay [16, 23]. In this assay, the consumption of ATP is coupled to the oxidation of NADH, which is monitored as a reduction in NADH absorption at 340 nm. Fluorescence Spectra and Iodide Quenching—Tryptophan fluorescence experiments were carried out in 100 mM Tris-HCl and 1 mM dithiothreitol using methods described for IRK [26]. Where F0 is the fluorescence intensity in the absence of quencher
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