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J. Biol. Chem., Vol. 281, Issue 33, 23785-23791, August 18, 2006

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Role of the Activation Loop Tyrosines in Regulation of the Insulin-like Growth Factor I Receptor-tyrosine Kinase^*

From the Department of Physiology and Biophysics, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794

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 V_max 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 k_cat/K_m 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.

Received for publication, June 1, 2006 , and in revised form, June 21, 2006.

^* This work was supported by National Institutes of Health Grant CA28146 (to W. T. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Physiology and Biophysics, Basic Science Tower, T-6, School of Medicine, SUNY at Stony Brook, Stony Brook, NY 11794-8661. Tel.: 631-444-3533; Fax: 631-444-3432; E-mail: todd.miller{at}stonybrook.edu.

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