Juxtamembrane autophosphorylation in the insulin receptor kinase.
Item
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Title
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Juxtamembrane autophosphorylation in the insulin receptor kinase.
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Identifier
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AAI9820518
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identifier
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9820518
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Creator
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Cann, Aaron Darius.
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Contributor
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Adviser: R. A. Kohanski
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Date
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1998
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry
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Abstract
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The insulin receptor is a {dollar}\alpha\sb2\beta\sb2{dollar} receptor tyrosine kinase which is activated by autophosphorylation of its kinase-bearing {dollar}\beta{dollar} subunits in response to insulin. The molecular mechanism of {dollar}\beta{dollar} subunit autophosphorylation has long been a matter of dispute, with some groups reporting intramolecular (cis) and others intermolecular (trans) autophosphorylation. We approached this question using site directed mutagenesis of the complete cytoplasmic kinase domain of the human insulin receptor (CKD) in conjunction with the standard criteria of enzyme concentration dependence. The apparent mechanism of autophosphorylation was dependent on the particular residues involved in the reaction. Thus, tyrosines in the juxtamembrane region of the receptor (Y{dollar}\sp{lcub}965{rcub}{dollar} and Y{dollar}\sp{lcub}972{rcub}{dollar}) autophosphorylate in cis, but tyrosine residues in the activation loop (Y{dollar}\sp{lcub}1158{rcub}{dollar}, Y{dollar}\sp{lcub}1162{rcub}{dollar}, and Y{dollar}\sp{lcub}1162{rcub}{dollar}) and carboxyl terminus (Y{dollar}\sp{lcub}1328{rcub}{dollar} and Y{dollar}\sp{lcub}1334{rcub}){dollar} autophosphorylate in trans. Moreover, in the presence of an intact unphosphorylated activation loop, juxtamembrane autophosphorylation was observed to correlate with activation of the CKD. This was measured with a new HPLC-based in vitro peptide phosphorylation assay using a peptide derived from insulin receptor substrate-1. Moreover, a mutant enzyme with both Y{dollar}\sp{lcub}965{rcub}{dollar} and Y{dollar}\sp{lcub}972{rcub}{dollar} mutated to F (but not a mutant with the carboxyl terminus tyrosines mutated to F) showed very little peptide phosphorylation activity. Thus, we have observed a novel cis activation process in the insulin receptor that appears to explain previous conflicts in the insulin receptor literature.;The crystal structure of a truncated CKD lacking the juxtamembrane region (Hubbard et al., Nature 372 746; 1994) has been solved, and identifies residues in the unphosphorylated activation loop that stabilize it for cis inhibition. We found that disruption of these residues caused an increased rate of juxtamembrane cis autophosphorylation, primarily due to a 30-fold drop in {dollar}K\sp{lcub}\rm MnATP{rcub}{dollar} or K{dollar}\sp{lcub}\rm MgATP{rcub}.{dollar} This implies that the wild-type unphosphorylated activation loop slows cis autophosphorylation by blocking the interaction with ATP. Although it was not recognized at the time, the converse observation has also been found: the truncated CKD, without the juxtamembrane region, autophosphorylates at the activation loop much more readily than the full-length enzyme (Wei et al., J. Biol. Chem. 270 8122; 1995). Taken together, these observations suggest a new model of the basal state of the insulin receptor's kinase. In this model, the unphosphorylated state of the insulin receptor's kinase is inhibited by reciprocal cis inhibition by the unphosphorylated juxtamembrane and activation loop regions. Either region can occupy the peptide binding site, but only the activation loop blocks ATP binding. This dual mode of autoinhibition may be required for prevention of trans activation in the constitutively tetrameric insulin receptor holomer.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
