Posttranslational Processing of the Insulin Proreceptor

Lane, .; Gv, Ronnett; Ra, Kohanski; Tl, Simpson

doi:10.1016/b978-0-12-152827-0.50031-1

Current Topics in Cellular Regulation

1985

DOI: 10.1016/b978-0-12-152827-0.50031-1

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Posttranslational Processing of the Insulin Proreceptor

. Lane

Ronnett Gv

Kohanski Ra

et al.

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Cited by 6 publications

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“…These modifications create high affinity binding sites for their substrates and/or result in conformational changes leading to high activity states (2). In the case of the insulin receptor, however, the receptor is already a disulfide-linked dimer in the absence of insulin (3), and signaling events still require insulin binding (4). Therefore, it has been of a great interest to establish the differences in catalytic properties of the insulin receptor between its basal and activated states.…”

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Activation of the Insulin Receptor's Kinase Domain Changes the Rate-Determining Step of Substrate Phosphorylation

Ablooglu

Kohanski

2000

Biochemistry

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The insulin receptor and many other protein kinases are activated by relief of intrasteric inhibition that is regulated by reversible phosphorylation. The changes accompanying activation of the insulin receptor's kinase domain were analyzed using steady-state kinetics, viscometric analysis, and equilibrium binding measurements. Peptide phosphorylation catalyzed by the unphosphorylated basal-state kinase is limited by a slow rate of the chemical step, and the activated enzyme is limited by product release rates. Underlying these changes were a 36-fold increase in the rate constant for the chemical step of the enzyme-catalyzed reaction, a 5-fold increase in the affinity for MgATP, and an 8-fold increase in the affinity for peptide substrate. This results in binding of substrates that is 2.2 kcal/mol more favorable and a free energy barrier for transition state formation that is lowered by 2.1 kcal/mol in the activated enzyme. Therefore, the change in conformational free energy inherent in the protein after autophosphorylation [Bishop, S. M., Ross, J. B. A., and Kohanski, R. A. (1999) Biochemistry 38, 3079-3089] is equally distributed between formation of the substrate ternary complex and formation of the transition state complex.

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confidence: 99%

Activation of the Insulin Receptor's Kinase Domain Changes the Rate-Determining Step of Substrate Phosphorylation

Ablooglu

Kohanski

2000

Biochemistry

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“…The insulin receptor is a disulfide-linked heterotetrameric R 2 β 2 transmembrane glycoprotein (1). The binding of insulin to the extracellular R-subunits causes autophosphorylation of the intracellular kinase domain of the β-subunits (2).…”

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“…This results in activation of signaling through increased kinase activity and substrate recruitment (). The insulin receptor kinase domain [IRKD; R 953 −S 1355 , as described by Villalba et al () but numbered according to Ebina et al ()] can be phosphorylated at up to seven sites within three distinct domains ( − ): Y 965 and Y 972 in the juxtamembrane domain, Y 1158 , Y 1162 , and Y 1163 in the activation loop (AL, residues D 1150 −P 1172 ), and Y 1328 and Y 1334 in the carboxy-terminal region. The AL tyrosines are the principle sites responsible for regulating the catalytic efficiency of the IRKD ( − ).…”

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Autophosphorylation Dependent Destabilization of the Insulin Receptor Kinase Domain: Tryptophan-1175 Reports Changes in the Catalytic Cleft

1999

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Protein kinases are regulated by conformational or chemical changes which facilitate access of substrates to the active site and promote correct orientations of catalytically essential residues and water molecules. The switch between basal and activated states of the insulin receptor's kinase domain (IRKD) results from autophosphorylation. We investigated the effects of IRKD autophosphorylation on the conformational stability by guanidine hydrochloride (GdnHCl) dependent denaturation and by iodide quenching of intrinsic fluorescence. Tryptophan residues of the recombinant soluble IRKD (residues R953-S1355) were excited at a lambdaex of 295 nm, and emission spectra were analyzed for centroid (a characteristic of average polarity of the indole rings' environments) and integrated fluorescence intensity over the lambdaem range of 310-420 nm. Denaturation profiles of both apo- and phospho-IRKD forms are complex with at least three distinct unfolding transitions. The first and last transitions were reversible and cooperative and had midpoints at 0.4 or 0.7 M GdnHCl and 2.4 or 2.7 M GdnHCl, respectively; transitions of phospho-IRKD occurred at lower GdnHCl concentrations. Calculations of free energy of unfolding suggested a loss of approximately 2.3 kcal/mol of stabilization for the first transition and approximately 1.5 kcal/mol for the third transition. Circular dichroism showed subtle changes in secondary structure over the first transition and global unfolding over the last transition. The first transition reports changes primarily in the local environment of W1175, which is near the catalytic loop and is conserved among protein tyrosine kinases. W1175 is also the dominant fluorophore of the native emission spectrum. Iodide quenching of W1175 was virtually undetectable in the apo-IRKD but significant in the phospho-IRKD, suggesting that W1175 exposure to small solutes is strongly dependent on the conformation of the activation loop. These studies indicate that autophosphorylation, while exposing the catalytic center, also produces a conformer less stable than the apoenzyme.

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“…The IR is a heterotetramer with an ␣ 2 ␤ 2 -subunit structure. The extracellular ␣-subunits are covalently linked to each other and to the membrane-spanning ␤-subunits by disulfide bonds (36). Nevertheless this dimerized RTK retains an unphosphorylated basal state and does not autophosphorylate or signal until insulin binds.…”

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Intrasteric Inhibition of ATP Binding Is Not Required To Prevent Unregulated Autophosphorylation or Signaling by the Insulin Receptor

Frankel

Ablooglu

Leone³

et al. 2001

Molecular and Cellular Biology

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Receptor tyrosine kinases may use intrasteric inhibition to suppress autophosphorylation prior to growth factor stimulation. To test this hypothesis we made an Asp1161Ala mutant in the activation loop that relieved intrasteric inhibition of the unphosphorylated insulin receptor (IR) and its recombinant cytoplasmic kinase domain (IRKD) without affecting the activated state. Solution studies with the unphosphorylated mutant IRKD demonstrated conformational changes and greater catalytic efficiency from a 10-fold increase in k cat and a 15-fold-lower K m ATP although K m peptide was unchanged. Kinetic parameters of the autophosphorylated mutant and wild-type kinase domains were virtually identical. The Asp1161Ala mutation increased the rate of in vitro autophosphorylation of the IRKD or IR at low ATP concentrations and in the absence of insulin. However, saturation with ATP (for the IRKD) or the presence of insulin (for the IR) yielded equivalent rates of autophosphorylation for mutant versus wild-type kinases. Despite a biochemically more active kinase domain, the mutant IR expressed in C2C12 myoblasts was not constitutively autophosphorylated. However, it displayed a 2.5-fold-lower 50% effective concentration for insulin stimulation of autophosphorylation and was dephosphorylated more slowly following withdrawal of insulin than wild-type IR. In tests of the regulation of the unphosphorylated basal state, these results demonstrate that neither intrasteric inhibition against ATP binding nor suppression of kinase activity is required to prevent premature autophosphorylation of the IR. Finally, the lower rate of dephosphorylation suggests invariant residues of the activation loop such as Asp1161 may function at multiple junctures in cellular regulation of receptor tyrosine kinases.Signal transduction through receptor tyrosine kinases (RTKs) is essential throughout the life of an organism. The tight control of RTK signaling required for normal cellular function is dependent on growth factor stimulation of autophosphorylation (50). Autophosphorylation is a unique regulatory motif among protein kinase signaling cascades because no other enzyme precedes or participates in autophosphorylation. Therefore the unphosphorylated RTK must have sufficient catalytic activity to perform the first phosphoryl transfer reaction while (i) avoiding autophosphorylation prior to growth factor binding and (ii) avoiding target phosphorylation prior to autophosphorylation. The first restriction is met by hormone-induced dimerization of monomeric RTKs (50), where the homodimer itself provides the enzyme-substrate pair. The second restriction is met by target or mediator recruitment sites which appear on the RTK as a consequence of autophosphorylation (44, 51). The logic of induced dimerization seems to fail when applied to the insulin receptor (IR). The IR is a heterotetramer with an ␣ 2 ␤ 2 -subunit structure. The extracellular ␣-subunits are covalently linked to each other and to the membrane-spanning ␤-subunits by disulfide bonds (36). Nev...

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Posttranslational Processing of the Insulin Proreceptor

Cited by 6 publications

References 26 publications

Activation of the Insulin Receptor's Kinase Domain Changes the Rate-Determining Step of Substrate Phosphorylation

Activation of the Insulin Receptor's Kinase Domain Changes the Rate-Determining Step of Substrate Phosphorylation

Autophosphorylation Dependent Destabilization of the Insulin Receptor Kinase Domain: Tryptophan-1175 Reports Changes in the Catalytic Cleft

Intrasteric Inhibition of ATP Binding Is Not Required To Prevent Unregulated Autophosphorylation or Signaling by the Insulin Receptor

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