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

Abstract

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...

Apoptosis of cancer cells is triggered by selective crosslinking and inhibition of receptor tyrosine kinases

“…As a typical example, the active and inactive conformation of INSR are mainly due to the orientations of the activation loop, which blocks substrate access to the catalytic cleft in the basal/inactive state while swings away from the orientation of the inactive conformation to form a platform for substrate binding 56 . As demonstrated by previous studies 23 and confirmed in the present study (Fig. 5c), this conformational change could be readily detected by limited trypsin digestion: the activation loop of INSR is susceptible for trypsin cleavage upon activation through ligand and ATP binding but only very weakly in its basal state.…”

“…5c, INSR was inaccessible to trypsin cleavage in the presence of insulin and ADP. However, the presence of insulin and AMP-PNP enabled the transition of INSR from inactive to active form, which was reported to be liable for trypsin digestion 23,24 and confirmed in the present study. We further demonstrated that the presence of ConA inhibited this conformational activation.

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“…The limited trypsin digestion was conducted as described before with minor modifications 23 . The membrane pellets prepared above were homogenized with reaction buffer (10 mM Tris, 10 mM MgCl 2 , 5 mM MnCl 2 , 0.1% TritonX-100, pH 7.4) and incubated with 20 μg/ml ConA for 20 min.…”

Multiple Activation Loop Conformations and Their Regulatory Properties in the Insulin Receptor's Kinase Domain

“…A D1161A mutation has been reported, showing that loss of three interactions (1, 2, and 3, Fig. 1) produced an unphosphorylated kinase with increased catalytic efficiency (13,14). The structure of this mutant IRKD displayed a "gate-open" conformation in which the ATP binding site was freed and pseudosubstrate binding of the A-loop was not observed; most residues in the A-loop were disordered.…”

Stepwise autophosphorylation regulates biased agonism of the insulin receptor

“…Similarly, alanine substitution of Asp1149 (D1149A), which plays a critical role in blocking substrate access to the active site of IR kinase, increases catalytic efficiency (~10-fold) even more strongly than Y972A. However, in contrast to catalytic efficiency, D1149A has a modest effect (~3-fold) on basal phosphorylation of IR relative to Y972A 22,23 . The difference between the two mutations in basal phosphorylation of the receptor and catalytic efficiency suggests that Tyr972 also plays an important role in conformational rearrangement of β-subunits, allowing trans-autophosphorylation of the receptor.…”