Insulin receptor autophosphorylation. I. Autophosphorylation kinetics of the native receptor and its cytoplasmic kinase domain

Abstract

Kinetic analysis of autophosphorylation was done using a non-Michaelis-Menten kinetic model. This model describes autophosphorylation in terms of a fast reaction phase, a slow reaction phase, and a partition function for the two phases. Kinetic parameters determined by this new approach show that insulin stimulates autophosphorylation by promoting (1) a 10-fold increase in the rate constant for the fast phase of the reaction and (2) a 2-fold increase in the partition function favoring the fast phase. Insulin did not significantly affect the binding constant for ATP in this fast phase. Kinetic parameters obtained for the cytoplasmic kinase domain were similar to those obtained for the native insulin receptor in the absence of insulin. The insulin receptor has three subdomains encompassing its seven autophosphorylation sites. The juxtamembrane sites react primarily in the slow kinetic phase, favored by the absence of stimulation and low ATP concentrations. The carboxy-terminal and central autophosphorylation subdomains react primarily in the fast kinetic phase, favored by raising the ATP concentration and/or the presence of insulin. These observations demonstrate that: (1) both ATP and insulin regulate reaction in each autophosphorylation subdomain, (2) insulin stimulation occurs predominantly in the central and carboxy-terminal regions, and (3) autophosphorylation observed with the cytoplasmic kinase domain was similar to native insulin receptor in the absence of insulin. These findings are consistent with conclusions based on the kinetic analysis of autophosphorylation.

Biochemical Basis for the Functional Switch That Regulates Hepatocyte Growth Factor Receptor Tyrosine Kinase Activation

“…Thus, the dominant role of ligand-mediated RTK oligomerization is thought to be promotion of autophosphorylation of tyrosine residues within the receptor's activation loop critical for receptor catalytic function. However, recent studies demonstrate that monomeric RTKs can also be rapidly phosphorylated on tyrosine residues involved in intracellular signal propagation (18)(19)(20), raising the question of exactly how ligand-dependent dimerization regulates RTK activation.Our work and that of others suggest that ligand-dependent oligomerization may rapidly and selectively switch a RTK between distinct inactive and active states (16)(17)(18)(21)(22)(23)(24), where the active state exists when a RTK is autophosphorylated and capable of binding to and signaling through immediate downstream effector substrates (e.g., PI3K, Shc, Gab1, and Grb2) (3,6,7,25,26 Activation of the hepatocyte growth factor receptor (c-MET) triggers complex intracellular signaling responses leading to cell proliferation, differentiation, branching morphogenesis, motility, and invasion (26,27). Prolonged c-MET activation correlates closely with tumor progression and metastasis.…”

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

“…Therefore, the gate-open conformation in this mutant IRKD shows a greater biochemical predisposition for trans autophosphorylation than WT-IRKD, based on its ability to maintain a high rate of autophosphorylation at the lower ATP concentration. Because previous studies showed that WT-IRKD mimics WT-IR in the absence of insulin (32,34), it is reasonable to expect that a full-length IR bearing the D1161A mutation also will be in a gate-open conformation and display greater autophosphorylation than WT-IR, at least in the absence of insulin.…”

“…However, previous studies with the native IR demonstrated that occupancy of the binding site by ATP or nonhydrolyzable analogs promoted trans interactions (57) and conformational changes in the receptor (39) and in particular enhanced the autophosphorylation that is responsible for kinase activation (41). Furthermore, kinetic evidence showed that ATP binding predisposes the unphosphorylated receptor for trans autophosphorylation (32). Given the appar-ent affinity of the kinase domain for MgATP of ϳ1 mM (1,7,18) and the intracellular ATP concentration of ϳ1 mM (possibly as high as 8 mM [37]), a significant fraction of the receptor's kinase domains should have nucleotide bound under physiological conditions and therefore should be nonlatent as an enzyme and nonlatent as a substrate (18).…”

“…It has kinetic features similar to the basal state of the IR (32,34), and it can be activated by autophosphorylation (11,53,55) so that its structure should reveal aspects of the kinase important for the receptor's function and regulation (24,26). The crystal structure of the catalytic core (residues 978 to 1283) (26) identified the activation loop (residues D1150 to P1172) as an intrasteric inhibitor which blocked entry of both substrates to the active site.…”

Is there an Association between Variants in Candidate Insulin Pathway Genes IGF-I, IGFBP-3, INSR, and IRS2 and Risk of Colorectal Cancer in the Iranian Population?

“…Independent of IGF-I, IGFBP-3 inhibits growth and promotes apoptosis (Renehan et al, 2004;Davies et al, 2006). Insulin binds to its cell surface receptor (INSR), which regulates its metabolic action through initiating a series of tyrosine residues phosphorylation (Kohanski, 1993;White, 1997;Zhang et al, 2010). Insulin receptor substrate-2 (IRS2) is one of the major substrate of INSR, given its key role as an adaptor to mediate insulin signals to the downstream molecules by binding to the p-85 subunit of phosphatidylinositol (PI)-3 kinase and activating the serine kinase PKB/Akt pathway (Biddinger et al, 2006;White, 2006).…”