Structural and Biochemical Evidence for an Autoinhibitory Role for Tyrosine 984 in the Juxtamembrane Region of the Insulin Receptor

Abstract

Tyrosine 984 in the juxtamembrane region of the insulin receptor, between the transmembrane helix and the cytoplasmic tyrosine kinase domain, is conserved among all insulin receptor-like proteins from hydra to humans. Crystallographic studies of the tyrosine kinase domain and proximal juxtamembrane region reveal that Tyr-984 interacts with several other conserved residues in the N-terminal lobe of the kinase domain, stabilizing a catalytically nonproductive position of ␣-helix C. Steady-state kinetics measurements on the soluble kinase domain demonstrate that replacement of Tyr-984 with phenylalanine results in a 4-fold increase in k cat in the unphosphorylated (basal state) enzyme. Moreover, mutation of Tyr-984 in the full-length insulin receptor results in significantly elevated receptor phosphorylation levels in cells, both in the absence of insulin and following insulin stimulation. These data demonstrate that Tyr-984 plays an important structural role in maintaining the quiescent, basal state of the insulin receptor. In addition, the structural studies suggest a possible target site for small molecule activators of the insulin receptor, with potential use in the treatment of noninsulin-dependent diabetes mellitus.The insulin receptor is a member of the receptor tyrosine kinase (RTK) 1 family of cell surface receptors. Unlike the majority of RTKs, which are monomeric in the absence of ligand, the insulin receptor is a disulfide-linked heterotetramer comprising two extracellular ␣ subunits and two membrane-spanning ␤ subunits (1, 2). Upon insulin binding to the ␣ subunits, the insulin receptor undergoes a poorly characterized structural rearrangement that facilitates autophosphorylation of specific tyrosine residues in the cytoplasmic portion of the ␤ subunits. Tyrosine autophosphorylation stimulates receptor catalytic (tyrosine kinase) activity (3) and creates recruitment sites for downstream signaling molecules such as the insulin receptor substrate (IRS) proteins (4) and APS (5).Three tyrosine residues in the cytoplasmic juxtamembrane region of the insulin receptor, Tyr-965, Tyr-972, and Tyr-984, are conserved to various extents in the insulin receptor subfamily of RTKs (Fig. 1). This subfamily includes the insulin receptor, the insulin-like growth factor-1 (IGF1) receptor, the insulin receptor-related receptor (IRR), and insulin receptorlike proteins in invertebrates such as Daf-2 in Caenorhabditis elegans and DIR in Drosophila melanogaster. Tyr-965, an autophosphorylation site that is not conserved in the invertebrate receptors, appears to be involved in receptor endocytosis (6, 7). Tyr-972, invariant in the insulin receptor subfamily, is an essential autophosphorylation site (NPXY motif) that recruits IRS proteins (4), Shc (8), and Stat-5b (9) via a phosphotyrosinebinding domain in these adapter proteins. Tyr-984 is also invariant in this subfamily, yet its role in insulin receptor signaling has not been determined. Biochemical studies indicate that Tyr-984 is not, to any appreciable extent, a site of a...

Imatinib sensitivity as a consequence of a CSF1R-Y571D mutation and CSF1/CSF1R signaling abnormalities in the cell line GDM1

“…In two reports, the alteration of an equivalent tyrosine residue has been shown to be activating: substitution of the equivalent tyrosine with alanine in PDGFRB (Y557) results in constitutive receptor phosphorylation and IL3 independence in Ba/F3 cells; 30 similarly, alanine substitution of an equivalent tyrosine (Y984) in the insulin receptor results in both increased basal phosphorylation and response to insulin. 27 In general, therefore, the association of constitutive kinase activity with CSF1R-Y571D, we have described, is consistent with the current understanding of the role of the juxtamembrane region and therefore shares similarities with other welldescribed activating TK receptor juxtamembrane mutations such as point mutations and tandem duplications in KIT and FLT3. 31 We did not find any CSF1R changes in patients who had been selected for possible imatinib sensitivity based on in vitro assays, and therefore we think it unlikely that CSF1R is mutated in the small number of patients who show imatinib sensitivity of unknown etiology.…”

“…Interaction of Y571 with a conserved glutamic acid (E576) within the kinase domain is thought to contribute to stabilization of the receptor in an inactive conformation, 26 and its disruption by Y571 phosphorylation contributes to kinase activation. In more distantly related TKs such as the insulin receptor 27 and ephrin B2 28 receptors, the disruption of equivalently placed tyrosines and glutamic acid residues by tyrosine phosphorylation has also been implicated in kinase activation.…”

Autoinhibition of the insulin‐like growth factor I receptor by the juxtamembrane region

“…In the IGF1R-0P and -2P structures, Y957 makes similar contacts with hydrophobic residues in the N-terminal lobe of the catalytic domain as seen in the IR juxtamembrane-catalytic structure. As noted previously by Li et al [20], there is a subtle repositioning of the Y957 sidechain in IGF1R relative to the sidechain of Y984 in IR. Y957 of IGF1R is hydrogen-bonded to K1025 in the N-lobe, whereas Y984 of IR is hydrogenbonded to E990.…”

“…3-5). A similar discrepancy was observed for the Y984F mutant form of IR [20]. This discrepancy is probably due to the additional steric constraints imposed on the cytoplasmic domain when it is assembled into the complete a 2 b 2 holoreceptor.…”

Drug binding and resistance mechanism of KIT tyrosine kinase revealed by hydrogen/deuterium exchange FTICR mass spectrometry

“…6,10 Without phosphorylation, KIT is in an autoinhibited conformation, in which the autoinhibitory JM domain inserts into the cleft between the N-and C-terminal lobes of KIT and interacts with the C-alpha helix, which also regulates the catalytic activity of the kinase. 11 The AspPhe-Gly (DFG) motif in the beginning of the activation loop (A-loop) [Fig. 1(b)] is in a ''DFG out'' conformation, with the Phe811 residue protruding out and preventing ATP binding.…”