Antiphosphotyrosine antibodies modulate insulin receptor kinase activity and insulin action

Insulin elicits the autophosphorylation of the beta-subunit of its receptor on tyrosine residues: this effect appears to be the earliest post-binding event involved in insulin action. In the present study we have raised highly specific antibodies to phosphotyrosine residues, and we have taken advantage of these antibodies to further evaluate the role of the insulin receptor tyrosine kinase in the generation of insulin's biological responses. Using a cell-free phosphorylation assay, we show here that these antibodies increase the tyrosine kinase activity of the receptor, and its phosphorylation on tyrosine residues. In contrast, the antibodies do not interfere with dephosphorylation of the insulin receptor. Introduction of the same antibodies in living Fao hepatoma cells enhances the effect of insulin on both glucose transport and aminoacid uptake. As a whole our data indicate that the insulin receptor kinase is involved in the generation of an early (glucose transport) and late (aminoacid uptake) response to insulin. Further, conformational changes in phosphotyrosine containing domains of the insulin receptor appear to modulate insulin's biological effects. Finally, the injection of antibodies in intact cells provides us with a novel and promising tool to search for cellular substrates for the insulin receptor tyrosine kinase.     

Early steps in insulin action.

The biological effects of insulin are initiated by the binding of insulin to the insulin receptor. Insulin binds to the extracellular domain of the insulin receptor and induces conformational changes in the receptor, leading to autophosphorylation of the receptor on intracellular tyrosine residues. These phosphorylated tyrosine residues act as binding sites for proteins which subsequently may be phosphorylated by the insulin receptor. As a result, yet other proteins can be recruited to form larger complexes and, in the case of enzymes, changes in their activity may take place. By a combination of these processes, the activated insulin receptor initiates cascades of biochemical events which are regulated mainly by specific phosphorylation or dephosphorylation reactions. Intermediates which are involved in the normal insulin signalling pathway are subjects of expanding research.     

Conformational stability of the epidermal growth factor (EGF) receptor as influenced by glycosylation,dimerization and EGF hormone binding

The epidermal growth factor receptor (EGFR) is an important transmembrane glycoprotein kinase involved the initiation or perpetuation of signal transduction cascades within cells. These processes occur after EGFR binds to a ligand [epidermal growth factor (EGF)], thus inducing its dimerization and tyrosine autophosphorylation. Previous publications have highlighted the importance of glycosylation and dimerization for promoting proper function of the receptor and conformation in membranes; however, the effects of these associations on the protein conformational stability have not yet been described. Molecular dynamics simulations were performed to characterize the conformational preferences of the monomeric and dimeric forms of the EGFR extracellular domain upon binding to EGF in the presence and absence of N‐glycan moieties. Structural stability analyses revealed that EGF provides the most conformational stability to EGFR, followed by glycosylation and dimerization, respectively. The findings also support that EGF–EGFR binding takes place through a large‐scale induced‐fitting mechanism. Proteins 2017; 85:561–570. © 2016 Wiley Periodicals, Inc.     

Evolution of Functional Diversity in the Holozoan Tyrosine Kinome

The emergence of multicellularity is strongly correlated with the expansion of tyrosine kinases, a conserved family of signaling enzymes that regulates pathways essential for cell-to-cell communication. Although tyrosine kinases have been classified from several model organisms, a molecular-level understanding of tyrosine kinase evolution across all holozoans is currently lacking. Using a hierarchical sequence constraint-based classification of diverse holozoan tyrosine kinases, we construct a new phylogenetic tree that identifies two ancient clades of cytoplasmic and receptor tyrosine kinases separated by the presence of an extended insert segment in the kinase domain connecting the D and E-helices. Present in nearly all receptor tyrosine kinases, this fast-evolving insertion imparts diverse functionalities, such as post-translational modification sites and regulatory interactions. Eph and EGFR receptor tyrosine kinases are two exceptions which lack this insert, each forming an independent lineage characterized by unique functional features. We also identify common constraints shared across multiple tyrosine kinase families which warrant the designation of three new subgroups: Src module (SrcM), insulin receptor kinase-like (IRKL), and fibroblast, platelet-derived, vascular, and growth factor receptors (FPVR). Subgroup-specific constraints reflect shared autoinhibitory interactions involved in kinase conformational regulation. Conservation analyses describe how diverse tyrosine kinase signaling functions arose through the addition of family-specific motifs upon subgroup-specific features and coevolving protein domains. We propose the oldest tyrosine kinases, IRKL, SrcM, and Csk, originated from unicellular premetazoans and were coopted for complex multicellular functions. The increased frequency of oncogenic variants in more recent tyrosine kinases suggests that lineage-specific functionalities are selectively altered in human cancers.     

Identification of a Frizzled-like cysteine rich domain in the extracellular region of developmental receptor tyrosine kinases.

In Drosophila, members of the Frizzled family of tissue-polarity genes encode proteins that appear to function as cell-surface receptors for Wnts. The Frizzled genes belong to the seven transmembrane class of receptors (7TMR) and have on their extracellular region a cysteine-rich domain that has been implicated as the Wnt binding domain. This region has a characteristic spacing of ten cysteines, which has also been identified in FrzB (a secreted antagonist of Wnt signaling) and Smoothened (another 7TMR, which is involved in suppression of the hedgehog pathway). We have identified, using BLAST, sequence similarity between the cysteine-rich domain of Frizzled and several receptor tyrosine kinases, which have roles in development. These include the muscle-specific receptor tyrosine kinase (MuSK), the neuronal specific kinase (NSK2), and ROR1 and ROR2. At present, the ligands for these developmental tyrosine kinases are unknown. Our results suggest that Wnt-like ligands may bind to these developmental tyrosine kinases.     

Identification of a Frizzled-like cysteine rich domain in the extracellular region of developmental receptor tyrosine kinases

In Drosophila, members of the Frizzled family of tissue-polarity genes encode proteins that appear to function as cell-surface receptors for Wnts. The Frizzled genes belong to the seven transmembrane class of receptors (7TMR) and have on their extracellular region a cysteine-rich domain that has been implicated as the Wnt binding domain. This region has a characteristic spacing of ten cysteines, which has also been identified in FrzB (a secreted antagonist of Wnt signaling) and Smoothened (another 7TMR, which is involved in the hedgehog signalling pathway). We have identified, using BLAST, sequence similarity between the cysteine-rich domain of Frizzled and several receptor tyrosine kinases, which have roles in development. These include the muscle-specific receptor tyrosine kinase (MuSK), the neuronal specific kinase (NSK2), and ROR1 and ROR2. At present, the ligands for these developmental tyrosine kinases are unknown. Our results suggest that Wnt-like ligands may bind to these developmental tyrosine kinases     

Regulation of tyrosine kinase cascades by G-protein-coupled receptors

Mitogenic signaling by G-protein-coupled receptors (GPCRs) involves tyrosine phosphorylation of adaptor proteins and assembly of multiprotein Ras activation complexes. Over the past three years, three types of scaffolds for GPCR-directed complex assembly have been identified: transactivated receptor tyrosine kinases (RTKs), integrin-based focal adhesions, and GPCRs themselves. Nonreceptor tyrosine kinases play an important role in each case. The processes of GPCR desensitization and sequestration via clathrin-coated pits are also involved in signaling through the RTK- and GPCR-based scaffolds.     

In vitro association of phosphatidylinositol 3-kinase activity with the activated insulin receptor tyrosine kinase.

We previously have shown that insulin treatment of cells greatly increases the activity of phosphatidylinositol (PI) 3-kinase in immunoprecipitates made with an antibody to phosphotyrosine. However, the association of PI 3-kinase activity with the activated insulin receptor is not significant under these conditions. In the present study, we have attempted to reconstitute the association of PI 3-kinase activity with the activated insulin receptor in vitro. PI 3-kinase activity does indeed associate with the autophosphorylated insulin receptor in our in vitro system. The autophosphorylation of the insulin receptor and/or its associated conformational change appear to be necessary for the association of PI 3-kinase activity with the receptor, since kinase negative receptor failed to bind PI 3-kinase activity. After binding, PI 3-kinase or its associated protein seems to be released from the activated receptor after the completion of its tyrosine phosphorylation by the receptor. Tyr960 in the juxtamembrane region of the insulin receptor beta-subunit seems to be involved in the association of PI 3-kinase activity with the receptor, but not C terminus region of the beta-subunit including two tyrosine autophosphorylation sites (Tyr1316 and Tyr1322). The in vitro assay system for the association of PI 3-kinase activity with the insulin receptor can be utilized to study the mechanism of interaction of these molecules and will be an useful method to detect other associated molecules with the insulin receptor.     

Effect of an induced conformational change on the physical properties of two chemotactic receptor molecules.

The physical properties and conformational dynamics of the Salmonella typhimurium ribose and galactose receptors have been examined. Studies involving circular dichroism, fluorescence, absorption spectroscopy, and sedimentation analysis show that the two receptor proteins have different morphologies and exhibit diverse responses to sugar binding. The ribose receptor lacks both tryptophan and disulfide residues, and the galactose receptor lacks disulfides and has only a single tryptophan residue. By virtue of these fortuitous properties, the conformational changes induced in these proteins by sugar binding can be dissected by utilizing a variety of physical probes. A ligand-induced conformational change in the ribose receptor is shown by circular dichroism and fluorescence spectroscopy, which reveal spectral changes assignable to tyrosine, phenylalanine, and methionine residues. A conformational change in the galactose receptor has been demonstrated by fluorescence spectroscopy involving the distant reporter group method, which shows changes assignable to tryptophan and methionine sites and which is corroborated by sedimentation analysis. It is clear that there are extensive conformational changes in the two receptor proteins and that the different physical methods provide complementary information on the nature of these changes.     

Control of ALK (wild type and mutated forms) phosphorylation: Specific role of the phosphatase PTP1B

Phosphorylation of proteins on tyrosine residues is regulated by the activities of protein tyrosine kinases and protein tyrosine phosphatases. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) essentially and transiently expressed during development of the central and peripheral nervous systems. ALK has been identified as a major neuroblastoma predisposition gene and activating mutations have been identified in a subset of sporadic neuroblastoma tumors. We previously established that the mutated receptors were essentially retained in the endoplasmic reticulum/Golgi compartments due to their constitutive activity. Intriguingly we demonstrated a stronger phosphorylation for the minor pool of receptor addressed to the plasma membrane. We decided to investigate the potential involvement of tyrosine phosphatase in dephosphorylation of this intracellular pool. In this study we first showed that general inhibition of tyrosine phosphatases resulted in a dramatic increase of the tyrosine phosphorylation of the wild type but also of the mutated receptors. This increase not only required the intrinsic kinase activity of the ALK receptor but also involved the Src tyrosine kinase family. Second we provided strong evidences that the endoplasmic reticulum associated phosphatase PTP1B is key player in the control of ALK phosphorylation. Our data shed a new light on the biological significance of the basal phosphorylation levels of both wild type and mutated ALK receptors and could be essential to further understand their roles in malignancies.     

Negative regulation of receptor tyrosine kinase signals

In Metazoans a number of cellular functions are controlled by receptor tyrosine kinases (RTKs) during development and in postnatal life. The execution of these programs requires that signals of adequate strength are delivered for the appropriate time within precise spatial boundaries. Several RTK inhibitors have been identified in invertebrate and mammalian organisms. Because they are involved in tuning and termination of receptor signals, negative regulators of RTK activity fulfill a fundamental function in the control of receptor signaling.     

Systemic analysis of tyrosine phosphorylated proteins in angiopoietin-1 induced signaling pathway of endothelial cells

Angiogenesis is an essential process in physiological and pathological processes and is well-regulated to maintain the cellular homeostasis by balancing the endothelial cells in proliferation and apoptosis. Angiopoietin-1 (Ang1) regulates angiogenesis as a ligand of Tie 2 receptor tyrosine kinase. However, the regulation pathways are not well-understood. To date, only a few of the signaling molecules involved in the Tie 2 receptor tyrosine kinase-mediated angiogenesis have been identified. In this study, we systematically identified tyrosine-phosphorylated proteins in Ang1-induced signaling cascade in human umbilical vein endothelial cells (HUVECs), employing proteomic analyses combining two-dimensional gel electrophoresis, Western analysis using phosphotyrosine antibody and mass spectrometry (MALDI-TOF MS and nanoLC-ESI-q-TOF tandem MS). We report here the identification, semiquantitative analysis, and kinetic changes of tyrosine-phosphorylated proteins in response to Ang1 in HUVECs and identified 66 proteins among 69 protein spots showing significant changes. Of these, p54nrb was validated as a molecule involved in cell migration. These results suggest that Ang1 induces stabilization of neo-vessel network by regulating the phosphorylations of metabolic and structural proteins.     

Conserved helix 7 tyrosine acts as a multistate conformational switch in the 5HT2C receptor. Identification of a novel "locked-on" phenotype and double revertant mutations

Studies in many rhodopsin-like G-protein-coupled receptors are providing a general scheme of the structural processes underlying receptor activation. Microdomains in several receptors have been identified that appear to function as activation switches. However, evidence is emerging that these receptor proteins exist in multiple conformational states. To study the molecular control of this switching process, we investigated the function of a microdomain involving the conserved helix 7 tyrosine in the serotonin 5HT2C receptor. This tyrosine of the NPXXY motif was substituted for all naturally occurring amino acids. Three distinct constitutively active receptor phenotypes were found: moderate, high, and "locked-on" constitutive activity. In contrast to the activity of the other receptor mutants, the high basal signaling of the locked-on Y7.53N mutant was neither increased by agonists nor decreased by inverse agonists. The Y7.53F mutant was uncoupled. Computational modeling based on the rhodopsin crystal structure suggested that Y7.53 interacts with the conserved aromatic ring at position 7.60 in the recently identified helix 8 domain. This provided a basis for seeking revertant mutations to correct the defective function of the Y7.53F receptor. When the Y7.53F receptor was mutated at position 7.60, the wild-type phenotype was restored. These results suggest that Y7.53 and Y7.60 contribute to a common functional microdomain connecting helices 7 and 8 that influences the switching of the 5HT2C receptor among multiple active and inactive conformations.     

Characterization of a 97-kDa phosphotyrosylprotein regulated by multiple cytokines.

We have examined the signal transduction pathways of a number of cytokines that interact with receptors that are members of the hematopoietin receptor superfamily. A 97-kDa protein was phosphorylated on tyrosine in response to stimulation of appropriate target cells with interleukin (IL)-2, IL-3, granulocyte-macrophage colony-stimulating factor (CSF), granulocyte-CSF, or erythropoietin. These data suggest that a 97-kDa phosphotyrosylprotein represents a point of convergence for signal transduction by a number of growth factor receptors that do not have homology with any known protein tyrosine kinase. To address the possibility that p97 may represent a tyrosine kinase involved in multiple signal transduction pathways, we tested the capacity of this protein to bind a tyrosine kinase substrate or ATP. Indeed, a 97-kDa phosphotyrosylprotein purified from IL-2-stimulated lymphoid cells as well as granulocyte-macrophage-CSF-stimulated myeloid cells bound to a polymer of glutamic acid and tyrosine which is a tyrosine kinase substrate. Further, a 97-kDa phosphotyrosylprotein present in both lineages also bound 8-azido-ATP. These data indicate that a 97-kDa phosphotyrosylprotein with properties consistent with those of a protein tyrosine kinase is involved in the signal transduction pathways of certain members of the newly identified hematopoietin receptor superfamily and may represent an early point of convergence in the stimulus-response coupling of multiple cytokine receptors.     

Drosophila protein tyrosine phosphatases

Seven protein tyrosine phosphatase (PTPase) genes have been identified in the fruit-fly Drosophila melanogaster. Four of these genes encode receptor-linked PTPases (R-PTPs) that are expressed on central nervous system axons in the embryo. Each axonal R-PTP has an extracellular domain that is homologous to vertebrate adhesion molecules and to identified mammalian R-PTPs. Two non-receptor PTPase genes have been isolated to date. One of these, corkscrew (csw), encodes an SH2 domain-containing PTPase that appears to be a homolog of mammalian PTP1D. Genetic evidence indicates that the csw PTPase is involved in the transduction of signals from receptor tyrosine kinases to their down-stream targets, which include Ras proteins.     

RTK mutations and human syndromeswhen good receptors turn bad

Mutations in receptor tyrosine kinases (RTKs) have been linked to an increasing number of inherited human disease syndromes, including dwarfism, craniosynostosis, heritable cancer susceptibility, venous malformation and Piebaldism. Both gain-of-function mutations resulting in constitutive receptor activation, and loss-of-function mutations resulting in non-functional or dominant negative receptors, have been observed. This review summarizes RTK families that are involved in inherited syndromes, describes the molecular consequences of the disease mutations, and predicts that many novel mutations remain to be identified.     

Modulation of the protein tyrosine kinase activity and autophosphorylation of the epidermal growth factor receptor by its juxtamembrane region

Using peptides epidermal growth factor receptor (EGFR)-13 and EGFR-14, which correspond to residues 645-657 and 679-692, respectively, in the juxtamembrane, cytosolic region of the epidermal growth factor receptor (EGFR) we have investigated the role of specific regions of the receptor in regulating its autophosphorylation and protein tyrosine kinase activity. EGFR-13, but not EGFR-14, increased autophosphorylation (by twofold) of the full-length and two truncated forms (Delta1022-1186 and a constitutively active receptor kinase domain) of the EGFR. EGFR-13 increased the stoichiometry of tyrosine phosphorylation of the full-length receptor from 4.2 to 10.1 mol Pi/mol EGFR and that of EGFRDelta1022-1186 from 1.0 to 2 mol Pi/mol receptor. Increased receptor autophosphorylation in the presence of EGFR-13 cannot solely be attributed to an increase in tyrosine kinase activity because EGFR-14 and polylysine increased tyrosine kinase activity of EGFRDelta1022-1186 and full-length EGFR, respectively, to the same extent as EGFR-13 without any effects on receptor autophosphorylation. Phosphorylation of EGFR-13 (P-EGFR-13) on the threonine residue corresponding to Thr654 in EGFR obliterated the ability of the peptide to increase autophosphorylation and markedly diminished its capacity to increase receptor tyrosine kinase activity. Additionally, EGFR-13, but not EGFR-14 or P-EGFR-13, decreased the migration of the receptor on nondenaturing gels, indicating that EGFR-13 induces some conformational change. Phosphopeptide maps of the EGFR phosphorylated in the presence of EGFR-13 or pp60(c-src) demonstrated that the additional sites phosphorylated in the presence of EGFR-13 were the same as those phosphorylated by pp60(c-src) (i.e., Y803, Y845, Y891, Y920, and Y1101). Thus, we conclude that EGFR-13, but not EGFR-14 or P-EGFR-13, competes to disrupt interactions between amino acids 645-657 and some other region(s) on the EGFR to either alleviate a conformational constraint or alter dimer conformation. This change increases the protein tyrosine kinase activity of the EGFR and provides access to additional tyrosine autophosphorylation sites in the receptor.     

Identification of the APS protein as a novel insulin receptor substrate

In order to identify novel substrates involved in insulin receptor signaling, a yeast two-hybrid 3T3-L1 adipocyte cDNA library was screened with the cytoplasmic domain of the human insulin receptor as bait. Here we describe the isolation and characterization of an interacting protein, APS, which contains pleckstrin homology and Src homology 2 domains and several potential tyrosine phosphorylation sites. APS mRNA and protein are expressed primarily in skeletal muscle, heart, and adipose tissue, and in differentiated 3T3-L1 adipocytes. We show that APS associates with phosphotyrosines situated within the activation loop of the insulin receptor via the APS Src homology 2 domain. Insulin stimulation of 3T3-L1 adipocytes resulted in rapid tyrosine phosphorylation of endogenous APS on tyrosine 618, whereas platelet-derived growth factor treatment resulted in no APS phosphorylation. In summary, we have identified a new insulin receptor substrate that is primarily expressed in insulin-responsive tissues and in 3T3-L1 adipocytes whose phosphorylation shows insulin receptor specificity. These findings suggest a potential role for APS in insulin-regulated metabolic signaling pathways.     

SAM as a protein interaction domain involved in developmental regulation.

More than 60 previously undetected SAM domain-containing proteins have been identified using profile searching methods. Among these are over 40 EPH-related receptor tyrosine kinases (RPTK), Drosophila bicaudal-C, a p53 from Loligo forbesi, and diacyglycerol-kinase isoform delta. This extended dataset suggests that SAM is an evolutionary conserved protein binding domain that is involved in the regulation of numerous developmental processes among diverse eukaryotes. A conserved tyrosine in the SAM sequences of the EPH related RPTKs is likely to mediate cell-cell initiated signal transduction via the binding of SH2 containing proteins to phosphotyrosine.     

MyD88 adapter-like (Mal) is phosphorylated by Bruton's tyrosine kinase during TLR2 and TLR4 signal transduction

Members of the Toll-like receptor (TLR) family are essential players in activating the host innate immune response against infectious microorganisms. All TLRs signal through Toll/interleukin 1 receptor domain-containing adapter proteins. MyD88 adapter-like (Mal) is one such adapter that specifically is involved in TLR2 and TLR4 signaling. When overexpressed we have found that Mal undergoes tyrosine phosphorylation. Three possible phospho-accepting tyrosines were identified at positions 86, 106, and 187, and two mutant forms of Mal in which tyrosines 86 and 187 were mutated to phenylalanine acted as dominant negative inhibitors of NF-kappaB activation by lipopolysaccharide (LPS). Activation of THP-1 monocytic cells with the TLR4 agonist LPS and the TLR2 agonist macrophage-activating lipopeptide-2 induced phosphorylation of Mal on tyrosine residues. We found that the Bruton's tyrosine kinase (Btk) inhibitor LFM-A13 could block the endogenous phosphorylation of Mal on tyrosine in cells treated with macrophage-activating lipopeptide-2 or LPS. Furthermore, Btk immunoprecipitated from THP-1 cells activated by LPS could phosphorylate Mal. Our study therefore provides the first demonstration of the key role of Mal phosphorylation on tyrosine during signaling by TLR2 and TLR4 and identifies a novel function for Btk as the kinase involved.