Delta-opioid receptors activate ERK/MAP kinase via integrin-stimulated receptor tyrosine kinases

Integrin-mediated cell adherence to extracellular matrix proteins results in stimulation of ERK1/2 activity, a mechanism involving focal adhesion tyrosine kinases (pp125FAK, Pyk-2) and epidermal growth factor receptors (EGFRs). G protein-coupled receptors (GPCRs) may also mediate ERK1/2 activation in an integrin-dependent manner, the underlying signaling mechanism of which still remains unclear. Here we demonstrate that the δ-opioid receptor (DOR), a typical GPCR, stimulates ERK1/2 activity in HEK293 cells via integrin-mediated transactivation of EGFR function. Inhibition of integrin signaling by RGDT peptides, cytochalasin, and by keeping the cells in suspension culture both blocked [D-Ala², D-Leu⁵]enkephalin (DADLE)- and etorphine-stimulated ERK1/2 activity. Integrin-dependent ERK1/2 activation does not involve FAK/Pyk-2, because over-expression of the FAK/Pyk-2 inhibitor SOCS-3 failed to attenuate DOR signaling. Exposure of the cells to the EGFR inhibitors AG1478 and BPIQ-I blocked DOR-mediated ERK1/2 activation. Because RGDT peptides also prevented DOR-mediated EGFR activation, the present findings indicate that in HEK293 cells DOR-stimulated ERK1/2 activity is mediated by integrin-stimulated EGFRs. Further studies with the phospholipase C (PLC) inhibitors U73122 and ET-18-OCH₃ revealed that opioid-stimulated integrin activation is sensitive to PLC. In contrast, integrin-mediated transactivation of EGFR function appears to be dependent on PKC-δ, as indicated by studies with rottlerin and siRNA knock-down. A similar ERK1/2 signaling pathway was observed for NG108-15 cells, a neuronal cell line endogenously expressing the DOR. In these cells, the nerve growth factor TrkA receptor replaces the EGFR in connecting DOR-activated integrins to the Ras/Raf/ERK1/2 pathway. Together, these data describe an alternative ERK1/2 signaling pathway in which the DOR transactivates the growth factor receptor associated mitogen-activated protein kinase cascade in an integrin-dependent manner.     

Basic polycations activate the insulin receptor kinase and a tightly associated serine kinase.

The effects of cationic polyamino acids on phosphorylation of the insulin and insulin-like growth factor 1 receptor kinases were studied and the following observations were made. (a) Polylysine stimulated both tyrosine and serine phosphorylation of the insulin receptor and of additional proteins present in lectin-purified membrane preparations from rat liver. (b) Polylysine synergized with insulin to enhance phosphorylation of the insulin receptor and of additional proteins (pp40 and pp110). (c) Polylysine effects were more pronounced upon increasing the polylysine chain length. (d) The effect of polylysine was biphasic with an optimum at 100 micrograms/ml. (e) Polylysine was found ineffective in stimulating the phosphorylation of immobilized insulin receptors. Taken together, these findings support the notion that the action of polylysine involves conformational changes and presumably aggregation of soluble receptors. The same effects of polylysine were obtained with highly purified insulin receptor preparations. Under these conditions polylysine enhanced both serine and tyrosine phosphorylation of the insulin receptor, suggesting that polylysine stimulates the activity of the insulin receptor kinase, and of a serine kinase that is tightly associated with the insulin receptor.     

Insulin receptor tyrosine kinase domain auto-dephosphorylation.

We have observed dephosphorylation of the soluble, 48 kDa insulin receptor tyrosine kinase domain following its tyrosine autophosphorylation. Dephosphorylation was associated with generation of inorganic phosphate, thereby making catalysis by reversal of the kinase reaction unlikely. The kinase domain preparations could not be shown to contain detectable, contaminating protein tyrosine phosphatase activity. In addition, dephosphorylation was insensitive to protein phosphatase inhibitors. However, it was blocked by the kinase inhibitor staurosporine. These results are consistent with insulin receptor kinase domain auto-dephosphorylation via catalysis involving the kinase itself. These findings raise the possibility of a novel mechanism for termination of the insulin receptor signal.     

Oncogenic receptor tyrosine kinase in leukemia.

Growth, survival and differentiation of hematopoietic cells are regulated by the interaction between hematopoietic growth factors and their receptors. While the defect in this interaction results in an insufficient hematopoiesis, the aberrantly elevated activation leads to the transformation of hematopoietic cells. The constitutive active mutations of receptor tyrosine kinase, such as c-Kit platelet-derived growth factor receptor (PDGFR) or fins-like tyrosine kinase 3 (Flt3), play a major role in the development of hematopoietic neoplasia. The constitutive activation is provoked by several mechanisms, such as making fusion genes by chromosomal translocations, or various mutations involving regulatory regions of the receptor. The chromosomal translocation brings the receptor intracytoplasmic domain juxtaposed to an unrelated molecule which has dimerization or multimerization motif, resulting in the constitutive dimerization of the receptor. The missense, insertion or deletion mutations in the regulatory regions, such as juxtamembrane domain, activation loop and extracellular domain, cause constitutive activation by releasing the respective auto-inhibitory functions of each regulatory region. Constitutive active receptors generate different signals quantitatively and qualitatively from wild type receptor, which mediate the oncogenic phenotype. Given the frequent involvement of constitutive active receptor tyrosine kinase in hematopoietic malignancies, targeted inhibitions of active tyrosine kinase and downstream aberrant signaling are rapidly developing novel therapeutic modality with much promise.     

Insulin action is mimicked by polyclonal antireceptor antibodies that activate the insulin receptor tyrosine kinase

The insulin-like properties of anti-insulin receptor antibodies (P95 Ab) that have been characterized as being directed against the receptor beta-subunit, were studied as probes to assess the interrelationship between insulin action and receptor phosphorylation. When tested on intact cells, P95 Ab mimicked insulin effects. On isolated fat cells, they stimulated 2-deoxyglucose (2-DG) transport and lipogenesis and the P95 antibody maximal effects (173 and 232% of the control values, respectively) represented about 50% of the maximal effects elicited by insulin (317 and 475% of the control values). On cultured Zajdela hepatoma cells (ZHC cells), P95 Ab also mimicked insulin action on the incorporation of [U-14C]glucose into glycogen (158 and 207% of the control value for antibody- and insulin-treated cells, respectively). In all cases the antibody effects were dose-dependent, specific and, when maximal, were not additive with those elicited by insulin. When tested in a cell-free system, P95 Ab faithfully reproduced insulin action on the phosphorylation of the receptor beta-subunit. The maximal antibody and insulin effects (317 and 328% of the control value, respectively) were not additive. P95 Ab were also equally potent as insulin to stimulate the receptor-mediated phosphorylation of an exogenous substrate (365 and 379% of the control value in P95 antibody- and insulin-treated receptors, respectively). As well, P95 Ab proved as able as insulin in stimulating the tyrosine kinase activity of the receptor (89% of the hormone effect) when the activation was carried out in vivo. Taken together, these results are consistent with a role for the kinase activity of the insulin receptor in mediating the action of insulin.     

Characterization of elk, a brain-specific receptor tyrosine kinase.

The elk gene encodes a novel receptorlike protein-tyrosine kinase, which belongs to the eph subfamily. We have previously identified a partial cDNA encompassing the elk catalytic domain (K. Letwin, S.-P. Yee, and T. Pawson, Oncogene 3:621-678, 1988). Using this cDNA as a probe, we have isolated cDNAs spanning the entire rat elk coding sequence. The predicted Elk protein contains all the hallmarks of a receptor tyrosine kinase, including an N-terminal signal sequence, a cysteine-rich extracellular domain, a membrane-spanning segment, a cytoplasmic tyrosine kinase domain, and a C-terminal tail. In both amino acid sequence and overall structure, Elk is most similar to the Eph and Eck protein-tyrosine kinases, suggesting that the eph, elk, and eck genes encode members of a new subfamily of receptorlike tyrosine kinases. Among rat tissues, elk expression appears restricted to brain and testes, with the brain having higher levels of both elk RNA and protein. Elk protein immunoprecipitated from a rat brain lysate becomes phosphorylated on tyrosine in an in vitro kinase reaction, consistent with the prediction that the mammalian elk gene encodes a tyrosine kinase capable of autophosphorylation. The characteristics of the Elk tyrosine kinase suggest that it may be involved in cell-cell interactions in the nervous system.     

Protein kinase C-associated kinase can activate NFkappaB in both a kinase-dependent and a kinase-independent manner

Protein kinase C-associated kinase (PKK, also known as RIP4/DIK) activates NFkappaB when overexpressed in cell lines and is required for keratinocyte differentiation in vivo. However, very little is understood about the factors upstream of PKK or how PKK activates NFkappaB. Here we show that certain catalytically inactive mutants of PKK can activate NFkappaB, although to a lesser degree than wild type PKK. The deletion of specific domains of wild type PKK diminishes the ability of this enzyme to activate NFkappaB; the same deletions made on a catalytically inactive PKK background completely ablate NFkappaB activation. PKK may be phosphorylated by two specific mitogen-activated protein kinase kinase kinases, MEKK2 and MEKK3, and this interaction may in part be mediated through a critical activation loop residue, Thr184. Catalytically inactive PKK mutants that block phorbol ester-induced NFkappaB activation do not interfere with, but unexpectedly enhance, the activation of NFkappaB by these two mitogen-activated protein kinase kinase kinases. Taken together, these data indicate that PKK may function in both a kinase-dependent as well as a kinase-independent manner to activate NFkappaB.     

Insulin receptor tyrosine kinase structure and function.

Six years have now elapsed since efforts to establish heterologous cell expression systems for studies of the human insulin receptor were begun. As is apparent from the results summarized in Figs. 1 and 2, a significant number of studies have been devoted to the analysis of receptor mutations, both experimentally derived (i.e. by mutagenesis) and those identified in human patients, as well as to the generation of soluble derivatives of the major functional domains of the receptor for use in biophysical studies. While it is certainly clear that these methods can be expected to yield an ever-increasing body of data concerning insulin receptor structure/function, it is equally apparent that attention to a number of basic experimental limitations inherent in these approaches will be required to resolve a number of fundamental questions and disagreements concerning particular receptor mutations. Given the level of interest in the insulin receptor that has persisted over the past several decades, one expects that these efforts will be forthcoming, and that our understanding of this complex transmembrane receptor will, with time, improve.     

Sprouty is a general inhibitor of receptor tyrosine kinase signaling.

Sprouty was originally identified as an inhibitor of Drosophila FGF receptor signaling during tracheal development. By following the capacity of ectopic Sprouty to abolish the pattern of activated MAP kinase in embryos, we show that Sprouty can inhibit other receptor tyrosine kinase (RTK) signaling pathways, namely the Heartless FGF receptor and the EGF receptor. Similarly, in wing imaginal discs, ectopic Sprouty abolishes activated MAP kinase induced by the EGF receptor pathway. Sprouty expression is induced by the EGFR pathway in some, but not all, tissues in which EGFR is activated, most notably in follicle cells of the ovary, the wing imaginal disc and the eye disc. In the ovary, induction of sprouty expression follows the pattern of EGFR activation in the follicle cells. Generation of homozygous sprouty mutant follicle-cell clones demonstrates an essential role for Sprouty in restricting EGFR activation throughout oogenesis. At the stage when dorso-ventral polarity of the follicle cells is established, Sprouty limits the ventral expansion of the activating Gurken signal. Later, when dorsal appendage fates are determined, reduction of signaling by Sprouty facilitates the induction of inter-appendage cell fates. The capacity of Sprouty to reduce or eliminate accumulation of activated MAP kinase indicates that in vivo it intersects with the pathway upstream to MAP kinase. The ability of ectopic Sprouty to rescue lethality caused by activated Raf suggests that it may impinge upon the pathway by interacting with Raf or downstream to it.     

The trkB tyrosine protein kinase is a receptor for neurotrophin-4.

Neurotrophin-4 is a novel member of the nerve growth factor family of neurotrophins recently isolated from Xenopus and viper DNA. We now report that the Xenopus NT-4 protein (XNT-4) can mediate some of its biological properties through gp145trkB, a murine tyrosine protein kinase previously identified as a primary receptor for the related brain-derived neurotrophic factor (BDNF). XNT-4 displaces 125I-labeled BDNF from binding to cells expressing gp145trkB receptors, induces their rapid phosphorylation on tyrosine residues, and causes the morphologic transformation of NIH 3T3 cells when coexpressed with gp145trkB. Moreover, XNT-4 induces the differentiation of PC12 cells into sympathetic-like neurons only if they ectopically express gp145trkB receptors. None of these biochemical or biological effects could be observed when XNT-4 was added to cells expressing the related receptors. Replacement of one of the extracellular cysteines (Cys-345) of gp145trkB by a serine residue prevents its activation by XNT-4 but not by BDNF. Therefore, XNT-4 and BDNF may interact with at least partially distinct domains within the gp145trkB receptor.     

Insulin receptor serine kinase activation by casein kinase 2 and a membrane tyrosine kinase

The insulin receptor (IR) tyrosine kinase can apparently directly phosphorylate and activate one or more serine kinases. The identities of such serine kinases and their modes of activation are still unclear. We have described a serine kinase (here designated insulin receptor serine (IRS) kinase) from rat liver membranes that co-purifies with IR on wheat germ agglutinin-agarose. The kinase was activated after phosphorylation of the membrane glycoproteins by casein kinase-1, casein kinase-2, or casein kinase-3 (Biochem Biophys Res Commun 171:75–83, 1990). In this study, IRS kinase was further characterized. The presence of vanadate or phosphotyrosine in reaction mixtures was required for activation to be observed. Phosphoserine and phosphothreonine are only about 25% as effective as phosphotyrosine, whereas sodium fluoride and molybdate were ineffective in supporting activation. Vanadate and phosphotyrosine support IRS kinase activation by apparently inhibiting phosphotyrosine protein phosphatases present among the membrane glycoproteins. IR -subunit, myelin basic protein, and microtubule-associated protein-2 are good substrates for IRS kinase. The kinase prefers Mn²⁺ (K_a=1.3 mM) as a metal cofactor. Mg²⁺ (K_a=3.3 mM) is only 30% as effective as Mn²⁺. The kinase activity is stimulated by basic polypeptides, with greater than 30-fold activation achieved with polylysine and protamine. Our results suggest that both serine/threonine and tyrosine phosphorylation are required for activation of IRS kinase. Serine phosphorylation is catalyzed by one of the casein kinases, whereas tyrosine phosphorylation is catalyzed by a membrane tyrosine kinase, possibly IR tyrosine kinase. (Mol Cell Biochem121: 167–174, 1993)     

Multiple angiopoietin recombinant proteins activate the Tie1 receptor tyrosine kinase and promote its interaction with Tie2

The Tie1 receptor tyrosine kinase was isolated over a decade ago, but so far no ligand has been found to activate this receptor. Here, we have examined the potential of angiopoietins, ligands for the related Tie2 receptor, to mediate Tie1 activation. We show that a soluble Ang1 chimeric protein, COMP-Ang1, stimulates Tie1 phosphorylation in endothelial cells with similar kinetics and angiopoietin dose dependence when compared with Tie2. The phosphorylation of overexpressed Tie1 was weakly induced by COMP-Ang1 also in transfected cells that do not express Tie2. When cotransfected, Tie2 formed heteromeric complexes with Tie1, enhanced Tie1 activation, and induced phosphorylation of a kinase-inactive Tie1 in a ligand-dependent manner. Tie1 phosphorylation was also induced by native Ang1 and Ang4, although less efficiently than with COMP-Ang1. In conclusion, we show that Tie1 phosphorylation is induced by multiple angiopoietin proteins and that the activation is amplified via Tie2. These results should be important in dissecting the signal transduction pathways and biological functions of Tie1.     

Phosphatidylinositol kinase or an associated protein is a substrate for the insulin receptor tyrosine kinase.

The tyrosine kinase activity intrinsic to the insulin receptor is thought to be important in eliciting the intracellular responses to insulin; however, it has been difficult to determine the biochemical functions of the proteins which are substrates for this receptor. Treatment of Chinese hamster ovary (CHO) cells overexpressing the human insulin receptor (CHO.T) with insulin results in a 38 +/- 11 (mean +/- S.E., n = 9)-fold increase in a phosphatidylinositol (PtdIns) kinase activity in anti-phosphotyrosine immunoprecipitates of whole cell lysates. One minute of treatment of cells with insulin causes a dramatic increase in the PtdIns kinase activity in the anti-phosphotyrosine immunoprecipitates; the activity peaks within 5 min and remains elevated for at least 60 min after addition of insulin to the cells. This response is only slightly delayed compared with the time course we observe for activation of the insulin receptor tyrosine kinase. The insulin dose-response curves are also very similar for the activation of the insulin receptor tyrosine kinase activity and for the appearance of PtdIns kinase in the anti-phosphotyrosine immunoprecipitates. Stimulation of the endogenous insulin receptor of CHO cells also results in the association of PtdIns kinase activity with phosphotyrosine-containing proteins. However, CHO cells are less sensitive to insulin than CHO.T cells, and the maximal PtdIns kinase activity in antiphosphotyrosine immunoprecipitates from CHO cells is one-sixth that of CHO.T cells. In contrast, immunoprecipitates from CHO.T cells made with anti-insulin receptor antibodies do not contain significant levels of PtdIns kinase activity. This demonstrates that the PtdIns kinase is either a substrate for the insulin receptor tyrosine kinase or is tightly associated with another tyrosine phosphoprotein, which is not the insulin receptor.     

Expression of the human epidermal growth factor receptor in a murine T-cell hybridoma. A transmembrane protein tyrosine kinase can synergize with the T-cell antigen receptor.

Although the T-cell receptor for antigen (TCR) lacks intrinsic kinase activity, stimulation of this receptor induces tyrosine phosphorylation of multiple substrates. In contrast, the epidermal growth factor receptor (EGFR) has intrinsic cytoplasmic tyrosine kinase catalytic activity that is activated upon EGF binding. To compare the functional effects of the TCR and a transmembrane protein tyrosine kinase (PTK), we used retrovirus-mediated gene transduction to express the human c-erbB proto-oncogene, encoding the EGFR, in a murine T-cell hybridoma. Tyrosine phosphorylation induced by the TCR and the EGFR occurred on substrates unique to each receptor as well as on several shared substrates, including the zeta chain of the TCR. Stimulation of the EGFR induced calcium ion flux in these cells, suggesting that the heterologous tyrosine kinase can couple to the T-cell phospholipase signal transduction pathway, but this stimulus did not lead to interleukin 2 production. However, EGF stimulation of transduced cells significantly enhanced TCR signaling, as assessed by interleukin 2 production, indicating that cross talk can occur between the TCR and a transmembrane PTK.     

Interaction between heterologous receptor tyrosine kinases. Hormone-stimulated insulin receptors activate unoccupied IGF-I receptors.

To determine whether heterologous receptor tyrosine kinases interact with each other we have investigated the ability of insulin receptors to transphosphorylate and transactivate IGF-I receptors. Using partially purified receptors we show that hormone-stimulated insulin receptors induced a 40% increase in IGF-I receptor phosphorylation. Remarkably, this transphosphorylation of IGF-I receptors by insulin receptors resulted in a 2.5-fold augmentation of the IGF-I receptor tyrosine kinase activity for substrates. Our findings demonstrate that transphosphorylation with transactivation can occur between insulin and IGF-I receptors. We would like to propose that such a phenomenon participates in the insulin-induced pleiotropic program by mediating the growth promoting effects of the hormone.     

Specific inhibition of NGF receptor tyrosine kinase activity by K-252a.

An involvement of protein tyrosine kinase in the transduction of the signals initiated by nerve growth factor (NGF) was investigated. A tyrosine kinase inhibitor, herbimycin, inhibited neurite outgrowth of rat pheochromocytoma PC12 cells induced by NGF but not that by dibutyryl-cAMP. Herbimycin and genistein blocked NGF-dependent activation of ras p21 whose essential function in neuronal differentiation has been reported. These observations suggested that tyrosine kinase activity is involved in the signaling pathways. K-252a, by contrast, inhibited NGF-induced but not EGF-dependent activation of ras p21. Tyrosine kinase activity of gp140trk, a constituent of NGF receptor, is activated by NGF for much a longer period compared to the activation of EGF receptor autokinase activity by EGF. We further demonstrated that autophosphorylation of gp140trk is selectively inhibited by K-252a.     

Calmodulin inhibits the epidermal growth factor receptor tyrosine kinase.

We demonstrate in this report that the epidermal growth factor (EGF) receptor from rat liver can be isolated by calmodulin affinity chromatography by binding in the presence of Ca2+ and elution with a Ca(2+)-chelating agent. The bulk of the EGF receptor is not eluted by a NaCl gradient in the presence of Ca2+. We ascertained the identity of the isolated receptor by immunoblot and immunoprecipitation using a polyclonal antibody against an EGF receptor from human origin. The purified receptor is autophosphorylated in tyrosine residues in an EGF-stimulated manner, and EGF-dependent phosphorylation of serine residues was also detected. Both the EGF and the transforming growth factor-alpha stimulate the tyrosine-directed protein kinase activity of the isolated receptor with similar affinities. Furthermore, we demonstrate that calmodulin inhibits the EGF-dependent tyrosine-directed protein kinase activity associated to the receptor in a concentration-dependent manner. This inhibition is partially Ca2+ dependent and is not displaced by increasing the concentration of EGF up to an EGF/calmodulin ratio of 10 (mol/mol). In addition, calmodulin was phosphorylated in an EGF-stimulated manner in the presence of a basic protein (histone) as cofactor and in the absence, but not in the presence, of Ca2+.     

Inhibition of the insulin receptor tyrosine kinase by sphingosine.

Sphingosine inhibits autophosphorylation of the insulin receptor tyrosine kinase in vitro and in situ. This lysosphingolipid has been shown previously to inhibit the Ca2+/lipid-dependent protein kinase C. Here we show that insulin-dependent autophosphorylation of partially purified insulin receptor is half-maximally inhibited by 145 microM sphingosine (9 mol %) in Triton X-100 micelles. Half-maximal inhibition of protein kinase C autophosphorylation occurs with 60 microM sphingosine (3.4 mol %) in Triton X-100 mixed micelles containing phosphatidylserine and diacylglycerol. Sphingomyelin does not inhibit significantly the insulin receptor, suggesting that, as with protein kinase C, the free amino group may be essential for inhibition. Similar to the effects observed for protein kinase C, inhibition of the insulin receptor kinase by sphingosine is reduced in the presence of other lipids. However, the reduction displays a marked dependence on the lipid species: phosphatidylserine, but not a mixture of lipids compositionally similar to the cell membrane, markedly reduces the potency of sphingosine inhibition. The inhibition occurs at the level of the protein/membrane interaction: a soluble form of the insulin receptor comprising the cytoplasmic kinase domain is resistant to sphingosine inhibition. Lastly, sphingosine inhibits the insulin-stimulated rate of tyrosine phosphorylation of the insulin receptor in NIH 3T3 cells expressing the human insulin receptor. These results suggest that sphingosine alters membrane function independently of protein kinase C.