Crystal structure of an angiogenesis inhibitor bound to the FGF receptor tyrosine kinase domain

Angiogenesis, the sprouting of new blood vessels from pre-existing ones, is an essential physiological process in development, yet also plays a major role in the progression of human diseases such as diabetic retinopathy, atherosclerosis and cancer. The effects of the most potent angiogenic factors, vascular endothelial growth factor (VEGF), angiopoietin and fibroblast growth factor (FGF) are mediated through cell surface receptors that possess intrinsic protein tyrosine kinase activity. In this report, we describe a synthetic compound of the pyrido[2,3-d]pyrimidine class, designated PD 173074, that selectively inhibits the tyrosine kinase activities of the FGF and VEGF receptors. We show that systemic administration of PD 173074 in mice can effectively block angiogenesis induced by either FGF or VEGF with no apparent toxicity. To elucidate the determinants of selectivity, we have determined the crystal structure of PD 173074 in complex with the tyrosine kinase domain of FGF receptor 1 at 2.5 A resolution. A high degree of surface complementarity between PD 173074 and the hydrophobic, ATP-binding pocket of FGF receptor 1 underlies the potency and selectivity of this inhibitor. PD 173074 is thus a promising candidate for a therapeutic angiogenesis inhibitor to be used in the treatment of cancer and other diseases whose progression is dependent upon new blood vessel formation.     

IRS-1-mediated inhibition of insulin receptor tyrosine kinase activity in TNF-alpha- and obesity-induced insulin resistance

Tumor necrosis factor-alpha (TNF-alpha) is an important mediator of insulin resistance in obesity and diabetes through its ability to decrease the tyrosine kinase activity of the insulin receptor (IR). Treatment of cultured murine adipocytes with TNF-alpha was shown to induce serine phosphorylation of insulin receptor substrate 1 (IRS-1) and convert IRS-1 into an inhibitor of the IR tyrosine kinase activity in vitro. Myeloid 32D cells, which lack endogenous IRS-1, were resistant to TNF-alpha-mediated inhibition of IR signaling, whereas transfected 32D cells that express IRS-1 were very sensitive to this effect of TNF-alpha. An inhibitory form of IRS-1 was observed in muscle and fat tissues from obese rats. These results indicate that TNF-alpha induces insulin resistance through an unexpected action of IRS-1 to attenuate insulin receptor signaling.     

PD98059 is an equipotent antagonist of the aryl hydrocarbon receptor and inhibitor of mitogen-activated protein kinase kinase

Striatal dopamine transporter function and dopamine D2 receptor status were evaluated in 15 patients with early untreated Parkinson's disease using single photon emission tomography (SPECT) with 123I-Iodo-2beta-carboxymethoxy-3beta-(4-idiophenyl)tropane (beta-CIT) and 123I-Iodobenzamide (IBZM) as pre- and postsynaptic ligands. Symptoms were unilateral in five patients and bilateral but asymmetric in 10 patients. Patients with bilateral symptoms had significantly lower 18-hour striatal/cerebellar beta-CIT binding ratios (3.59 +/- 0.79) than hemiparkinsonian patients (5.76 +/- 1.48, p < 0.05) reflecting more advanced disease in this subgroup. Patients with bilateral parkinsonism were also found to have a significant side-to-side difference in striatal beta-CIT binding with more marked reduction contralateral to the presenting limb (18-hour striatal/cerebellar ratio: 4.13 +/- 0.78 [ipsilateral] versus 3.59 +/- 0.79 [contralateral], p < 0.05). Dopamine D2 receptor binding as measured by IBZM was significantly elevated contralateral to the affected side in hemiparkinsonian patients (striatal/cerebellar ratio: 2.42 +/- 0.90 [contralateral] versus 2.19 +/- 0.80 [ipsilateral], p < 0.05). This asymmetric upregulation was absent in the patients with bilateral parkinsonism (striatal/cerebellar ratio: 1.85 +/- 0.43 [contralateral to more severely affected side] versus 1.83 +/- 0.34 [ipsilateral], p > 0.05). Our data suggest that postsynaptic dopamine receptor upregulation contralateral to the presenting side occurs in untreated unilateral PD and disappears in untreated bilateral (asymmetric) PD despite a greater loss of dopamine transporter function. Combined beta-CIT and IBZM SPECT studies may be helpful to monitor the progression of nigrostriatal dysfunction in early PD.     

Megalin is an endocytic receptor for insulin

Renal clearance is a major pathway for regulating the levels of insulin and other low molecular weight polypeptide hormones in the systemic circulation. Previous studies have shown that the reabsorption of insulin from the glomerular filtrate occurs by binding to as yet unidentified sites on the luminal surface of proximal tubule cells followed by endocytosis and degradation in lysosomes. In this study, an insulin binding site was identified in renal microvillar membranes by chemical cross-linking procedures. By immunoprecipitation it was demonstrated that this binding site is megalin, the large multiligand binding endocytic receptor that is abundantly expressed in clathrin-coated pits on the apical surface of proximal tubule cells. Moreover, using cytochemical procedures, it was also shown that megalin is able to internalize insulin into endocytic vesicles. In ligand blotting assays, megalin also bound several other low molecular weight polypeptides, including beta2-microglobulin, epidermal growth factor, prolactin, lysozyme, and cytochrome c. These data suggest that megalin may play a significant role as a renal reabsorption receptor for the uptake of insulin and other low molecular weight polypeptides from the glomerular filtrate.     

Protein kinase C modulation of insulin receptor substrate-1 tyrosine phosphorylation requires serine 612

Activation of the endogenous protein kinase Cs in human kidney fibroblast (293) cells was found in the present study to inhibit the subsequent ability of insulin to stimulate the tyrosine phosphorylation of an expressed insulin receptor substrate-1. This inhibition was also observed in an in vitro phosphorylation reaction if the insulin receptor and its substrate were both isolated from cells in which the protein kinase C had been activated. To test whether serine phosphorylation of the insulin receptor substrate-1 was contributing to this process, serine 612 of this molecule was changed to an alanine. The insulin-stimulated tyrosine phosphorylation and the associated phosphatidylinositol 3-kinase activity of the expressed mutant were found to be comparable to those of the expressed wild-type substrate. However, unlike the wild-type protein, activation of protein kinase C did not inhibit the insulin-stimulated tyrosine phosphorylation of the S612A mutant nor its subsequent association with phosphatidylinositol 3-kinase. Tryptic peptide mapping of in vivo labeled IRS-1 and the S612A mutant revealed that PMA stimulates the phosphorylation of a peptide from wild-type IRS-1 that is absent from the tryptic peptide maps of the S612A mutant. Moreover, a synthetic peptide containing this phosphoserine and its nearby tyrosine was found to be phosphorylated by the insulin receptor to a much lower extent than the same peptide without the phosphoserine. Activation of protein kinase C was found to stimulate by 10-fold the ability of a cytosolic kinase to phosphorylate this synthetic peptide as well as the intact insulin receptor substrate-1. Finally, cytosolic extracts from the livers of ob/ob mice showed an 8-fold increase in a kinase activity capable of phosphorylating this synthetic peptide, compared to extracts of livers from lean litter mates. These results indicate that activation of protein kinase C stimulates a kinase which can phosphorylate insulin receptor substrate-1 at serine 612, resulting in an inhibition of insulin signaling in the cell, posing a potential mechanism for insulin resistance in some models of obesity.     

Impact of mutations at different serine residues on the tyrosine kinase activity of the insulin receptor

Insulin binding to its receptor activates a cascade of signaling events which are initiated by tyrosine autophosphorylation of the receptor and activation of the tyrosine kinase activity towards the insulin receptor substrates. In addition to phosphorylation at tyrosine residues a serine phosphorylation of the insulin receptor is observed. Neither the functional significance of serine phosphorylation of the receptor nor the location of relevant regulatory sites has been determined exactly so far. We studied potential functions of serine residues in human insulin receptor (HIR) with respect to its ability to undergo insulin stimulated autophosphorylation. Using site directed mutagenesis of HIR we exchanged serine to alanine at 13 different positions in the HIR beta-subunit. Sites were chosen according to the criteria of known serine phosphorylation sites (1023/25, 1293/94, 1308/09), conserved positions in hIR, hIGF-1 receptor, hIRR, and dIR (962, 994, 1037, 1055, 1074/78, 1168, 1177/78/82, 1202, 1263, 1267). All HIR mutants were expressed in HEK 293 cells and basal and insulin stimulated autophosphorylation were determined. We found that the exchange of serine to alanine at position 994 and at position 1023/25 increased insulin stimulated receptor autophosphorylation significantly (147% +/- 12% and 129% +/- 6% of control, p < 0.01, n = 7), while all other exchanges did not significantly alter insulin stimulated HIR autophosphorylation. The data suggest that the serine residues at position 994 as well as 1023/25 might be part of inhibitory domains of the insulin receptor.     

p125Fak focal adhesion kinase is a substrate for the insulin and insulin-like growth factor-I tyrosine kinase receptors

The focal adhesion kinase p125(Fak) is a widely expressed cytosolic tyrosine kinase, which is involved in integrin signaling and in signal transduction of a number of growth factors. In contrast to tyrosine kinase receptors such as the platelet-derived growth factor and the hepatocyte growth factor receptors, which induce p125(Fak) phosphorylation, insulin has been shown to promote its dephosphorylation. In this study, we compared p125(Fak) phosphorylation in insulin-stimulated cells maintained in suspension or in an adhesion state. We found that, in nonattached cells, insulin promotes p125(Fak) phosphorylation, whereas dephosphorylation occurred in attached cells. This was observed in Rat-1 fibroblasts overexpressing the insulin receptor, as well as in Hep G2 hepatocytes and in 3T3-L1 adipocytes expressing more natural levels of insulin receptors. Insulin-induced p125(Fak) phosphorylation correlated with an increase in paxillin phosphorylation, indicating that p125(Fak) kinase activity may be stimulated by insulin. Mixing of purified insulin or insulin-like growth factor-I (IGF-I) receptors with p125(Fak) resulted in an increase in p125(Fak) phosphorylation. Using a kinase-deficient p125(Fak) mutant, we found that this protein is a direct substrate of the insulin and IGF-I receptor tyrosine kinases. This view is supported by two additional findings. (i) A peptide corresponding to p125(Fak) sequence comprising amino acids 568-582, which contains tyrosines 576 and 577 of the kinase domain regulatory loop, is phosphorylated by the insulin receptor; and (ii) p125(Fak) phosphorylation by the insulin receptor is prevented by addition of this peptide. Finally, we observed that p125(Fak) phosphorylation by the receptor results in its activation. Our results show that the nature of the cross-talk between the insulin/IGF-I receptors and p125(Fak) is dependent on the cell architecture, and hence the interaction of the insulin/IGF-I signaling system with the integrin system will vary accordingly.     

Reduced tyrosine kinase activity of the insulin receptor in obesity-diabetes. Central role of tumor necrosis factor-alpha

Insulin resistance is an important metabolic abnormality often associated with infections, cancer, obesity, and especially non-insulin-dependent diabetes mellitus (NIDDM). We have previously demonstrated that tumor necrosis factor-alpha produced by adipose tissue is a key mediator of insulin resistance in animal models of obesity-diabetes. However, the mechanism by which TNF-alpha interferes with insulin action is not known. Since a defective insulin receptor (IR) tyrosine kinase activity has been observed in obesity and NIDDM, we measured the IR tyrosine kinase activity in the Zucker (fa/fa) rat model of obesity and insulin resistance after neutralizing TNF-alpha with a soluble TNF receptor (TNFR)-lgG fusion protein. This neutralization resulted in a marked increase in insulin-stimulated autophosphorylation of the IR, as well as phosphorylation of insulin receptor substrate 1 (IRS-1) in muscle and fat tissues of the fa/fa rats, restoring them to near control (lean) levels. In contrast, no significant changes were observed in insulin-stimulated tyrosine phosphorylations of IR and IRS-1 in liver. The physiological significance of the improvements in IR signaling was indicated by a concurrent reduction in plasma glucose, insulin, and free fatty acid levels. These results demonstrate that TNF-alpha participates in obesity-related systemic insulin resistance by inhibiting the IR tyrosine kinase in the two tissues mainly responsible for insulin-stimulated glucose uptake: muscle and fat.     

Inhibition of experimental autoimmune encephalomyelitis by a tyrosine kinase inhibitor

Migration of lymphocytes from the blood into the brain is a critical event in the pathogenesis of experimental autoimmune encephalomyelitis. Lymphocyte adhesion to brain endothelium is the first step in lymphocyte entry into the central nervous system, leading subsequently to myelin damage and paralysis. In this paper we show that the tyrosine kinase inhibitor, tyrphostin AG490, prevents binding of freshly isolated mouse lymph node cells and of in vivo activated lymphocytes to endothelium of inflamed brain in Stamper-Woodruff adhesion assays. Moreover, AG490 inhibits adhesion of encephalitogenic T cell lines to purified ICAM-1 and VCAM-1, molecules implicated in T cell recruitment into the central nervous system. In contrast, 2-h treatment of T cell lines with high doses of tyrphostin AG490 have no effect on the viability, intracellular calcium elevation induced by Con A or TCR cross-linking, proliferation, or TNF production by Ag-stimulated T cell lines. Systemic administration of AG490 prevents the accumulation of leukocytes in the brain and the development of experimental autoimmune encephalomyelitis induced by proteolipid protein, peptide 139-151-specific T cell lines in SJL/J mice. Blood leukocytes isolated from mice treated with tyrphostin AG490 are less adhesive on purified very late Ag-4 ligands compared with adhesion of leukocytes from control animals. Our results suggest that inhibition of signaling pathways involved in lymphocyte adhesion may represent a novel therapeutic approach for demyelinating diseases.     

Growth hormone stimulates the tyrosine kinase activity of JAK2 and induces tyrosine phosphorylation of insulin receptor substrates and Shc in rat tissues

Each year, acute myocardial infarctions (AMI) account for more than half a million deaths in the United States. Complicating treatment of AMI is the difficulty in accurately diagnosing the event, for patients have nondiagnostic electrocardiograms (ECG) more than 50% of the time. In this population, cardiac markers are essential to confirm the diagnosis. The new bedside cardiac markers, which use eight drops of whole blood and require 15 minutes to be read negative, make it possible to shorten time needed to diagnose AMI. One hundred twenty-seven consecutive patients presented to the emergency department complaining of atypical chest pain. All had ECGs that were nondiagnostic for myocardial infarction. Serial cardiac markers were performed: myoglobin, troponin I, rapid myoglobin, and rapid troponin I. One hundred eighteen patients with negative serial cardiac markers had exercise treadmill tests in the emergency department. Nine patients with positive serial cardiac markers received emergent primary angioplasty. Six of the nine patients were treated based on the positive results of the rapid bedside cardiac markers. A 100% correlation existed between the quantitative serum results and the rapid bedside markers. With the availability of rapid bedside assays, dependency on the laboratory can be minimized, since quantitative cardiac markers require at least 1 hour of turnaround time. Rapidly and correctly diagnosing AMIs in patients with ECGs nondiagnostic for AMI has always been a dilemma. Rapid bedside assays enable the physician to accurately diagnose myocardial infarction and safely decrease the time in evaluating chest pain, thus maximizing the benefits of early reperfusion.     

Vanadyl sulfate-stimulated glycogen synthesis is associated with activation of phosphatidylinositol 3-kinase and is independent of insulin receptor tyrosine phosphorylation

Salts of the trace element vanadium, such as sodium orthovanadate and vanadyl sulfate (VS), exhibit a myriad of insulin-like effects, including stimulation of glycogen synthesis and improvement of glucose homeostasis in type I and type II animal models of diabetes mellitus. However, the cellular mechanism by which these effects are mediated remains poorly characterized. We have shown earlier that different vanadium salts stimulate the MAP kinase pathway and ribosomal-S-6-kinase (p70s6k) in chinese hamster ovary cells overexpressing human insulin receptor (CHO-HIR cells) [Pandey, S. K., Chiasson, J.-L., and Srivastava, A. K. (1995) Mol. Cell. Biochem. 153, 69-78]. In the present studies, we have investigated if similar to insulin, VS also activates phosphatidylinositol 3-kinase (PI3-k) activity, and whether VS-induced activation of the PI3-k, MAP kinase, and p70s6k pathways contributes to glycogen synthesis. Treatment of CHO-HIR cells with VS resulted in increased glycogen synthesis and PI3-k activity which were blocked by pretreatment of the cells with wortmannin and LY294002, two specific inhibitors of PI3-k. On the other hand, PD98059 and rapamycin, specific inhibitors of the MAP kinase pathway and p70s6k, respectively, were unable to inhibit VS-stimulated glycogen synthesis. Moreover, VS-stimulated glycogen synthesis and PI3-k were observed without any change in the tyrosine phosphorylation of insulin receptor (IR) beta-subunit but were associated with increased tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). In addition, PI3-k activation was detected in IRS-1 immunoprecipitates from VS-stimulated cells, indicating that tyrosine-phosphorylated IRS-1 was able to interact and thereby activate PI3-k in response to VS. Taken together, these results provide evidence that tyrosine phosphorylation of IRS-1 and activation of PI3-k play a key role in mediating the insulinomimetic effect of VS on glycogen synthesis independent of IR-tyrosine phosphorylation.     

Neurturin responsiveness requires a GPI-linked receptor and the Ret receptor tyrosine kinase

Neurturin (NTN) is a recently identified homologue of glial-cell-line-derived neurotrophic factor (GDNF). Both factors promote the survival of a variety of neurons, and GDNF is required for the development of the enteric nervous system and kidney. GDNF signals through a receptor complex consisting of the receptor tyrosine kinase Ret and a glycosyl-phosphatidylinositol (GPI)-linked receptor termed GDNFR-alpha. Here we report the cloning of a new GPI-linked receptor termed NTNR-alpha that is homologous with GDNFR-alpha and is widely expressed in the nervous system and other tissues. By using microinjection to introduce expression plasmids into neurons, we show that coexpression of NTNR-alpha with Ret confers a survival response to neurturin but not GDNF, and that coexpression of GDNFR-alpha with Ret confers a survival response to GDNF but not neurturin. Our findings indicate that GDNF and neurturin promote neuronal survival by signalling through similar multicomponent receptors that consist of a common receptor tyrosine kinase and a member of a GPI-linked family of receptors that determines ligand specificity.     

Phosphatidylinositol-3' kinase is not required for mitogenesis or internalization of the Flt3/Flk2 receptor tyrosine kinase

Flt3/Flk2 is a receptor tyrosine kinase that is expressed on early hematopoietic progenitor cells. Flt3/Flk2 belongs to a family of receptors, including Kit and colony-stimulating factor-1R, which support growth and differentiation within the hematopoietic system. The Flt3/Flk2 ligand, in combination with other growth factors, stimulates the proliferation of hematopoietic progenitors of both lymphoid and myeloid lineages in vitro. We report that phosphatidylinositol 3'-kinase (PI3K) binds to a unique site in the carboxy tail of murine Flt3/Flk2. In distinction to Kit and colony-stimulating factor-1R, mutant receptors unable to couple to PI3K and expressed in rodent fibroblasts or in the interleukin 3-dependent cell line Ba/F3 provide a mitogenic signal comparable to wild-type receptors. Flt3/Flk2 receptors that do not bind to PI3K also normally down-regulate, a function ascribed to PI3K in the context of other receptor systems. These data point to the existence of other unidentified pathways that, alone or in combination with PI3K, transduce these cellular responses following the activation of Flt3/Flk2.     

Identification of the major autophosphorylation sites of Nyk/Mer, an NCAM-related receptor tyrosine kinase

Nyk/Mer receptor tyrosine kinase is a new member of the Ufo/Axl tyrosine kinase family and is characterized by its neural cell adhesion molecule-like extracellular domain. By using a vaccinia virus expression system to express a constitutively activated form of Nyk, we identified the major sites of Nyk autophosphorylation in tryptic peptide IY749SGDY753Y754R. Tyr-749, Tyr-753, and Tyr-754 in this peptide lie in the activation loop of the kinase domain. We also studied a series of Nyk mutants in which the three tyrosine residues were replaced individually, in pairs, or all together by phenylalanine. Single mutations of Tyr-749 or Tyr-753 to phenylalanine reduced Nyk kinase activity toward exogenous substrate to 39 or 10% of that of the wild type Nyk, respectively, whereas the Tyr-754 mutant is completely inactive. All of the double and triple Tyr-Phe mutants reduced Nyk kinase activity to a level below the background. Similar results were obtained when Nyk autophosphorylation levels were examined. Our studies suggest that full activity of Nyk/Mer kinase requires phosphorylation of all three tyrosine residues in the kinase domain (Tyr-749, Tyr-753, and Tyr-754) and that Nyk kinase activity is modulated by the level of autophosphorylation in the kinase domain. Given the highly conserved nature of this region among the Ufo/Axl receptor family members, the information presented in this report may provide insight to the biochemical properties of other members of this family.     

Structure of malhamensilipin A, an inhibitor of protein tyrosine kinase, from the cultured chrysophyte Poterioochromonas malhamensis

A new chlorosulfolipid, malhamensilpin A [1] was isolated from the cultured chrysophyte Poterioochromonas malhamensis. Malhamensilipin A was demonstrated to be a modest inhibitor of pp60v-src protein tyrosine kinase. The structure was determined by detailed spectral analysis to be a novel C24 hexachloro lipid containing a vinyl sulfate ester (2,11,12,13,15,16-hexachloro-14-hydroxy-n-tetracos-1E-enol-1-sulfa te).     

Cutting edge: the CD45 tyrosine phosphatase is an inhibitor of Lck activity in thymocytes

A widely accepted model for regulation of the Lck tyrosine kinase is that it is activated by CD45-mediated dephosphorylation of its COOH-terminal negative regulatory tyrosine (Tyr505). Previous work from our laboratory, however, found that despite hyperphosphorylation of Tyr505, the activity of Lck from CD45- T cell lines was actually increased due to hyperphosphorylation of the positive regulatory tyrosine, residue 394. To avoid potential complications introduced by transformed cells, in this study we have characterized the effect of CD45 on Lck activity in normal cells. Lck in thymocytes from CD45-/- mice was hyperphosphorylated on tyrosine residues. Importantly, and in disagreement with the model that CD45 only activates Lck in vivo, the kinase activity of Lck from cells lacking CD45 was substantially increased. These results support a model in which CD45 dephosphorylates both Tyr505 and Tyr394, the net effect in normal thymocytes being a decrease in enzymatic activity.     

Rapsyn clusters and activates the synapse-specific receptor tyrosine kinase MuSK

Nerve-induced clustering of the nicotinic acetylcholine receptor (AChR) requires rapsyn, a synaptic peripheral membrane protein, as well as protein-tyrosine kinase activity. Here, we show that rapsyn induces the clustering of the synapse-specific receptor-tyrosine kinase MuSK in transfected QT-6 fibroblasts. Furthermore, rapsyn stimulates the autophosphorylation of MuSK, leading to a subsequent MuSK-dependent increase in cellular tyrosine phosphorylation. Moreover, rapsyn-activated MuSK specifically phosphorylated the AChR beta subunit, the same subunit that is tyrosine phosphorylated during innervation or agrin treatment of muscle. These results suggest rapsyn may mediate the synaptic localization of MuSK in muscle and that MuSK may play an important role in the agrin-induced clustering of the AChR.     

The epidermal growth factor receptor tyrosine kinase phosphorylates connexin32

To elucidate the role of excessive nitric oxide (NO) via the inducible nitric oxide synthase (iNOS) in experimental allergic encephalomyelitis (EAE), the effect of a selective iNOS inhibitor, aminoguanidine, was investigated using mice with actively induced EAE. Administration of aminoguanidine by intraperitoneal or intracisternal injection from day 2 to day 12 after immunization produced a significant delay in the onset of EAE. On the other hand, administration of aminoguanidine by intraperitoneal or intracisternal injection for 10 days after the onset of EAE enhanced the clinical severity and mortality rate and hastened the onset of relapse significantly. The histological study at day 11 after the onset revealed that more inflammatory cells were present in the central nervous system of mice treated with aminoguanidine as compared with mice without aminoguanidine treatment. These results suggested that NO via iNOS was a pathogenetic factor in the induction phase of EAE, but had an inhibitory role in the progression phase of EAE. Although the effect of NO synthase inhibitors on EAE has been controversial, the present study suggested that the timing of administration might be an important consideration and might explain the previous contradictory reports.