Directing traffic in neural cells: determinants of receptor tyrosine kinase localization and cellular responses

The trafficking of receptor tyrosine kinases (RTKs) to distinct subcellular locations is essential for the specificity and fidelity of signal transduction and biological responses. This is particularly important in the PNS and CNS in which RTKs mediate key events in the development and maintenance of neurons and glia through a wide range of neural processes, including survival, proliferation, differentiation, neurite outgrowth, and synaptogenesis. The mechanisms that regulate the targeting of RTKs to their subcellular destinations for appropriate signal transduction, however, are still elusive. In this review, we discuss evidence for the spatial organization of signaling machinery into distinct subcellular compartments, as well as the role for ligand specificity, receptor sorting signals, and lipid raft microdomains in RTK targeting and the resultant cellular responses in neural cells.     

Vps22/EAP30 in ESCRT-II mediates endosomal sorting of growth factor and chemokine receptors destined for lysosomal degradation

The ubiquitin-binding protein Hrs and endosomal sorting complex required for transport (ESCRT)-I and ESCRT-III are involved in sorting endocytosed and ubiquitinated receptors to lysosomes for degradation and efficient termination of signaling. In this study, we have investigated the role of the ESCRT-II subunit Vps22/EAP30 in degradative protein sorting of ubiquitinated receptors. Vps22 transiently expressed in HeLa cells was detected in endosomes containing endocytosed epidermal growth factor receptors (EGFRs) as well as Hrs and ESCRT-I and ESCRT-III. Depletion of Vps22 by small interfering RNA, which was accompanied by decreased levels of other ESCRT-II subunits, greatly reduced degradation of EGFR and its ligand EGF as well as the chemokine receptor CXCR4. EGFR accumulated on the limiting membranes of early endosomes and aberrantly small multivesicular bodies in Vps22-depleted cells. Phosphorylation and nuclear translocation of extracellular-signal-regulated kinase1/2 downstream of the EGF-activated receptor were sustained by depletion of Hrs or the ESCRT-I subunit Tsg101. In contrast, this was not the case when Vps22 was depleted. These results indicate an important role for Vps22 in ligand-induced EGFR and CXCR4 turnover and suggest that termination of EGF signaling occurs prior to ESCRT-II engagement.     

Activation mechanism of solubilized epidermal growth factor receptor tyrosine kinase.

Dimerization of epidermal growth factor receptor (EGFR) leads to the activation of its tyrosine kinase. To elucidate whether dimerization is responsible for activation of the intracellular tyrosine kinase domain or just plays a role in the stabilization of the active form, the activated status of wild-type EGFR moiety in the heterodimer with kinase activity-deficient mutant receptors was investigated. The kinase activity of the wild-type EGFR was partially activated by EGF in the heterodimer with intracellular domain deletion (sEGFR) or ATP binding-deficient mutant (K721A) EGFRs, while the wild-type EGFR in the heterodimer of wild-type and phosphate transfer activity-deficient mutant receptor D813N could be fully activated. After treatment with EGF, the ATP binding affinity and the V(max) of the wild-type EGFR increased. In the presence of sEGFR, a similar increase in the affinity for ATP was observed, but V(max) did not change. A two-step activation mechanism for EGFR was proposed: upon binding of EGF, the affinity for ATP increased and then, as a result of interaction between the neighboring tyrosine kinase domain, V(max) increased.     

A novel bispecific diabody targeting both vascular endothelial growth factor receptor 2 and epidermal growth factor receptor for enhanced antitumor activity

Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2) are receptor tyrosine kinases known to play critical roles in the development and progression of tumors. Based on the cross‐talk between EGFR and VEGFR2 signal pathways, we designed and produced a bispecific diabody (bDAb) targeting both EGFR and VEGFR2 simultaneously. The bispecific molecule (EK‐02) demonstrated that it could bind to HUVEC (VEGFR2 high‐expressing) and A431 (EGFR overexpressing) cells. Additionally, similar to the parental antibodies, it was able to inhibit proliferation and migration, and induced apoptosis in these cells (HUVECs and A431), demonstrating that it had retained the functional properties of its parental antibodies. Furthermore, the efficacy of EK‐02 was evaluated using the human colon adenocarcinoma cell line HT29 (VEGFR2 and EGFR coexpressing). In vitro assay showed that EK‐02 could bind to HT29 cells, restrain cell growth and migration, and induce apoptosis with enhanced efficacy compared to both parental antibodies. Further, it inhibited the neovascularization and tumor formation on an HT29 cell bearing chicken chorioallantoic membrane (CAM) tumor model in vivo. In conclusion, these data suggest that the novel bDAb (EK‐02) has antiangiogenesis and antitumor capacity both in vitro and in vivo, and can possibly be used as cotargeted therapy for the treatment of EGFR and VEGFR2 overexpressing tumors. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:294–302, 2016     

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)     

Characterization and development of a peptide substrate-based phosphate transfer assay for the human vascular endothelial growth factor receptor-2 tyrosine kinase

Vascular endothelial growth factor (VEGF), a critical regulator in angiogenesis, exerts its angiogenic effect via binding to its receptor, VEGF receptor-2 tyrosine kinase (VEGFR2) or kinase insert domain-containing receptor (Kdr), on the surface of endothelial cells. Kdr-mediated signaling plays an important role in the proliferation, migration, differentiation, and survival of endothelial cells. Therefore, the inhibition of this signaling pathway represents a promising therapeutic approach for the discovery of novel anticancer agents by destabilizing the progression of solid tumors via abrogating tumor-induced angiogenesis. To explore Kdr as an anticancer target and further characterize the enzyme, we purified a cytoplasmic domain of human Kdr (Kdr-CD) and characterized its autophosphorylation activity. We also designed and synthesized peptides containing amino acid sequences corresponding to the autophosphorylation sites of Kdr and developed a simple, robust, high-throughput assay for measuring the phosphate transfer activity of the enzyme. This assay was validated by the experiments showing that the phosphate transfer activity of the purified Kdr-CD required Mg2+ or Mn2+ and preactivation by adenosine 5'-triphosphate (ATP) and was inhibited by known Kdr inhibitors. Using this assay, we examined effects of Mg2+ and Mn2+ on the enzyme activity; optimized the concentrations of Kdr-CD, peptide and ATP substrates, and metal ions in the assay; and determined the kinetic properties of the enzyme for the peptide and ATP as well as IC50 values of two known Kdr inhibitors. Thus, the results of these studies have validated the utilities of this assay for biochemical characterizations of the enzyme and its inhibitors. This approach of designing peptides corresponding to the autophosphorylation sites of Kdr as substrates for the enzyme has general practical implications to other kinases.     

The receptor tyrosine kinase TIE is required for integrity and survival of vascular endothelial cells.

Vascular endothelial cells are critical for the development and function of the mammalian circulatory system. We have analyzed the role of the endothelial cell-specific receptor tyrosine kinase TIE in the mouse vasculature. Mouse embryos homozygous for a disrupted Tie allele developed severe edema, their microvasculature was ruptured and they died between days 13.5 and 14.5 of gestation. The major blood vessels of the homozygous embryos appeared normal. Cells lacking a functional Tie gene were unable to contribute to the adult kidney endothelium in chimeric animals, further demonstrating the intrinsic requirement for TIE in endothelial cells. We conclude that TIE is required during embryonic development for the integrity and survival of vascular endothelial cells, particularly in the regions undergoing angiogenic growth of capillaries. TIE is not essential, however, for vasculogenesis, the early differentiation of endothelial cells.     

mTORC2 promotes type I insulin-like growth factor receptor and insulin receptor activation through the tyrosine kinase activity of mTOR

Mammalian target of rapamycin (mTOR) is a core component of raptor-mTOR (mTORC1) and rictor-mTOR (mTORC2) complexes that control diverse cellular processes. Both mTORC1 and mTORC2 regulate several elements downstream of type I insulin-like growth factor receptor (IGF-IR) and insulin receptor (InsR). However, it is unknown whether and how mTOR regulates IGF-IR and InsR themselves. Here we show that mTOR possesses unexpected tyrosine kinase activity and activates IGF-IR/InsR. Rapamycin induces the tyrosine phosphorylation and activation of IGF-IR/InsR, which is largely dependent on rictor and mTOR. Moreover, mTORC2 promotes ligand-induced activation of IGF-IR/InsR. IGF- and insulin-induced IGF-IR/InsR phosphorylation is significantly compromised in rictor-null cells. Insulin receptor substrate (IRS) directly interacts with SIN1 thereby recruiting mTORC2 to IGF-IR/InsR and promoting rapamycin- or ligand-induced phosphorylation of IGF-IR/InsR. mTOR exhibits tyrosine kinase activity towards the general tyrosine kinase substrate poly(Glu-Tyr) and IGF-IR/InsR. Both recombinant mTOR and immunoprecipitated mTORC2 phosphorylate IGF-IR and InsR on Tyr1131/1136 and Tyr1146/1151, respectively. These effects are independent of the intrinsic kinase activity of IGF-IR/InsR, as determined by assays on kinase-dead IGF-IR/InsR mutants. While both rictor and mTOR immunoprecitates from rictor^+/+ MCF-10A cells exhibit tyrosine kinase activity towards IGF-IR and InsR, mTOR immunoprecipitates from rictor^−/− MCF-10A cells do not induce IGF-IR and InsR phosphorylation. Phosphorylation-deficient mutation of residue Tyr1131 in IGF-IR or Tyr1146 in InsR abrogates the activation of IGF-IR/InsR by mTOR. Finally, overexpression of rictor promotes IGF-induced cell proliferation. Our work identifies mTOR as a dual-specificity kinase and clarifies how mTORC2 promotes IGF-IR/InsR activation.     

Mesenchymal cells potentiate vascular endothelial growth factor-induced angiogenesis in vitro

The role of soluble factors (including angiogenic cytokines) and extracellular matrix components in the regulation of angiogenesis is clearly established. However, the interrelationship between these factors and perivascular mesenchymal cells is not well understood. Here we have used a three-dimensional collagen gel coculture system to assess the effect of mesenchymal C3H10T1/2 cells on vascular endothelial growth factor-A (VEGF-A)- and fibroblast growth factor-2 (FGF-2)-induced angiogenesis in vitro. We found that coculture markedly potentiated the angiogenic activity of VEGF-A, irrespective of whether or not direct cell-to-cell contact occurred. In contrast, under conditions in which cell-to-cell contact was possible, FGF-2-induced angiogenesis was inhibited by cocultured 10T1/2 cells; this effect was not seen when cell-to-cell contact was prevented. Attempts to identify the molecules responsible for this effect allowed us to exclude FGF-2, transforming growth factorbeta1, platelet derived growth factor-BB, angiopoietin-1, and NO as possible mediators of the potentiating effect of coculture on VEGF-A-induced invasion. In the living organism, angiogenesis occurs in a three-dimensional microenvironment. Contrary to the inhibitory effect of 10T1/2 cells previously reported by others in two-dimensional cultures, our data demonstrate that the paracrine interaction between endothelial and mesenchymal cells potentiates angiogenesis in vitro and that this is cytokine-specific, i.e., it occurs with VEGF-A but not with FGF-2.     

Differential response of the epidermal growth factor receptor tyrosine kinase activity to several plant and mammalian lectins

Biosignalling via lectins may involve modulation of protein kinase activities. This aspect of the biological action of mammalian and plant lectins has been investigated for their effect on the activity of the isolated epidermal growth factor receptor (EGFR). The constitutive tyrosine kinase activity of the epidermal growth factor receptor from rat liver, isolated by calmodulin-affinity chromatography, was activated by concanavalin A (ConA), and wheat germ agglutinin (WGA) to a similar extent as the measured enhancement induced by EGF. In contrast, two mannose-specific lectins, the mannan-binding protein (MBP) and serum amyloid P component (SAP), isolated from human serum, have inhibitory effects, both in the absence and presence of EGF. The differential effects of these lectins were tested using as phosphorylatable substrates a co-polymer of glutamic acid-tyrosine, as well as calmodulin. However, two galactoside-specific lectins, the laminin-binding -galactoside-binding 14 kDa lectin, isolated from bovine heart (14K-BHL), and the /-galactoside-binding lectin, isolated from mistletoe (Viscum album L.) leaves (VAA), do not inhibit the EGFR tyrosine kinase activity. The sugar dependence of the lectin-mediated action was studied by inhibition assays. Mannose and a mannose-containing neoglycoprotein prevent the activating effect of ConA, and N-acetyl-D-glucosamine partially prevents the activation produced by WGA. However, mannose and mannose-containing neoglycoprotein were ineffective to reduce the inhibitory effect of MBP or SAP. Although the response to binding of ConA and WGA was different to that of MBP or SAP with respect to the tyrosine kinase activity of the EGFR, it should be noted that the four lectins inhibited the binding of [¹²⁵I]EGF to its receptor with similar efficiency.Abbreviations EGF epidermal growth factor - EGFR epidermal growth factor receptor - ConA concanavalin A - MBP mannan-binding protein - SAP serum amyloid P component - WGA wheat germ agglutinin - 14K-BHL bovine heart 14 kDa lectin - VAA Viscum album L. (mistletoe) agglutinin - EGTA [ethylenebis(oxyethylenenitrilo)]-tetraacetic acid; poly(Glu:Tyr)-co-polymer of L-glutamic acid and L-tyrosine - Hepes 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid - Tris tris(hydroxymethyl)-aminomethane - DSS suberic acid bis(N-hydroxy-succinimide ester) - PMSF phenylmethanesulfonyl fluoride - Man mannose - Gal galactose - BSA bovine serum albumin - Man-BSA neoglycoprotein containing -D-mannose - Lac-BSA neoglycoprotein containing -lactose - Gal-BSA neoglycoprotein containing galactose     

Endosomes,receptor tyrosine kinase internalization and signal transduction

Upon the binding of insulin or epidermal growth factor to their cognate receptors on the liver parenchymal plasmalemma, signal transduction and receptor internalization are near co-incident. Indeed, the rapidity and extent; of ligand mediated receptor internalization into endosomes in liver as well as other organs predicts that signal transduction is regulated at this intracellular locus. Although internalization has been thought as a mechanism to attenuate ligand mediated signal transduction responses, detailed studies of internalized receptors in isolated liver endosomes suggest an alternative scenario whereby selective signal transduction pathways can be accessed at this locus.     

Hrs regulates the endocytic sorting of the fibroblast growth factor receptor 2b

The keratinocyte growth factor receptor or fibroblast growth factor receptor 2b (KGFR/FGFR2b) is activated by the specific interaction with the keratinocyte growth factor (KGF/FGF7), which targets the receptor to the degradative pathway, and the fibroblast growth factor 10 (FGF10/KGF2), which drives the receptor to the juxtanuclear recycling route. Hrs plays a key role in the regulation of the endocytic degradative transport of ubiquitinated receptor tyrosine kinases, but the direct involvement of this protein in the regulation of FGFR endocytosis has not been investigated yet. We investigated here the possible role of Hrs in the alternative endocytic pathways of KGFR. Quantitative immunofluorescence microscopy and biochemical analysis showed that both overexpression and siRNA interference of Hrs inhibit the KGF-triggered KGFR degradation, blocking receptor transport to lysosomes and causing its rapid reapparance at the plasma membrane. In contrast, the FGF10-induced KGFR targeting to the recycling compartment is not affected by Hrs overexpression or depletion. Coimmunoprecipitation approaches indicated that Hrs is recruited to KGFR only after KGF treatment, although it is not tyrosine phosphorylated by the ligand. In conclusion, Hrs regulates the KGFR degradative pathway, but not its juxtanuclear recycling transport. In addition, the results suggest that Hrs recruitment to the receptor, but not its ligand-induced phosphorylation, could be required for its function.     

Identification of an autoinhibitory domain in the insulin receptor tyrosine kinase

We have tested the hypothesis that activation of the insulin receptor tyrosine kinase is due to autophosphorylation of tyrosines 1146, 1150 and 1151 within a putative autoinhibitory domain. A synthetic peptide corresponding to residues 1134–1162, with tyrosines substituted by alanine or phenylalanine, of the insulin receptor subunit was tested for its inhibitory potency and specificity towards the tyrosine kinase activity. This synthetic peptide gave inhibition of the insulin receptor tyrosine kinase autophosphorylation and phosphorylation of the exogenous substrate poly(Glu, Tyr) with an approximate IC₅₀ of 100 M. Inhibition appeared to be independent of the concentrations of insulin or the substrate poly(Glu, Tyr) but was decreased by increasing concentrations of ATP. This same peptide also inhibited the EGF receptor tyrosine kinase but not a serine/threonine protein kinase. These results are consistent with the hypothesis that this autophosphorylation domain contains an autoinhibitory sequence. (Mol Cell Biochem120: 103–110, 1993)Abbreviations IR Insulin Receptor - SDS/PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis - CaM Calmodulin - HEPES 4-(2-Hydroxyethyl)-Piperazineethane-Sulfonic Acid - DMEM Dulbecco's Modified Eagle' Medium - PMSF Phenylmethyl-Sulfonyl Fluoride - HPLC High Performance Liquid Chromatography - PKC Protein Kinase C - PKI Inhibitory Peptide for cAMP-Kinase - CaMK II Ca²⁺/Calmodulin-Dependent Protein Kinase II - CaN A A Subunit of Calcineurin     

Selection and growth regulation of genetically modified cells with hapten‐specific antibody/receptor tyrosine kinase chimera

Although receptor tyrosine kinases (RTKs) play a pivotal role in the development and maintaining the homeostasis of the body, overexpression or mutation of RTKs often induces tumorigenesis or metastasis. To mimic the function of RTKs, we developed two fusion receptors consisting of anti‐fluorescein antibody single‐chain Fv, extracellular D2 domain of erythropoietin receptor and transmembrane/intracellular domains of epidermal growth factor receptor or c‐fms based on previously constructed antibody/cytokine receptor chimeras. The expression of these chimeric receptors in the hematopoietic cell line Ba/F3 and non‐hematopoietic cell line NIH/3T3 resulted in the activation of receptors themselves, downstream signaling molecules and cell proliferation in response to fluorescein‐conjugated BSA, leading to selective expansion of transduced cells up to almost 100%. These results indicate that the cognate antigen could activate the chimeric receptors even though the wild‐type extracellular domains were switched to the antibody fragment. This is the first study to show that our antigen‐mediated genetically modified cell amplification (AMEGA) system could be applied to non‐hematopoietic cells by utilizing antibody/RTK chimeras. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

TGF-alpha-driven tumor growth is inhibited by an EGF receptor tyrosine kinase inhibitor.

The simultaneous presence of the EGFR and its ligand TGF-alpha in human tumor tissues suggests that autocrine TGF-alpha stimulation drives tumor growth. Here we show that autocrine TGF-alpha stimulation does cause increased tumor growth in vivo, an effect that was proven to be mediated via EGFR activation, and that this TGF-alpha/EGFR autocrine loop was accessible to an EGFR specific tyrosine kinase inhibitor. Clones of the EGFR expressing glioma cell line U-1242 MG were transfected with TGF-alpha cDNA using a tetracycline-inhibitory system for gene expression. TGF-alpha expression was inhibited by the presence of tetracycline, and subcutaneous tumors forming from cell lines injected into nude mice could be inhibited by feeding mice tetracycline. We confirmed that TGF-alpha mRNA and protein were present in these tumors and that, subsequently, the endogenous EGFR was activated. Tumor growth could be inhibited by an EGFR specific tyrosine kinase inhibitor of the type 4-(3-chloroanilino)-6,7-dimethoxy-quinazoline, administered daily by intraperitoneal injection, thereby interrupting the autocrine loop.     

Regulation of heart insulin receptor tyrosine kinase activity by magnesium and spermine

Insulin action and aspects of the insulin-signaling pathway have been studied in the heart although the direct regulation of the heart’s insulin receptor has not been explored. This study describes the first purification and characterization of the mammalian (rabbit, rat and bovine) heart insulin receptor. The rabbit heart IR showed maximum insulin binding of 18 μg/mg (~1 mole insulin/mole (α₂β₂) receptor) and a curvilinear Scatchard plot with a high affinity K_D for insulin binding of ~4 nM at optimal pH (7.8) and NaCl concentration (150 mM). The insulin receptor tyrosine kinase activity was stimulated by insulin, Mg²⁺ (half-maximum response at ~5.6–10.6 nM and ~8.5 mM, respectively) and by the physiological polyamines, spermine and spermidine. The stimulation by Mg²⁺ and the polyamines occurred with and without insulin. These characteristics of the heart insulin receptor provide a mechanism for regulating the activity of the receptor’s tyrosine kinase activity by the intracellular free Mg²⁺ concentration and the polyamines in the absence and presence of insulin.     

Tyrphostin AG556 increases the activity of large conductance Ca2+‐activated K+ channels by inhibiting epidermal growth factor receptor tyrosine kinase

The present study was designed to investigate whether large conductance Ca²⁺‐activated K⁺ (BK) channels were regulated by epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase. BK current and channel tyrosine phosphorylation level were measured in BK‐HEK 293 cells expressing both functional α‐subunits and the auxiliary β1‐subunits using electrophysiology, immunoprecipitation and Western blotting approaches, respectively, and the function of rat cerebral basilar arteries was determined with a wire myography system. We found that BK current in BK‐HEK 293 cells was increased by the broad spectrum protein tyrosine kinase (PTK) inhibitor genistein and the selective EGFR tyrosine kinase inhibitor AG556, one of the known tyrphostin. The effect of genistein or AG556 was antagonized by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate. On the other hand, orthovanadate or EGF decreased BK current, and the effect was counteracted by AG556. The tyrosine phosphorylation level of BK channels (α‐ and β1‐subunits) was increased by EGF and orthovanadate, while decreased by genistein and AG556, and the reduced tyrosine phosphorylation of BK channels by genistein or AG556 was reversed by orthovanadate. Interestingly, AG556 induced a remarkable enhancement of BK current in rat cerebral artery smooth muscle cells and relaxation of pre‐contracted rat cerebral basilar arteries with denuded endothelium, and these effects were antagonized by the BK channel blocker paxilline or orthovanadate. These results demonstrate that tyrosine phosphorylation of BK channels by EGFR kinase decreases the channel activity, and inhibition of EGFR kinase by AG556 enhances the channel activity and dilates rat cerebral basilar arteries.     

Negative regulation of Ros receptor tyrosine kinase signaling. An epithelial function of the SH2 domain protein tyrosine phosphatase SHP-1

Male "viable motheaten" (me(v)) mice, with a naturally occurring mutation in the gene of the SH2 domain protein tyrosine phosphatase SHP-1, are sterile. Known defects in sperm maturation in these mice correlate with an impaired differentiation of the epididymis, which has similarities to the phenotype of mice with a targeted inactivation of the Ros receptor tyrosine kinase. Ros and SHP-1 are coexpressed in epididymal epithelium, and elevated phosphorylation of Ros in the epididymis of me(v) mice suggests that Ros signaling is under control of SHP-1 in vivo. Phosphorylated Ros strongly and directly associates with SHP-1 in yeast two-hybrid, glutathione S-transferase pull-down, and coimmunoprecipitation experiments. Strong binding of SHP-1 to Ros is selective compared to six other receptor tyrosine kinases. The interaction is mediated by the SHP-1 NH(2)-terminal SH2 domain and Ros phosphotyrosine 2267. Overexpression of SHP-1 results in Ros dephosphorylation and effectively downregulates Ros-dependent proliferation and transformation. We propose that SHP-1 is an important downstream regulator of Ros signaling.     

The interplay of cyclic stretch and vascular endothelial growth factor in regulating the initial steps for angiogenesis

Angiogenesis is regulated by chemical and mechanical factors in vivo. The regulatory role of mechanical factors and how chemical and mechanical angiogenic regulators work in concert remains to be explored. We investigated the effect of cyclic uniaxial stretch (20%, 1 Hz), with and without the stimulation of vascular endothelial growth factor (VEGF), on sprouting angiogenesis by employing a stretchable three‐dimensional cell culture model. When compared to static controls, stretch alone significantly increased the density of endothelial sprouts, and these sprouts aligned perpendicular to the direction of stretch. The Rho‐associated kinase (ROCK) inhibitor Y27632 suppressed stretch‐induced sprouting angiogenesis and associated sprout alignment. While VEGF is a potent angiogenic stimulus through ROCK‐dependent pathways, the combination of VEGF and stretch did not have an additive effect on angiogenesis. In the presence of VEGF stimulation, the ROCK inhibitor suppressed stretch‐induced sprout alignment but did not affect stretch‐induced sprout density; in contrast, the receptor tyrosine kinase (RTK) inhibitor sunitinib had no effect on stretch‐induced alignment but trended toward suppressed stretch‐induced sprout density. Our results suggest that the formation of sprouts and their directionality do not have completely identical regulatory pathways, and thus it is possible to separately manipulate the number and pattern of new sprouts. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:248–257, 2015