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J M Backer

Publications and source records attributed to J M Backer.

At least 19 recordsLinked to original sources
PubMed2001

Human VPS34 is required for internal vesicle formation within multivesicular endosomes.

After internalization from the plasma membrane, activated EGF receptors (EGFRs) are delivered to multivesicular bodies (MVBs). Within MVBs, EGFRs are removed from the perimeter membrane to internal vesicles, thereby being sorted from transferrin receptors, which recycle back to the plasma membrane. The phosphatidylinositol (PI) 3'-kinase inhibitor, wortmannin, inhibits internal vesicle formation within MVBs and causes EGFRs to remain in clusters on the perimeter membrane. Microinjection of isotype-specific inhibitory antibodies demonstrates that the PI 3'-kinase required for internal vesicle formation is hVPS34. In the presence of wortmannin, EGFRs continue to be delivered to lysosomes, showing that their removal from the recycling pathway and their delivery to lysosomes does not depend on inward vesiculation. We showed previously that tyrosine kinase-negative EGFRs fail to accumulate on internal vesicles of MVBs but are recycled rather than delivered to lysosomes. Therefore, we conclude that selection of EGFRs for inclusion on internal vesicles requires tyrosine kinase but not PI 3'-kinase activity, whereas vesicle formation requires PI 3'-kinase activity. Finally, in wortmannin-treated cells there is increased EGF-stimulated tyrosine phosphorylation when EGFRs are retained on the perimeter membrane of MVBs. Therefore, we suggest that inward vesiculation is involved directly with attenuating signal transduction.

Androstadienes↗
PubMed2001

Distinct roles of class I and class III phosphatidylinositol 3-kinases in phagosome formation and maturation.

Phagosomes acquire their microbicidal properties by fusion with lysosomes. Products of phosphatidylinositol 3-kinase (PI 3-kinase) are required for phagosome formation, but their role in maturation is unknown. Using chimeric fluorescent proteins encoding tandem FYVE domains, we found that phosphatidylinositol 3-phosphate (PI[3]P) accumulates greatly but transiently on the phagosomal membrane. Unlike the 3'-phosphoinositides generated by class I PI 3-kinases which are evident in the nascent phagosomal cup, PI(3)P is only detectable after the phagosome has sealed. The class III PI 3-kinase VPS34 was found to be responsible for PI(3)P synthesis and essential for phagolysosome formation. In contrast, selective ablation of class I PI 3-kinase revealed that optimal phagocytosis, but not maturation, requires this type of enzyme. These results highlight the differential functional role of the two families of kinases, and raise the possibility that PI(3)P production by VPS34 may be targeted during the maturation arrest induced by some intracellular parasites.

Androstadienes↗
PubMed2001

Vps34p differentially regulates endocytosis from the apical and basolateral domains in polarized hepatic cells.

Using a microinjection approach to study apical plasma membrane protein trafficking in hepatic cells, we found that specific inhibition of Vps34p, a class III phosphoinositide 3 (PI-3) kinase, nearly perfectly recapitulated the defects we reported for wortmannin-treated cells (Tuma, P.L., C.M. Finnegan, J.-H Yi, and A.L. Hubbard. 1999. J. Cell Biol. 145:1089-1102). Both wortmannin and injection of inhibitory Vps34p antibodies led to the accumulation of resident apical proteins in enlarged prelysosomes, whereas transcytosing apical proteins and recycling basolateral receptors transiently accumulated in basolateral early endosomes. To understand how the Vps34p catalytic product, PI3P, was differentially regulating endocytosis from the two domains, we examined the PI3P binding protein early endosomal antigen 1 (EEA1). We determined that EEA1 distributed to two biochemically distinct endosomal populations: basolateral early endosomes and subapical endosomes. Both contained rab5, although the latter also contained late endosomal markers but was distinct from the transcytotic intermediate, the subapical compartment. When PI3P was depleted, EEA1 dissociated from basolateral endosomes, whereas it remained on subapical endosomes. From these results, we conclude that PI3P, via EEA1, regulates early steps in endocytosis from the basolateral surface in polarized WIF-B cells. However, PI3P must use different machinery in its regulation of the apical endocytic pathway, since later steps are affected by Vps34p inhibition.

Androstadienes↗
PubMed2001

Role of phosphatidylinositol 3-kinase and Rab5 effectors in phagosomal biogenesis and mycobacterial phagosome maturation arrest.

Phagosomal biogenesis is a fundamental biological process of particular significance for the function of phagocytic and antigen-presenting cells. The precise mechanisms governing maturation of phagosomes into phagolysosomes are not completely understood. Here, we applied the property of pathogenic mycobacteria to cause phagosome maturation arrest in infected macrophages as a tool to dissect critical steps in phagosomal biogenesis. We report the requirement for 3-phosphoinositides and acquisition of Rab5 effector early endosome autoantigen (EEA1) as essential molecular events necessary for phagosomal maturation. Unlike the model phagosomes containing latex beads, which transiently recruited EEA1, mycobacterial phagosomes excluded this regulator of vesicular trafficking that controls membrane tethering and fusion processes within the endosomal pathway and is recruited to endosomal membranes via binding to phosphatidylinositol 3-phosphate (PtdIns[3]P). Inhibitors of phosphatidylinositol 3'(OH)-kinase (PI-3K) activity diminished EEA1 recruitment to newly formed latex bead phagosomes and blocked phagosomal acquisition of late endocytic properties, indicating that generation of PtdIns(3)P plays a role in phagosomal maturation. Microinjection into macrophages of antibodies against EEA1 and the PI-3K hVPS34 reduced acquisition of late endocytic markers by latex bead phagosomes, demonstrating an essential role of these Rab5 effectors in phagosomal biogenesis. The mechanism of EEA1 exclusion from mycobacterial phagosomes was investigated using mycobacterial products. Coating of latex beads with the major mycobacterial cell envelope glycosylated phosphatidylinositol lipoarabinomannan isolated from the virulent Mycobacterium tuberculosis H37Rv, inhibited recruitment of EEA1 to latex bead phagosomes, and diminished their maturation. These findings define the generation of phosphatidylinositol 3-phosphate and EEA1 recruitment as: (a) important regulatory events in phagosomal maturation and (b) critical molecular targets affected by M. tuberculosis. This study also identifies mycobacterial phosphoinositides as products with specialized toxic properties, interfering with discrete trafficking stages in phagosomal maturation.

Androstadienes↗
PubMed2001

Molecular vehicle for target-mediated delivery of therapeutics and diagnostics.

Selective targeting of therapeutic and diagnostic agents improves their efficacy and minimizes potentially adverse side effects. Existing methods for selective targeting are based on chemical conjugation of therapeutics and diagnostics, or their carriers, to cell-specific targeting molecules (e.g., growth factors, antibodies). These methods are limited by potential damage to targeting molecules that can be inflicted by the conjugation procedure. In addition, conjugation procedures have to be developed on a case-by-case basis. In order to avoid these problems we have developed a new approach to constructing molecular vehicles for target-mediated delivery of therapeutics and diagnostics. In this approach, the targeting molecule is expressed as a fusion protein containing a recognition tag. The recognition tag is defined as a peptide or protein that can bind non-covalently another peptide or protein (adapter). In turn, the adapter is chemically conjugated to a carrier of therapeutics or diagnostics. The assembled molecular delivery vehicle contains a carrier-adapter conjugate bound non-covalently to a recognition tag fused to the targeting protein. The advantages of this technology are: (i) no chemical modification of targeting molecules, and (ii) universal, 'off-the-shelf' carrier-adapter constructs that can be combined with different fusion targeting proteins. To obtain a proof-of-principle we have constructed VEGF fusion proteins containing a 15-aa S-peptide fragment of RNase A as a recognition tag. Using the S-protein fragment of RNase A as an adapter and polyethylenimine as a DNA carrier we have achieved selective gene delivery to cells overexpressing VEGFR-2.

Animals↗
PubMed2001

Shiga-like toxin-VEGF fusion proteins are selectively cytotoxic to endothelial cells overexpressing VEGFR-2.

Growing endothelial cells at sites of angiogenesis may be more sensitive than quiescent endothelial cells to toxin-VEGF fusion proteins, because they express higher numbers of VEGF receptors. We have constructed, expressed and purified a protein containing the catalytic A-subunit of Shiga-like toxin I fused to VEGF(121) (SLT-VEGF/L). SLT-VEGF/L inhibits protein synthesis in a cell-free translation system and induces VEGFR-2 tyrosine autophosphorylation in cells overexpressing VEGFR-2 indicating that both SLT and VEGF moieties are properly folded in the fusion protein. SLT-VEGF/L selectively inhibits growth of porcine endothelial cells expressing 2-3x10(5) VEGFR-2/cell with an IC(50) of 0.1 nM, and rapidly induces apoptosis at concentrations >1 nM. Similar results are observed with human transformed embryonic kidney cells, 293, engineered to express 2.5x10(6) VEGFR-2/cell. In contrast, SLT-VEGF/L does not affect three different types of endothelial cells (PAE/KDR(low), HUVE, MS1) expressing between 5x10(3) and 5x10(4) VEGFR-2/cell, and quiescent endothelial cells overexpressing VEGFR-2. Growth inhibition and induction of apoptosis by SLT-VEGF/L require intrinsic N-glycosidase activity of the SLT moiety, but occur without significant inhibition of protein synthesis. The selective cytotoxicity of SLT-VEGF proteins against growing endothelial cells overexpressing VEGFR-2 suggests that they may be useful in targeting similar cells at sites of angiogenesis.

Animals↗
PubMed2001

Kinase insert domain receptor (KDR) extracellular immunoglobulin-like domains 4-7 contain structural features that block receptor dimerization and vascular endothelial growth factor-induced signaling.

The vascular endothelial growth factor (VEGF) receptor tyrosine kinase subtype kinase insert domain receptor (KDR) contains seven extracellular Ig-like domains, of which the three most amino-terminal contain the necessary structural features required for VEGF binding. To clarify the functional role of KDR Ig-like domains 4-7, we compared VEGF-induced signaling in human embryonic kidney and porcine aortic endothelial cells expressing native versus mutant receptor proteins in which Ig-like domains 4-7, 4-6, or 7 had been deleted. Western blotting using an anti-receptor antibody indicated equivalent expression levels for each of the recombinant proteins. As expected, VEGF treatment robustly augmented native receptor autophosphorylation. In contrast, receptor autophosphorylation, as well as downstream signaling events, were VEGF-independent for cells expressing mutant receptors. (125)I-VEGF(165) bound with equal or better affinity to mutant versus native receptor, although the number of radioligand binding sites was significantly reduced because a significant percentage of mutant, but not native, receptors were localized to the cell interior. As was the case for native KDR, (125)I-VEGF(165) binding to the mutant receptors was dependent upon cell surface heparan sulfate proteoglycans, and (125)I-VEGF(121) bound with an affinity equal to that of (125)I-VEGF(165) to the native and mutant receptors. It is concluded that KDR Ig-like domains 4-7 contain structural features that inhibit receptor signaling by a mechanism that is independent of neuropilin-1 and heparan sulfate proteoglycans. We speculate that this provides a cellular mechanism for blocking unwanted signaling events in the absence of VEGF.

Adaptor Proteins, Signal Transducing↗
PubMed2001

p110beta and p110delta phosphatidylinositol 3-kinases up-regulate Fc(epsilon)RI-activated Ca2+ influx by enhancing inositol 1,4,5-trisphosphate production.

Fc(epsilon)RI-induced Ca2+ signaling in mast cells is initiated by activation of cytosolic tyrosine kinases. Here, in vitro phospholipase assays establish that the phosphatidylinositol 3-kinase (PI 3-kinase) lipid product, phosphatidylinositol 3,4,5-triphosphate, further stimulates phospholipase Cgamma2 that has been activated by conformational changes associated with tyrosine phosphorylation or low pH. A microinjection approach is used to directly assess the consequences of inhibiting class IA PI 3-kinases on Ca2+ responses after Fc(epsilon)RI cross-linking in RBL-2H3 cells. Injection of antibodies to the p110beta or p110delta catalytic isoforms of PI 3-kinase, but not antibodies to p110alpha, lengthens the lag time to release of Ca2+ stores and blunts the sustained phase of the calcium response. Ca2+ responses are also inhibited in cells microinjected with recombinant inositol polyphosphate 5-phosphatase I, which degrades inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), or heparin, a competitive inhibitor of the Ins(1,4,5)P3 receptor. This indicates a requirement for Ins(1,4,5)P3 to initiate and sustain Ca2+ responses even when PI 3-kinase is fully active. Antigen-induced cell ruffling, a calcium-independent event, is blocked by injection of p110beta and p110delta antibodies, but not by injection of 5-phosphatase I, heparin, or anti-p110alpha antibodies. These results suggest that the p110beta and p110delta isoforms of PI 3-kinase support Fc(epsilon)RI-induced calcium signaling by modulating Ins(1,4,5)P3 production, not by directly regulating the Ca2+ influx channel.

Animals↗
PubMed2001

N-terminal domains of the class ia phosphoinositide 3-kinase regulatory subunit play a role in cytoskeletal but not mitogenic signaling.

Phosphoinositide (PI) 3-kinases are required for the acute regulation of the cytoskeleton by growth factors. We have shown previously that in the MTLn3 rat adenocarcinoma cells line, the p85/p110alpha PI 3-kinase is required for epidermal growth factor (EGF)-stimulated lamellipod extension and formation of new actin barbed ends at the leading edge of the cell. We have now examined the role of the p85alpha regulatory subunit in greater detail. Microinjection of recombinant p85alpha into MTLn3 cells blocked both EGF-stimulated mitogenic signaling and lamellipod extension. In contrast, a truncated p85(1-333), which lacks the SH2 and iSH2 domains and does not bind p110, had no effect on EGF-stimulated mitogenesis but still blocked EGF-stimulated lamellipod extension. Additional deletional analysis showed that the SH3 domain was not required for inhibition of lamellipod extension, as a construct containing only the proline-rich and breakpoint cluster region (BCR) homology domains was sufficient for inhibition. Although the BCR domain of p85 binds Rac, the effects of the p85 constructs were not because of a general inhibition of Rac signaling, because sorbitol-induced JNK activation in MTLn3 cells was not inhibited. These data show that the proline-rich and BCR homology domains of p85 are involved in the coupling of p85/p110 PI 3-kinases to regulation of the actin cytoskeleton. These data provide evidence of a distinct cellular function for the N-terminal domains of p85.

Adenocarcinoma↗
PubMed2001

Functionally active VEGF fusion proteins.

Angiogenesis is stimulated by vascular endothelial growth factor (VEGF) acting via endothelial cell-specific receptors, such as VEGFR-2, that are overexpressed at the sites of angiogenesis. If VEGF retains activity as a fusion protein with a large N-terminal extension, it would facilitate development of VEGF-based vehicles for receptor-mediated delivery of therapeutic and diagnostic agents to the sites of angiogenesis. We have constructed, expressed in Escherichia coli, and purified VEGF fusion proteins containing a 158-amino acid N-terminal extension fused to human VEGF(121), VEGF(165), and VEGF(189). We report here that VEGF fusion proteins induce tyrosine autophosphorylation of VEGFR-2 and its downstream targets, as well as cell contraction in cells overexpressing VEGFR-2. Although N-terminal extensions decrease the affinity of VEGF fusion proteins to VEGFR-2, at saturating concentrations these proteins are as efficient as correct size VEGF(165). We hypothesize that VEGF fusion proteins may be employed for targeting endothelial cells at the sites of angiogenesis.

Amino Acid Sequence↗
PubMed2000

Expression of a catalytically inactive H118Y mutant of nm23-H2 suppresses the metastatic potential of line IV Cl 1 human melanoma cells.

Nm23-H1 and nm23-H2 are putative metastasis suppressor genes that encode nucleoside diphosphate kinase (NDPK) A and B. NDPKs form oligomers distributed between soluble and particulate fractions of cells and therefore may exert their effects as either soluble or bound proteins. To determine whether metastasis-related functions of NDPKs are mediated by their catalytic activity in membrane bound or soluble complexes, we have stably transfected highly metastatic human melanoma Line IV Cl 1 cells with wild-type and catalytically inactive (H118Y) nm23-H1 and nm23-H2 genes and assayed their metastatic potential in nude mice. Transfection with wild-type nm23-H1 and nm23-H2 genes and catalytically inactive nm23-H1 did not significantly (all p > 0.10) alter the metastatic potential of Line IV Cl 1 cells while transfection with catalytically inactive nm23-H2 significantly (p < 0.01) reduced their metastatic potential. The lack of effect of transfection with wild-type and catalytically inactive nm23-H1 suggests that neither soluble nor membrane bound NDPK A affect the metastatic potential of Line IV Cl 1 cells. The metastasis suppressive effect of catalytically inactive NDPK B overexpression suggests that competition with bound complexes containing catalytically active NDPK B inhibits metastasis of Line IV Cl 1 cells. These results imply that bound NDPK B promotes metastasis and suggest that inhibition of its function or of its binding to critical sites may be a useful approach to limit the development of metastases in human melanoma.

Amino Acid Substitution↗
PubMed2000

Inhibition of phosphatidic acid synthesis alters the structure of the Golgi apparatus and inhibits secretion in endocrine cells.

In mammalian cells, activation of a Golgi-associated phospholipase D by ADP-ribosylation factor results in the hydrolysis of phosphatidylcholine to form phosphatidic acid (PA). This reaction stimulates the release of nascent secretory vesicles from the trans-Golgi network of endocrine cells. To understand the role of PA in mediating secretion, we have exploited the transphosphatidylation activity of phospholipase D. Rat anterior pituitary GH3 cells, which secrete growth hormone and prolactin, were treated with 1-butanol resulting in the synthesis of phosphatidylbutanol rather than PA. Under these conditions transport from the ER through the Golgi apparatus and secretion of polypeptide hormones were inhibited quantitatively. Furthermore, the in vitro synthesis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) by Golgi membranes was inhibited quantitatively. Most significantly, in the presence of 1-butanol the architecture of the Golgi apparatus was disrupted, resulting in its disassembly and fragmentation. Removal of the alcohol resulted in the rapid restoration of Golgi structure and secretion of growth hormone and prolactin. Our results suggest that PA stimulation of PtdIns(4,5)P(2) synthesis is required for maintaining the structural integrity and function of the Golgi apparatus.

1-Butanol↗
PubMed2000

Specific requirement for the p85-p110alpha phosphatidylinositol 3-kinase during epidermal growth factor-stimulated actin nucleation in breast cancer cells.

We have studied the role of phosphatidylinositol 3-kinases (PI 3-kinases) in the regulation of the actin cytoskeleton in MTLn3 rat adenocarcinoma cells. Stimulation of MTLn3 cells with epidermal growth factor (EGF) induced a rapid increase in actin polymerization, with production of lamellipodia within 3 min. EGF-stimulated lamellipodia were blocked by 100 nM wortmannin, suggesting the involvement of a class Ia PI 3-kinase. MTLn3 cells contain equal amounts of p110alpha and p110beta, and do not contain p110delta. Injection of specific inhibitory antibodies to p110alpha induced cell rounding and blocked EGF-stimulated lamellipod extension, whereas control or anti-p110beta antibodies had no effect. In contrast, both antibodies inhibited EGF-stimulated DNA synthesis. An in situ assay for actin nucleation showed that EGF-stimulated formation of new barbed ends was blocked by injection of anti-p110alpha antibodies. In summary, the p110alpha isoform of PI 3-kinase is specifically required for EGF-stimulated actin nucleation during lamellipod extension in breast cancer cells.

Actins↗
PubMed1999

Discordant effects of glucosamine on insulin-stimulated glucose metabolism and phosphatidylinositol 3-kinase activity.

The impact of increased GlcN availability on insulin-stimulated p85/p110 phosphatidylinositol 3-kinase (PI3K) activity in skeletal muscle was examined in relation to GlcN-induced defects in peripheral insulin action. Primed continuous GlcN infusion (750 micromol/kg bolus; 30 micromol/kg.min) in conscious rats limited both maximal stimulation of muscle PI3K by acute insulin (I) (1 unit/kg) bolus (I + GlcN = 1.9-fold versus saline = 3.3-fold above fasting levels; p < 0.01) and chronic activation of PI3K following 3-h euglycemic, hyperinsulinemic (18 milliunits/kg.min) clamp studies (I + GlcN = 1.2-fold versus saline = 2.6-fold stimulation; p < 0.01). To determine the time course of GlcN-induced defects in insulin-stimulated PI3K activity and peripheral insulin action, GlcN was administered for 30, 60, 90, or 120 min during 2-h euglycemic, hyperinsulinemic clamp studies. Activation of muscle PI3K by insulin was attenuated following only 30 min of GlcN infusion (GlcN 30 min = 1.5-fold versus saline = 2.5-fold stimulation; p < 0.05). In contrast, the first impairment in insulin-mediated glucose uptake (Rd) developed following 110 min of GlcN infusion (110 min = 39.9 +/- 1.8 versus 30 min = 42.8 +/- 1.4 mg/kg.min, p < 0.05). However, the ability of insulin to stimulate phosphatidylinositol 3,4, 5-trisphosphate production and to activate glycogen synthase in skeletal muscle was preserved following up to 180 min of GlcN infusion. Thus, increased GlcN availability induced (a) profound and early inhibition of proximal insulin signaling at the level of PI3K and (b) delayed effects on insulin-mediated glucose uptake, yet (c) complete sparing of insulin-mediated glycogen synthase activation. The pattern and time sequence of GlcN-induced defects suggest that the etiology of peripheral insulin resistance may be distinct from the rapid and marked impairment in insulin signaling.

Animals↗
PubMed1999

Nm23-transfected MDA-MB-435 human breast carcinoma cells form tumors with altered phospholipid metabolism and pH: a 31P nuclear magnetic resonance study in vivo and in vitro.

Nm23 genes are involved in the control of the metastatic potential of breast carcinoma cells. To understand the impact of nm23 genes on tumor physiology and metabolism, a 31P nuclear magnetic resonance (NMR) spectroscopic study was performed on tumors formed in the mammary fat pad of severe combined immunodeficiency mice by MDA-MB-435 human breast carcinoma cells transfected with cDNA encoding wild type nm23-H1 and nm23-H2 proteins. Tumors formed by MDA-MB-435 cells transfected with vector alone were used as controls. All transgene tumors exhibited significantly higher levels of phosphodiester (PDE) compounds relative to phosphomonoester (PME) compounds in vivo compared with control tumors. Similar differences in PDE and PME also were observed for spectra obtained from cells growing in culture. Intracellular pH was significantly lower and extracellular pH was significantly higher for transgene tumors compared with control tumors. Histologic analysis of lung sections confirmed reductions in incidence, number, and size of metastatic nodules for animals bearing transgene tumors. These results suggest that nm23 genes may affect suppression of metastasis through phospholipid-mediated signaling and cellular pH regulation.

Animals↗
PubMed1999

Rab5 regulates motility of early endosomes on microtubules.

The small GTPase Rab5 regulates membrane docking and fusion in the early endocytic pathway. Here we reveal a new role for Rab5 in the regulation of endosome interactions with the microtubule network. Using Rab5 fused to green fluorescent protein we show that Rab5-positive endosomes move on microtubules in vivo. In vitro, Rab5 stimulates both association of early endosomes with microtubules and early-endosome motility towards the minus ends of microtubules. Moreover, similarly to endosome membrane docking and fusion, Rab5-dependent endosome movement depends on the phosphatidylinositol-3-OH kinase hVPS34. Thus, Rab5 functionally links regulation of membrane transport, motility and intracellular distribution of early endosomes.

Biological Transport↗