RESUMO
Many malignancies show increased expression of the epidermal growth factor (EGF) receptor family member ErbB3 (HER3). ErbB3 binds heregulin ß-1 (HRGß1) and forms a heterodimer with other ErbB family members, such as ErbB2 (HER2) or EGF receptor (EGFR; HER1), enhancing phosphorylation of specific C-terminal tyrosine residues and activation of downstream signaling pathways. ErbB3 contains six YXXM motifs that bind the p85 subunit of phosphoinositide 3 (PI3)-kinase. Previous studies demonstrated that overexpression of ErbB3 in mammary tumor cells can significantly enhance chemotaxis to HRGß1 and overall metastatic potential. We tested the hypothesis that ErbB3-mediated PI3-kinase signaling is critical for heregulin-induced motility, and therefore crucial for ErbB3-mediated invasion, intravasation and metastasis. The tyrosines in the six YXXM motifs on the ErbB3 C-terminus were replaced with phenylalanine. In contrast to overexpression of the wild-type ErbB3, overexpression of the mutant ErbB3 did not enhance chemotaxis towards HRGß1 in vitro or in vivo. We also observed reduced tumor cell motility in the primary tumor by multiphoton microscopy, as well as a dramatically reduced ability of these cells to cross the endothelium and intravasate into the circulation. Moreover, whereas mutation of the ErbB3 C-terminus had no effect on tumor growth, it had a dramatic effect on spontaneous metastatic potential. Treatment with the PI3-kinase inhibitor PIK-75 similarly inhibited motility and invasion in vitro and in vivo. Our results indicate that stimulation of the early metastatic steps of motility and invasion by ErbB3 requires activation of the PI3-kinase pathway by the ErbB3 receptor.
Assuntos
Movimento Celular/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Receptor ErbB-3/metabolismo , Transdução de Sinais/fisiologia , Animais , Sítios de Ligação/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Feminino , Humanos , Hidrazonas/farmacologia , Imunoprecipitação , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos SCID , Microscopia de Fluorescência por Excitação Multifotônica , Mutação , Invasividade Neoplásica , Metástase Neoplásica , Neuregulina-1/metabolismo , Neuregulina-1/farmacologia , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Ligação Proteica , Ratos , Receptor ErbB-3/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sulfonamidas/farmacologia , Transplante Heterólogo , Tirosina/genética , Tirosina/metabolismoRESUMO
The class III PI3K (phosphoinositide 3-kinase), Vps34 (vacuolar protein sorting 34), was first identified as a regulator of vacuolar hydrolase sorting in yeast. Unlike other PI3Ks, the Vps34 lipid kinase specifically utilizes phosphatidylinositol as a substrate, producing the single lipid product PtdIns3P. While Vps34 has been studied for some time in the context of endocytosis and vesicular trafficking, it has more recently been implicated as an important regulator of autophagy, trimeric G-protein signalling, and the mTOR (mammalian target of rapamycin) nutrient-sensing pathway. The present paper will focus on studies that describe the regulation of hVps34 (human Vps34) intracellular targeting and enzymatic activity in yeast and mammalian cells.
Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Animais , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Cinética , Mamíferos , Estado Nutricional , Transporte ProteicoRESUMO
Class IA PI3Ks (phosphoinositide 3-kinases) regulate a wide range of cellular responses through the production of PI(3,4,5)P(3) (phosphatidylinositol 3,4,5-trisphosphate) in cellular membranes. They are activated by receptor tyrosine kinases, by Ras and Rho family GTPases, and in some cases by G(betagamma) subunits from trimeric G-proteins. Crystallographic studies on the related class IB PI3Kgamma, and biochemical and structural studies on the class IA PI3Ks, have led to new insights into how these critical enzymes are regulated in normal cells and how mutations can lead to their constitutive activation in transformed cells. The present paper will discuss recent studies on the regulation of class I (p85/p110) PI3Ks, with a focus on the role of SH2 domains (Src homology 2 domains) in the p85 regulatory subunit in modulating PI3K activity.
Assuntos
Fosfatidilinositol 3-Quinases/classificação , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Membrana Celular/metabolismo , Cinética , Lipídeos de Membrana/metabolismo , Modelos Moleculares , Mutação , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositóis/metabolismo , Subunidades Proteicas/metabolismo , Domínios de Homologia de srcRESUMO
Growing endothelial cells at the sites of angiogenesis express high numbers of VEGF receptors and therefore may be particularly sensitive to VEGF-mediated drug delivery. To test this hypothesis we have constructed a protein containing the catalytic A-subunit of Shiga-like toxin I fused to VEGF121 (SLT-VEGF/L). Wild-type A-subunit is a site-specific N-glycosidase of 28S rRNA that inhibits protein synthesis after being delivered into cells by separate cell-binding B-subunits. SLT-VEGF/L retains functional activities of both SLT and VEGF121 moieties, since it inhibits protein synthesis in a cell-free translation system and induces VEGFR-2 tyrosine autophosphorylation. SLT-VEGF/L selectively inhibits growth of porcine endothelial cells expressing 2.5 x 10(5) VEGFR-2/cell with an IC50 of 0.2 nM and rapidly induces apoptosis at concentrations >1 nM. We found that sensitivity of VEGFR-2 transfected PAE cells to SLT-VEGF/L declined as the cellular VEGFR-2 density decreased; PAE cells expressing 25000 VEGFR-2/cell were as sensitive as parental cells lacking the receptor. Growth inhibition and induction of apoptosis by SLT-VEGF/L require intrinsic N-glycosidase activity of the SLT moiety, but take place without significant inhibition of protein synthesis. Selective cytotoxicity of SLT-VEGF/L against growing endothelial cells overexpressing VEGFR-2 suggests that it may be useful in targeting similar cells at the sites of angiogenesis.
Assuntos
Sistemas de Liberação de Medicamentos , Endotélio Vascular/efeitos dos fármacos , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Proteínas Recombinantes de Fusão/toxicidade , Inibidores da Angiogênese/farmacocinética , Inibidores da Angiogênese/toxicidade , Animais , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Fatores de Crescimento Endotelial/metabolismo , Endotélio Vascular/metabolismo , Linfocinas/metabolismo , Ligação Proteica , Inibidores da Síntese de Proteínas/farmacologia , Receptores Proteína Tirosina Quinases/biossíntese , Receptores de Fatores de Crescimento/biossíntese , Receptores de Fatores de Crescimento do Endotélio Vascular , Proteínas Recombinantes de Fusão/biossíntese , Toxina Shiga I/toxicidade , Suínos , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
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.
Assuntos
Fagocitose/fisiologia , Fagossomos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Androstadienos/farmacologia , Animais , Células Cultivadas , Inibidores Enzimáticos/metabolismo , Fibroblastos/metabolismo , Genes Reporter , Humanos , Imunoglobulina G/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Lisossomos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Microinjeções , Fagossomos/ultraestrutura , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , WortmaninaRESUMO
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.
Assuntos
Fatores de Crescimento Endotelial/farmacologia , Linfocinas/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Sequência de Aminoácidos , Linhagem Celular Transformada , Tamanho Celular/efeitos dos fármacos , Desenho de Fármacos , Fatores de Crescimento Endotelial/biossíntese , Fatores de Crescimento Endotelial/genética , Humanos , Linfocinas/biossíntese , Linfocinas/genética , Dados de Sequência Molecular , Fosforilação/efeitos dos fármacos , Ligação Proteica , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Isoformas de Proteínas/farmacologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
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.
Assuntos
Polaridade Celular/fisiologia , Endocitose/efeitos dos fármacos , Fígado/citologia , Fosfatidilinositol 3-Quinases/farmacologia , Androstadienos/farmacologia , Animais , Anticorpos Monoclonais/farmacologia , Membrana Celular/metabolismo , Endossomos/efeitos dos fármacos , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Fígado/enzimologia , Lisossomos/efeitos dos fármacos , Lisossomos/ultraestrutura , Proteínas de Membrana/metabolismo , Microinjeções , Fosfatidilinositol 3-Quinases/imunologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas/metabolismo , Ratos , Células Tumorais Cultivadas , Vacúolos/química , Proteínas de Transporte Vesicular , WortmaninaRESUMO
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.
Assuntos
Macrolídeos , Mycobacterium tuberculosis , Fagossomos/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Tuberculose Pulmonar/metabolismo , Proteínas de Transporte Vesicular , Proteínas rab5 de Ligação ao GTP/metabolismo , Androstadienos/farmacologia , Animais , Antibacterianos/farmacologia , Anticorpos/farmacologia , Proteínas de Transporte/metabolismo , Linhagem Celular , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Glicosilação , Lipopolissacarídeos/farmacologia , Lisofosfolipídeos/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Microinjeções , Microesferas , Monoglicerídeos , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/imunologia , Proteínas Qa-SNARE , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida , Vesículas Transportadoras/metabolismo , Tuberculose Pulmonar/imunologia , WortmaninaRESUMO
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.
Assuntos
Sistemas de Liberação de Medicamentos , Fatores de Crescimento Endotelial/administração & dosagem , Linfocinas/administração & dosagem , Neoplasias/diagnóstico , Neoplasias/terapia , Animais , Fatores de Crescimento Endotelial/farmacocinética , Terapia Genética , Humanos , Luciferases/biossíntese , Luciferases/genética , Linfocinas/farmacocinética , Polietilenoimina , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/farmacocinética , Ribonuclease Pancreático/metabolismo , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
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.
Assuntos
Fatores de Crescimento Endotelial/farmacologia , Endotélio Vascular/citologia , Linfocinas/farmacologia , Receptores Proteína Tirosina Quinases/biossíntese , Receptores de Fatores de Crescimento/biossíntese , Toxina Shiga/farmacologia , Animais , Western Blotting , Caspase 6 , Caspases/biossíntese , Sobrevivência Celular/efeitos dos fármacos , DNA/administração & dosagem , DNA/genética , Fatores de Crescimento Endotelial/administração & dosagem , Endotélio Vascular/efeitos dos fármacos , Humanos , Linfocinas/administração & dosagem , Receptores de Fatores de Crescimento do Endotélio Vascular , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/farmacologia , Toxina Shiga/administração & dosagem , Suínos , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
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.
Assuntos
Fatores de Crescimento Endotelial/farmacologia , Endotélio Vascular/fisiologia , Imunoglobulinas/química , Linfocinas/farmacologia , Receptores Proteína Tirosina Quinases/química , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/química , Receptores de Fatores de Crescimento/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Aorta , Sítios de Ligação , Linhagem Celular , Membrana Celular/metabolismo , Células Cultivadas , Dimerização , Fatores de Crescimento Endotelial/metabolismo , Endotélio Vascular/citologia , Proteoglicanas de Heparan Sulfato/metabolismo , Humanos , Isoenzimas/metabolismo , Cinética , Linfocinas/metabolismo , Proteínas Oncogênicas/metabolismo , Fosfolipase C gama , Fosforilação , Ensaio Radioligante , Receptores de Fatores de Crescimento do Endotélio Vascular , Proteínas Recombinantes/metabolismo , Suínos , Transfecção , Fosfolipases Tipo C/metabolismo , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
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.
Assuntos
Cálcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Receptores de IgE/metabolismo , Animais , Anticorpos/farmacologia , Clonagem Molecular , Reagentes de Ligações Cruzadas , Ativação Enzimática , Heparina/farmacologia , Isoenzimas/metabolismo , Cinética , Camundongos , Microinjeções , Fosfolipase C gama , Fosfotirosina/metabolismo , Ratos , Proteínas Recombinantes/metabolismo , Células Tumorais Cultivadas , Fosfolipases Tipo C/metabolismo , Regulação para CimaRESUMO
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.
Assuntos
Citoesqueleto/fisiologia , Fator de Crescimento Epidérmico/farmacologia , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/metabolismo , Pseudópodes/fisiologia , Transdução de Sinais/fisiologia , Adenocarcinoma , Animais , Proteínas Quinases JNK Ativadas por Mitógeno , Cinética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Subunidades Proteicas , Pseudópodes/ultraestrutura , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Transdução de Sinais/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
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.
Assuntos
Vesículas Revestidas/ultraestrutura , Endossomos/ultraestrutura , Fosfatidilinositol 3-Quinases/metabolismo , Androstadienos/farmacologia , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacologia , Comunicação Autócrina , Carcinoma Hepatocelular , Vesículas Revestidas/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Humanos , Lisossomos/ultraestrutura , Microinjeções , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Fosfotirosina/metabolismo , Transporte Proteico , Células Tumorais Cultivadas , WortmaninaRESUMO
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.
Assuntos
Genes Supressores de Tumor , Isoenzimas/genética , Melanoma/genética , Melanoma/patologia , Proteínas Monoméricas de Ligação ao GTP/genética , Metástase Neoplásica/genética , Núcleosídeo-Difosfato Quinase/genética , Fatores de Transcrição/genética , Substituição de Aminoácidos , Animais , Biomarcadores Tumorais/genética , Domínio Catalítico , Humanos , Camundongos , Camundongos Nus , Proteínas Monoméricas de Ligação ao GTP/fisiologia , Nucleosídeo NM23 Difosfato Quinases , Fosforilação , Mutação Puntual , Proteínas Recombinantes/biossíntese , Fatores de Transcrição/fisiologia , Transfecção , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Aggressiveness of neuroblastoma is associated with increased expression of the putative metastasis suppressor genes, nm23-1 and nm23-2. These genes encode nucleoside diphosphate kinases A and B that form free or bound homo- and heteromers, which are distributed between soluble and particulate fractions of cells and display catalytic and non-catalytic activities. MATERIALS AND METHODS: In order to establish which forms and activities of nm23 proteins are operative in neuroblastoma we stably transfected IMR-32 human neuroblastoma cells with constructs encoding wild type and catalytically inactive nm23-1 and nm23-2 proteins. RESULTS: Overexpression of wild type nm23-1 proteins stimulated spontaneous neurite outgrowth and enhanced differentiation in response to serum starvation and retinoic acid. In contrast, overexpression of the catalytically inactive nm23-1T mutant enhanced TPA-mediated inhibition of differentiation. CONCLUSION: Our findings suggest that differentiation associated functions of nm23 proteins in IMR-32 neuroblastoma cells are carried out by bound nm23-1 proteins docked in a limited number of nm23-1 specific sites.
Assuntos
Proteínas Monoméricas de Ligação ao GTP/biossíntese , Neuritos/patologia , Neuroblastoma/metabolismo , Núcleosídeo-Difosfato Quinase , Fatores de Transcrição/biossíntese , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Humanos , Proteínas Monoméricas de Ligação ao GTP/genética , Nucleosídeo NM23 Difosfato Quinases , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neuroblastoma/patologia , Estaurosporina/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Transcrição/genética , Transfecção , Tretinoína/farmacologia , Células Tumorais CultivadasAssuntos
Fosfatidilinositol 3-Quinases/metabolismo , Vacúolos/metabolismo , Animais , Vesículas Revestidas/enzimologia , Vesículas Revestidas/metabolismo , Endocitose , Endossomos/enzimologia , Endossomos/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/classificação , Isoenzimas/metabolismo , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/classificação , Fosfatidilinositóis/química , Fosfatidilinositóis/metabolismo , Pinocitose , Transdução de Sinais , Vacúolos/enzimologiaRESUMO
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.
Assuntos
Complexo de Golgi/ultraestrutura , Ácidos Fosfatídicos/biossíntese , Adeno-Hipófise/metabolismo , 1-Butanol/farmacologia , Animais , Linhagem Celular , Cricetinae , Eletroforese em Gel de Poliacrilamida , Complexo de Golgi/efeitos dos fármacos , Hormônio do Crescimento/metabolismo , Haplorrinos , Hexosaminidases/metabolismo , Adeno-Hipófise/efeitos dos fármacos , Prolactina/metabolismo , RatosRESUMO
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.