scholarly journals Enforced C-Src Activation Causes Compartmental Dysregulation of PI3K and PTEN Molecules in Lipid Rafts of Tongue Squamous Carcinoma Cells by Attenuating Rac1-Akt-GLUT-1-Mediated Sphingolipid Synthesis

2020 ◽  
Vol 21 (16) ◽  
pp. 5812
Author(s):  
Chien-Wei Wu ◽  
Shyang-Guang Wang ◽  
Ching-Hsiao Lee ◽  
Wen-Ling Chan ◽  
Meng-Liang Lin ◽  
...  
Keyword(s):  
Complex Formation ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Cell Invasion ◽  
Ectopic Expression ◽  
Squamous Carcinoma ◽  
Membrane Targeting ◽  
Glut 1 ◽  
Src Activation ◽  
Tongue Squamous Carcinoma

Pharmacologic intervention to affect the membrane lipid homeostasis of lipid rafts is a potent therapeutic strategy for cancer. Here we showed that gallic acid (GA) caused the complex formation of inactive Ras-related C3 botulinum toxin substrate 1 (Rac1)-phospho (p)-casein kinase 2 α (CK2α) (Tyr 255) in human tongue squamous carcinoma (TSC) cells, which disturbed the lipid raft membrane-targeting of phosphatidylinositol 3-kinase (PI3K)-Rac1-protein kinase B (Akt) signal molecules by inducing the association of p110α-free p85α with unphosphorylated phosphatase tensin homolog deleted on chromosome 10 (PTEN) in lipid rafts. The effects on induction of inactive Rac1-p-CK2α (Tyr 255) complex formation and attenuation of p-Akt (Ser 473), GTP-Rac1, glucose transporter-1 (GLUT-1) lipid raft membrane-targeting, and cell invasive activity by GA were counteracted either by CK2α short hairpin RNA or cellular-Src (c-Src) inhibitor PP1. PP1 treatment, GLUT-1 or constitutively active Rac1 ectopic-expression blocked GA-induced decreases in cellular glucose, sphingolipid and cholesterol of lipid raft membranes, p85α-p110α-GTP-Rac1 complexes, glucosylceramide synthase activity and increase in ceramide and p110α-free p85α-PTEN complex levels of lipid raft membranes, which reversed the inhibition on matrix metalloproteinase (MMP)-2/-9-mediated cell invasion induced by GA. Using transient ectopic expression of nuclear factor-kappa B (NF-κB) p65, MMP-2/-9 promoter-driven luciferase, and NF-κB-dependent luciferase reporter genes and NF-κB specific inhibitors or Rac1 specific inhibitor NSC23766, we confirmed that an attenuation of Rac1 activity by GA confers inhibition of NF-κB-mediated MMP-2/-9 expression and cell invasion. In conclusion, GA-induced c-Src activation is a key inductive event for the formation of inactive Rac1-p-CK2α (Tyr 255) complexes, which disturbed lipid raft compartment of PI3K and PTEN molecules by impairing Akt-regulated GLUT-1-mediated sphingolipid synthesis, and finally resulting in inhibition of TSC cell invasion.

scholarly journals Impairment of Membrane Lipid Homeostasis by Bichalcone Analog TSWU-BR4 Attenuates Function of GRP78 in Regulation of the Oxidative Balance and Invasion of Cancer Cells

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 371
Author(s):  
Tsung-Lin Lee ◽  
Shyang-Guang Wang ◽  
Wen-Ling Chan ◽  
Ching-Hsiao Lee ◽  
Tian-Shung Wu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Complex Formation ◽  
Cancer Cells ◽  
Cancer Cell ◽  
Lipid Raft ◽  
Cell Invasion ◽  
Membrane Trafficking ◽  
Ectopic Expression ◽  
Cancer Cell Invasion ◽  
Oxidative Balance

The specialized cholesterol/sphingolipid-rich membrane domains termed lipid rafts are highly dynamic in the cancer cells, which rapidly assemble effector molecules to form a sorting platform essential for oncogenic signaling transduction in response to extra- or intracellular stimuli. Density-based membrane flotation, subcellular fractionation, cell surface biotinylation, and co-immunoprecipitation analyses of bichalcone analog ((E)-1-(4-Hydroxy-3-((4-(4-((E)-3-(pyridin-3-yl)acryloyl)phenyl)piperazin-1-yl)methyl)phenyl)-3-(pyridin-3-yl)prop-2-en-1-one (TSWU-BR4)-treated cancer cells showed dissociation between GRP78 and p85α conferring the recruitment of PTEN to lipid raft membranes associated with p85α. Ectopic expression of GRP78 could overcome induction of lipid raft membrane-associated p85α–unphosphorylated PTEN complex formation and suppression of GRP78−PI3K−Akt−GTP-Rac1-mediated and GRP78-regulated PERK−Nrf2 antioxidant pathway and cancer cell invasion by TSWU-BR4. Using specific inducer, inhibitor, or short hairpin RNA for ASM demonstrated that induction of the lipid raft membrane localization and activation of ASM by TSWU-BR4 is responsible for perturbing homeostasis of cholesterol and ceramide levels in the lipid raft and ER membranes, leading to alteration of GRP78 membrane trafficking and subsequently inducing p85α–unphosphorylated PTEN complex formation, causing disruption of GRP78−PI3K−Akt−GTP-Rac1-mediated signal and ER membrane-associated GRP78-regulated oxidative stress balance, thus inhibiting cancer cell invasion. The involvement of the enrichment of ceramide to lipid raft membranes in inhibition of NF-κB-mediated MMP-2 expression was confirmed through attenuation of NF-κB activation using C2-ceramide, NF-κB specific inhibitors, ectopic expression of NF-κB p65, MMP-2 promoter-driven luciferase, and NF-κB-dependent reporter genes. In conclusion, localization of ASM in the lipid raft membranes by TSWU-BR4 is a key event for initiating formation of ceramide-enriched lipid raft membrane platforms, which causes delocalization of GRP78 from the lipid raft and ER membranes to the cytosol and formation of p85α–unphosphorylated PTEN complexes to attenuate the GRP78-regulated oxidative stress balance and GRP78−p85α−Akt−GTP-Rac1−NF-κB−MMP-2-mediated cancer cell invasion.

Requirement of Lipid Raft Integrity for Signaling by JAK2-V617F

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4191-4191
Author(s):  
Lori N. Griner ◽  
Kathy L. McGraw ◽  
Joseph O. Johnson ◽  
Alan F. List ◽  
Gary W. Reuther
Keyword(s):  
Signal Transduction ◽  
Plasma Membrane ◽  
Complex Formation ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Kinase Activity ◽  
Jak2 V617f ◽  
Cytokine Receptors ◽  
Downstream Signaling ◽  
Stat Signaling

Abstract Abstract 4191 JAK2 is a cytoplasmic tyrosine kinase that plays an important role in signaling following activation of various cytokine receptors. JAK2 activation promotes growth, survival, and differentiation of various cell types. Mutation of JAK2 is seen in numerous hematopoietic diseases, most notably in myeloproliferative neoplasms (MPNs). JAK2-V617F is a frequent mutation found in the classical MPNs: polycythemia vera, essential thrombocythemia, and primary myelofibrosis. The single amino acid change of valine to phenylalanine occurs in the pseudokinase domain of JAK2, relieving auto-inhibition of the kinase domain and allowing constitutive kinase activity. Numerous mouse models have demonstrated that JAK2-V617F can induce MPN-like disorders in mice. Thus, this point mutation, as well as other less common JAK2 mutations, is believed to play an important etiologic role in the development of MPNs in humans. The development and use of JAK2 inhibitors in clinical trials has shown promising results, again demonstrating the important role JAK2 plays in MPNs. While the JAK2-V617F mutation, as well as other JAK2 mutations, decreases auto-inhibition of JAK2 kinase activity, it is clear that mutated JAK2 still requires the expression of cytokine receptors to induce activation of transforming signals in hematopoietic cells. Normally, JAK2 binds to homodimeric and heterodimeric cytokine receptors through specific receptor motifs and is activated by various structural changes induced by cytokine stimulation. Following activation it utilizes receptor tyrosines as substrates for phosphorylation, leading to recruitment of downstream signaling molecules, such as STAT5, among others. JAK2 then activates STAT5 via phosphorylation and STAT5 then translocates to the nucleus to regulate transcription of target genes. JAK2-V617F does not require ligand for activation, but still requires the scaffolding function of cytokine receptors to facilitate its full activation and activation of downstream signaling via phosphorylation. Lipid rafts are microdomains of the plasma membrane that are enriched in cholesterol and sphingolipids. They have gained appreciation in signal transduction as sites of localization of signaling mediators, including membrane-bound receptors. Congregation of signaling proteins in lipid rafts within the plasma membrane promotes complex formation and signaling cascade activation. We have recently demonstrated that JAK2 is present in lipid rafts during erythropoietin signaling and that lipid raft integrity is required for erythropoietin-mediated signal transduction (Blood 2009, 114: 292). In our current study, we demonstrate that constitutive JAK-STAT signaling driven by JAK2-V617F is sensitive to lipid raft disruption. Human erythroleukemia (HEL) cells express constitutive activation of JAK-STAT signaling due to the presence of JAK2-V617F. Treatment of these cells with methyl-beta-cyclodextrin to disrupt lipid rafts abolished JAK2, STAT5, and STAT3 activation. Similar results are obtained in other cell lines harboring JAK2-V617F and that exhibit JAK-STAT activation that is dependent on this activated form of JAK2. We also demonstrate that JAK2-V617F co-localizes with lipid rafts, as shown by immunofluorescence, and that this co-localization is abolished by lipid raft disruption. This suggests the loss of JAK2-V617F-mediated JAK-STAT activation we observe following lipid raft disruption may be due to an inhibition of properly localized protein complex formation in the plasma membrane that is necessary for JAK2-V617F signaling. Lipid rafts may provide a site for an accumulation of JAK2-V617F-containing signaling complexes and may be necessary for the cellular signals initiated by JAK2-V617F. Our data show JAK2-V617F-driven JAK-STAT pathway activation is vulnerable to lipid raft disrupting agents and suggest lipid raft integrity as a potential therapeutic target for JAK2-V617F positive neoplasms. Targeting lipid rafts in combination with JAK2 kinase inhibitors may allow for more effective kinase inhibition at lower doses, potentially decreasing undesirable side effects associated with kinase inhibitor treatment. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Nef Associates with p21-Activated Kinase 2 in a p21-GTPase-Dependent Dynamic Activation Complex within Lipid Rafts

2004 ◽  
Vol 78 (23) ◽  
pp. 12773-12780 ◽  
Author(s):  
Kati Pulkkinen ◽  
G. Herma Renkema ◽  
Frank Kirchhoff ◽  
Kalle Saksela
Keyword(s):  
Catalytic Activity ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Kinase Activity ◽  
Highly Active ◽  
Membrane Compartment ◽  
Per Se ◽  
P21 Activated Kinase ◽  
Autoregulatory Mechanism ◽  
Pak2 Activation

ABSTRACT We have previously reported that Nef specifically interacts with a small but highly active subpopulation of p21-activated kinase 2 (PAK2). Here we show that this is due to a transient association of Nef with a PAK2 activation complex within a detergent-insoluble membrane compartment containing the lipid raft marker GM1. The low abundance of this Nef-associated kinase (NAK) complex was found to be due to an autoregulatory mechanism. Although activation of PAK2 was required for assembly of the NAK complex, catalytic activity of PAK2 also promoted dissociation of this complex. Testing different constitutively active PAK2 mutants indicated that the conformation associated with p21-mediated activation rather than kinase activity per se was required for PAK2 to become NAK. Although association with PAK2 is one of the most conserved properties of Nef, we found that the ability to stimulate PAK2 activity differed markedly among divergent Nef alleles, suggesting that PAK2 association and activation are distinct functions of Nef. However, mutations introduced into the p21-binding domain of PAK2 revealed that p21-GTPases are involved in both of these Nef functions and, in addition to promoting PAK2 activation, also help to physically stabilize the NAK complex.

scholarly journals Lipid raft–dependent plasma membrane repair interferes with the activation of B lymphocytes

2015 ◽  
Vol 211 (6) ◽  
pp. 1193-1205 ◽  
Author(s):  
Heather Miller ◽  
Thiago Castro-Gomes ◽  
Matthias Corrotte ◽  
Christina Tam ◽  
Timothy K. Maugel ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
B Cell ◽  
B Lymphocytes ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Cell Activation ◽  
Dependent Manner ◽  
B Cell Activation ◽  
Membrane Repair ◽  
Membrane Injury

Cells rapidly repair plasma membrane (PM) damage by a process requiring Ca2+-dependent lysosome exocytosis. Acid sphingomyelinase (ASM) released from lysosomes induces endocytosis of injured membrane through caveolae, membrane invaginations from lipid rafts. How B lymphocytes, lacking any known form of caveolin, repair membrane injury is unknown. Here we show that B lymphocytes repair PM wounds in a Ca2+-dependent manner. Wounding induces lysosome exocytosis and endocytosis of dextran and the raft-binding cholera toxin subunit B (CTB). Resealing is reduced by ASM inhibitors and ASM deficiency and enhanced or restored by extracellular exposure to sphingomyelinase. B cell activation via B cell receptors (BCRs), a process requiring lipid rafts, interferes with PM repair. Conversely, wounding inhibits BCR signaling and internalization by disrupting BCR–lipid raft coclustering and by inducing the endocytosis of raft-bound CTB separately from BCR into tubular invaginations. Thus, PM repair and B cell activation interfere with one another because of competition for lipid rafts, revealing how frequent membrane injury and repair can impair B lymphocyte–mediated immune responses.

scholarly journals CD95 death-inducing signaling complex formation and internalization occur in lipid rafts of type I and type II cells

2004 ◽  
Vol 34 (7) ◽  
pp. 1930-1940 ◽  
Author(s):  
Adriana Eramo ◽  
Massimo Sargiacomo ◽  
Lucia Ricci-Vitiani ◽  
Matilde Todaro ◽  
Giorgio Stassi ◽  
...  
Keyword(s):  
Complex Formation ◽  
Lipid Rafts ◽  
Type I ◽  
Type Ii Cells ◽  
Type Ii ◽  
Signaling Complex

Mediators of innate immune recognition of bacteria concentrate in lipid rafts and facilitate lipopolysaccharide-induced cell activation

2002 ◽  
Vol 115 (12) ◽  
pp. 2603-2611 ◽  
Author(s):  
Martha Triantafilou ◽  
Kensuke Miyake ◽  
Douglas T. Golenbock ◽  
Kathy Triantafilou
Keyword(s):  
Lipid Rafts ◽  
Lipid Raft ◽  
Cell Activation ◽  
Imaging Techniques ◽  
Recognition System ◽  
Innate Immune ◽  
Membrane Microdomains ◽  
Immune Recognition ◽  
Cellular Activation ◽  
Signalling Molecules

The plasma membrane of cells is composed of lateral heterogeneities,patches and microdomains. These membrane microdomains or lipid rafts are enriched in glycosphingolipids and cholesterol and have been implicated in cellular processes such as membrane sorting and signal transduction. In this study we investigated the importance of lipid raft formation in the innate immune recognition of bacteria using biochemical and fluorescence imaging techniques. We found that receptor molecules that are implicated in lipopolysaccharide (LPS)-cellular activation, such as CD14, heat shock protein(hsp) 70, 90, Chemokine receptor 4 (CXCR4), growth differentiation factor 5(GDF5) and Toll-like receptor 4 (TLR4), are present in microdomains following LPS stimulation. Lipid raft integrity is essential for LPS-cellular activation, since raft-disrupting drugs, such as nystatin or MCD, inhibit LPS-induced TNF-α secretion. Our results suggest that the entire bacterial recognition system is based around the ligation of CD14 by bacterial components and the recruitment of multiple signalling molecules, such as hsp70, hsp90, CXCR4, GDF5 and TLR4, at the site of CD14-LPS ligation, within the lipid rafts.

scholarly journals Lipid rafts mediate internalization of β1-integrin in migrating intestinal epithelial cells

2008 ◽  
Vol 295 (5) ◽  
pp. G965-G976 ◽  
Author(s):  
Elena V. Vassilieva ◽  
Kirsten Gerner-Smidt ◽  
Andrei I. Ivanov ◽  
Asma Nusrat
Keyword(s):  
Epithelial Cells ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Intestinal Epithelial Cells ◽  
Focal Adhesions ◽  
Endocytic Pathway ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Caveolin 1 ◽  
Cell Matrix

Intestinal mucosal inflammation is associated with epithelial wounds that rapidly reseal by migration of intestinal epithelial cells (IECs). Cell migration involves cycles of cell-matrix adhesion/deadhesion that is mediated by dynamic turnover (assembly and disassembly) of integrin-based focal adhesions. Integrin endocytosis appears to be critical for deadhesion of motile cells. However, mechanisms of integrin internalization during remodeling of focal adhesions of migrating IECs are not understood. This study was designed to define the endocytic pathway that mediates internalization of β1-integrin in migrating model IECs. We observed that, in SK-CO15 and T84 colonic epithelial cells, β1-integrin is internalized in a dynamin-dependent manner. Pharmacological inhibition of clathrin-mediated endocytosis or macropinocytosis and small-interfering RNA (siRNA)-mediated knock down of clathrin did not prevent β1-integrin internalization. However, β1-integrin internalization was inhibited following cholesterol extraction and after overexpression of lipid raft protein, caveolin-1. Furthermore, internalized β1-integrin colocalized with the lipid rafts marker cholera toxin, and siRNA-mediated knockdown of caveolin-1 and flotillin-1/2 increased β1-integrin endocytosis. Our data suggest that, in migrating IEC, β1-integrin is internalized via a dynamin-dependent lipid raft-mediated pathway. Such endocytosis is likely to be important for disassembly of integrin-based cell-matrix adhesions and therefore in regulating IEC migration and wound closure.

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