Regulation of ecto-5′-nucleotidase activity via Ca2+-dependent, annexin 2-mediated membrane rearrangement?

2006 ◽  
Vol 34 (3) ◽  
pp. 374-376 ◽  
Author(s):  
E.B. Babiychuk ◽  
A. Draeger
Keyword(s):  
Signal Transduction ◽  
Plasma Membrane ◽  
Lipid Rafts ◽  
Binding Proteins ◽  
Extracellular Atp ◽  
Membrane Rafts ◽  
Nucleotidase Activity ◽  
Annexin 2 ◽  
Associated Proteins ◽  
A Cell

The spatial segregation of the plasma membrane plays a prominent role in distinguishing and sorting a large number of signals a cell receives simultaneously. The plasma membrane comprises regions known as lipid rafts, which serve as signal-transduction hubs and platforms for sorting membrane-associated proteins. Ca2+-binding proteins of the annexin family have been ascribed a role in the regulation of raft dynamics. Glycosylphosphatidylinositol-anchored 5′-nucleotidase is an extracellular, raft-associated enzyme responsible for conversion of extracellular ATP into adenosine. Our results point to a regulation of ecto-5′-nucleotidase activity by Ca2+-dependent, annexin-mediated stabilization of membrane rafts.

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.

The Neuronal Protein GAP-43 Associates with Microtubules and Lipid Rafts in Platelets and Megakaryocytes and Is Involved in Platelet Genesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2201-2201
Author(s):  
Antonija Jurak Begonja ◽  
Lilly Nguyen ◽  
Joseph E. Italiano ◽  
Herve Falet ◽  
Karina F. Meiri ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Flow Cytometry ◽  
Lipid Rafts ◽  
Neurotransmitter Release ◽  
Conflicts Of Interest ◽  
Membrane Rafts ◽  
Pleckstrin Homology Domain ◽  
Flow Cytometry Analysis ◽  
Neuronal Protein ◽  
Negative Role

Abstract Abstract 2201 GAP-43 (neuromodulin) is a membrane-associated protein involved in neurite outgrowth, pathfinding and branching during development where it regulates neurotransmitter release and endocytosis, and plays a role in cytoskeletal signal transduction at the nerve ending. We have found GAP-43 to be expressed in platelets and megakaryocytes (MK) as evidenced by immunoblot and flow cytometry analysis. Immunofluorescence shows that GAP-43 localizes intracellularly as discrete punctuate structures mostly near the marginal microtubule (MT) coil of platelets and along the MT bundles in MKs and proplatelets. In contrast to neuronal cells where GAP-43 is associated with their detergent insoluble F-actin cytoskeleton, it is mostly soluble in platelets unless the platelets are first treated with taxol, a drug that stabilizes MTs. MT disruption with nocodazole or by chilling decreases the amount of GAP-43 in the detergent-insoluble cytoskeleton and disturbs its localization in proplatelets. GAP-43 colocalizes in platelets and MKs with the glycosylphosphatidylinositol-linked lipid raft marker Thy-1 and Cherry-PH PLC-delta1 (pleckstrin homology domain of phospholipase C-delta1) that stains membrane rafts. Since GAP-43 overexpression in MKs diminishes proplatelet formation, it appears to play a negative role in thrombopoiesis. Together, the data shows that GAP-43 is associated with MT and lipid rafts and suggests a role in signaling reactions between these two components to modulate proplatelet elaboration. Disclosures: No relevant conflicts of interest to declare.

Membrane cholesterol and the regulation of signal transduction

2004 ◽  
Vol 32 (1) ◽  
pp. 65-69 ◽  
Author(s):  
C.J. Fielding ◽  
P.E. Fielding
Keyword(s):  
Signal Transduction ◽  
Plasma Membrane ◽  
Lipid Rafts ◽  
Mammalian Cells ◽  
Protein Composition ◽  
Cholesterol Content ◽  
Membrane Cholesterol ◽  
Transmembrane Signalling

The plasma membrane of mammalian cells consists of microdomains differing in lipid and protein composition. Two distinct classes of cholesterol/sphingolipid microdomain (caveolae and lipid rafts) are assembly points for transmembrane signalling complexes. Recent evidence suggests that transient changes in cholesterol content may be important in regulating signal transduction.

scholarly journals Dynamic ergosterol- and ceramide-rich domains in the peroxisomal membrane serve as an organizing platform for peroxisome fusion

2005 ◽  
Vol 168 (5) ◽  
pp. 761-773 ◽  
Author(s):  
Tatiana Boukh-Viner ◽  
Tong Guo ◽  
Alex Alexandrian ◽  
André Cerracchio ◽  
Christopher Gregg ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Rafts ◽  
Binding Proteins ◽  
Lateral Movement ◽  
Membrane Bilayer ◽  
Peroxisomal Membrane ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Unusual Distribution ◽  
Key Features

We describe unusual ergosterol- and ceramide-rich (ECR) domains in the membrane of yeast peroxisomes. Several key features of these detergent-resistant domains, including the nature of their sphingolipid constituent and its unusual distribution across the membrane bilayer, clearly distinguish them from well characterized detergent-insoluble lipid rafts in the plasma membrane. A distinct set of peroxisomal proteins, including two ATPases, Pex1p and Pex6p, as well as phosphoinositide- and GTP-binding proteins, transiently associates with the cytosolic face of ECR domains. All of these proteins are essential for the fusion of the immature peroxisomal vesicles P1 and P2, the earliest intermediates in a multistep pathway leading to the formation of mature, metabolically active peroxisomes. Peroxisome fusion depends on the lateral movement of Pex1p, Pex6p, and phosphatidylinositol-4,5-bisphosphate–binding proteins from ECR domains to a detergent-soluble portion of the membrane, followed by their release to the cytosol. Our data suggest a model for the multistep reorganization of the multicomponent peroxisome fusion machinery that transiently associates with ECR domains.

scholarly journals Lipid rafts and pathogens: the art of deception and exploitation

2019 ◽  
Vol 61 (5) ◽  
pp. 601-610 ◽  
Author(s):  
Michael I. Bukrinsky ◽  
Nigora Mukhamedova ◽  
Dmitri Sviridov
Keyword(s):  
Immune Response ◽  
Plasma Membrane ◽  
Host Cell ◽  
Lipid Rafts ◽  
Therapeutic Approach ◽  
Host Cells ◽  
Infectious Agents ◽  
Systematic Overview ◽  
Intracellular Parasites ◽  
A Cell

Lipid rafts, solid regions of the plasma membrane enriched in cholesterol and glycosphingolipids, are essential parts of a cell. Functionally, lipid rafts present a platform that facilitates interaction of cells with the outside world. However, the unique properties of lipid rafts required to fulfill this function at the same time make them susceptible to exploitation by pathogens. Many steps of pathogen interaction with host cells, and sometimes all steps within the entire lifecycle of various pathogens, rely on host lipid rafts. Such steps as binding of pathogens to the host cells, invasion of intracellular parasites into the cell, the intracellular dwelling of parasites, microbial assembly and exit from the host cell, and microbe transfer from one cell to another all involve lipid rafts. Interaction also includes modification of lipid rafts in host cells, inflicted by pathogens from both inside and outside the cell, through contact or remotely, to advance pathogen replication, to utilize cellular resources, and/or to mitigate immune response. Here, we provide a systematic overview of how and why pathogens interact with and exploit host lipid rafts, as well as the consequences of this interaction for the host, locally and systemically, and for the microbe. We also raise the possibility of modulation of lipid rafts as a therapeutic approach against a variety of infectious agents.

Plasma membrane associated proteins and glycoproteins of a temperature-sensitive hamster cell line (ts 3)

1978 ◽  
Vol 56 (11) ◽  
pp. 1061-1068
Author(s):  
Vijai N. Nigam ◽  
Carlos A. Brailovsky
Keyword(s):  
Plasma Membrane ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cell Phenotype ◽  
Temperature Sensitive ◽  
Major Significance ◽  
Sds Page ◽  
C Distribution ◽  
Associated Proteins ◽  
A Cell

Hamster ts 3 cells grown at 32 and at 39 °C were compared for (a) cell phenotype and growth rates, (b) sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) patterns of plasma membrane polypeptides, and (c) distribution of radioactivity in SDS–PAGE of plasma membrane glycopeptides metabolically labeled with [3H]glucosamine and [3H]fucose. It was observed that ts 3 cells growing at 39 °C and having a normal phenotype contained increased amounts of a glycopeptide of about 250 000 daltons and a polypeptide of about 200 000 daltons, as compared with ts 3 cells grown at 32 °C and having a transformed phenotype. No other alterations of major significance were observed.

scholarly journals Phosphorylation of mRNA-Binding Proteins Puf1 and Puf2 by TORC2-Activated Protein Kinase Ypk1 Alleviates Their Repressive Effects

Membranes ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 500
Author(s):  
Henri A. Galez ◽  
Françoise M. Roelants ◽  
Sarah M. Palm ◽  
Kendra K. Reynaud ◽  
Nicholas T. Ingolia ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Binding Proteins ◽  
Rna Binding ◽  
Fluorescent Protein ◽  
Rna Binding Proteins ◽  
Physiological Role ◽  
Rna Recognition Motif ◽  
Associated Proteins

Members of the Puf family of RNA-binding proteins typically associate via their Pumilio homology domain with specific short motifs in the 3’-UTR of an mRNA and thereby influence the stability, localization and/or efficiency of translation of the bound transcript. In our prior unbiased proteome-wide screen for targets of the TORC2-stimulated protein kinase Ypk1, we identified the paralogs Puf1/Jsn1 and Puf2 as high-confidence substrates. Earlier work by others had demonstrated that Puf1 and Puf2 exhibit a marked preference for interaction with mRNAs encoding plasma membrane-associated proteins, consistent with our previous studies documenting that a primary physiological role of TORC2-Ypk1 signaling is maintenance of plasma membrane homeostasis. Here, we show, first, that both Puf1 and Puf2 are authentic Ypk1 substrates both in vitro and in vivo. Fluorescently tagged Puf1 localizes constitutively in cortical puncta closely apposed to the plasma membrane, whereas Puf2 does so in the absence of its Ypk1 phosphorylation, but is dispersed in the cytosol when phosphorylated. We further demonstrate that Ypk1-mediated phosphorylation of Puf1 and Puf2 upregulates production of the protein products of the transcripts to which they bind, with a concomitant increase in the level of the cognate mRNAs. Thus, Ypk1 phosphorylation relieves Puf1- and Puf2-mediated post-transcriptional repression mainly by counteracting their negative effect on transcript stability. Using a heterologous protein-RNA tethering and fluorescent protein reporter assay, the consequence of Ypk1 phosphorylation in vivo was recapitulated for full-length Puf1 and even for N-terminal fragments (residues 1-340 and 143-295) corresponding to the region upstream of its dimerization domain (an RNA-recognition motif fold) encompassing its two Ypk1 phosphorylation sites (both also conserved in Puf2). This latter result suggests that alleviation of Puf1-imposed transcript destabilization does not obligatorily require dissociation of Ypk1-phosphorylated Puf1 from a transcript. Our findings add new insight about how the TORC2-Ypk1 signaling axis regulates the content of plasma membrane-associated proteins to promote maintenance of the integrity of the cell envelope.

scholarly journals Dictyostelium discoideumflotillin homologues are essential for phagocytosis and participate in plasma membrane recycling and lysosome biogenesis

2019 ◽  
Author(s):  
Cristina Bosmani ◽  
Frauke Bach ◽  
Florence Leuba ◽  
Nabil Hanna ◽  
Frédéric Burdet ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Rafts ◽  
Membrane Trafficking ◽  
Membrane Recycling ◽  
Lysosome Biogenesis ◽  
Wide Range ◽  
Particle Recognition ◽  
Associated Proteins ◽  
Soil Amoeba ◽  
Early Recruitment

ABSTRACTThe metazoan flotillins are lipid rafts residents involved in membrane trafficking and recycling of plasma membrane proteins.Dictyostelium discoideum, a social soil amoeba, uses phagocytosis to digest, kill and feed on bacteria.D. discoideumpossesses three flotillin-like proteins, termed VacA, VacB and the recently identified VacC. All three vacuolins gradually accumulate on postlysosomes and, like flotillins, are strongly associated with membranes and partly with lipid rafts. Vacuolins are absolutely required for uptake of various particles. Their absence impairs particle recognition possibly because of defective recycling of plasma membrane or cortex-associated proteins. In addition, vacuolins are involved in phagolysosome biogenesis, although this does not impact digestion and killing of a wide range of bacteria. Furthermore, vacuolin knockout affects early recruitment of the WASH complex on phagosomes, suggesting that vacuolins may be involved in the WASH-dependent plasma membrane recycling. Altogether, these results indicate that vacuolins act as the functional homologues of flotillins inD. discoideum.

Involvement of Lipid Rafts in Impaired fMLP-Stimulated ROS Production of GM-CSF-Primed Neutrophils from Patients with Myelodysplasia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3863-3863
Author(s):  
Gwenny M. Fuhler ◽  
A. Lyndsay Drayer ◽  
Paul J. Coffer ◽  
Edo Vellenga
Keyword(s):  
Signal Transduction ◽  
Plasma Membrane ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Bacterial Infections ◽  
Signal Transduction Pathways ◽  
Ros Production ◽  
Gm Csf ◽  
Healthy Donors ◽  
Pre Treatment

Abstract Neutrophils from MDS patients are generally dysfunctional, resulting in increased susceptibility to bacterial infections. An important bactericidal activity of neutrophils is their production of reactive oxygen species (ROS) upon stimulation with the bacterial product fMLP. ROS production by fMLP can be enhanced by pre-treatment of neutrophils with pro-inflammatory cytokines such as GM-CSF. In MDS patients, this GM-CSF priming of ROS production is severely impaired. Furthermore, activation of the extracellular signal-regulated kinase (ERK1/2) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/PKB) signal transduction pathways, involved in ROS production, are impaired in MDS (Fuhler et al, Blood, 101, 2003). In the current study we investigated the involvement of lipid rafts in neutrophil ROS production, and show that treatment of healthy neutrophils with the lipid-raft disrupting agent methyl-b-cyclodextrin (CD) abrogates fMLP-induced ROS production in unprimed cells (mean fluorescence intensity [MFI] of 16±2 vs 4±0.3, n=5, P=0.03) and GM-CSF-primed neutrophils (MFI 50±14 vs 9±1, P=0.04). We further demonstrate that activation of ERK and PKB by fMLP was abolished by CD treatment of unprimed or GM-CSF-primed neutrophils. In contrast, GM-CSF-stimulated phosphorylation of STAT5 was not affected by CD pre-treatment of healthy neutrophils. These results indicate the importance of lipid rafts in fMLP-stimulated ROS production and in activation of signal transduction pathways involved in this process. The expression of the ganglioside GM1 at the plasma membrane of cells has been described to be a marker for the presence of lipid rafts. We analyzed the expression of GM1 on healthy neutrophils by FACS analysis and show that stimulation of cells with fMLP increased the expression of GM1 at the plasma membrane (MFI 102±20 vs 161±38, n=6, P=0.03). Furthermore, pretreatment of neutrophils with GM-CSF further increased the fMLP-induced GM1 expression (MFI 161 ±38 vs 193±36, P=0.03). We next assessed the expression of GM1 at the plasma membrane, in conjunction with ROS production, in 8 MDS patients compared to healthy donors. As described previously, ROS production in response to fMLP was normal in unprimed MDS neutrophils. In contrast, in 6 patients, fMLP-induced ROS production in GM-CSF primed neutrophils was significantly impaired when compared to their healthy controls (mean MFI 128±54 vs 191±72, P=0.03). When investigating the GM1 expression in these patients, we found that although fMLP-triggered GM1 expression was normal in unprimed neutrophils from MDS patients, fMLP-induced GM1 expression on GM-CSF-primed cells was significantly lower on MDS neutrophils compared to their healthy counterpart (mean MFI 174±76 vs 214±78, P=0.03). GM-CSF primed neutrophils from one patient with normal ROS production also exhibited normal GM1 expression. Taken together, these data indicate an involvement of lipid rafts in ROS production, and suggest that in MDS neutrophils, an impaired lipid raft formation in GM-CSF primed cells correlates with impaired fMLP-induced ROS production.

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