Article

Contribution of PIP-5 kinase Iα to raft-based FcγRIIA signaling

Laboratory of Plasma Membrane Receptors, Nencki Institute of Experimental Biology, 3 Pasteur St., 02-093 Warsaw, Poland; INSERM U563, Département Lipoprotéines et Médiateurs Lipidiques, Site Purpan, BP3028, Toulouse F-31024, France
Experimental Cell Research DOI:10.1016/j.yexcr.2009.01.023 pp.981-995

ABSTRACT Receptor FcγIIA (FcγRIIA) associates with plasma membrane rafts upon activation to trigger signaling cascades leading to actin polymerization. We examined whether compartmentalization of PI(4,5)P2 and PI(4,5)P2-synthesizing PIP5-kinase Iα to rafts contributes to FcγRIIA signaling. A fraction of PIP5-kinase Iα was detected in raft-originating detergent-resistant membranes (DRM) isolated from U937 monocytes and other cells. The DRM of U937 monocytes contained also a major fraction of PI(4,5)P2. PIP5-kinase Iα bound PI(4,5)P2, and depletion of the lipid displaced PIP5-kinase Iα from the DRM. Activation of FcγRIIA in BHK transfectants led to recruitment of the kinase to the plasma membrane and enrichment of DRM in PI(4,5)P2. Immunofluorescence studies revealed that in resting cells the kinase was associated with the plasma membrane, cytoplasmic vesicles and the nucleus. After FcγRIIA activation, PIP5-kinase Iα and PI(4,5)P2 co-localized transiently with the activated receptor at distinct cellular locations. Immunoelectron microscopy studies revealed that PIP5-kinase Iα and PI(4,5)P2 were present at the edges of electron-dense assemblies containing activated FcγRIIA in their core. The data suggest that activation of FcγRIIA leads to membrane rafts coalescing into signaling platforms containing PIP5-kinase Iα and PI(4,5)P2.

0 0
 · 
0 Bookmarks
 · 
25 Views
  • Source
    Article: Biophysical mechanism for ras-nanocluster formation and signaling in plasma membrane.
    Thomas Gurry, Ozan Kahramanoğullari, Robert G Endres
    [show abstract] [hide abstract]
    ABSTRACT: Ras GTPases are lipid-anchored G proteins, which play a fundamental role in cell signaling processes. Electron micrographs of immunogold-labeled Ras have shown that membrane-bound Ras molecules segregate into nanocluster domains. Several models have been developed in attempts to obtain quantitative descriptions of nanocluster formation, but all have relied on assumptions such as a constant, expression-level independent ratio of Ras in clusters to Ras monomers (cluster/monomer ratio). However, this assumption is inconsistent with the law of mass action. Here, we present a biophysical model of Ras clustering based on short-range attraction and long-range repulsion between Ras molecules in the membrane. To test this model, we performed Monte Carlo simulations and compared statistical clustering properties with experimental data. We find that we can recover the experimentally-observed clustering across a range of Ras expression levels, without assuming a constant cluster/monomer ratio or the existence of lipid rafts. In addition, our model makes predictions about the signaling properties of Ras nanoclusters in support of the idea that Ras nanoclusters act as an analog-digital-analog converter for high fidelity signaling.
    PLoS ONE 02/2009; 4(7):e6148. · 4.09 Impact Factor
  • Source
    Article: Sphingomyelin-rich domains are sites of lysenin oligomerization: implications for raft studies.
    Magdalena Kulma, Monika Hereć, Wojciech Grudziński, Gregor Anderluh, Wiesław I Gruszecki, Katarzyna Kwiatkowska, Andrzej Sobota
    [show abstract] [hide abstract]
    ABSTRACT: Lysenin is a self-assembling, pore-forming toxin which specifically recognizes sphingomyelin. Mutation of tryptophan 20 abolishes lysenin oligomerization and cytolytic activity. We studied the interaction of lysenin WT and W20A with sphingomyelin in membranes of various lipid compositions which, according to atomic force microscopy studies, generated either homo- or heterogeneous sphingomyelin distribution. Liposomes composed of SM/DOPC, SM/DOPC/cholesterol and SM/DPPC/cholesterol could bind the highest amounts of GST-lysenin WT, as shown by surface plasmon resonance analysis. These lipid compositions enhanced the release of carboxyfluorescein from liposomes induced by lysenin WT, pointing to the importance of heterogeneous sphingomyelin distribution for lysenin WT binding and oligomerization. Lysenin W20A bound more weakly to sphingomyelin-containing liposomes than did lysenin WT. The same amounts of lysenin W20A bound to sphingomyelin mixed with either DOPC or DPPC, indicating that the binding was not affected by sphingomyelin distribution in the membranes. The mutant lysenin had a limited ability to penetrate hydrophobic region of the membrane as indicated by measurements of surface pressure changes. When applied to detect sphingomyelin on the cell surface, lysenin W20A formed large conglomerates on the membrane, different from small and regular clusters of lysenin WT. Only lysenin WT recognized sphingomyelin pool affected by formation of raft-based signaling platforms. During fractionation of Triton X-100 cell lysates, SDS-resistant oligomers of lysenin WT associated with membrane fragments insoluble in Triton X-100 while monomers of lysenin W20A partitioned to Triton X-100-soluble membrane fractions. Altogether, the data suggest that oligomerization of lysenin WT is a prerequisite for its docking in raft-related domains.
    Biochimica et Biophysica Acta 12/2009; 1798(3):471-81. · 4.66 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.

Keywords

actin polymerization
 
activated FcγRIIA
 
activated receptor
 
BHK transfectants
 
cytoplasmic vesicles
 
distinct cellular locations
 
FcγRIIA activation
 
FcγRIIA signaling
 
Immunoelectron microscopy studies
 
membrane rafts coalescing
 
PI(4,5)P2 co-localized transiently
 
PI(4,5)P2-synthesizing PIP5-kinase Iα
 
PIP5-kinase Iα
 
plasma membrane rafts
 
raft-originating detergent-resistant membranes
 
rafts contributes
 
Receptor FcγIIA
 
resting cells
 
signaling cascades
 
signaling platforms