Contribution of PIP-5 kinase Ialpha to raft-based FcgammaRIIA signaling.

Receptor FcgammaIIA (FcgammaRIIA) associates with plasma membrane rafts upon activation to trigger signaling cascades leading to actin polymerization. We examined whether compartmentalization of PI(4,5)P(2) and PI(4,5)P(2)-synthesizing PIP5-kinase Ialpha to rafts contributes to FcgammaRIIA signaling. A fraction of PIP5-kinase Ialpha 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)P(2). PIP5-kinase Ialpha bound PI(4,5)P(2), and depletion of the lipid displaced PIP5-kinase Ialpha from the DRM. Activation of FcgammaRIIA in BHK transfectants led to recruitment of the kinase to the plasma membrane and enrichment of DRM in PI(4,5)P(2). Immunofluorescence studies revealed that in resting cells the kinase was associated with the plasma membrane, cytoplasmic vesicles and the nucleus. After FcgammaRIIA activation, PIP5-kinase Ialpha and PI(4,5)P(2) co-localized transiently with the activated receptor at distinct cellular locations. Immunoelectron microscopy studies revealed that PIP5-kinase Ialpha and PI(4,5)P(2) were present at the edges of electron-dense assemblies containing activated FcgammaRIIA in their core. The data suggest that activation of FcgammaRIIA leads to membrane rafts coalescing into signaling platforms containing PIP5-kinase Ialpha and PI(4,5)P(2).