Cell surface ceramide generation precedes and controls FcgammaRII clustering and phosphorylation in rafts.
Despite the role of sphingolipid/cholesterol rafts as signaling platforms for Fcgamma receptor II (FcgammaRII), the mechanism governing translocation of an activated receptor toward the rafts is unknown. We show that at the onset of FcgammaRII cross-linking acid sphingomyelinase is rapidly activated. This enzyme is extruded from intracellular compartments to the cell surface, and concomitantly, exofacially oriented ceramide is produced. Both non-raft and, to a lesser extent, raft sphingomyelin pools were hydrolyzed at the onset of FcgammaRII cross-linking. The time course of ceramide production preceded the recruitment of FcgammaRII to rafts and the receptor phosphorylation. Exogenous C(16)-ceramide facilitated clustering of FcgammaRII and its association with rafts. In contrast, inhibition of acid sphingomyelinase diminished both the ceramide generation and clustering of cross-linked FcgammaRII. Under these conditions, tyrosine phosphorylation of FcgammaRII and receptor-accompanying proteins was also reduced. All the inhibitory effects were bypassed by treatment of cells with exogenous ceramide. These data provide evidence that the generation of cell surface ceramide is a prerequisite for fusion of cross-linked FcgammaRII and rafts, which triggers the receptor tyrosine phosphorylation and signaling.
Abdel Shakor
Abo Bakr
AB
Nencki Institute of Experimental Biology, the Department of Cell Biology, 3 Pasteur St., 02-093 Warsaw, Poland.
Kwiatkowska
Katarzyna
K
Sobota
Andrzej
A
D000818:Animals; D015703:Antigens, CD / Q000737:chemistry*; D019921:Biotinylation; D018958:CD55 Antigens / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002518:Ceramides / Q000737:chemistry* / Q000494:pharmacology; D006224:Cricetinae; D006801:Humans; D006868:Hydrolysis; D015151:Immunoblotting; D021962:Membrane Microdomains / Q000737:chemistry*; D018613:Microscopy, Confocal; D010766:Phosphorylation; D017452:Receptors, IgG / Q000737:chemistry*; D011990:Receptors, Transferrin / Q000378:metabolism; D015398:Signal Transduction; D013108:Sphingomyelin Phosphodiesterase / Q000494:pharmacology; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry; D020298:U937 Cells
D015703:Antigens, CD; D018958:CD55 Antigens; D002518:Ceramides; C080568:Fc gamma receptor IIA; D017452:Receptors, IgG; D011990:Receptors, Transferrin; C097760:N-palmitoylsphingosine; D014443:Tyrosine; D013108:Sphingomyelin Phosphodiesterase