Visualization of negative signaling in B cells by quantitative confocal microscopy.
Numerous biochemical experiments have invoked a model in which B-cell antigen receptor (BCR)-Fc receptor for immunoglobulin (Ig) G (FcgammaRII) coclustering provides a dominant negative signal that blocks B-cell activation. Here, we tested this model using quantitative confocal microscopic techniques applied to ex vivo splenic B cells. We found that FcgammaRII and BCR colocalized with intact anti-Ig and that the SH2 domain-containing inositol 5'-phosphatase (SHIP) was recruited to the same site. Colocalization of BCR and SHIP was inefficient in FcgammaRII-/- but not gamma chain-/- splenic B cells. We also examined the subcellular location of a variety of enzymes and adapter proteins involved in signal transduction. Several proteins (CD19, CD22, SHP-1, and Dok) and a lipid raft marker were co-recruited to the BCR, regardless of the presence or absence of FcgammaRII and SHIP. Other proteins (Btk, Vav, Rac, and F-actin) displayed reduced colocalization with BCR in the presence of FcgammaRII and SHIP. Colocalization of BCR and F-actin required phosphatidylinositol (PtdIns) 3-kinase and was inhibited by SHIP, because the block in BCR/F-actin colocalization was not seen in B cells of SHIP-/- animals. Furthermore, BCR internalization was inhibited with intact anti-Ig stimulation or by expression of a dominant-negative mutant form of Rac. From these results, we propose that SHIP recruitment to BCR/FcgammaRII and the resulting hydrolysis of PtdIns-3,4,5-trisphosphate prevents the appropriate spatial redistribution and activation of enzymes distal to PtdIns 3-kinase, including those that promote Rac activation, actin polymerization, and receptor internalization.
Phee
H
H
Immunobiology and Cancer Program, The Oklahoma Medical Research Foundation, 825 N.E. 13th St., Oklahoma City, OK 73104, USA.
Rodgers
W
W
Coggeshall
K M
KM
D000199:Actins / Q000378:metabolism; D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism*; D006868:Hydrolysis; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods*; D008954:Models, Biological; D015233:Models, Statistical; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D017452:Receptors, IgG / Q000737:chemistry; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000378:metabolism
D000199:Actins; D017452:Receptors, IgG; C080476:ShPI proteinase inhibitor; D014360:Trypsin Inhibitor, Kunitz Soybean; D019869:Phosphatidylinositol 3-Kinases