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Molecular and Cellular Biology, December 2001, p. 8615-8625, Vol. 21, No. 24
Immunobiology and
Cancer1 and Molecular
Immunogenetics3 Programs, The Oklahoma
Medical Research Foundation, Oklahoma City, Oklahoma 73104, and
Departments of Biochemistry2 and
Microbiology,4 The Ohio State
University, Columbus, Ohio 43210
Received 26 June 2001/Accepted 13 September 2001
Numerous biochemical experiments have invoked a model in which
B-cell antigen receptor (BCR)-Fc receptor for immunoglobulin (Ig) G
(Fc
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.24.8615-8625.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Visualization of Negative Signaling in B Cells
by Quantitative Confocal Microscopy
RII) 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 Fc
RII 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
Fc
RII
/
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
corecruited to the BCR, regardless of the presence or absence of
Fc
RII and SHIP. Other proteins (Btk, Vav, Rac, and F-actin)
displayed reduced colocalization with BCR in the presence of Fc
RII
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/Fc
RII 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.
*
Corresponding author. Mailing address: The Oklahoma
Medical Research Foundation, Immunobiology and Cancer Program, 825 N.E. 13th. St., Oklahoma City, OK 73104. Phone: (405) 271-7905. Fax: (405)
271-8568. E-mail: mark-coggeshall{at}omrf.ouhsc.edu.
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