Swiprosin-1/EFhd2 controls B cell receptor signaling through the assembly of the B cell receptor, Syk, and phospholipase C gamma2 in membrane rafts.
Compartmentalization of the BCR in membrane rafts is important for its signaling capacity. Swiprosin-1/EFhd2 (Swip-1) is an EF-hand and coiled-coil-containing adaptor protein with predicted Src homology 3 (SH3) binding sites that we identified in membrane rafts. We showed previously that Swip-1 amplifies BCR-induced apoptosis; however, the mechanism of this amplification was unknown. To address this question, we overexpressed Swip-1 and found that Swip-1 amplified the BCR-induced calcium flux in WEHI231, B62.1, and Bal17 cells. Conversely, the BCR-elicited calcium flux was strongly attenuated in Swip-1-silenced WEHI231 cells, and this was due to a decreased calcium mobilization from intracellular stores. Complementation of Swip-1 expression in Swip-1-silenced WEHI231 cells restored the BCR-induced calcium flux and enhanced spleen tyrosine kinase (Syk) tyrosine phosphorylation and activity as well as SLP65/BLNK/BASH and phospholipase C gamma2 (PLCgamma2) tyrosine phosphorylation. Furthermore, Swip-1 induced the constitutive association of the BCR itself, Syk, and PLCgamma2 with membrane rafts. Concomitantly, Swip-1 stabilized the association of BCR with tyrosine-phosphorylated proteins, specifically Syk and PLCgamma2, and enhanced the constitutive interaction of Syk and PLCgamma2 with Lyn. Interestingly, Swip-1 bound to the rSH3 domains of the Src kinases Lyn and Fgr, as well as to that of PLCgamma. Deletion of the predicted SH3-binding region in Swip-1 diminished its association and that of Syk and PLCgamma2 with membrane rafts, reduced its interaction with the SH3 domain of PLCgamma, and diminished the BCR-induced calcium flux. Hence, Swip-1 provides a membrane scaffold that is required for the Syk-, SLP-65-, and PLCgamma2-dependent BCR-induced calcium flux.
Kroczek
Carmen
C
Division of Molecular Immunology, Department of Medicine III, Nikolaus Fiebiger Center, University of Erlangen-Nürnberg, Erlangen, Germany.
Lang
Christiane
C
Brachs
Sebastian
S
Grohmann
Marcus
M
Dütting
Sebastian
S
Schweizer
Astrid
A
Nitschke
Lars
L
Feller
Stephan M
SM
Jäck
Hans-Martin
HM
Mielenz
Dirk
D
D048868:Adaptor Proteins, Signal Transducing / Q000276:immunology* / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002135:Calcium-Binding Proteins; D002460:Cell Line; D002469:Cell Separation; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000276:immunology* / Q000378:metabolism; D008213:Lymphocyte Activation / Q000276:immunology; D021962:Membrane Microdomains / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D051966:Phospholipase C gamma / Q000276:immunology* / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000276:immunology* / Q000378:metabolism; D011947:Receptors, Antigen, B-Cell / Q000276:immunology* / Q000378:metabolism; D015398:Signal Transduction / Q000276:immunology*; D000072377:Syk Kinase
D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; C530752:EFHD2 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D011947:Receptors, Antigen, B-Cell; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase; C000606897:Syk protein, mouse; D051966:Phospholipase C gamma