Sos, Vav, and C3G participate in B cell receptor-induced signaling pathways and differentially associate with Shc-Grb2, Crk, and Crk-L adaptors.
B cell antigen receptor (BCR)-mediated signal transduction controls B cell proliferation and differentiation. The BCR activates Ras, presumably by the formation of a Shc-Grb2 adaptor complex, which recruits the Grb2-associated guanine nucleotide exchange factor Sos to the plasma membrane. In order to reveal additional BCR-induced signaling events involving the Grb2 adaptor, we undertook the isolation of Grb2-binding proteins. Using the yeast two-hybrid system and bacterial fusion proteins, Vav and C3G were identified as Grb2 binders. Vav is a putative nucleotide exchange factor and a target for BCR-induced tyrosine phosphorylation. C3G exerts nucleotide exchange activity on the Ras-related Rap1 protein. While Sos binds to both Grb2 Src homology-3 (SH3) domains, Vav was found to associate selectively with the carboxyl-terminal SH3 domain, while C3G bound selectively to the amino-terminal SH3 domain of bacterially expressed Grb2. Despite the association of Vav with Grb2 in vitro, we could not demonstrate an interaction between endogenous Vav and Grb2 molecules in primary B cells. Instead, Vav was found to inducibly associate with the Grb2-related adaptor protein Crk upon BCR stimulation. C3G did not bind to either Grb2, Shc, or Crk in vivo. Instead, C3G was found in association with the Crk-L adaptor, both before and after BCR stimulation. We show that Crk-L also participates in BCR signaling, since it inducibly interacts with tyrosine-phosphorylated Cbl. We conclude that, in addition to Sos, Vav and C3G play a role in BCR-mediated signal transduction. These guanine nucleotide exchange factors selectively associate with Grb2, Crk, and Crk-L, respectively, which may serve to direct them to different target molecules. Since Cbl binds to Grb2, Crk, as well as Crk-L, we hypothesize that Cbl may affect the function of all three exchangers.
Smit
L
L
Division of Cellular Biochemistry, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
van der Horst
G
G
Borst
J
J
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D001402:B-Lymphocytes / Q000502:physiology*; D001483:Base Sequence; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D051380:GRB2 Adaptor Protein; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism* / Q000502:physiology; D050737:Proto-Oncogene Proteins c-crk; D051567:Proto-Oncogene Proteins c-vav; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism*; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction; D020664:Son of Sevenless Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D020663:ras Guanine Nucleotide Exchange Factors
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; C082869:CRKL protein; D018797:Cell Cycle Proteins; D017931:DNA Primers; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D020662:Guanine Nucleotide Exchange Factors; D008565:Membrane Proteins; D009687:Nuclear Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D051567:Proto-Oncogene Proteins c-vav; D011947:Receptors, Antigen, B-Cell; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; D020664:Son of Sevenless Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; C496618:VAV1 protein, human; D020663:ras Guanine Nucleotide Exchange Factors