Stimulation through the T cell receptor induces Cbl association with Crk proteins and the guanine nucleotide exchange protein C3G.
We and others have recently identified Cbl, the protein product of the c-cbl protooncogene, as an early tyrosine kinase substrate upon T cell activation and have shown that Cbl forms in vivo complexes with Src family tyrosine kinases, Grb2 adaptor protein, and the p85 subunit of PI-3 kinase. Here we show that Cbl associates with all three forms of the human Crk protein, predominantly CrkL, following T cell receptor activation of Jurkat T cells. Association between Cbl and Crk proteins was confirmed in normal human peripheral blood-derived T cells. In vitro, Cbl was able to interact with the Crk SH2 domain but not the SH3 domain. A phosphopeptide corresponding to a potential Crk SH2 domain-binding motif in Cbl (pYDVP) specifically inhibited binding between Cbl and Crk SH2 domain. Anti-Cbl antibody completely immunodepleted the CrkL-associated 120kDa phosphotyrosyl polypeptide, suggesting that the recently described p130cas-related Crk-associated p116 of T cells may be Cbl. Consistent with this possibility, the 4F4 antibody used to characterize the p116 polypeptide cross-reacted with Cbl protein when it was resolved on one- or two-dimensional gels. CrkL was constitutively associated with a substantial amount of the guanine nucleotide exchange protein C3G, and a fraction of the C3G protein was coimmunoprecipitated with Cbl in activated Jurkat T cells. These results suggest the possibility that Cbl may participate in a signaling pathway that regulates guanine nucleotide exchange on small G-proteins in T cells.
Reedquist
K A
KA
Lymphocyte Biology Section, Division of Rheumatology and Immunology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Fukazawa
T
T
Panchamoorthy
G
G
Langdon
W Y
WY
Shoelson
S E
SE
Druker
B J
BJ
Band
H
H
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D050719:Crk-Associated Substrate Protein; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D016350:Leucine Zippers; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl; D011948:Receptors, Antigen, T-Cell / Q000502:physiology*; D011994:Recombinant Proteins; D050717:Retinoblastoma-Like Protein p130; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000378:metabolism; D014407:Tumor Cells, Cultured; D044767:Ubiquitin-Protein Ligases; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; C493841:BCAR1 protein, human; C082869:CRKL protein; D050719:Crk-Associated Substrate Protein; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D011948:Receptors, Antigen, T-Cell; D011994:Recombinant Proteins; D050717:Retinoblastoma-Like Protein p130; D019000:Phosphotyrosine; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; C067419:CBL protein, human