Rapid T-cell receptor-mediated tyrosine phosphorylation of p120, an Fyn/Lck Src homology 3 domain-binding protein.
Tyrosine phosphorylation of cellular proteins is the earliest identifiable event following T-cell antigen receptor (TCR) stimulation and is essential for activating downstream signaling machinery. Two Src-family protein-tyrosine kinases, the TCR-associated p59fyn (Fyn) and the CD4/8-associated p56lck (Lck), have emerged as the likely mediators of early tyrosine phosphorylation in T cells. Here, we show direct binding of a 120-kDa TCR-induced phosphotyrosyl polypeptide, p120, to glutathione S-transferase fusion proteins of the Src homology 3 (SH3) domains of Fyn, Lck, and p60src (Src) but not other proteins. While binding of p120 to Fyn SH2 domain was phosphotyrosine-dependent as expected, its binding to the SH3 domain was independent of tyrosine phosphorylation, as shown by lack of competition with a phosphotyrosyl competitor peptide. In contrast, an SH3-specific proline-rich peptide completely abolished p120 binding to SH3. p120 was tyrosine-phosphorylated within 10 sec following stimulation of Jurkat cells with anti-CD3 monoclonal antibody, with maximal phosphorylation at 30 sec. Importantly, p120 was found associated with Fyn and Lck proteins in unstimulated Jurkat cells and served as an in vitro substrate for these kinases. These results provide evidence for a role of the SH3 domains of Fyn and Lck in the recruitment of early tyrosine-phosphorylation substrates to the TCR-associated tyrosine kinases.
Reedquist
K A
KA
Department of Rheumatology and Immunology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115.
Fukazawa
T
T
Druker
B
B
Panchamoorthy
G
G
Shoelson
S E
SE
Band
H
H
D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000096:biosynthesis / Q000378:metabolism*; D002460:Cell Line; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D015458:Leukemia, T-Cell; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008969:Molecular Sequence Data; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism
D002352:Carrier Proteins; D011518:Proto-Oncogene Proteins; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; C494957:FYN protein, human; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051076:Proto-Oncogene Proteins c-fyn