Related Experiment Videos
Rapid T-cell receptor-mediated tyrosine phosphorylation of p120, an Fyn/Lck Src homology 3 domain-binding protein
K A Reedquist1, T Fukazawa, B Druker
1Department of Rheumatology and Immunology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115.
Abstract:
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.
Insights
Early T-cell signaling involves tyrosine phosphorylation. A 120-kDa protein (p120) binds to Fyn and Lck kinases via their SH3 domains, acting as an early substrate for T-cell receptor activation.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- T-cell antigen receptor (TCR) stimulation initiates early signaling via protein tyrosine phosphorylation.
- Src-family kinases, Fyn and Lck, are key mediators of this early phosphorylation in T cells.
Purpose of the Study:
- To investigate the interaction of TCR-induced phosphotyrosyl polypeptides with Src-family kinases.
- To elucidate the role of specific kinase domains in substrate recruitment during T-cell activation.
Main Methods:
- Utilized glutathione S-transferase (GST) fusion proteins of Src homology (SH) domains (SH2 and SH3) to test protein binding.
- Employed phosphotyrosyl competitor peptides and SH3-specific proline-rich peptides to analyze binding interactions.
- Stimulated Jurkat cells with anti-CD3 antibody to assess tyrosine phosphorylation kinetics and protein association.
Main Results:
- A 120-kDa TCR-induced phosphotyrosyl polypeptide (p120) directly bound to the SH3 domains of Fyn, Lck, and Src kinases.
- p120 binding to SH3 domains was independent of tyrosine phosphorylation but competed by proline-rich peptides.
- p120 was rapidly tyrosine-phosphorylated upon TCR stimulation and associated with Fyn and Lck, serving as their in vitro substrate.
Conclusions:
- The SH3 domains of Fyn and Lck play a crucial role in recruiting early tyrosine-phosphorylation substrates like p120.
- This interaction is vital for initiating downstream signaling cascades following T-cell receptor activation.