Reedquist K A, Fukazawa T, Druker B, Panchamoorthy G, Shoelson S E, Band H
Department of Rheumatology and Immunology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115.
Proc Natl Acad Sci U S A. 1994 May 10;91(10):4135-9. doi: 10.1073/pnas.91.10.4135.
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.
细胞蛋白的酪氨酸磷酸化是T细胞抗原受体（TCR）刺激后最早可识别的事件，对于激活下游信号传导机制至关重要。两种Src家族蛋白酪氨酸激酶，即TCR相关的p59fyn（Fyn）和CD4/8相关的p56lck（Lck），已成为T细胞早期酪氨酸磷酸化的可能介质。在此，我们展示了一种120 kDa的TCR诱导的磷酸酪氨酸多肽p120与Fyn、Lck和p60src（Src）的Src同源3（SH3）结构域的谷胱甘肽S-转移酶融合蛋白直接结合，但不与其他蛋白结合。正如预期的那样，p120与Fyn SH2结构域的结合依赖于磷酸酪氨酸，但其与SH3结构域的结合不依赖于酪氨酸磷酸化，这通过与磷酸酪氨酸竞争肽缺乏竞争得以证明。相反，一种SH3特异性富含脯氨酸的肽完全消除了p120与SH3的结合。在用抗CD3单克隆抗体刺激Jurkat细胞后10秒内，p120被酪氨酸磷酸化，在30秒时达到最大磷酸化。重要的是，在未刺激的Jurkat细胞中发现p120与Fyn和Lck蛋白相关，并作为这些激酶的体外底物。这些结果为Fyn和Lck的SH3结构域在将早期酪氨酸磷酸化底物募集到TCR相关酪氨酸激酶中所起的作用提供了证据。
