van Oers N S, Tao W, Watts J D, Johnson P, Aebersold R, Teh H S
Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada.
Mol Cell Biol. 1993 Sep;13(9):5771-80. doi: 10.1128/mcb.13.9.5771-5780.1993.
The T-cell receptor (TCR) zeta subunit is an important component of the TCR complex, involved in signal transduction events following TCR engagement. In this study, we showed that the TCR zeta chain is constitutively tyrosine phosphorylated to similar extents in thymocytes and lymph node T cells. Approximately 35% of the tyrosine-phosphorylated TCR zeta (phospho zeta) precipitated from total cell lysates appeared to be surface associated. Furthermore, constitutive phosphorylation of TCR zeta in T cells occurred independently of antigen stimulation and did not require CD4 or CD8 coreceptor expression. In lymph node T cells that constitutively express tyrosine-phosphorylated TCR zeta, there was a direct correlation between surface TCR-associated protein tyrosine kinase (PTK) activity and expression of phospho zeta. TCR stimulation of these cells resulted in an increase in PTK activity that coprecipitated with the surface TCR complex and a corresponding increase in the levels of phospho zeta. TCR ligations also contributed to the detection of several additional phosphoproteins that coprecipitated with surface TCR complexes, including a 72-kDa tyrosine-phosphorylated protein. The presence of TCR-associated PTK activity also correlated with the binding of a 72-kDa protein, which became tyrosine phosphorylated in vitro kinase assays, to tyrosine phosphorylated TCR zeta. The cytoplasmic region of the TCR zeta chain was synthesized, tyrosine phosphorylated, and conjugated to Sepharose beads. Only tyrosine-phosphorylated, not nonphosphorylated, TCR zeta beads were capable of immunoprecipitating the 72-kDa protein from total cell lysates. This 72-kDa protein is likely the murine equivalent of human PTK ZAP-70, which has been shown to associate specifically with phospho zeta. These results suggest that TCR-associated PTK activity is regulated, at least in part, by the tyrosine phosphorylation status of TCR zeta.
T细胞受体（TCR）ζ亚基是TCR复合物的重要组成部分，参与TCR结合后的信号转导事件。在本研究中，我们发现TCR ζ链在胸腺细胞和淋巴结T细胞中组成性酪氨酸磷酸化程度相似。从总细胞裂解物中沉淀出的约35%的酪氨酸磷酸化TCR ζ（磷酸化ζ）似乎与表面相关。此外，T细胞中TCR ζ的组成性磷酸化独立于抗原刺激，且不需要CD4或CD8共受体表达。在组成性表达酪氨酸磷酸化TCR ζ的淋巴结T细胞中，表面TCR相关蛋白酪氨酸激酶（PTK）活性与磷酸化ζ的表达之间存在直接相关性。这些细胞的TCR刺激导致与表面TCR复合物共沉淀的PTK活性增加以及磷酸化ζ水平相应升高。TCR连接还有助于检测与表面TCR复合物共沉淀的几种额外的磷酸化蛋白，包括一种72 kDa的酪氨酸磷酸化蛋白。TCR相关PTK活性的存在也与一种72 kDa蛋白的结合相关，该蛋白在体外激酶测定中被酪氨酸磷酸化，并与酪氨酸磷酸化的TCR ζ结合。合成了TCR ζ链的胞质区域，使其酪氨酸磷酸化，并与琼脂糖珠偶联。只有酪氨酸磷酸化而非未磷酸化的TCR ζ珠能够从总细胞裂解物中免疫沉淀72 kDa蛋白。这种72 kDa蛋白可能是人类PTK ZAP - 70的小鼠等同物，已证明其与磷酸化ζ特异性结合。这些结果表明，TCR相关PTK活性至少部分受TCR ζ酪氨酸磷酸化状态的调节。
