Department of Biochemistry, Iowa State University, Ames, IA 50011, USA.
J Immunol. 2010 Apr 15;184(8):4228-35. doi: 10.4049/jimmunol.0901908. Epub 2010 Mar 17.
The Tec family tyrosine kinase (Itk), is a key component of the TCR signaling pathway. Biochemical studies have shown that Itk activation requires recruitment of Itk to the membrane via its pleckstrin homology domain, phosphorylation of Itk by the Src kinase, Lck, and binding of Itk to the SLP-76/LAT adapter complex. However, the regulation of Itk enzymatic activity by Itk domain interactions is not yet well understood. In this study, we show that full-length Itk self-associates in an intermolecular fashion. Using this information, we have designed an Itk variant that exhibits reduced self-association but maintains normal binding to exogenous ligands via each of its regulatory domains. When expressed in insect cells, the Itk substrate phospholipase Cgamma1 is phosphorylated more efficiently by the Itk variant than by wild-type Itk. Furthermore, expression of the Itk variant in primary murine T cells induced higher ERK activation and increased calcium flux following TCR stimulation compared with that of wild-type Itk. Our results indicate that the Tec kinase Itk is negatively regulated by intermolecular clustering and that disruption of this clustering leads to increased Itk kinase activity following TCR stimulation.
Tec 家族酪氨酸激酶（Itk）是 TCR 信号通路的关键组成部分。生化研究表明，Itk 的激活需要通过其 pleckstrin 同源结构域将 Itk 募集到膜上，Src 激酶 Lck 对 Itk 的磷酸化，以及 Itk 与 SLP-76/LAT 衔接子复合物的结合。然而，Itk 结构域相互作用对 Itk 酶活性的调节尚不清楚。在本研究中，我们表明全长 Itk 以分子间方式自我缔合。利用这一信息，我们设计了一种 Itk 变体，它的自我缔合减少，但通过其每个调节结构域仍能正常结合外源性配体。当在昆虫细胞中表达时，Itk 底物磷脂酶 Cgamma1 被 Itk 变体磷酸化的效率比野生型 Itk 更高。此外，与野生型 Itk 相比，Itk 变体在原代小鼠 T 细胞中的表达在 TCR 刺激后诱导更高的 ERK 激活和增加的钙流。我们的结果表明 Tec 激酶 Itk 受分子间聚集的负调节，并且这种聚集的破坏导致 TCR 刺激后 Itk 激酶活性增加。
