Department of Medicine, Division of Nephrology, McGill University Health Centre, Montreal, Quebec, Canada H3A 2B4.
Biochem Biophys Res Commun. 2012 Oct 12;427(1):67-72. doi: 10.1016/j.bbrc.2012.08.148. Epub 2012 Sep 11.
The seven members of the Dok adapter protein family share a highly conserved phosphotyrosine-binding (PTB) domain. In the case of Dok-1, 2 and 3, the PTB domain binds to the lipid phosphatase Ship1, a key component of their inhibitory signaling mechanisms in immune cells. In contrast to most other Dok family members, Dok-4 is expressed widely but is poorly understood, largely because of limited knowledge of its partner molecules. We previously showed that, in contrast to the Dok-1 PTB domain (defined as aa 107-260), the homologous sequence in Dok-4 (aa 100-233) bound very poorly to Ret, a known Dok-4 partner. In the current study, we show that binding of Dok-4 to Ret requires residues C-terminal to the previously defined PTB domain boundaries (up to aa 246). These residues are predicted to form an extension in a critical C-terminal α-helix. We show that the Dok-4 PTB domain also binds the phosphorylated NPXY motifs in Ship1 but not Ship2. Finally, we found that a rare human single nucleotide polymorphism causing a R186H substitution in the PTB domain abolishes tyrosine phosphorylation of Dok-4 by Ret. In addition to providing a clearer understanding of Dok-4 PTB domain structure and function, our findings point to a potential mechanism for Dok-4 inhibitory signaling in T-cells and to the possibility of a rare Dok-4-related phenotype in humans.
Dok 衔接蛋白家族的 7 个成员拥有高度保守的磷酸酪氨酸结合（PTB）结构域。在 Dok-1、2 和 3 的情况下，PTB 结构域与脂质磷酸酶 Ship1 结合，后者是其在免疫细胞中抑制信号机制的关键组成部分。与大多数其他 Dok 家族成员不同，Dok-4 广泛表达，但知之甚少，主要是因为对其伴侣分子的了解有限。我们之前曾表明，与 Dok-1 的 PTB 结构域（定义为 aa 107-260）不同，Dok-4 的同源序列（aa 100-233）与已知的 Dok-4 伴侣 Ret 结合得很差。在当前的研究中，我们表明 Dok-4 与 Ret 的结合需要在先前定义的 PTB 结构域边界（最多至 aa 246）之后的残基。这些残基预测形成关键 C 末端α螺旋中的延伸。我们表明 Dok-4 的 PTB 结构域还结合了 Ship1 中的磷酸化 NPXY 基序，但不结合 Ship2。最后，我们发现导致 PTB 结构域 R186H 取代的罕见人类单核苷酸多态性会阻止 Ret 对 Dok-4 的酪氨酸磷酸化。除了更清楚地了解 Dok-4 PTB 结构域的结构和功能外，我们的发现还指出了 T 细胞中 Dok-4 抑制信号的潜在机制，以及人类中罕见的 Dok-4 相关表型的可能性。
