Tyrosines in the Carboxyl Terminus Regulate Syk Kinase Activity and Function
Tyrosines in the Carboxyl Terminus Regulate Syk Kinase Activity and Function
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DOI：
10.1074/jbc.m110.134262
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发表时间：
2010-08-20
影响因子：
4.8
通讯作者：
Siraganian, Reuben P.
中科院分区：
文献类型：
--
作者：
de Castro, Rodrigo O.;Zhang, Juan;Siraganian, Reuben P.
Syk酪氨酸激酶家族在基于免疫受体酪氨酸的激活基序（ITAM）信号传导中起重要作用。Syk与免疫受体的酪氨酸磷酸化ITAM亚基（如肥大细胞上的Fc ε RI）的结合导致构象变化，并增加Syk的酶活性。这种构象变化暴露了Syk的COOH末端尾，其具有三个保守的Tyr残基（大鼠Syk的Tyr-623、Tyr-624和Tyr-625）。为了理解这些残基在信号传导中的作用，在Syk缺陷型肥大细胞中表达具有这三个Tyr突变为Phe的野生型和突变体Syk。突变的Syk降低了Fc γ RI诱导的脱粒、T细胞活化的核因子和NF κ B活化，同时降低了MAP激酶p38和p42/44 ERK的磷酸化。在非刺激的细胞中，突变的Syk主要由于自磷酸化而被更多的酪氨酸磷酸化。在体外，由于这种增加的磷酸化，突变的Syk与磷酸化的ITAM的结合减少。来自非刺激细胞的这种突变的Syk对外源底物的激酶活性显著降低，而其自磷酸化能力不受影响。然而，当蛋白质在体外激酶反应之前被去磷酸化时，这种突变的Syk的激酶活性和自磷酸化能力显著降低。此外，Syk的COOH末端区域中的这些酪氨酸的突变将其转化为类似于其同源物ZAP-70的酶，其依赖于其他酪氨酸激酶以获得最佳活化。在测试Syk在每个酪氨酸处单独突变时，Tyr-624但尤其是Tyr-625在这些反应中具有主要作用。因此，这些结果表明，在尾部区域的这些酪氨酸在调节Syk的激酶活性和功能中起关键作用。
The Syk tyrosine kinase family plays an essential role in immunoreceptor tyrosine-based activation motif (ITAM) signaling. The binding of Syk to tyrosine-phosphorylated ITAM subunits of immunoreceptors, such as Fc epsilon RI on mast cells, results in a conformational change, with an increase of enzymatic activity of Syk. This conformational change exposes the COOH-terminal tail of Syk, which has three conserved Tyr residues (Tyr-623, Tyr-624, and Tyr-625 of rat Syk). To understand the role of these residues in signaling, wild-type and mutant Syk with these three Tyr mutated to Phe was expressed in Syk-deficient mast cells. There was decreased Fc epsilon RI-induced degranulation, nuclear factor for T cell activation and NF kappa B activation with the mutated Syk together with reduced phosphorylation of MAP kinases p38 and p42/44 ERK. In non-stimulated cells, the mutated Syk was more tyrosine phosphorylated predominantly as a result of autophosphorylation. In vitro, there was reduced binding of mutated Syk to phosphorylated ITAM due to this increased phosphorylation. This mutated Syk from non-stimulated cells had significantly reduced kinase activity toward an exogenous substrate, whereas its autophosphorylation capacity was not affected. However, the kinase activity and the autophosphorylation capacity of this mutated Syk were dramatically decreased when the protein was dephosphorylated before the in vitro kinase reaction. Furthermore, mutation of these tyrosines in the COOH-terminal region of Syk transforms it to an enzyme, similar to its homolog ZAP-70, which depends on other tyrosine kinases for optimal activation. In testing Syk mutated singly at each one of the tyrosines, Tyr-624 but especially Tyr-625 had the major role in these reactions. Therefore, these results indicate that these tyrosines in the tail region play a critical role in regulating the kinase activity and function of Syk.