Src binds cortactin through an SH2 domain cystine-mediated linkage
Src binds cortactin through an SH2 domain cystine-mediated linkage
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DOI：
10.1242/jcs.121046
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发表时间：
2012-12-15
影响因子：
4
通讯作者：
Weed, Scott A.
中科院分区：
文献类型：
--
作者：
Evans, Jason V.;Ammer, Amanda G.;Weed, Scott A.
模块蛋白结构域介导的基于酪氨酸激酶的信号转导对细胞功能至关重要。Src同源(SH)2结构域是细胞内信号传导的重要导体，它与受体蛋白上的磷酸化酪氨酸结合，产生负责信号传递的分子复合体。Cortactin是一种细胞骨架蛋白和酪氨酸激酶底物，通过与含有SH2结构域的蛋白相互作用来调节基于肌动蛋白的运动。Src激酶SH2结构域参与调节皮质蛋白结合和酪氨酸磷酸化，但其与皮质蛋白的相互作用机制尚不清楚。在这里，我们证明了Src通过磷酸酪氨酸结合口袋中SH2结构域的Src C185和Cortactin重复序列结构域的Cortactin C112/246之间的半胱氨酸键与Cortactin结合，而不依赖于酪氨酸磷酸化。相互作用研究表明，还原剂的存在消除了Src-Cortactin的结合，消除了Src对Cortactin的磷酸化，并阻止了Src SH2结构域与Cortactin的结合。串联MS/MS测序表明，Src C185和Cortactin C112/246之间形成了半胱氨酸键。突变研究表明，一个完整的半胱氨酸结合界面对于Src介导的皮质动蛋白磷酸化、细胞迁移和侵袭前的形成是必需的。我们的结果确定了一种新的不依赖磷酸酪氨酸的Src SH2结构域与Cortactin的结合模式。除Src外，四分之一的SH2结构域在类似的Src C185位置或附近含有半胱氨酸。这为含半胱氨酸的SH2结构域提供了一种潜在的替代酪氨酸磷酸化的机制来结合同源配体，这种配体可能广泛存在于调节不同细胞功能的信号传播中。
Tyrosine-kinase-based signal transduction mediated by modular protein domains is critical for cellular function. The Src homology (SH)2 domain is an important conductor of intracellular signaling that binds to phosphorylated tyrosines on acceptor proteins, producing molecular complexes responsible for signal relay. Cortactin is a cytoskeletal protein and tyrosine kinase substrate that regulates actin-based motility through interactions with SH2-domain-containing proteins. The Src kinase SH2 domain mediates cortactin binding and tyrosine phosphorylation, but how Src interacts with cortactin is unknown. Here we demonstrate that Src binds cortactin through cystine bonding between Src C185 in the SH2 domain within the phosphotyrosine binding pocket and cortactin C112/246 in the cortactin repeats domain, independent of tyrosine phosphorylation. Interaction studies show that the presence of reducing agents ablates Src-cortactin binding, eliminates cortactin phosphorylation by Src, and prevents Src SH2 domain binding to cortactin. Tandem MS/MS sequencing demonstrates cystine bond formation between Src C185 and cortactin C112/246. Mutational studies indicate that an intact cystine binding interface is required for Src-mediated cortactin phosphorylation, cell migration, and pre-invadopodia formation. Our results identify a novel phosphotyrosine-independent binding mode between the Src SH2 domain and cortactin. Besides Src, one quarter of all SH2 domains contain cysteines at or near the analogous Src C185 position. This provides a potential alternative mechanism to tyrosine phosphorylation for cysteine-containing SH2 domains to bind cognate ligands that may be widespread in propagating signals regulating diverse cellular functions.