Phosphoproteomics of collagen receptor networks reveals SHP-2 phosphorylation downstream of wild-type DDR2 and its lung cancer mutants.
Phosphoproteomics of collagen receptor networks reveals SHP-2 phosphorylation downstream of wild-type DDR2 and its lung cancer mutants.
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DOI：
10.1042/bj20121750
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发表时间：
2013-09-15
期刊：
影响因子：
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通讯作者：
Huang PH
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作者：
Iwai LK;Payne LS;Luczynski MT;Chang F;Xu H;Clinton RW;Paul A;Esposito EA;Gridley S;Leitinger B;Naegle KM;Huang PH
胶原蛋白是一种重要的细胞外基质成分，指导许多基本的细胞过程，包括分化、增殖和运动。驱动这些过程的信号网络由胶原蛋白受体（例如 β1 整联蛋白和 DDR（盘状蛋白结构域受体））传播。为了深入了解胶原蛋白受体信号传导的分子机制，我们对整合素和 DDR2 的胶原蛋白激活下游的磷酸化网络进行了定量分析。七个时间点的时间分析鉴定出 424 个磷酸化蛋白质。不同的 DDR2 酪氨酸磷酸化位点显示出独特的时间激活曲线，与体外激酶数据一致。磷酸化蛋白质组数据的多重聚类分析揭示了几个 DDR2 候选下游信号转导节点，包括 SHP-2（含 Src 同源 2 结构域的蛋白酪氨酸磷酸酶 2）、NCK1（酪氨酸激酶接头蛋白 1 的非催化区域）、LYN、SHIP-2 [SH2（Src 同源 2）结构域含肌醇磷酸酶 2]、PIK3C2A （磷脂酰肌醇-4-磷酸 3-激酶，催化亚基类型 2α）和 PLCL2（磷脂酶 C 样 2）。生化验证表明 SHP-2 酪氨酸磷酸化依赖于 DDR2 激酶活性。对一组肺 SCC（鳞状细胞癌）DDR2 突变体进行的靶向蛋白质组分析表明，SHP-2 被 L63V 和 G505S 突变体酪氨酸磷酸化。相比之下，I638F 激酶结构域突变体表现出 DDR2 和 SHP-2 酪氨酸磷酸化水平降低，这与克隆形成潜力呈反比关系。总而言之，本研究的结果表明，SHP-2 是 DDR2 受体下游的关键信号传导节点，这可能对最近在全基因组肺 SCC 测序筛选中发现的 DDR2 突变子集具有治疗意义。本研究表征了胶原蛋白激活的整合素和 DDR2 信号网络。使用聚类方法，识别出 DDR2 特定的信号组件，例如 SHP-2。我们进一步证明 SHP-2 被 DDR2 肺癌突变体的一个子集磷酸化。
Collagen is an important extracellular matrix component that directs many fundamental cellular processes including differentiation, proliferation and motility. The signalling networks driving these processes are propagated by collagen receptors such as the β1 integrins and the DDRs (discoidin domain receptors). To gain an insight into the molecular mechanisms of collagen receptor signalling, we have performed a quantitative analysis of the phosphorylation networks downstream of collagen activation of integrins and DDR2. Temporal analysis over seven time points identified 424 phosphorylated proteins. Distinct DDR2 tyrosine phosphorylation sites displayed unique temporal activation profiles in agreement with in vitro kinase data. Multiple clustering analysis of the phosphoproteomic data revealed several DDR2 candidate downstream signalling nodes, including SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2), NCK1 (non-catalytic region of tyrosine kinase adaptor protein 1), LYN, SHIP-2 [SH2 (Src homology 2)-domain-containing inositol phosphatase 2], PIK3C2A (phosphatidylinositol-4-phosphate 3-kinase, catalytic subunit type 2α) and PLCL2 (phospholipase C-like 2). Biochemical validation showed that SHP-2 tyrosine phosphorylation is dependent on DDR2 kinase activity. Targeted proteomic profiling of a panel of lung SCC (squamous cell carcinoma) DDR2 mutants demonstrated that SHP-2 is tyrosine-phosphorylated by the L63V and G505S mutants. In contrast, the I638F kinase domain mutant exhibited diminished DDR2 and SHP-2 tyrosine phosphorylation levels which have an inverse relationship with clonogenic potential. Taken together, the results of the present study indicate that SHP-2 is a key signalling node downstream of the DDR2 receptor which may have therapeutic implications in a subset of DDR2 mutations recently uncovered in genome-wide lung SCC sequencing screens. The present study characterizes integrin and DDR2 signalling networks activated by collagen. Using clustering approaches, DDR2-specific signalling components such as SHP-2 were identified. We further demonstrate that SHP-2 is phosphorylated by a subset of DDR2 lung cancer mutants.