Identification of Endogenous Kinase Substrates by Proximity Labeling Combined with Kinase Perturbation and Phosphorylation Motifs.
Identification of Endogenous Kinase Substrates by Proximity Labeling Combined with Kinase Perturbation and Phosphorylation Motifs.
复制标题
内源性激酶底物的接近标记结合激酶扰动和磷酸化基序鉴定。
DOI：
10.1016/j.mcpro.2021.100119
复制
发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Ishihama Y
中科院分区：
文献类型：
--
作者：
Niinae T;Imami K;Sugiyama N;Ishihama Y
基于质谱的磷酸化蛋白质组学可以在一次实验中识别 10,000 多个磷酸化位点。但是，尽管公共存储库中已经积累了大量的磷酸位点信息，但蛋白激酶与底物的关系在很大程度上仍然未知。在这里，我们描述了一种通过结合使用邻近依赖性生物素识别方法（称为 BioID）和其他两种独立方法（激酶扰动的磷酸蛋白质组学和磷酸化基序匹配）来识别激酶的内源底物的方法。为了证明概念，该方法应用于酪蛋白激酶 2 (CK2) 和蛋白激酶 A (PKA)，我们分别鉴定了 24 种和 35 种推定底物。我们还表明，底物磷酸位点附近已知的与癌症相关的错义突变会影响 CK2 或 PKA 的磷酸化，因此可能会改变携带这些突变的癌细胞中的下游信号传导。这种方法通过提供大规模鉴定激酶内源底物的新方法，扩展了我们探测生理激酶底物网络的能力。通过 BioID 鉴定新型激酶相互作用因子。应用两个正交过滤器、激酶扰动和磷酸化基序。新型 CK2 和 PKA 底物的鉴定。鉴定所有激酶内源底物的通用方法。通过使用 BioID 邻近标记与其他两种独立方法（激酶扰动的磷酸蛋白质组学和磷酸化基序匹配）的组合，描述了一种鉴定激酶内源底物的方法。该方法分别鉴定了 24 种和 35 种推定的 CK2 和 PKA 底物。
Mass-spectrometry-based phosphoproteomics can identify more than 10,000 phosphorylated sites in a single experiment. But, despite the fact that enormous phosphosite information has been accumulated in public repositories, protein kinase–substrate relationships remain largely unknown. Here, we describe a method to identify endogenous substrates of kinases by using a combination of a proximity-dependent biotin identification method, called BioID, with two other independent methods, kinase-perturbed phosphoproteomics and phosphorylation motif matching. For proof of concept, this approach was applied to casein kinase 2 (CK2) and protein kinase A (PKA), and we identified 24 and 35 putative substrates, respectively. We also show that known cancer-associated missense mutations near phosphosites of substrates affect phosphorylation by CK2 or PKA and thus might alter downstream signaling in cancer cells bearing these mutations. This approach extends our ability to probe physiological kinase–substrate networks by providing new methodology for large-scale identification of endogenous substrates of kinases. Identification of novel kinase interactors by BioID. Applying two orthogonal filters, kinase perturbation and phosphorylation motif. Identification of novel CK2 and PKA substrates. A universal method for the identification of endogenous substrates for all kinases. A method to identify endogenous substrates of kinases was described by using a combination of a BioID proximity labeling, with two other independent methods, kinase-perturbed phosphoproteomics, and phosphorylation motif matching. This approach identified 24 and 35 putative CK2 and PKA substrates, respectively.
登录
查看更多内容
影响因子：
7.3
作者：
Gao J;Aksoy BA;Dogrusoz U;Dresdner G;Gross B;Sumer SO;Sun Y;Jacobsen A;Sinha R;Larsson E;Cerami E;Sander C;Schultz N
通讯作者：
Schultz N
DOI：
10.3390/ph10010018
发表时间：
2017-01-28
期刊：
Pharmaceuticals (Basel, Switzerland)
影响因子：
--
作者：
Chua MM;Ortega CE;Sheikh A;Lee M;Abdul-Rassoul H;Hartshorn KL;Dominguez I
通讯作者：
Dominguez I
影响因子：
14.9
作者：
Dinkel H;Chica C;Via A;Gould CM;Jensen LJ;Gibson TJ;Diella F
通讯作者：
Diella F
影响因子：
14.9
作者：
Hornbeck PV;Zhang B;Murray B;Kornhauser JM;Latham V;Skrzypek E
通讯作者：
Skrzypek E
影响因子：
7.3
作者：
Bodenmiller B;Wanka S;Kraft C;Urban J;Campbell D;Pedrioli PG;Gerrits B;Picotti P;Lam H;Vitek O;Brusniak MY;Roschitzki B;Zhang C;Shokat KM;Schlapbach R;Colman-Lerner A;Nolan GP;Nesvizhskii AI;Peter M;Loewith R;von Mering C;Aebersold R
通讯作者：
Aebersold R