Combining docking site and phosphosite predictions to find new substrates: Identification of smoothelin-like-2 (SMTNL2) as a c-Jun N-terminal kinase (JNK) substrate
Combining docking site and phosphosite predictions to find new substrates: Identification of smoothelin-like-2 (SMTNL2) as a c-Jun N-terminal kinase (JNK) substrate
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DOI：
10.1016/j.cellsig.2013.08.004
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发表时间：
2013-12-01
影响因子：
4.8
通讯作者：
Bardwell, Lee
中科院分区：
文献类型：
--
作者：
Gordon, Elizabeth A.;Whisenant, Thomas C.;Bardwell, Lee
有丝分裂原活化蛋白激酶（MAPK），其调节剂和其下游底物之间的特异性对接相互作用对于有效和准确的信号传递至关重要。为了鉴定MAPK的c-Jun N-末端激酶（JNK）家族的新底物，我们在人类基因组中搜索含有（1）预测的JNK对接位点（D-位点）和（2）位于D-位点附近的一簇推定的JNK靶磷酸化位点的蛋白质。在这里，我们描述了一种新的JNK底物，出现从这个分析，功能不明的蛋白质smoothelin样2（SMTNL 2）。SMTNL 2蛋白与包括JNK 1 -3和ERK 2在内的多个MAPK高亲和力结合;此外，预测的对接位点（残基180-193）中保守氨基酸的同一性对于这种高亲和力结合是必需的。此外，纯化的全长SMTNL 2蛋白在体外被JNK 1 -3磷酸化，这需要D-位点的完整性。使用质谱和诱变，我们确定了四个D-位点依赖性的磷酸受体位点在紧密接近的对接位点，在S217，S241，T236和T239。由SMTNL 2 D位点组成的短肽抑制JNK介导的ATF 2转录因子磷酸化，表明SMTNL 2可以与其他底物竞争JNK结合。此外，当转染到HEK 293细胞中时，SMTNL 2在体外鉴定的相同残基上以D位点依赖性方式被内源性JNK磷酸化。SMTNL 2蛋白在多种哺乳动物组织中表达，在骨骼肌中的表达量显著较高。与SMTNL 2在骨骼肌中具有功能的假设一致，SMTNL 2蛋白表达在分化的C2 C12细胞中从成肌细胞向肌管的转变期间被强烈诱导。(C)2013 Elsevier Inc. All rights reserved.
Specific docking interactions between mitogen-activated protein kinases (MAPKs), their regulators, and their downstream substrates, are crucial for efficient and accurate signal transmission. To identify novel substrates of the c-Jun N-terminal kinase (JNK) family of MAPKs, we searched the human genome for proteins that contained (1), a predicted JNK-docking site (D-site): and (2), a cluster of putative JNK target phosphosites located close to the D-site. Here we describe a novel JNK substrate that emerged from this analysis, the functionally uncharacterized protein smoothelin-like 2 (SMTNL2). SMTNL2 protein bound with high-affinity to multiple MAPKs including JNK1-3 and ERK2; furthermore, the identity of conserved amino acids in the predicted docking site ( residues 180-193) was necessary for this high-affinity binding. In addition, purified full-length SMTNL2 protein was phosphorylated by JNK1-3 in vitro, and this required the integrity of the D-site. Using mass spectrometry and mutagenesis, we identified four D-site-dependent phosphoacceptor sites in close proximity to the docking site, at S217, S241, T236 and T239. A short peptide comprised of the SMTNL2 D-site inhibited JNK-mediated phosphorylation of the ATF2 transcription factor, showing that SMTNL2 can compete with other substrates for JNK binding. Moreover, when transfected into HEK293 cells, SMTNL2 was phosphorylated by endogenous JNK in a D-site dependent manner, on the same residues identified in vitro. SMTNL2 protein was expressed in many mammalian tissues, with a notably high expression in skeletal muscle. Consistent with the hypothesis that SMTNL2 has a function in skeletal muscle, SMTNL2 protein expression was strongly induced during the transition from myoblasts to myotubes in differentiating C2C12 cells. (C) 2013 Elsevier Inc. All rights reserved.