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Regulation of CaMKII by phospho-Thr253 or phospho-Thr286 sensitive targeting alters cellular function

机译:通过磷酸化Thr253或磷酸化Thr286敏感靶向调控CaMKII改变细胞功能

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摘要

Calcium/calmodulin-stimulated protein kinase II (CaMKII) is an important mediator of synaptic function that is regulated by multi-site phosphorylation and targeting through interactions with proteins. A new phosphorylation site at Thr253 has been identified in vivo, that does not alter CaMKII activity, but does alter CaMKII function through interactions with binding proteins. To identify these proteins, as well as to examine the specific effects following Thr253 or Thr286 phosphorylation on these interactions, we developed an in vitro overlay binding assay. We demonstrated that the interaction between CaMKII and its binding proteins was altered by the phosphorylation state of both the CaMKII and the partner, and identified a CaMKII-specific sequence that was responsible for the interaction between CaMKII and two interacting proteins. By comparing CaMKII binding profiles in tissue and cell extracts, we demonstrated that the CaMKII binding profiles varied with cell type, and also showed that overexpression of a CaMKII Thr253 phospho-mimic mutant in human neuroblastoma and breast cancer cells dramatically altered the morphology and growth rates when compared to overexpression of non-phosphorylated CaMKII. This data highlights the importance of the microenvironment in regulating CaMKII function, and describes a potentially new mechanism by which the functions of CaMKII can be regulated.
机译:钙/钙调蛋白刺激的蛋白激酶II(CaMKII)是突触功能的重要介体,其通过多位磷酸化和与蛋白质的相互作用来靶向来调节。在体内已鉴定出Thr253处一个新的磷酸化位点,该位点不会改变CaMKII的活性,但会通过与结合蛋白的相互作用来改变CaMKII的功能。为了鉴定这些蛋白质,以及检查Thr253或Thr286磷酸化后对这些相互作用的特异性作用,我们开发了一种体外覆盖结合测定法。我们证明了CaMKII和其结合蛋白之间的磷酸化状态改变了CaMKII及其结合蛋白之间的相互作用,并确定了负责CaMKII与两个相互作用蛋白之间相互作用的CaMKII特异性序列。通过比较组织和细胞提取物中的CaMKII结合图谱,我们证明了CaMKII结合图谱随细胞类型而变化,并且还表明了CaMKII Thr253磷酸模拟突变体在人神经母细胞瘤和乳腺癌细胞中的过表达显着改变了形态和生长速率与非磷酸化CaMKII的过表达相比。该数据突出了微环境在调节CaMKII功能中的重要性,并描述了潜在的新机制,可通过该机制调节CaMKII的功能。

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