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首页> 外文期刊>Journal of cellular biochemistry. >Identification of PSF as a protein kinase Calpha-binding protein in the cell nucleus.
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Identification of PSF as a protein kinase Calpha-binding protein in the cell nucleus.

机译:将PSF鉴定为细胞核中的蛋白激酶Calpha结合蛋白。

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摘要

Protein kinase C (PKC) isoforms are present in the cell nucleus in diverse cell lines and tissues. Since little is known about proteins interacting with PKC inside the cell nucleus, we used Neuro-2a neuroblastoma cells, in which PKCalpha is present in the nucleus, to screen for nuclear binding partners for PKC. Applying overlay assays, we detected several nuclear proteins which bind to PKCalpha. Specificity of binding was shown by its dependence on PKC activation by phorbol ester, calcium, and phosphatidylserine. The PKC-binding proteins were partially purified and analyzed by microsequencing and mass spectrometry. Four proteins could be identified: PTB-associated splicing factor (PSF), p68 RNA helicase, and the heterogeneous nuclear ribonucleoprotein (hnRNP) proteins A3 and L. In the case of PSF, binding to PKC could also be demonstrated in a GST-pull-down assay using GST-PKCalpha, expressed in insect cells. Phosphorylation experiments revealed that PSF is a weak in vitro substrate for PKCalpha.
机译:蛋白激酶C(PKC)亚型存在于各种细胞系和组织的细胞核中。由于对与细胞核内的PKC相互作用的蛋白质知之甚少,因此我们使用Neuro-2a神经母细胞瘤细胞(其中PKCalpha存在于细胞核中)筛选PKC的核结合伴侣。应用覆盖分析,我们检测到了几种与PKCalpha结合的核蛋白。结合的特异性通过佛波酯,钙和磷脂酰丝氨酸对PKC活化的依赖性来显示。 PKC结合蛋白被部分纯化,并通过微测序和质谱分析。可以鉴定出四种蛋白:PTB相关剪接因子(PSF),p68 RNA解旋酶以及异源核糖核蛋白(hnRNP)蛋白A3和L。在PSF的情况下,也可以在GST-pull中证明与PKC的结合在昆虫细胞中表达的使用GST-PKCalpha的落定量分析。磷酸化实验表明,PSF是PKCalpha的弱体外底物。

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