Photo-crosslinking of human protein kinase regulatory subunit CK2β for the identification of CK2 binding partners

A. Nickelsen, J. Jose
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引用次数: 0

Abstract

Human protein kinase CK2 is a heterotetrameric Ser/Thr kinase, consisting of two catalytic (CK2α/α’) and two regulatory (CK2β) subunits. CK2 plays a key role in several physiological and pathological processes. Moreover in cancer cells it was shown that CK2 is upregulated [1]. Although the number of more than 300 substrates is still increasing, the regulation of CK2 remains unclear [2]. It is assumed that several proteinprotein interactions are involved in the regulation of CK2. Thereby CK2β modulates the substrate specificity of CK2 and also functions as a docking platform for regulators and substrates. This study aims for the identification of binding partners by photo-crosslinking coupled with mass spectrometry. Therefore the unnatural amino acid p-azidophenylalanine (pAzF) is incorporated into CK2β [3]. Here we report the establishment of the photo-crosslinking procedure with purified CK2β-pAzF with its strongest binding partner CK2α as a proof of principle. The photo-crosslinking product of CK2β-pAzF and CK2α was detected by SDS-PAGE analysis and immunostaining. Furthermore it was shown, that the photocrosslink reaction is specific for interaction partners and is not affected by other proteins. The site directed photo-crosslinking reaction was compared to the common used homo-bifunctional NHS-ester disuccinimidyl suberate (DSS) that crosslinks primary amino groups. References: [1] Tawfic, S. et al.: Histol Histopathol. 2001, 16:573-582. [2] Meggio, F.and Pinna, L.A.: FASEB J. 2003, 17:349-368. [3] Chin, J.W. et al.: J. Am. Chem. Soc. 2002, 124, 9026-9027.
人蛋白激酶调控亚基CK2β的光交联鉴定CK2结合伙伴
人蛋白激酶CK2是一种异四聚体丝氨酸/苏氨酸激酶,由两个催化亚基(CK2α/α′)和两个调节亚基(CK2β)组成。CK2在许多生理和病理过程中起关键作用。此外,在癌细胞中,CK2表达上调[1]。虽然超过300种底物的数量仍在增加,但CK2的调控机制尚不清楚[2]。据推测,多种蛋白相互作用参与了CK2的调控。因此,CK2β调节CK2的底物特异性,并作为调节因子和底物的对接平台。本研究旨在利用光交联-质谱联用技术鉴定结合伙伴。因此,非天然氨基酸对叠氮苯丙氨酸(pAzF)被掺入CK2β中[3]。在这里,我们报道了用纯化的CK2β-pAzF与其最强的结合伙伴CK2α建立光交联程序作为原理证明。通过SDS-PAGE分析和免疫染色检测CK2β-pAzF和CK2α的光交联产物。此外,光交联反应对相互作用伙伴具有特异性,不受其他蛋白质的影响。将该反应与常用的双官能团苯磺酸二琥珀酰亚酸酯(DSS)进行了比较。[1]王晓明,王晓明,等。中国生物医学工程学报,2003,16(4):573-582。[2]张建军,张建军,张建军,等。中国生物医学工程学报,2003,17(5):369 - 369。[3]陈建伟等。化学。社会科学学报,2002,24,9026-9027。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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