Tyrosine 656 in topoisomerase IIβ is important for the catalytic activity of the enzyme: Identification based on artifactual +80-Da modification at this site.
Tyrosine 656 in topoisomerase IIβ is important for the catalytic activity of the enzyme: Identification based on artifactual +80-Da modification at this site.
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DOI：
10.1002/pmic.201000194
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发表时间：
2011-03
期刊：
影响因子：
3.4
通讯作者：
Ganapathi, Mahrukh K.
中科院分区：
文献类型：
--
作者：
Grozav, Adrian G.;Willard, Belinda B.;Kozuki, Toshiyuki;Chikamori, Kenichi;Micluta, Marius A.;Petrescu, Andrei-Jose;Kinter, Michael;Ganapathi, Ram;Ganapathi, Mahrukh K.
拓扑异构酶II催化DNA中的拓扑变化。尽管人类同工酶，topo IIα和β都是磷酸化的，但topo IIβ的位点特异性磷酸化特征很差。用胰蛋白酶、精氨酸C或溴化氰加胰蛋白酶切割topo IIβ，用LC-MS/MS分析，我们检测到4个+80-Da修饰位点：tyr 656、ser 1395、thr 1426和ser 1545。基于CID光谱中H3 PO 4（−98 Da）的中性丢失以及32 P标记的野生型（WT）和S1395 A或T1426 A/S1545 A突变体topo IIβ的2-D-磷酸肽图谱的差异，确定了ser 1395、thr 1426和ser 1545处的磷酸化。然而，磷酸化在tyr 656不能验证2-D-磷酸肽映射的32 P标记的WT和Y 656 F突变体蛋白或蛋白质印迹与磷酸酪氨酸特异性抗体。由于仅在用CNBr和胰蛋白酶切割期间观察到tyr 656上的+80-Da修饰，因此该修饰可能代表溴化，其发生在CNBr切割期间。CID光谱的重新评价在含有tyr 656和tyr 711的两种肽的CID光谱中鉴定了+78/+80-Da碎片离子，证实了溴化。有趣的是，只有tyr 656的突变，而不是ser 1395，thr 1326或ser 1545，降低topo IIβ活性，表明tyr 656的功能作用。这些结果，同时确定了一个重要的酪氨酸拓扑IIβ，强调了重要的是要仔细解释的修饰具有相同的标称质量。
Topoisomerase (topo) II catalyzes topological changes in DNA. Although both human isozymes, topo IIα and β are phosphorylated, site-specific phosphorylation of topo IIβ is poorly characterized. Using LC-MS/MS analysis of topo IIβ, cleaved with trypsin, Arg C or cyanogen bromide (CNBr) plus trypsin, we detected four +80-Da modified sites: tyr656, ser1395, thr1426 and ser1545. Phosphorylation at ser1395, thr1426 and ser1545 was established based on neutral loss of H3PO4 (−98 Da) in the CID spectra and on differences in 2-D-phosphopeptide maps of 32P-labeled wild-type (WT) and S1395A or T1426A/S1545A mutant topo IIβ. However, phosphorylation at tyr656 could not be verified by 2-D-phosphopeptide mapping of 32P-labeled WT and Y656F mutant protein or by Western blotting with phosphotyrosine-specific antibodies. Since the +80-Da modification on tyr656 was observed exclusively during cleavage with CNBr and trypsin, this modification likely represented bromination, which occurred during CNBr cleavage. Re-evaluation of the CID spectra identified +78/+80-Da fragment ions in CID spectra of two peptides containing tyr656 and tyr711, confirming bromination. Interestingly, mutation of only tyr656, but not ser1395, thr1326 or ser1545, decreased topo IIβ activity, suggesting a functional role for tyr656. These results, while identifying an important tyrosine in topo IIβ, underscore the importance of careful interpretation of modifications having the same nominal mass.
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拓扑异构酶iialpha和IIBETA的C末端区域确定体内酶的同工型特异性功能。
影响因子：
14.9
作者：
Linka, Rene M;Porter, Andrew C G;Volkov, Arsen;Mielke, Christian;Boege, Fritz;Christensen, Morten O
通讯作者：
Christensen, Morten O
影响因子：
3.8
作者：
Lee, Ming-Ta;Bachant, Jeff
通讯作者：
Bachant, Jeff
影响因子：
11.4
作者：
Chikamori, K.;Hill, J. E.;Ganapathi, R.
通讯作者：
Ganapathi, R.
影响因子：
4.8
作者：
Mao, Y;Desai, SD;Liu, LF
通讯作者：
Liu, LF
DOI：
10.1016/0167-4781(92)90050-a
发表时间：
1992-08-17
期刊：
BIOCHIMICA ET BIOPHYSICA ACTA
影响因子：
--
作者：
CAPRANICO, G;TINELLI, S;ZUNINO, F
通讯作者：
ZUNINO, F