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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >CONFORMATION-DEPENDENT PHOSPHORYLATION OF P53
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CONFORMATION-DEPENDENT PHOSPHORYLATION OF P53

机译：P53的构象依赖性磷酸化

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Phosphorylation of the p53 tumor suppressor protein is known to modulate its functions, Using bacterially produced glutathione S-transferase (GST)-p53 fusion protein and baculovirus-expressed histidine-tagged p53 (Hi,p53), we have determined human p53 phosphorylation by purified forms of jun-N-kinase (JNK), protein kinase A (PKA), and beta subunit of casein kinase II (CKII beta) as well as by kinases present in whole cell extracts (WCEs), We demonstrate that PKA is potent p53 kinase, albeit, in a conformation- and concentration-dependent manner, as concluded by comparing full-length with truncated forms of p53, We further demonstrate JNK interaction with GST;p53 and the ability of JNK to phosphorylate truncated forms of GST-p53 or full-length (His)p53. Dependence of phosphorylation on conformation of p53 is further supported by the finding that the wild-type form of p53 (p53(wt)) undergoes better phosphorylation by CKII beta and by WCE kinases than mutant forms of p53 at amino acid 249 (p53(249)) or 273 (p53(273)), Moreover, shifting the kinase reaction's temperature from 37 degrees C to 18 degrees C reduces the phosphorylation of mutant p53 to a greater extent than of p53(wt)., Comparing truncated forms of p53 revealed that the ability of CKII beta, PKA, or WCE kinases to phosphorylate p53 requires amino acids 97-155 within the DNA-binding domain region, Among three 20-aa peptides spanning this region we have identified residues 97-117 that increase p53 phosphorylation by CKII beta while inhibiting p53 phosphorylation by PKA or WCE kinases, The importance of this region is further supported by computer modeling studies, which demonstrated that mutant p53(249) exhibits significant changes to the conformation of p53 within amino acids 97-117, In summary, phosphorylation-related analysis of different p53 forms in vitro indicates that conformation of p53 is a key determinant in its availability as a substrate for different kinases, as for the phosphorylation pattern generated by the same kinase. [References: 38]
机译：已知p53抑癌蛋白的磷酸化可调节其功能，使用细菌产生的谷胱甘肽S-转移酶（GST）-p53融合蛋白和杆状病毒表达的组氨酸标记的p53（Hi，p53），我们通过纯化确定了人p53的磷酸化-N激酶（JNK），蛋白激酶A（PKA）和酪蛋白激酶II（CKII beta）的β亚基的形式以及存在于全细胞提取物中（WCEs）的激酶的形式，我们证明PKA是有效的p53激酶，尽管以构象和浓度依赖性方式，通过比较全长和p53的截短形式而得出结论，我们进一步证明了JNK与GST，p53的相互作用以及JNK磷酸化GST-p53或截短形式的能力。全长（His）p53。磷酸化对p53构象的依赖性进一步得到了以下发现的支持：发现p53（p53（wt））的野生型形式在249位氨基酸（p53（249 ））或273（p53（273）），此外，将激酶反应的温度从37摄氏度降低到18摄氏度，突变体p53的磷酸化程度比p53（wt）更大。比较p53的截短形式CKII beta，PKA或WCE激酶磷酸化p53的能力需要DNA结合结构域区域内的氨基酸97-155。在跨越该区域的三个20-aa肽中，我们已经鉴定出通过增加p53磷酸化的残基97-117 CKII beta抑制PKA或WCE激酶引起的p53磷酸化，该区域的重要性进一步得到计算机建模研究的支持，该研究表明，突变体p53（249）对氨基酸内p53的构象表现出显着变化97-117，总而言之，体外对不同p53形式的磷酸化相关分析表明，p53的构象是作为不同激酶底物的可用性的关键决定因素，就像同一激酶产生的磷酸化模式一样。 [参考：38]

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