Adler V, Pincus M R, Minamoto T, Fuchs S Y, Bluth M J, Brandt-Rauf P W, Friedman F K, Robinson R C, Chen J M, Wang X W, Harris C C, Ronai Z
Molecular Carcinogenesis Program, American Health Foundation, Valhalla, NY 10595, USA.
Proc Natl Acad Sci U S A. 1997 Mar 4;94(5):1686-91. doi: 10.1073/pnas.94.5.1686.
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 ((His)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 (CKIIbeta) 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 (p53wt) undergoes better phosphorylation by CKIIbeta 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 p53wt. Comparing truncated forms of p53 revealed that the ability of CKIIbeta, 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 CKIIbeta 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.
已知p53肿瘤抑制蛋白的磷酸化可调节其功能。我们利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（（His）p53），通过纯化形式的Jun-N-激酶（JNK）、蛋白激酶A（PKA）和酪蛋白激酶II的β亚基（CKIIβ）以及全细胞提取物（WCE）中的激酶来测定人p53的磷酸化。我们证明PKA是一种有效的p53激酶，尽管是以构象和浓度依赖的方式，这是通过比较全长p53和截短形式的p53得出的结论。我们进一步证明JNK与GST-p53相互作用，以及JNK磷酸化截短形式的GST-p53或全长（His）p53的能力。p53构象对磷酸化的依赖性进一步得到以下发现的支持：野生型p53（p53wt）比p53在氨基酸249（p53（249））或273（p53（273））处的突变形式更容易被CKIIβ和WCE激酶磷酸化。此外，将激酶反应温度从37℃降至18℃时，突变型p53的磷酸化程度比p53wt降低得更多。比较p53的截短形式发现，CKIIβ、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个氨基酸的肽段中，我们鉴定出氨基酸97-117，它可增加CKIIβ对p53的磷酸化，同时抑制PKA或WCE激酶对p53的磷酸化。计算机建模研究进一步支持了该区域的重要性，该研究表明突变型p53（249）在氨基酸97-117范围内p53的构象有显著变化。总之，体外对不同p53形式的磷酸化相关分析表明，p53的构象是其作为不同激酶底物可用性的关键决定因素，对于同一激酶产生的磷酸化模式也是如此。
