Burge Sarah, Teufel Daniel P, Townsley Fiona M, Freund Stefan M V, Bycroft Mark, Fersht Alan R
Medical Research Council Centre for Protein Engineering, Hills Road, Cambridge CB2 0QH, United Kingdom.
Proc Natl Acad Sci U S A. 2009 Mar 3;106(9):3142-7. doi: 10.1073/pnas.0900383106. Epub 2009 Feb 13.
The transcription factor p73 belongs to the p53 family of proteins and can transactivate a number of target genes in common with p53. Here, we characterized the interaction of the p73 N terminus with four domains of the transcriptional coactivator p300 and with the negative regulator Mdm2 by using biophysical and cellular measurements. We found that, like p53, the N terminus of p73 contained two distinct transactivation subdomains, comprising residues 10-30 and residues 46-67. The p73 N terminus bound weakly to the Taz1, Kix, and IBiD domains of p300 but with submicromolar affinity for Taz2, in contrast to previous reports. We found weaker binding of the p73 N terminus to the p300 domains in vitro correlated with a significant decrease in transactivation activity in a cell line for the QS and T14A mutants, and tighter binding of the phosphomimetic T14D in vitro correlated with an increase in vivo. Further, we found that phosphorylation of T14 increased the affinity of the p73 N terminus for Taz2 10-fold. The phosphomimetic p73alpha T14D caused increased levels of transactivation.
转录因子p73属于p53蛋白家族，能够反式激活许多与p53相同的靶基因。在此，我们通过生物物理和细胞检测方法，对p73的N端与转录共激活因子p300的四个结构域以及负调节因子Mdm2之间的相互作用进行了表征。我们发现，与p53一样，p73的N端包含两个不同的反式激活亚结构域，分别由第10 - 30位氨基酸残基和第46 - 67位氨基酸残基组成。与之前的报道相反，p73的N端与p300的Taz1、Kix和IBiD结构域结合较弱，但与Taz2具有亚微摩尔亲和力。我们发现，在体外，p73的N端与p300结构域的较弱结合与QS和T14A突变体在细胞系中的反式激活活性显著降低相关，而磷酸模拟物T14D在体外的较强结合与体内活性增加相关。此外，我们发现T14的磷酸化使p73的N端与Taz2的亲和力增加了10倍。磷酸模拟物p73α T14D导致反式激活水平升高。
