【原创】Cell：p53泛素化的新机制被发现

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生物谷报道：p53的功能被多种翻译后修饰机制所调控，包括Hdm2介导的泛素化作用，该作用可使其蛋白酶体降解。目前，来自法国，德国，西班牙等国家的学者发现，P53相关因子E4F1可以作为一个不典型的E3泛素链接酶，调控p53效应，并且该过程不依赖于P53的降解。这项新发现发表于最新一期的《Cell》杂志上。

E4F1是一种广泛表达的锌指蛋白，最初被发现是病毒癌蛋白E1A的靶作用蛋白。它可引起p53铰链区的赖氨酸发生少量的泛素化，其作用机制不同于Hdm2，后者是通过乙酰转移酶PCAF乙酰化特定的靶蛋白。因而，E4F1与PCAF对p53的调控作用是互斥的。同时，依赖于E4F1的p53泛素化，伴随着染色体的相关，以及p53信号通路的激活，引起细胞周期停滞，而非凋亡。

    该研究证实了E4F1是p53的一个重要的翻译后修饰调控因子，并通过对p53功能的调控，影响着细胞的命运：究竟是生长停滞还是凋亡。这一发现对于未来进一步研究p53的功能具有重要意义。



Figure 1. E4F1 Exhibits Intrinsic Ubiquitin E3 Ligase Activity on p53 In Vitro and In Vivo

(A) Autoubiquitylation activity of E4F1. Autoradiograms of in vitro ubiquitylation assays performed under standard conditions in presence of in vitro translated (IVT) E4F1 labeled by 35S-methionine.

(B) Recombinant E4F1 stimulates p53 ubiquitylation in vitro. Autoradiograms of in vitro ubiquitylation assays performed with IVT 35S-labeled p53 and GST-E4F1 or GST.

(C) Autoradiograms of in vitro ubiquitylation assays performed under standard conditions with IVT 35S-labeled p53 and cellular E4F1 immunoprecipitated from E4F1- or mock-transfected U2OS cells.

(D) E4F1 stimulates K48-Ub branching. Autoradiograms of in vitro ubiquitylation assays performed with IVT 35S-labeled p53, baculovirus expressed E4F1 and Ub (WT), or Ub mutants bearing mutations of all (K0) or all but one of its lysine residues at the indicated position. Equal amounts of Ub mutants were used in each assay (Figure S1F).

(E) Cre-induced E4F1-GFP expression in U2OS cells stably expressing the LSL-E4F1 construct. (Upper panels) Western blot **yses of nuclear extracts prepared from LSL-E4F1 cells at the indicated time points after infection with Cre retrovirus, probed with anti-GFP, -E4F1, -p53 (DO1), -Cre, and -TBP (loading control) antibodies (Abs). (Lower panels) Analysis of E4F1-GFP expression by fluorescence microscopy 3 days after infection with Cre (+) or control (−) retroviruses. Cells are shown at 40× magnification.

(F) E4F1 stimulates ubiquitylation of endogenous p53 in vivo. Western blot **yses of Ub-conjugated proteins in LSL-E4F1 cells transiently transfected for 24 hr with a 6XHis-Ub expression vector 4 days after infection by Cre (+) or control (−) retroviruses. One percent of cellular extracts were probed for the presence of E4F1-GFP and total endogenous p53 using anti-GFP and anti-p53 (DO1) Abs (input). These cellular extracts, normalized to contain equal amounts of total p53, were loaded on nickel (Ni+)-NTA columns. Ni+-purified 6XHis-Ub-conjugated proteins were probed for the presence of Ub-p53 forms using an anti-p53 (DO1) Ab (Ni-purified). Neither cells nor cellular extracts were treated with proteasome inhibitors.

(G) Depletion of endogenous E4F1 impacts on endogenous p53 ubiquitylation in vivo. Western blot **yses of Ub-conjugated proteins in U2OS cells treated for 72 hr with control scramble or E4F1 SiRNAs. His-Ub-conjugated forms of endogenous p53 present in these cells were purified and **yzed as described in Figure 1F.



原文下载：

Cell    November 17, 2006: 127 (4)

E4F1 Is an Atypical Ubiquitin Ligase that Modulates p53 Effector Functions Independently of Degradationp775
Laurent Le Cam, Laëtitia K. Linares, Conception Paul, Eric Julien, Matthieu Lacroix, Elodie Hatchi, Robinson Triboulet, Guillaume Bossis, Ayelet Shmueli, Manuel S. Rodriguez, Olivier Coux, and Claude Sardet
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