Sun Kai, Montana Vedrana, Chellappa Karthikeyani, Brelivet Yann, Moras Dino, Maeda Yutaka, Parpura Vladimir, Paschal Bryce M, Sladek Frances M
Environmental Toxicology Graduate Program, University of California, Riverside, California 92521, USA.
Mol Endocrinol. 2007 Jun;21(6):1297-311. doi: 10.1210/me.2006-0300. Epub 2007 Mar 27.
Nuclear receptors (NRs) are a superfamily of transcription factors whose genomic functions are known to be activated by lipophilic ligands, but little is known about how to deactivate them or how to turn on their nongenomic functions. One obvious mechanism is to alter the nuclear localization of the receptors. Here, we show that protein kinase C (PKC) phosphorylates a highly conserved serine (Ser) between the two zinc fingers of the DNA binding domain of orphan receptor hepatocyte nuclear factor 4alpha (HNF4alpha). This Ser (S78) is adjacent to several positively charged residues (Arg or Lys), which we show here are involved in nuclear localization of HNF4alpha and are conserved in nearly all other NRs, along with the Ser/threonine (Thr). A phosphomimetic mutant of HNF4alpha (S78D) reduced DNA binding, transactivation ability, and protein stability. It also impaired nuclear localization, an effect that was greatly enhanced in the MODY1 mutant Q268X. Treatment of the hepatocellular carcinoma cell line HepG2 with PKC activator phorbol 12-myristate 13-acetate also resulted in increased cytoplasmic localization of HNF4alpha as well as decreased endogenous HNF4alpha protein levels in a proteasome-dependent fashion. We also show that PKC phosphorylates the DNA binding domain of other NRs (retinoic acid receptor alpha, retinoid X receptor alpha, and thyroid hormone receptor beta) and that phosphomimetic mutants of the same Ser/Thr result in cytoplasmic localization of retinoid X receptor alpha and peroxisome proliferator-activated receptor alpha. Thus, phosphorylation of this conserved Ser between the two zinc fingers may be a common mechanism for regulating the function of NRs.
核受体（NRs）是一类转录因子超家族，其基因组功能已知可被亲脂性配体激活，但对于如何使其失活或如何开启其非基因组功能却知之甚少。一种明显的机制是改变受体的核定位。在此，我们表明蛋白激酶C（PKC）使孤儿受体肝细胞核因子4α（HNF4α）DNA结合域的两个锌指之间的一个高度保守的丝氨酸（Ser）发生磷酸化。这个丝氨酸（S78）与几个带正电荷的残基（精氨酸或赖氨酸）相邻，我们在此表明这些残基参与HNF4α的核定位，并且与丝氨酸/苏氨酸（Thr）一起在几乎所有其他核受体中都是保守的。HNF4α的一个拟磷酸化突变体（S78D）降低了DNA结合、反式激活能力和蛋白质稳定性。它还损害了核定位，在MODY1突变体Q268X中这种效应大大增强。用PKC激活剂佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯处理肝癌细胞系HepG2也导致HNF4α的细胞质定位增加以及内源性HNF4α蛋白水平以蛋白酶体依赖的方式降低。我们还表明PKC使其他核受体（视黄酸受体α、维甲酸X受体α和甲状腺激素受体β）的DNA结合域发生磷酸化，并且相同丝氨酸/苏氨酸的拟磷酸化突变体导致维甲酸X受体α和过氧化物酶体增殖物激活受体α的细胞质定位。因此，两个锌指之间这个保守丝氨酸的磷酸化可能是调节核受体功能的一种常见机制。
