Porte D, Oertel-Buchheit P, John M, Granger-Schnarr M, Schnarr M
Institut de Biologie Moléculaire et Cellulaire, UPR 9002 du CNRS, F-67084 Strasbourg Cedex, France.
Nucleic Acids Res. 1997 Aug 1;25(15):3026-33. doi: 10.1093/nar/25.15.3026.
The mammalian Fos and Fos-related proteins are unable to form homodimers and to bind DNA in the absence of a second protein, like c-Jun for example. In order to study the implications of hydrophobic point mutations in the c-Fox leucine zipper on DNA binding of the entire c-Fos protein, we have constructed and purified a set of Fos mutant proteins harboring one or several isoleucine or leucine residues in the five Fos zipper a positions. We show that a single point mutation in the hydrophobic interface of the c-Fos leucine zipper enables the c-Fos mutant protein to bind specifically to an oligonucleotide duplex harboring the TRE consensus sequence TGA(C/G)TCA. This point mutation (Thr196-->Ile) is situated in the a position of the second heptade (a2) of the Fos zipper. The introduction of additional isoleucine residues in the other a positions progressively increases the DNA binding affinity of these homodimerizing Fos zipper variants. Heterodimerization of these c-Fos variants with c-Jun reveals a complex behavior, in that the DNA binding affinity of these heterodimers does not simply increase with the number of isoleucine side chains in position a. For example, a c-Fos variant harboring a wild-type Thr in position a1 aad Ile in the four other a positions (c-Fos4I) interacts more tightly with c-Jun than a variant harboring Ile in all five a positions (c-Fos5I). The same holds true for the corresponding leucine variants, suggesting that the wild-type a1 residue of the Fox zipper (Thr162) is thermodynamically relevant for Fos-Jun heterodimer formations and DNA binding. The c-Fos4I variant forms heterodimers with c-Jun slightly better than the wild-type zipper protein, suggesting that the driving force for Fos-Jun heterodimerization is not the simple fact that the Fos protein is unable to form homodimers. These c-Fos variants were further tested for their transactivation properties in F9 and NIH3T3 cells. At low expression levels the most efficiently homodimerizing variant (c-Fos5I) activates transcription in F9 cells about 6-fold. However part of this activation may be due to the formation of heterodimers with a member of the Jun family (like JunD for example), since a wild type c-Fos expression vector confers a 3-fold activation under these conditions. In the case of the homodimerizing c-Fos variants however, this activation is abrogated at higher expression levels due to a strong inhibition of basal transcription activity.
哺乳动物的Fos及Fos相关蛋白在没有第二种蛋白（如c-Jun）存在时，无法形成同二聚体并结合DNA。为了研究c-Fos亮氨酸拉链中疏水点突变对整个c-Fos蛋白DNA结合的影响，我们构建并纯化了一组Fos突变蛋白，这些蛋白在Fos拉链的5个a位置含有一个或几个异亮氨酸或亮氨酸残基。我们发现，c-Fos亮氨酸拉链疏水界面的单点突变使c-Fos突变蛋白能够特异性结合含有TRE共有序列TGA(C/G)TCA的寡核苷酸双链体。这个点突变（Thr196→Ile）位于Fos拉链第二个七肽（a2）的a位置。在其他a位置引入额外的异亮氨酸残基会逐渐增加这些能形成同二聚体的Fos拉链变体的DNA结合亲和力。这些c-Fos变体与c-Jun的异二聚化表现出复杂的行为，即这些异二聚体的DNA结合亲和力并不简单地随着a位置异亮氨酸侧链数量的增加而增加。例如，在a1位置含有野生型苏氨酸且在其他4个a位置含有异亮氨酸的c-Fos变体（c-Fos4I）与c-Jun的相互作用比在所有5个a位置都含有异亮氨酸的变体（c-Fos5I）更紧密。相应的亮氨酸变体也是如此，这表明Fos拉链的野生型a1残基（Thr162）在热力学上与Fos-Jun异二聚体的形成和DNA结合相关。c-Fos4I变体与c-Jun形成异二聚体的能力略优于野生型拉链蛋白，这表明Fos-Jun异二聚化的驱动力并非仅仅是Fos蛋白无法形成同二聚体这一事实。我们进一步测试了这些c-Fos变体在F9和NIH3T3细胞中的反式激活特性。在低表达水平下，最有效地形成同二聚体的变体（c-Fos5I）在F9细胞中激活转录的能力约为6倍。然而，这种激活的一部分可能是由于与Jun家族成员（如JunD）形成异二聚体所致，因为在这些条件下，野生型c-Fos表达载体可导致3倍的激活。然而，对于能形成同二聚体的c-Fos变体来说，由于基础转录活性受到强烈抑制，在较高表达水平时这种激活作用会被消除。
