Sato T, Xiao D M, Li H, Huang F L, Huang K P
Section on Metabolic Regulation, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 1995 Apr 28;270(17):10314-22. doi: 10.1074/jbc.270.17.10314.
A 13-kilobase pair genomic DNA encoding a 78-amino acid brain-specific calmodulin-binding protein kinase C (PKC) substrate, neurogranin (Ng/RC3; also known as RC3 or p17), has been sequenced. The Ng/RC3 gene is composed of four exons and three introns, with the protein-coding region located in the first and second exons. This gene was found to have multiple transcriptional start sites clustered within 20 base pairs (bp); it lacks the TATA, GC, and CCAAT boxes in the proximal upstream region of the start sites. The promoter activity was characterized by transfection of 293 cells with nested deletion mutants of the 5'-flanking region fused to the luciferase reporter gene. A minimal construct containing bp +11 to +256 was nearly as active as that covering bp -1508 to +256, whereas a shorter one covering bp +40 to +256 had a greatly reduced activity. Between bp +11 and +40 lies a 12-nucleotide sequence (CCCCGCCCACCC) containing overlapping binding sites for AP2 (CCGCCCACCC) and SP1 (CCCGCC); this region may be important for conferring the basal transcriptional activity of the Ng/RC3 gene. The expression of a Ng/RC3-luciferase fusion construct (-1508/+256) in transfected 293 cells was stimulated by phorbol 12-myristate 13-acetate (PMA), but not by cAMP, arachidonic acid, vitamin D, retinoic acid, or thyroxines T3 and T4. PMA caused a 2-4-fold stimulation of all the reporter gene constructs ranging from +11/+256 to -1508/+256. The stimulatory effects of PMA could be magnified by cotransfection with both Ca(2+)-dependent and -independent phorbol ester-binding PKC-alpha, -beta I, -beta II, -gamma, -delta, and -epsilon cDNAs, but not by non-phorbol ester-binding PKC-zeta cDNA. The Ng/RC3 and PKC-gamma genes have a similar expression pattern in the brain during development. These two genes share at least four conserved sequence segments 1.5 kilobase pair upstream from their transcriptional start sites and a gross similarity in that they possess several AT-rich segments within bp -550 to -950. A near homogeneous 20-kDa DNA-binding protein purified from rat brain was able to bind to these AT-rich regions of both Ng/RC3 and PKC-gamma genes with footprints containing ATTA, ATAA, and AATA sequences.
一个编码78个氨基酸的脑特异性钙调蛋白结合蛋白激酶C（PKC）底物——神经颗粒素（Ng/RC3；也称为RC3或p17）的13千碱基对基因组DNA已被测序。Ng/RC3基因由四个外显子和三个内含子组成，蛋白质编码区位于第一和第二个外显子中。发现该基因有多个转录起始位点聚集在20个碱基对（bp）内；它在起始位点近端上游区域缺乏TATA、GC和CCAAT框。通过用与荧光素酶报告基因融合的5'侧翼区域的嵌套缺失突变体转染293细胞来表征启动子活性。一个包含bp +11至+256的最小构建体的活性几乎与覆盖bp -1508至+256的构建体相同，而一个较短的覆盖bp +40至+256的构建体活性大大降低。在bp +11和+40之间有一个12个核苷酸的序列（CCCCGCCCACCC），包含AP2（CCGCCCACCC）和SP1（CCCGCC）的重叠结合位点；该区域可能对赋予Ng/RC3基因的基础转录活性很重要。在转染的293细胞中，Ng/RC3-荧光素酶融合构建体（-1508/+256）的表达受到佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）的刺激，但不受cAMP、花生四烯酸、维生素D、视黄酸或甲状腺素T3和T4的刺激。PMA对从+11/+256到-1508/+256的所有报告基因构建体都有2至4倍的刺激作用。PMA的刺激作用可以通过与钙依赖性和非钙依赖性佛波酯结合的PKC-α、-βI、-βII、-γ、-δ和-ε cDNA共转染而放大，但不能通过非佛波酯结合的PKC-ζ cDNA放大。Ng/RC3和PKC-γ基因在发育过程中在脑中具有相似的表达模式。这两个基因在其转录起始位点上游1.5千碱基对处至少共享四个保守序列片段，并且在bp -550至-950内具有几个富含AT的片段，总体相似。从大鼠脑中纯化的一种近乎同质的20 kDa DNA结合蛋白能够与Ng/RC3和PKC-γ基因的这些富含AT的区域结合，其足迹包含ATTA、ATAA和AATA序列。
