Regulation of transcriptional factors by phosphorylation

cANP反应元件（CRE）是一种诱导增强基因，如pre - proproenkephaun和somatostatin的基因，可以在cAMP水平升高时转录。我们之前通过CDNA克隆鉴定了CRE-BP1结合蛋白。CRE- bpi具有“B-ZIP”结构域作为dna结合域，与CRE结合为同二聚体或异二聚体。我们研究了CRE-BP1的磷酸化。我们还分析了CRE-BPL的功能域，以检查含有磷酸化位点的蛋白质的作用。人重组CRE-BPl在体外被camp依赖性蛋白idase和蛋白激酶C磷酸化。这两种蛋白激酶修饰了CRE-BP1不同的半氨酸残基。Ser-62是camp依赖性蛋白激酶的磷酸化位点，而Ser-340和Ser-367是蛋白激酶C的主要磷酸化位点。通过检测CRE-BP1的一系列缺失和点突变体的活性，确定了CRE-BP1的两个功能位点：氨基末端的金属指结构和羧基末端与一簇碱性氨基酸相连的亮氨酸拉链基序。前者是一个转录激活域，后者是一个dna结合域。因此，被camp依赖性蛋白激酶磷酸化的Ser-62位于转录激活域附近，而蛋白Idnase C磷酸化位点Ser-340和Ser-367位于dna结合域的基本区域。这些结果表明，CRE-BPL的转录和dna结合活性受这些蛋白激酶的磷酸化调节。我们还研究了CK-H磷酸化对c-myb癌基因产物（c-myb）活性的影响。c-Myb是一种转录激活剂，可以与特定的DNA序列结合。已知酪蛋白激酶H（CK-II）磷酸化c-Myb的Ser- 11和Ser- 12。为了检验这些丝氨酸残基磷酸化对c-Myb活性的影响，我们将这些丝氨酸残基替换为丙氨酸。丙氨酸突变体的反激活能力比正常的c-Myb高约2倍。这些结果表明CK-H对c-Myb的磷酸化抑制了c-Myb的活性。由于CK-H活性与细胞生长控制有关，这些结果可能为理解转录因子与细胞生长控制之间的联系提供线索。少

The cANP response element(CRE)is an inducible enhancer of genes such as those of preproenkephaun and somatostatin, with can be transcribed in response to increased cAMP levels. We previously identified the CRE-binding protein, CRE-BP1, by CDNA cloning. CRE-BPI has a "B-ZIP" structure as a DNA-binidng domain and binds to CRE as a homodimer or a heterodimer with c-Jun. We have investigated the phosphorylation of CRE-BP1. We also analyzed the functional domains of CRE-BPL to examine the role of protein containing the phosphorylation sites. The human recombinant CRE-BPl was phosphorylated by cAMP-dependent protein Idnase ard protein kinase C, in vitro. These two protein kinases modified distinct semine residues of CRE-BP1. Ser-62 was the phosphorylation site by cAMP-dependent protein kinase, whereas two semine residues, Ser-340 and Ser-367 were the major protein kinase C phophorylation sites. By examining the activity of a series of deletion and point mutants of CRE-BP1, two functional dom … More ains of CRE-BPL were identified : the putative metal finger structure in the amino-terininal region and the leucine zipper motif linked to a cluster of basic amino acids in the carboxyl-terminal region. The former was a transcriptional activation domain, and the latter was a DNA-binding domain. Therefore, Ser-62 which is phosphorylated by cAMP-dependent protein kinase is close to the transcriptional activation domain, whereas Ser-340 and Ser-367, the protein Idnase C phosphorylation sites, are located in the basic region of the DNA-binding domain. These results suggest that transcriptional and DNA-binding activities of CRE-BPL are regulated by phosphorylation with these protein kinases.We have also examined the effect of phosphorylation by CK-H on the activity of c-myb oncogene product(c-Myb). c-Myb is a transcriptional activator that can bind to the specific DNA sequence. Casein kinase H(CK-II)is known to phosphorylate Ser- 1 1 and Ser- 12 of c-Myb. To examine the effect of phosphorylation of these Ser residues on c-Myb activity, we replaced these Ser residues to alanine. Alanine mutant had about 2-fold higher transactivating capacity than the normal c-Myb. These results indicate that phophorylation of c-Myb by CK-H represses the c-Myb activity. Since CK-H activity is liked to the cellular growth control, these results may give a clue to understand a linkage between transcriptional factors and cellular growth control. Less