REGULATION OF SOMATOSTATIN GENE EXPRESSION

环磷酸腺苷(CAMP)通过调节多种基因的转录 蛋白激酶A(PK-A)介导的转录磷酸化 CREB因子在第133位。尽管磷酸化可能会刺激 转录激活剂通过调节它们的核运输或DNA- 结合亲和力，CREB属于一类激活剂，其 磷酸化似乎特别地增强了它们的反式激活 潜力。在CREB蛋白中，一个60个氨基酸的激酶被诱导 结构域(KID)与构成富含谷氨酰胺的结构域(Q2)在 CREB刺激cAMP反应基因转录。现有证据 提示CREB的KID和Q2结构域与不同的 蛋白质，这两个蛋白质都是转录因子组装的关键 对cAMP的反应中的启蒙情结。这样做的总体目标是 建议阐明cAMP刺激的机制 靶基因的转录，重点是PK-A的假设 CREB介导的磷酸化促进蛋白质-蛋白质相互作用 最终导致一般转录因子被招募到 阵营响应型推动者。 I.我们将测试结构性激活之间的相互作用 CREB中的结构域称为Q2，是一般转录的一个组成部分 TFIID因子(dTAF-II 110)是PK-A诱导转录的关键因子。 我们将在CREB和dTAF-II 110中划定参与 复合体的形成，我们将监测CREB dTAF-II 110结合突变体 用于在体外和体内丢失PK诱导的转录。 II.我们将定义最近表征的CREB结合的区域 CAMP反应所需的蛋白质(CBP) 利用表达载体进行瞬时转染法转录 编码野生型和突变型CBP。 III.我们将鉴定磷酸化所需的序列 CREB称为KID的蛋白激酶诱导结构域之间的依赖相互作用 和CBP采用体外结合试验。 IV.我们将确定相互作用的通用转录因子 CBP刺激cAMP反应基因转录的功能 PK-A介导的CREB在Ser133位的磷酸化。 CAMP介导多种细胞对激素和生长的反应 各种因素。在内分泌靶器官中，如甲状腺和脑下垂体， CAMP第二信使通路的病理性激活导致 内分泌肿瘤和功能亢进的证候。这项研究建议 这里将定义转录中间体，它可能会 在这些疾病和其他疾病的发病机制中很重要，cAMP 不适当地被激活。

Cyclic AMP (cAMP) regulates the transcription of numerous genes through the protein kinase-A (PK-A) mediated phosphorylation of transcription factor CREB at Ser133. Although phosphorylation may stimulate transcriptional activators by modulating their nuclear transport or DNA- binding affinity, CREB belongs to a class of activators whose phosphorylation appears to specifically enhance their trans-activation potential. Within the CREB protein, a 60 amino acid Kinase Inducible Domain (KID) cooperates with a constitutive glutamine rich domain (Q2) in CREB to stimulate transcription of cAMP responsive genes. Current evidence suggests that the KID and Q2 domains of CREB interact with distinct proteins, both of which are critical for assembly of transcriptional initiation complex in response to cAMP. The overall objective of this proposal is to elucidate the mechanism by which cAMP stimulates transcription of target genes, focusing on the hypothesis that PK-A mediated phosphorylation of CREB stimulates protein-protein interactions which culminate in the recruitment of general transcription factors to cAMP responsive promoters. I. We will test whether interaction between a constitutive activation domain in CREB termed Q2 and a component of the general transcription factor TFIID (dTAF-II 110) is critical for PK-A inducible transcription. We will delineate regions in CREB and dTAF-II 110 which participate in complex formation, and we will monitor CREB dTAF-II 110-binding mutants for loss of PK-inducible transcription in vitro and in vivo. II. We will define regions in a recently characterized CREB binding protein (CBP) which are functionally required for cAMP responsive transcription by transient transfection assay with expression vectors encoding wild-type and mutant forms of CBP. III. We will characterize sequences which are required for phosphorylation dependent interaction between a kinase inducible domain in CREB termed KID and CBP using in vitro binding assays. IV. We will identify general transcription factors which interact functionally with CBP to stimulate transcription of cAMP responsive genes following PK-A mediated phosphorylation of CREB at Ser133. cAMP mediates a number of cellular responses to hormones and growth factors. In endocrine target organs such as thyroid and pituitary, pathologic activation of the cAMP second messenger pathway causes syndromes of endocrine neoplasia and hyperfunction. The studies proposed herein will define transcriptional intermediates which may figure importantly in the pathogenesis of these and other diseases where cAMP is inappropriately activated.