NEUROENDOCRINE REGULATION OF CREB BY CAMP AND CALCIUM

CAMP和Ca~(2+)信号通路贯穿于 神经内分泌系统调节分泌和基因表达。它有 最近的研究表明，cAMP和钙离子可以汇聚在一起，调节细胞内 单个转录因子CREB的活性。这种趋同很可能 允许CREB在调节中整合多个细胞外信号 抄写。推测钙离子对CREB激活的影响是 由钙/钙调蛋白依赖的蛋白激酶(CaM激酶)介导。 该实验室最近的研究表明，特定的CaM 激酶对CREB的激活可能有非常不同的影响。在……里面 特别是，我们发现CaM Kinase II可以抑制CREB的激活 而CaM Kinase IV可以激活CREB。拟议的研究旨在 进一步鉴定特定的CaM蛋白的调节能力 激活CREB/ATF转录因子家族。具体目标 包括：i)检验CREB的Ser142的磷酸化是 生理调节的。这将涉及到抗体的制备 它检测到Ser142的磷酸化。2)确定抑制性是否 CaMKII对一种相关的亮氨酸拉链atf1也有影响。 转录因子，也已被证明对 CAMP和钙离子。CREB负性调控部位周围区域 在Ser142上是保守的，但在atf1上不是相同的。3)考查能力 CaMKI激活CREB。有可能是CaM激酶而不是 CaMKIV可激活CREB。我们将使用转染法来确定 CaMKI可以激活CREB。4)检验以下假设：抑制性 CaMKII对CREB激活的影响是由于 Ser142处的磷酸化以阻断CREB与辅酶-2之间的相互作用 激活蛋白，CREB结合蛋白(CBP)。5)绘制CREB各部分地图 和Cl3P，它们是高亲和力、依赖PKA和CaM所必需的 激活剂诱导的相互作用。6)形成一种对cAMP有反应的上体 报告基因检测cAMP与Ca~(2+)相互作用 具有染色质结构的CREB。这些研究应该会提供新的见解 探讨cAMP和Ca~(2+)调节血管内皮细胞 CREB/ATF家族。

The cAMP and Ca2+ signaling pathways are used throughout the neuroendocrine system to regulate secretion and gene expression. It has recently been shown that cAMP and Ca2+ can converge to regulate the activity of a single transcription factor, CREB. This convergence likely permits CREB to integrate multiple extracellular signals in the regulation of transcription. Presumably the effects of Ca2+ on CREB activation are mediated by Ca2+/calmodulin-dependent protein kinases (CaM Kinases). Recent studies from this laboratory have demonstrated that specific CaM Kinases can have very different effects on the activation of CREB. In particular, we found that CaM Kinase II can inhibit activation of CREB while CaM Kinase IV can activate CREB. The proposed studies seek to further characterize the ability of specific CaM Kinases to regulate activation of the CREB/ATF transcription factor family. The specific aims include: i) Test the hypothesis that phosphorylation of Ser142 of CREB is physiologically regulated. This will involve preparation of an antibody which detects phosphorylation of Ser142. 2) Determine if the inhibitory effects of CaMKII are also observed for ATF1, a related leucine-zipper transcription factor, which has also been shown to respond to changes in cAMP and Ca2+. The region surrounding the negative regulatory site of CREB at Ser142 is conserved, but not identical in ATF1. 3) Examine the ability of CaMKI to activate CREB. It is possible that CaM Kinases other than CaMKIV can activate CREB. We will use transfection assays to determine if CaMKI can activate CREB. 4) Test the hypothesis that the inhibitory effects of CaMKII on activation of CREB are due to the effects of phosphorylation at Ser142 to block interaction between CREB and the co- activator protein, CREB binding protein (CBP). 5) Map the portions of CREB and Cl3P which are required for high affinity, PKA-dependent and CaM Kinase-induced interactions. 6) Develop an episomal, cAMP-responsive reporter gene to examine effects of cAMP and Ca2+ on the interaction of CREB with chromatin structure. These studies should provide new insight into the mechanisms which permit cAMP and Ca2+ to regulate members of the CREB/ATF family.