GASTROINTESTINAL CALMODULIN-DEPENDENT PROTEIN KINASE II

胃肠道钙调蛋白依赖性蛋白激酶 II

• 批准号: 6190728
• 负责人: Brian A Perrino
• 金额: $ 14.43万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6190728
• 关键词: biological signal transduction; calcium flux; calmodulin dependent protein kinase; gastrointestinal motility /pressure; isozymes; laboratory mouse; polymerase chain reaction; protein structure function; smooth muscle

The broad long-term objective of this project is to characterize calmodulin-dependent protein kinase II from smooth muscle cells of the proximal colon and fundus. Calmodulin-dependent protein kinase II is a multifunctional Ser/Thr protein kinase having an Oligomeric structure of 10-12 subunits. The brain enzyme is composed of the alpha and beta isoforms, while the delta and gamma isoforms are predominantly expressed in smooth muscle. Cytosolic Ca2+ levels in phasic smooth muscle oscillate, but remain elevated in tonic smooth muscle. The isoform composition of calmodulin-dependent protein kinase II modulates its response to Ca2+ oscillations. The calmodulin-dependent protein kinase II isoforms expressed in fundus and proximal colon smooth muscle are unknown. A major goal of this project is to test the hypothesis that different calmodulin-dependent protein kinase II isoforms are expressed in proximal colon and fundus smooth muscle cells. First strand cDNAs generated from purified mRNA of fundus or proximal colon smooth muscle cells will be used in PCR reactions to amplify calmodulin-dependent protein kinase II cDNAs. Isoform-specific oligonucleotides will be designed using the nucleotide sequences of the known isoforms. The enzymatic characteristics of colonic and fundic smooth muscle cell calmodulin-dependent protein kinase II will be investigated by using holoenzymes obtained by baculovirus expression [and endogenous holoenzymes purified from cultured fundus and proximal colon smooth muscle cells.] The kinetics of generation of Ca2+/calmodulin-independent activity levels will be determined using in vitro kinase assays. Calmodulin-dependent protein kinase II plays a pivotal role in transducing Ca2+ signals into physiological vents. The studies from this project will identify and elucidate the enzymatic characteristics of calmodulin-dependent protein kinase II expressed in fundus and proximal colon smooth muscle. These findings will further our understanding of the role of calmodulin-dependent protein kinase II in regulating the Ca2+- dependent behavior of tonic and phasic gastrointestinal smooth muscle cells.

该项目的广泛长期目标是表征近端结肠和眼底平滑肌细胞的钙调蛋白依赖性蛋白激酶 II。 钙调蛋白依赖性蛋白激酶 II 是一种多功能 Ser/Thr 蛋白激酶，具有 10-12 个亚基的寡聚结构。 脑酶由α和β亚型组成，而δ和γ亚型主要在平滑肌中表达。相位平滑肌中的胞浆 Ca2+ 水平振荡，但强直平滑肌中的胞质 Ca2+ 水平保持升高。 钙调蛋白依赖性蛋白激酶 II 的亚型组成可调节其对 Ca2+ 振荡的反应。 在眼底和近端结肠平滑肌中表达的钙调蛋白依赖性蛋白激酶 II 亚型尚不清楚。 该项目的一个主要目标是检验以下假设：不同的钙调蛋白依赖性蛋白激酶 II 亚型在近端结肠和眼底平滑肌细胞中表达。从眼底或近端结肠平滑肌细胞的纯化 mRNA 产生的第一链 cDNA 将用于 PCR 反应，以扩增钙调蛋白依赖性蛋白激酶 II cDNA。将使用已知同种型的核苷酸序列来设计同种型特异性寡核苷酸。将使用杆状病毒表达获得的全酶[以及从培养的眼底和近端结肠平滑肌细胞纯化的内源全酶]研究结肠和胃底平滑肌细胞钙调蛋白依赖性蛋白激酶II的酶学特征。Ca2+/钙调蛋白独立活性水平的产生动力学将使用体外激酶测定来确定。钙调蛋白依赖性蛋白激酶 II 在将 Ca2+ 信号转导至生理孔中起着关键作用。 该项目的研究将鉴定并阐明在眼底和近端结肠平滑肌中表达的钙调蛋白依赖性蛋白激酶 II 的酶学特征。 这些发现将进一步了解钙调蛋白依赖性蛋白激酶 II 在调节强直性和阶段性胃肠道平滑肌细胞的 Ca2+ 依赖性行为中的作用。