GASTROINTESTINAL CALMODULIN-DEPENDENT PROTEIN KINASE II

胃肠道钙调蛋白依赖性蛋白激酶 II

• 批准号: 6402598
• 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/6402598
• 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.

该项目的长期目标是表征近端结肠和眼底平滑肌细胞中的钙调素依赖性蛋白激酶II。钙调素依赖性蛋白激酶II是一种多功能丝氨酸/苏氨酸蛋白激酶，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。细胞内Ca2+水平在相位平滑肌振荡，但在强直平滑肌保持升高。钙调素依赖性蛋白激酶II的异构体组成调节其对Ca2+振荡的反应。在眼底和近端结肠平滑肌中表达的钙调素依赖性蛋白激酶II亚型是未知的。本项目的主要目的是验证不同钙调素依赖性蛋白激酶II亚型在近端结肠和眼底平滑肌细胞中表达的假设。从眼底或近端结肠平滑肌细胞纯化的mRNA中产生的第一链cdna将用于PCR反应，以扩增钙调素依赖性蛋白激酶II cdna。同种异构体特异性寡核苷酸将使用已知同种异构体的核苷酸序列来设计。利用杆状病毒表达获得的全酶和从培养的眼底和近端结肠平滑肌细胞中纯化的内源性全酶，研究结肠和基底平滑肌细胞钙调素依赖性蛋白激酶II的酶学特性。生成Ca2+/钙调素独立活性水平的动力学将通过体外激酶测定来确定。钙调素依赖性蛋白激酶II在Ca2+信号转导到生理通道中起关键作用。本项目的研究将确定和阐明在眼底和近端结肠平滑肌中表达的钙调素依赖性蛋白激酶II的酶学特性。这些发现将进一步加深我们对钙调素依赖性蛋白激酶II在调节强直和相位胃肠道平滑肌细胞Ca2+依赖性行为中的作用的理解。