Lukas Thomas J
Department of Molecular Pharmacology and Drug Discovery Program, Northwestern University, Chicago, Illinois 60611, USA.
Biophys J. 2004 Sep;87(3):1417-25. doi: 10.1529/biophysj.104.042721.
An agonist-initiated Ca(2+) signaling model for calmodulin (CaM) coupled to the phosphorylation of myosin light chains was created using a computer-assisted simulation environment. Calmodulin buffering was introduced as a module for directing sequestered CaM to myosin light chain kinase (MLCK) through Ca(2+)-dependent release from a buffering protein. Using differing simulation conditions, it was discovered that CaM buffering allowed transient production of more Ca(2+)-CaM-MLCK complex, resulting in elevated myosin light chain phosphorylation compared to nonbuffered control. Second messenger signaling also impacts myosin light chain phosphorylation through the regulation of myosin light chain phosphatase (MLCP). A model for MLCP regulation via its regulatory MYPT1 subunit and interaction of the CPI-17 inhibitor protein was assembled that incorporated several protein kinase subsystems including Rho-kinase, protein kinase C (PKC), and constitutive MYPT1 phosphorylation activities. The effects of the different routes of MLCP regulation depend upon the relative concentrations of MLCP compared to CPI-17, and the specific activities of protein kinases such as Rho and PKC. Phosphorylated CPI-17 (CPI-17P) was found to dynamically control activity during agonist stimulation, with the assumption that inhibition by CPI-17P (resulting from PKC activation) is faster than agonist-induced phosphorylation of MYPT1. Simulation results are in accord with literature measurements of MLCP and CPI-17 phosphorylation states during agonist stimulation, validating the predictive capabilities of the system.
利用计算机辅助模拟环境创建了一个与肌球蛋白轻链磷酸化偶联的钙调蛋白（CaM）的激动剂启动的Ca(2+)信号模型。引入钙调蛋白缓冲作为一个模块，通过从缓冲蛋白中Ca(2+)依赖性释放，将隔离的CaM导向肌球蛋白轻链激酶（MLCK）。使用不同的模拟条件，发现钙调蛋白缓冲允许短暂产生更多的Ca(2+)-CaM-MLCK复合物，与非缓冲对照相比，导致肌球蛋白轻链磷酸化升高。第二信使信号也通过调节肌球蛋白轻链磷酸酶（MLCP）影响肌球蛋白轻链磷酸化。组装了一个通过其调节性MYPT1亚基和CPI-17抑制蛋白相互作用来调节MLCP的模型，该模型纳入了几个蛋白激酶子系统，包括 Rho激酶、蛋白激酶C（PKC）和组成型MYPT1磷酸化活性。MLCP调节的不同途径的影响取决于MLCP与CPI-17的相对浓度，以及Rho和PKC等蛋白激酶的比活性。发现磷酸化的CPI-17（CPI-17P）在激动剂刺激期间动态控制活性，假设CPI-17P的抑制（由PKC激活导致）比激动剂诱导的MYPT1磷酸化更快。模拟结果与激动剂刺激期间MLCP和CPI-17磷酸化状态的文献测量结果一致，验证了该系统的预测能力。
