Asthagiri A R, Nelson C M, Horwitz A F, Lauffenburger D A
Department of Chemical Engineering, Division of Bioengineering and Environmental Health, and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
J Biol Chem. 1999 Sep 17;274(38):27119-27. doi: 10.1074/jbc.274.38.27119.
Because integrin-mediated signals are transferred through a physical architecture and synergistic biochemical network whose properties are not well defined, quantitative relationships between extracellular integrin-ligand binding events and key intracellular responses are poorly understood. We begin to address this by quantifying integrin-mediated FAK and ERK2 responses in CHO cells for varied alpha(5)beta(1) expression level and substratum fibronectin density. Plating cells on fibronectin-coated surfaces initiated a transient, biphasic ERK2 response, the magnitude and kinetics of which depended on integrin-ligand binding properties. Whereas ERK2 activity initially increased with a rate proportional to integrin-ligand bond number for low fibronectin density, the desensitization rate was independent of integrin and fibronectin amount but proportional to the ERK2 activity level with an exponential decay constant of 0.3 (+/- 0.08) min(-1). Unlike the ERK2 activation time course, FAK phosphorylation followed a superficially disparate time course. However, analysis of the early kinetics of the two signals revealed them to be correlated. The initial rates of FAK and ERK2 signal generation exhibited similar dependence on fibronectin surface density, with both rates monotonically increasing with fibronectin amount until saturating at high fibronectin density. Because of this similar initial rate dependence on integrin-ligand bond formation, the disparity in their time courses is attributed to differences in feedback regulation of these signals. Whereas FAK phosphorylation increased to a steady-state level as new integrin-ligand bond formation continued during cell spreading, ERK2 activity was decoupled from the integrin-ligand stimulus and decayed back to a basal level. Accordingly, we propose different functional metrics for representing these two disparate dynamic signals: the steady-state tyrosine phosphorylation level for FAK and the integral of the pulse response for ERK2. These measures of FAK and ERK2 activity were found to correlate with short term cell-substratum adhesivity, indicating that signaling via FAK and ERK2 is proportional to the number of integrin-fibronectin bonds.
由于整合素介导的信号是通过一种物理结构和协同生化网络进行传递的，而该网络的特性尚未明确界定，因此细胞外整合素 - 配体结合事件与关键细胞内反应之间的定量关系仍知之甚少。我们通过量化不同α(5)β(1)表达水平和基质纤连蛋白密度下CHO细胞中整合素介导的FAK和ERK2反应来着手解决这一问题。将细胞接种在纤连蛋白包被的表面上会引发一种瞬时、双相的ERK2反应，其幅度和动力学取决于整合素 - 配体结合特性。对于低纤连蛋白密度，ERK2活性最初以与整合素 - 配体键数量成比例的速率增加，而脱敏速率与整合素和纤连蛋白的量无关，但与ERK2活性水平成比例，指数衰减常数为0.3（±0.08）min(-1)。与ERK2激活时间进程不同，FAK磷酸化遵循表面上不同的时间进程。然而，对这两种信号早期动力学的分析表明它们是相关的。FAK和ERK2信号产生的初始速率对纤连蛋白表面密度表现出相似的依赖性，两种速率均随纤连蛋白量单调增加，直到在高纤连蛋白密度下达到饱和。由于对整合素 - 配体键形成的这种相似的初始速率依赖性，它们时间进程的差异归因于这些信号反馈调节的差异。在细胞铺展过程中，随着新的整合素 - 配体键形成持续，FAK磷酸化增加到稳态水平，而ERK2活性与整合素 - 配体刺激解耦并衰减回基础水平。因此，我们提出了不同的功能指标来表示这两种不同的动态信号：FAK的稳态酪氨酸磷酸化水平和ERK2脉冲反应的积分。发现这些FAK和ERK2活性测量值与短期细胞 - 基质粘附性相关，表明通过FAK和ERK2的信号传导与整合素 - 纤连蛋白键的数量成正比。
