Quantitative Relationship among Integrin-Ligand Binding, Adhesion, and Signaling via Focal Adhesion Kinase and Extracellular Signal-regulated Kinase 2

Abstract

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 ␣ 5 ␤ 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.Integrins are adhesion receptors that not only provide the mechanical link between the cell and the extracellular matrix (ECM) 1 that is essential for adhesion, spreading, and migration (1, 2), but also generate intracellular signals that affect multiple cell functions (3-5). Altered cell behavior due to aberrant regulation of these signals results in pathologies, such as cancer, in which loss of integrin signaling-based control of cell cycle progression leads to anchorage-independent cell growth and tumor formation (6). Because of these significant and wideranging regulatory roles, modulating integrin-mediated signals may provide powerful targets for disease therapy. Furthermore, since integrins interface cells to biomaterials, biomimetic surfaces may be designed to instigate appropriate integrin-mediated signals to elicit desired cell behavior on...

Integrin-mediated signalling through the MAP-kinase pathway

“…L t ), thus requiring a high assumed receptor density. Furthermore, the linear correlation between the ligand density and initial rates of FAK and ERK activation observed by Asthagiri et al [20] was also seen in simulations (Fig. 3b).…”

“…Asthagiri et al [20] have shown that activation of ERK by adhesion occurs before maximal FAK activation.…”

“…Fibronectin density is one of the most readily controllable experimental parameters in the system and has been shown to affect both the magnitude and kinetics of ERK activation [20]. As ligand density increases, the magnitude of the ERKPP peak also increases, whereas the peak occurs faster.…”

“…The model successfully simulates experimental FAK and ERK activation time courses. Semi-quantitative data on FAK and ERK activation after adhesion [20] are used to verify the results of the model. After comparing simulated signals with experimental results, the pathways were explored to gain insight into specific characteristics that contribute to ERK activation dynamics.…”

Rapid alterations in cortical protein profiles underlie spontaneous sleep and wake bouts

“…1C,D). As changes in protein phosphorylation occur within minutes and are associated with alterations in cellular activities [Shaw et al, 1995;Asthagiri et al, 1999;King et al, 2006;Morii et al, 2006;Nunez Rodriguez et al, 2006], our results provide evidence that such alterations occur within the time frames associated with individual sleep-wake bouts in rats [Timo-Iaria et al, 1970;Tobler and Borbely, 1986]. In addition, these data are consistent with previous reports that examined regional phosphorylated protein expression by immunohistochemical methods [Cirelli and Tononi, 1998], levels of specific phosphoproteins [Guan et al, 2004;Basheer et al, 2005;Naidoo et al, 2005;Bandyopadhya et al, 2006;Davis et al, 2006], or enzymatic activities [Ramanathan et al, 2002;Mackiewicz et al, 2003;Naidoo et al, 2005] following SD.…”