A Rapid and Sensitive Quantitative Kinase Activity Assay Using a Convenient 96-Well Format

Asthagiri, Anand R.; Horwitz, Alan F.; Lauffenburger, Douglas A.

doi:10.1006/abio.1999.4055

Cited by 21 publications

(18 citation statements)

References 9 publications

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“…ERK2 Kinase Activity and Phosphorylation Assay-ERK2 kinase activity was measured using a sensitive in vitro assay performed in a 96-well format as described previously (25). Briefly, anti-ERK2 antibody was coated on the surface of Reacti-Bind TM protein A-coated wells (Pierce) by incubating wells with 10 g/ml sc-154 antibody overnight at 4 o C. After the wells were washed, 25 g of cell lysate was incubated for 3 h at 4 o C. To measure background, an extra well was incubated with just lysis buffer and was carried through the assay in the same manner as other samples.…”

Section: Methodsmentioning

confidence: 99%

“…In total, this involved ERK2 activity measurement of ϳ60 samples per trial. To handle such a large quantity of samples, we developed and utilized a modified microtiter ERK2 activity assay that allowed for convenient and concurrent processing of multiple samples (25). Using this method, the measured time course of ERK2 activity revealed quantitative variations in response to changes in integrin-ligand binding properties (Fig.…”

Section: Magnitude and Kinetics Of Erk2 Response Depend On Integrin-lmentioning

confidence: 99%

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

Asthagiri¹,

Nelson²,

Horwitz³

et al. 1999

Journal of Biological Chemistry

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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...

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Section: Methodsmentioning

confidence: 99%

Section: Magnitude and Kinetics Of Erk2 Response Depend On Integrin-lmentioning

confidence: 99%

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

Asthagiri¹,

Nelson²,

Horwitz³

et al. 1999

Journal of Biological Chemistry

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“…3,15 Briefly, HUVECs were lysed in ice-cold 50 mM Tris-HCl buffer pH 7.5 containing 150 mM NaCl, 2 mM EGTA, 1 mM DTT, 20 mM sodium pyrophosphate, 30 mM sodium fluoride, 200 lM sodium orthovanadate, 1 mM benzamidine, 1% triton X-100, and 10 lL mL À1 protease inhibitor cocktail. Cell debris was cleared by centrifuging at 130009g for 15 min at 4°C.…”

Section: Immunoprecipitation and Kinase Assaysmentioning

confidence: 99%

Substrates Elicit Different Patterns of Intracellular Signaling Which in Turn Cause Differences in Cell Adhesion

2010

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Although intracellular signal transduction is relatively well understood, how the cells can distinguish among thousands of micro-environments is not. This study proposes that a systems level view of intracellular signaling is necessary to interpret differences in cell interactions with different substrates. Human umbilical vein endothelial cells were exposed to 10 substrates (9 adsorbed extracellular matrix proteins and uncoated polystyrene), and the activities of 4 intracellular signaling kinases were quantified for cells on each substrate as a function of time. Principal component analysis demonstrates that each substrate elicits a different pattern of signaling. Mean activity of the 4 kinases can distinguish 44 of 45 possible pair-wise comparisons among the substrates. Partial Least Squares Regression Analysis is used to hypothesize causal relationships between signaling activity and cell adhesion or b 1 -integrin expression. Inhibition studies generally confirm that ERK (extracellular signalrelated kinase) and JNK (c-Jun N-terminal kinase) cause increased adhesion and b 1 -integrin expression. Inhibition of IKK (IjB kinase), on the other hand, showed no statistically significant effect on b 1 -integrin expression and led to significant decreases in cell adhesion-confirming a causal link but opposite of the hypothesized relationship (that inhibition of IKK would increase adhesion).

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“…We have found this double stain to be the most sensitive, quantitatively reproducible measure of apoptosis in HT-29 cells. 3 When LY294002 was added 1 h before cytokine addition and tonically maintained to block Akt activity over the entire time course, a dramatic increase in TNF-induced cell death was observed (Fig. 8B, red).…”

Section: Fig 3 Purification Of Endogenous Kinases Is Linear On Antimentioning

confidence: 99%

A High-throughput Quantitative Multiplex Kinase Assay for Monitoring Information Flow in Signaling Networks

Janes¹,

Albeck²,

Peng³

et al. 2003

Molecular & Cellular Proteomics

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To treat complex human diseases effectively, a systemslevel approach is needed to understand the interplay of environmental cues, intracellular signals, and cellular behaviors that underlie disease states. This approach requires high-throughput, multiplex techniques that measure quantitative temporal variations of multiple protein activities in the intracellular signaling network. Here, we describe a single microtiter-based format that simultaneously quantifies protein kinase activities in the phosphatidylinositol 3-kinase pathway (Akt), nuclear factor-B pathway (IKK), and three core mitogen-activated protein kinase pathways (ERK, JNK1, MK2). These parallel highthroughput assays are stringently linear, redundantly specific, reproducible, and sensitive compared with classical low-throughput techniques. When applied to a model of sepsis-induced colon epithelial apoptosis, this approach identified a late phase of Akt activity as a critical mediator of cell survival that quantitatively contributed to the efficacy of insulin as an anti-apoptotic cue. Thus, sampling parallel nodes in the intracellular signaling network identified part of the molecular mechanism underlying the efficacy of insulin in the treatment of human sepsis. Molecular & Cellular Proteomics 2:463-473, 2003.Complex patterns of signal transduction arise when cells are exposed to combinations of extracellular cues that vary in onset, duration, origin, and synchrony. Cells process these cues through an interconnected network of multifunctional, redundant molecules to elicit a set of phenotypic responses that subsequently impact function at the cell, tissue, and organ level. In order to develop a molecular understanding of the complex pathophysiology underlying human diseases and utilize this information for prognosis and therapy, a systemslevel, network-biology approach should be applied to the signaling networks governing the relevant cell responses (1). This approach will require frequent temporal sampling of protein activity at critical nodes within parallel signaling pathways inside the cell in a quantitative manner to characterize the flow of information accurately. Such functional measurements are likely to be as valuable, or more valuable, than measurements of simple protein abundance. By quantitatively exploring the functional response of the signaling network to distinct extracellular cues and correlating these molecular events with phenotypic responses, one can construct predictive models of cue-signal and signal-response relationships.Evolving proteomic approaches to network biology have largely focused on measuring abundances of many proteins at only a few time points or under a limited number of experimental conditions (2). Complementary information on functional protein characteristics, such as enzyme activity, has been lacking in these systematic analyses, in large part because there do not exist quantitatively robust, high-throughput techniques that simultaneously measure multiple protein activities in cells. Initially, this type of dat...

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A Rapid and Sensitive Quantitative Kinase Activity Assay Using a Convenient 96-Well Format

Cited by 21 publications

References 9 publications

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

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

Substrates Elicit Different Patterns of Intracellular Signaling Which in Turn Cause Differences in Cell Adhesion

A High-throughput Quantitative Multiplex Kinase Assay for Monitoring Information Flow in Signaling Networks

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