Biomedical subjects

A R Asthagiri

Publications and source records attributed to A R Asthagiri.

6 recordsLinked to original sources

PubMed2001

A role for NF-kappa B in the induction of beta-R1 by interferon-beta.

Previous experiments have suggested that induction of the beta-R1 gene by interferon (IFN)-beta required transcription factor ISGF-3 (IFN-stimulated gene factor-3) and an additional component. We now provide evidence that nuclear factor-kappaB (NF-kappaB) can serve as this component. Site-directed mutagenesis of an NF-kappaB binding site in the beta-R1 promoter or over-expression of an IkappaBalpha super-repressor abrogated IFN-beta-mediated induction of a beta-R1 promoter-reporter. IFN-beta treatment did not augment abundance of NF-kappaB but did lead to phosphorylation of the p65 NF-kappaB subunit. It is proposed that IFN-beta-mediated enhancement of the transactivation competence of NF-kappaB components is required for inducible transcription of the beta-R1 promoter. These results provide a novel insight into the role of NF-kappaB in the transcriptional response to IFN-beta.

Binding Sites↗

PubMed2000

Bioengineering models of cell signaling.

Strategies for rationally manipulating cell behavior in cell-based technologies and molecular therapeutics and understanding effects of environmental agents on physiological systems may be derived from a mechanistic understanding of underlying signaling mechanisms that regulate cell functions. Three crucial attributes of signal transduction necessitate modeling approaches for analyzing these systems: an ever-expanding plethora of signaling molecules and interactions, a highly interconnected biochemical scheme, and concurrent biophysical regulation. Because signal flow is tightly regulated with positive and negative feedbacks and is bidirectional with commands traveling both from outside-in and inside-out, dynamic models that couple biophysical and biochemical elements are required to consider information processing both during transient and steady-state conditions. Unique mathematical frameworks will be needed to obtain an integrated perspective on these complex systems, which operate over wide length and time scales. These may involve a two-level hierarchical approach wherein the overall signaling network is modeled in terms of effective "circuit" or "algorithm" modules, and then each module is correspondingly modeled with more detailed incorporation of its actual underlying biochemical/biophysical molecular interactions.

Algorithms↗

PubMed2000

The role of transient ERK2 signals in fibronectin- and insulin-mediated DNA synthesis.

Both the extracellular matrix and growth factors jointly regulate cell cycle progression via a complex network of signaling pathways. Applying quantitative assays and analysis, we demonstrate here that concurrent stimulation of Chinese hamster ovary (CHO) cells with fibronectin (Fn) and insulin elicits a DNA synthesis response that reveals a synergy far more complex than a simple additive enhancement of response magnitude. CHO cell adhesion to higher Fn density shifts the sensitivity of the DNA synthesis response to insulin concentration from smoothly graded to sharply 'switch-like' and dramatically decreases the insulin concentration required for half-maximal response by about 1000-fold. Conversely, treatment with insulin has a milder and less complex effect on the response to varying Fn concentrations. Governing this DNA synthesis response is a common requirement for a transient, cell area-independent extracellular signal-regulated kinase 2 (ERK2) signal. Moreover, we show that the time-integrated value of this 'pulse' signal provides an appropriate metric for quantifying the dependence of DNA synthesis on the degree of ERK2 activation. Indeed, in the absence of insulin, the adhesion-mediated response is linearly proportional to ERK2 activation over a broad range of stimulatory Fn and MEK inhibitor amounts. However, in the presence of both Fn and insulin, total integrated ERK2 activity (the sum of Fn- and insulin-mediated signals) no longer serves as a predictor of DNA synthesis, demonstrating that the signaling crosstalk underlying response synergism does not converge at ERK2 activation. Instead, adhesion to higher Fn density enhances insulin stimulation of DNA synthesis, not by increasing insulin-mediated ERK2 activation, but via parallel elevation of at least one other insulin-mediated signal such as IRS-1 phosphorylation.

Animals↗

PubMed1999

Quantitative relationship among integrin-ligand binding, adhesion, and signaling via focal adhesion kinase and extracellular signal-regulated kinase 2.

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.

Animals↗

PubMed1999

A rapid and sensitive quantitative kinase activity assay using a convenient 96-well format.

Activation of protein kinases in response to growth factor and extracellular matrix stimulation has been implicated in regulating a number of cell functions including differentiation, gene expression, migration, and proliferation. An improved quantitative assay for measuring protein kinase activity is crucial to the detailed study of this important category of signaling proteins and their role in regulating cell behavior. We describe a modified in vitro kinase activity assay that is both sensitive and quantitative. It offers several advantages when compared to the traditional immunoprecipitation/kinase assay: (i) high sensitivity that reduces the required amount of cell lysate by an order of magnitude, (ii) an immunoseparation technique utilizing antibody immobilization onto the surface of microtiter wells that replaces the cumbersome immunoprecipitation method, (iii) a 96-well plate configuration that eases handling of multiple samples and increases throughput of the assay, and (iv) the use of 96-well filter plates that greatly reduces radioactive liquid waste generation. While we implement this technique in a case study for measuring the activity of extracellular signal-regulated kinase 2 (ERK2), this assay can be extended to studying other protein kinases by using an appropriate antibody and in vitro substrate for the kinase of interest.

Animals↗

PubMedDate not supplied

A computational study of feedback effects on signal dynamics in a mitogen-activated protein kinase (MAPK) pathway model.

Exploiting signaling pathways for the purpose of controlling cell function entails identifying and manipulating the information content of intracellular signals. As in the case of the ubiquitously expressed, eukaryotic mitogen-activated protein kinase (MAPK) signaling pathway, this information content partly resides in the signals' dynamical properties. Here, we utilize a mathematical model to examine mechanisms that govern MAPK pathway dynamics, particularly the role of putative negative feedback mechanisms in generating complete signal adaptation, a term referring to the reset of a signal to prestimulation levels. In addition to yielding adaptation of its direct target, feedback mechanisms implemented in our model also indirectly assist in the adaptation of signaling components downstream of the target under certain conditions. In fact, model predictions identify conditions yielding ultra-desensitization of signals in which complete adaptation of target and downstream signals culminates even while stimulus recognition (i.e., receptor-ligand binding) continues to increase. Moreover, the rate at which signal decays can follow first-order kinetics with respect to signal intensity, so that signal adaptation is achieved in the same amount of time regardless of signal intensity or ligand dose. All of these features are consistent with experimental findings recently obtained for the Chinese hamster ovary (CHO) cell lines (Asthagiri et al., J. Biol. Chem. 1999, 274, 27119-27127). Our model further predicts that although downstream effects are independent of whether an enzyme or adaptor protein is targeted by negative feedback, adaptor-targeted feedback can "back-propagate" effects upstream of the target, specifically resulting in increased steady-state upstream signal. Consequently, where these upstream components serve as nodes within a signaling network, feedback can transfer signaling through these nodes into alternate pathways, thereby promoting the sort of signaling cross-talk that is becoming more widely appreciated.

Animals↗