Ingrid Remy

PKB/Akt modulates TGF-beta signalling through a direct interaction with Smad3

Transforming growth factor β (TGF-β) has a major role in cell proliferation, differentiation and apoptosis in many cell types. Integration of the TGF-β pathway with other signalling cascades that control the same cellular processes may modulate TGF-β responses. Here we report the discovery of a new functional link between TGF-β and growth factor signalling pathways, mediated by a physical interaction between the serine-threonine kinase PKB (protein kinase B)/Akt and the transcriptional activator Smad3. Formation of the complex is induced by insulin, but inhibited by TGF-β stimulation, placing PKB–Smad3 at a point of convergence between these two pathways. PKB inhibits Smad3 by preventing its phosphorylation, binding to Smad4 and nuclear translocation. In contrast, Smad3 does not inhibit PKB. Inhibition of Smad3 by PKB occurs through a kinase-activity-independent mechanism, resulting in a decrease in Smad3-mediated transcription and protection of cells against TGF-β-induced apoptosis. Consistently, knockdown of the endogenous PKB gene with small-interfering RNA (siRNA) has the opposite effect. Our results suggest a very simple mechanism for the integration of signals arising from growth-factor- and TGF-β-mediated pathways.

A highly sensitive protein-protein interaction assay based on Gaussia luciferase

Protein-fragment complementation assays (PCAs) provide a general strategy to study the dynamics of protein-protein interactions in vivo and in vitro. The full potential of PCA requires assays that are fully reversible and sensitive at subendogenous protein expression levels. We describe a new assay that meets these criteria, based on the Gaussia princeps luciferase enzyme, demonstrating chemical reversal, and induction and inhibition of a key interaction linking insulin and TGFβ signaling.

Universal strategies in research and drug discovery based on protein-fragment complementation assays.

Changes in the interactions among proteins that participate in a biochemical pathway can reflect the immediate regulatory responses to intrinsic or extrinsic perturbations of the pathway. Thus, methods that allow for the direct detection of the dynamics of protein–protein interactions can be used to probe the effects of any perturbation on any pathway of interest. Here we describe experimental strategies — based on protein-fragment complementation assays (PCAs) — that can achieve this. PCA-based strategies can be used with or instead of traditional target-based drug discovery strategies to identify novel pathway-component proteins of therapeutic interest, to increase the quantity and quality of information about the actions of potential drugs, and to gain insight into the intricate networks that make up the molecular machinery of living cells.

[14] Detection of protein-protein interactions by protein fragment complementation strategies

Publisher Summary This chapter presents the basic concept of protein fragment complementation assays (PCAs) and how they are designed, with particular attention to the system developed based on murine dihydrofolate reductase (mDHFR). It then discusses several applications of the assay, including a simple, large-scale library-versus-library screening strategy in Escherichia coli . The implementation of mammalian assays is discussed, including applications to the quantitative detection of induced protein interactions and allosteric transitions in intact cells. Finally, the generality of the PCA strategy is demonstrated with examples of assays that are designed on the basis of other enzymes including glycinamide ribonucleotide transformylase, aminoglycoside kinase, and hygromycin B kinase.

Regulation of apoptosis by the Ft1 protein, a new modulator of protein kinase B/Akt.

ABSTRACT The serine/threonine kinase protein kinase B (PKB)/Akt plays a central role in many cellular processes, including cell growth, glucose metabolism, and apoptosis. However, the identification and validation of novel regulators or effectors is key to future advances in understanding the multiple functions of PKB. Here we report the identification of a novel PKB binding protein, called Ft1, from a cDNA library screen using a green fluorescent protein-based protein-fragment complementation assay. We show that the Ft1 protein interacts directly with PKB, enhancing the phosphorylation of both of its regulatory sites by promoting its interaction with the upstream kinase PDK1. Further, the modulation of PKB activity by Ft1 has a strong effect on the apoptosis susceptibility of T lymphocytes treated with glucocorticoids. We demonstrate that this phenomenon occurs via a PDK1/PKB/GSK3/NF-ATc signaling cascade that controls the production of the proapoptotic hormone Fas ligand. The wide distribution of Ft1 in adult tissues suggests that it could be a general regulator of PKB activity in the control of differentiation, proliferation, and apoptosis in many cell types.

Visualization of biochemical networks in living cells

Functional annotation of novel genes can be achieved by detection of interactions of their encoded proteins with known proteins followed by assays to validate that the gene participates in a specific cellular function. We report an experimental strategy that allows for detection of protein interactions and functional assays with a single reporter system. Interactions among biochemical network component proteins are detected and probed with stimulators and inhibitors of the network. In addition, the cellular location of the interacting proteins is determined. We used this strategy to map a signal transduction network that controls initiation of translation in eukaryotes. We analyzed 35 different pairs of full-length proteins and identified 14 interactions, of which five have not been observed previously, suggesting that the organization of the pathway is more ramified and integrated than previously shown. Our results demonstrate the feasibility of using this strategy in efforts of genomewide functional annotation.

Application of protein-fragment complementation assays in cell biology

We have developed a general experimental strategy that enables the quantitative detection of dynamic protein-protein interactions in intact living cells, based on protein-fragment complementation assays (PCAs). In this method, protein-protein interactions are coupled to refolding of enzymes from cognate fragments where reconstitution of enzyme activity acts as the detector of a protein interaction. Here we discuss the application of PCA to different aspects of cell biology.

Mapping Biochemical Networks With Protein-Fragment Complementation Assays

Cellular biochemical machineries, what we call pathways, consist of dynamically assembling and disassembling macromolecular complexes. Although our models for the organization of biochemical machines are derived largely from in vitro experiments, do they reflect their organization in intact, living cells? We have developed a general experimental strategy that addresses this question by allowing the quantitative probing of molecular interactions in intact, living cells. The experimental strategy is based on Protein fragment Complementation Assays (PCA), a method whereby protein interactions are coupled to refolding of enzymes from cognate fragments where reconstitution of enzyme activity acts as the detector of a protein interaction. A biochemical machine or pathway is defined by grouping interacting proteins into those that are perturbed in the same way by common factors (hormones, metabolites, enzyme inhibitors, etc.). In this chapter we review some of the essential principles of PCA and provide details and protocols for applications of PCA, particularly in mammalian cells, based on three PCA reporters, dihydrofolate reductase, green fluorescent protein, and β-lactamase.

Using the |[beta]|-lactamase protein-fragment complementation assay to probe dynamic protein|[ndash]|protein interactions

We have developed a general experimental strategy that enables the quantitative detection of dynamic protein–protein interactions in intact living cells, based on protein-fragment complementation assays (PCAs). In this method, protein interactions are coupled to refolding of enzymes from cognate fragments where reconstitution of enzyme activity acts as the detector of a protein interaction. We have described a number of assays with different reporter readouts, but of particular value to studies of protein interaction dynamics are assays based on enzyme reporters that catalyze the creation of products, thus taking advantage of the amplification of signal afforded. Here we describe protocols for one such PCA based on the enzyme TEM β-lactamase as a reporter in mammalian cells. The β-lactamase PCA consists of fusing complementary fragments of β-lactamase to two proteins of interest. If the proteins interact, the fragments are brought together and fold into active β-lactamase. Here we describe a protocol for this PCA that can be completed in a few hours, using two different substrates that are converted to fluorescent or colored products by β-lactamase.

Dynamic visualization of expressed gene networks

Cellular biochemical machineries, what we call pathways, consist of dynamically assembling and disassembling macromolecular complexes. While our models for the organization of biochemical machines are derived largely from in vitro experiments, do they reflect their organization in living cells? We have developed a general experimental strategy that addresses this question by allowing the quantitative probing of molecular interactions in intact living cells. The experimental strategy is based on protein fragment complementation assays (PCA), a method whereby protein interactions are coupled to refolding of enzymes from cognate fragments where reconstitution of enzyme activity acts as the detector of a protein interaction. A biochemical machine or pathway is defined by grouping interacting proteins into those that are perturbed in the same way by common factors (hormones, metabolites, enzyme inhibitors, etc). In this review, we describe how we go from descriptive to quantitative representations of biochemical networks at an individual to whole genome level and how our approach will lead ultimately to better descriptions of the biochemical machineries that underlie living processes. J. Cell. Physiol. 196: 419–429, 2003.