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David S Lawrence

Publications and source records attributed to David S Lawrence.

At least 19 recordsLinked to original sources
PubMed2006

Light-regulated sampling of protein tyrosine kinase activity.

Protein tyrosine kinases serve as key mediators of signaling pathways, biochemical highways that control various aspects of cell behavior. Although fluorescent reporters of tyrosine kinases have been described, these species can suffer immediate phosphorylation upon exposure to the cellular milieu, thereby hindering a detailed analysis of kinase activity as a function of the cell cycle or exposure to environmental stimuli. The first example of a light-regulated tyrosine kinase reporter is described herein, which allows the investigator to control when kinase activity is sampled. In addition, the set of sensors created in this study contain different fluorophores, each with its own unique photophysical properties, thereby furnishing an array of choices that can be used in combination with other intracellular probes.

Cell Line↗
PubMed2006

Increased hypothalamic protein tyrosine phosphatase 1B contributes to leptin resistance with age.

Animals at advanced ages exhibit a reduction in central leptin sensitivity. However, changes in growth, metabolism, and obesity risk occur much earlier in life, particularly during the transition from youth to middle age. To determine when initial decreases in central leptin sensitivity occur, leptin-dependent suppression of food intake was tested in 8-, 12-, and 20-wk-old male, chow-fed Sprague Dawley rats. Intracerebroventricular leptin injection (3 microg) suppressed 24-h food intake in 8- and 12-wk-old rats (P < 0.05) but not 20-wk-old rats. To identify potential cellular mediators of this resistance, we focused on protein tyrosine phosphatase 1B (PTP1B), a recently described inhibitor of leptin signaling. PTP1B protein levels, as determined by Western blot, were significantly higher in mediobasal hypothalamic punches collected from 20-wk-old rats, compared with 8-wk-old rats (P < 0.05). When 20-wk-old rats were fasted for 24 h, levels of hypothalamic PTP1B decreased (P < 0.05), coincident with a restoration of leptin sensitivity. To directly test whether inhibition of PTP1B restores leptin sensitivity, 20-wk-old chow-fed rats were pretreated with a pharmacological PTP1B inhibitor 1 h before leptin, and 24-h food intake was recorded. As expected, leptin alone produced a small but nonsignificant reduction in food intake. However, pretreatment with the PTP1B inhibitor resulted in a marked improvement in leptin-dependent suppression of food intake (P < 0.05). These data are consistent with the hypothesis that increases in PTP1B contribute to hypothalamic leptin resistance as rats transition into middle age.

Aging↗
PubMed2006

Acquisition of a "Group A"-selective Src kinase inhibitor via a global targeting strategy.

A "global" strategy for the acquisition of selective high affinity inhibitors for the Src kinase subfamily of tyrosine kinases is described. Members of the Src family exhibit a strong amino acid sequence homology. However, recent studies have revealed differences in the relative spatial relationships of the three distinct protein-binding domains present in these enzymes. We have constructed an inhibitor, using an amalgamation of combinatorial methods and directed design, which simultaneously associates with the active site and an ancillary protein-binding region (SH2 domain). The inhibitor exhibits high inhibitory potency and selectivity for the Group A versus Group B subset of Src kinases.

Binding Sites↗
PubMed2006

Self-reporting fluorescent substrates of protein tyrosine kinases.

A new mechanistic principle by which protein tyrosine kinase substrates fluorescently report the introduction of a phosphate moiety has been developed. NMR was used to establish that tyrosine phosphorylation induces the disruption of pi-pi stacking interactions of the tyrosine moiety with a proximal fluorophore on the peptide substrate. We have demonstrated that (1) the peptide substrates described in this study are useful for a wide variety of different tyrosine kinases, (2) physiological concentrations of ATP can be employed (unlike the standard radioactive ATP kinase assays), thus providing a more realistic assessment of inhibitor potency, and (3) protein kinase self-activation can be observed in real-time.

Amino Acid Sequence↗
PubMed2006

Identification of phosphocaveolin-1 as a novel protein tyrosine phosphatase 1B substrate.

Protein tyrosine phosphatase 1B (PTP1B) is implicated in a number of signaling pathways including those mediated by insulin, epidermal growth factor (EGF), and the Src family kinases. The scaffolding protein caveolin-1 is also a participant in these pathways and is specifically phosphorylated on tyrosine 14, when these pathways are activated. Here, we provide evidence that PTP1B can efficiently catalyze the removal of the phosphoryl group from phosphocaveolin-1. Overexpression of PTP1B decreases tyrosine 14 phosphorylation in caveolin-1, while expression of the substrate-trapping mutant PTP1B/D181A causes the accumulation of phosphocaveolin-1 and prevents its dephosphorylation by endogenous PTPs. We further demonstrate that PTP1B physically associates with caveolin-1. Finally, we show that inhibition of PTP1B activity with a potent and specific small molecule PTP1B inhibitor blocks the PTP1B-catalyzed caveolin-1 dephosphorylation both in vitro and in vivo. Taken together, the results strongly suggest that caveolin-1 is a specific substrate for PTP1B. Identification of caveolin-1 as a PTP1B substrate represents an important new step in further understanding the signaling pathways regulated by PTP1B.

Animals↗
PubMed2005

The preparation and in vivo applications of caged peptides and proteins.

Cellular behavior, such as mitosis and motility, are controlled by both when and where specific intracellular signaling pathways are activated in response to environmental cues. Analogous temporally and spatially controlled events occur throughout the lifetime of an organism (e.g. embryogenesis). Consequently, reagents that can be switched on (or off) at any time or at any place in a cell, a tissue, or a living animal, represent the means by which the biochemical basis of spatially and temporally sensitive biological behavior can be evaluated. This review summarizes recent advances in the design and synthesis of light-activated ('caged') peptides and proteins as well as the application of these caged reagents to unanswered questions in biology.

Peptide Biosynthesis↗
PubMed2005

Signaling protein inhibitors via the combinatorial modification of peptide scaffolds.

Compounds that selectively interfere with protein-protein interactions are not only invaluable as biological reagents, but may ultimately serve as therapeutically useful drugs for the treatment of a wide variety of disease states. However, unlike active site directed inhibitors that bind to a relatively small, well-defined, hydrophobic pocket, reagents that disrupt protein-protein interactions must contend with a protein surface that is comparatively large, ill defined, and solvent exposed. We have developed a straightforward method for the acquisition of protein-protein interaction inhibitors. The library-based strategy starts with low affinity consensus sequence peptides, which are then transformed in a stepwise fashion into high affinity inhibitors. The approach has been used to create potent ligands for SH2 and SH3 domains, as well as powerful and highly selective inhibitors for protein kinases and phosphatases. The protocol is easily automated and therefore has the potential to be routinely applied, in a high throughput fashion.

Animals↗
PubMed2005

Phosphorylation-driven protein-protein interactions: a protein kinase sensing system.

A highly flexible protein kinase sensing system is described that furnishes severalfold changes in fluorescence in response to phosphorylation. A library of Src kinase peptide substrates was prepared that contained different environmentally sensitive fluorophores positioned at various sites on the active site directed sequence. Robust changes in fluorescent intensity were observed in the presence of a phosphotyrosine binding domain protein (Lck SH2 domain), which furnishes a hydrophobic environment for the fluorophore. This protein kinase sensing system has the advantages that the fluorescent indicator can be unobtrusively positioned on the peptide substrate, and that different environmentally sensitive fluorophores with distinct photophysical properties can be employed.

Aminobutyrates↗
PubMed2005

The role of protein-tyrosine phosphatase 1B in integrin signaling.

Protein-tyrosine phosphatase 1B (PTP1B) is a key negative regulator of insulin and leptin signaling and a novel therapeutic target for the treatment of type 2 diabetes, obesity, and other associated metabolic syndromes. Because PTP1B regulates multiple signal pathways and it can both enhance and antagonize a cellular event, it is important to establish the physiological relevance of PTP1B in these processes. In this study, we utilize potent and selective PTP1B inhibitors to delineate the role of PTP1B in integrin signaling. We show that down-regulation of PTP1B activity with small molecule inhibitors suppresses cell spreading and migration to fibronectin, increases Tyr(527) phosphorylation in Src, and decreases phosphorylation of FAK, p130(Cas), and ERK1/2. In addition, PTP1B "substrate-trapping" mutants bind Tyr(527)-phosphorylated Src and protect it from dephosphorylation by endogenous PTP1B. These results establish that PTP1B promotes integrin-mediated responses in fibroblasts by dephosphorylating the inhibitory pTyr(527) and thereby activating the Src kinase. We also show that PTP1B forms a complex with Src and p130(Cas), and that the proline-rich motif PPRPPK (residues 309-314) in PTP1B is essential for the complex formation. We suggest that the specificity of PTP1B for Src pTyr(527) is mediated by protein-protein interactions involving the docking protein p130(Cas) with both Src and PTP1B in addition to the interactions between the PTP1B active site and the pTyr(527) motif.

Amino Acid Motifs↗
PubMed2005

Acquisition of Fyn-selective SH3 domain ligands via a combinatorial library strategy.

A stepwise library-based strategy has been employed to acquire a potent ligand for the SH3 domain of Fyn, a Src kinase family member that plays a key role in T cell activation. The easily automated methodology is designed to identify potential interaction sites that circumscribe the protein/peptide binding region on the SH3 domain. The library protocol creates peptide/nonpeptide chimeras that are able to bind to these interaction sites that are otherwise inaccessible to natural amino acid residues. The peptide-derived lead and the Fyn-SH3 domain form a complex that exhibits a K(D) of 25 +/- 5 nM, approximately 1000-fold more potent than that displayed by the corresponding conventional peptide ligand. Furthermore, the lead ligand exhibits selectivity against SH3 domains derived from other Src kinases, in spite of a sequence identity of approximately 80%.

Amino Acid Sequence↗
PubMed2005

Small molecule approach to studying protein tyrosine phosphatase.

Understanding the function of protein tyrosine phosphatases (PTPs) is crucial to deciphering cellular signaling in higher organisms. Of the 100 putative PTPs in human genome, only a little is known about their precise biological functions. Thus establishing novel ways to study PTP function remains a top priority among researchers. Classical genetics and more recently the use of RNA interference (RNAi) for gene silencing remains a popular choice to study function. However, the one gene-one function hypothesis is now recognized as an oversimplified scenario, especially among the signaling proteins such as PTPs. Therefore, there is a need to understand gene function in an appropriate cellular context. Since proteins are the work horses of the cell, alteration of protein function by various means is a particularly attractive strategy. In this context, the chemical approach, where a small molecule is used to affect the function of the desired protein is increasingly being recognized as a method of choice. In this review, we describe how small molecules can be used to study the function of a prototypical PTP, PTP1B, which is a negative regulator in insulin signaling. This includes our initial strategies for finding the most potent and specific PTP1B inhibitor to date, synthesizing cell permeable analogues suitable for cellular studies, and using them to dissect the role of PTP1B in the insulin signaling pathway. This approach is potentially general and thus could be utilized to study the function of other PTPs.

Biological Assay↗
PubMed2004

Enhanced production of functional proteins from defective genes.

Nonsense mutations are associated with a host of genetic disorders. The protein products encoded by genes containing these mutations are either truncated or completely missing. Recently, members of the aminoglycoside family of antibiotics have been found to induce the ribosome to read past these inappropriately inserted stop codons, albeit at extremely modest efficiencies. We report herein a strategy that enhances the production of functional proteins from the corresponding mutated genes.

Aminoglycosides↗
PubMed2004

Cofilin promotes actin polymerization and defines the direction of cell motility.

A general caging method for proteins that are regulated by phosphorylation was used to study the in vivo biochemical action of cofilin and the subsequent cellular response. By acute and local activation of a chemically engineered, light-sensitive phosphocofilin mimic, we demonstrate that cofilin polymerizes actin, generates protrusions, and determines the direction of cell migration. We propose a role for cofilin that is distinct from its role as an actin-depolymerizing factor.

Actin Depolymerizing Factors↗
PubMed2004

A highly potent and selective PKCalpha inhibitor generated via combinatorial modification of a peptide scaffold.

A potent and highly selective inhibitor of protein kinase C alpha has been generated via the combinatorial modification of a consensus sequence peptide. The inhibitor displays a Ki of 800 pM versus variable peptide substrate and good selectivity versus other members of the PKC family, including PKCbeta (385-fold), PKCgamma (580-fold), PKCdelta (2730-fold), PKCepsilon (600-fold), PKCeta (1310-fold), PKCtheta (1210-fold), PKCiota (940-fold), and PKCzeta (640-fold). The parallel synthesis strategy employed is easily automated and straightforward to implement.

Adenosine Triphosphate↗
PubMed2004

Biosensors of protein kinase action: from in vitro assays to living cells.

Protein kinases, and the signal transduction pathways in which they participate, are now recognized to be medicinally attractive targets of opportunity. Inhibitors of the protein kinase family not only hold great promise as therapeutic agents, but are also of profound utility in the characterization of signaling pathways. The direct visualization of protein kinase activity in living cells provides a genuine assessment of the efficacy and selectivity of these inhibitors in a physiological setting. In addition, the ability to visualize the activity of a protein kinase in real time furnishes a direct measurement of the activation of specific signaling pathways in response to extracellular stimuli. We have developed two series of fluorescent substrates for protein kinase C (PKC) using a strategy that positions the reporter-group directly on the residue undergoing phosphorylation. The first series of PKC substrates is based, in part, on the Ca(+2) indicators developed by Tsien and his collaborators during the 1980s. In this case, phosphorylation of the substrate creates a divalent metal ion binding site. Upon metal ion coordination, a fluorescence change transpires via a mechanism analogous to that described for the Ca(+2) indicators. The second series of PKC sensors was identified via the preparation and subsequent screen of a library of fluorescently-labeled PKC peptide substrates. The lead derivative displays a phosphorylation-induced fluorescence change that allows the visualization of real-time PKC activity in both cell lysates and living cells. Furthermore, immunodepletion experiments demonstrate that the fluorescently-tagged peptide is selectively, if not exclusively, phosphorylated by the conventional PKCs. Both of the protein kinase biosensor strategies take advantage of the ease with which peptides can be modified to create libraries of structurally altered analogs. However, the inherent synthetic mutability of peptides is not just limited to library construction. For example, it may ultimately be possible to simultaneously monitor multiple protein kinases by affixing fluorophores with distinct photophysical properties to appropriately designed active site-directed peptides.

Amino Acid Sequence↗
PubMed2003

Cellular effects of small molecule PTP1B inhibitors on insulin signaling.

Protein tyrosine phosphatase 1B (PTP1B) is implicated as a negative regulator of insulin receptor (IR) signaling and a potential drug target for the treatment of type 2 diabetes and other associated metabolic syndromes. To further define the role of PTP1B in insulin signaling and to test the hypothesis that blocking the activity of PTP1B would augment the action of insulin, we prepared several cell permeable, potent and selective, small molecule PTP1B inhibitors, and evaluated their biological effects in several insulin sensitive cell lines. Our data indicate that PTP1B inhibitors bind to and colocalize with PTP1B on the surface of the endoplasmic reticulum and PTP1B exerts its negative effect on insulin signaling upstream of phosphatidylinositol 3-kinase and MEK1. Treatment of cells with PTP1B inhibitors, both in the presence and in the absence of insulin, markedly enhances IRbeta and IRS-1 phosphorylation, Akt and ERK1/2 activation, Glut4 translocation, glucose uptake, and Elk1 transcriptional activation and cell proliferation. These results indicate that small molecule inhibitors targeted to PTP1B can act as both insulin mimetics and insulin sensitizers. Taken together, our findings combined with results from PTP1B knockout, antisense, and biochemical studies provide strong evidence that PTP1B negatively regulates insulin signaling and that small molecule PTP1B inhibitors have the ability to potentiate and augment the action of insulin.

Animals↗
PubMed2003

A light-activated probe of intracellular protein kinase activity.

The first example of a photoactivated probe of intracellular enzymatic activity is described. The caged derivative of a fluorescent protein kinase C peptide-based sensor was prepared by modifying the free hydroxyl group of a phosphorylatable serine moiety with a photolabile appendage that blocks phosphoryl transfer. We have demonstrated that the caged sensor allows one to (1) sample PKC activity with exquisite temporal precision, (2) control the relative amount of active sensor available for phosphorylation, and (3) examine protein kinase activity at multiple time points.

Acetamides↗