An improved cerulean fluorescent protein with enhanced brightness and reduced reversible photoswitching.

Cyan fluorescent proteins (CFPs), such as Cerulean, are widely used as donor fluorophores in Förster resonance energy transfer (FRET) experiments. Nonetheless, the most widely used variants suffer from drawbacks that include low quantum yields and unstable flurorescence. To improve the fluorescence properties of Cerulean, we used the X-ray structure to rationally target specific amino acids for optimization by site-directed mutagenesis. Optimization of residues in strands 7 and 8 of the ß-barrel improved the quantum yield of Cerulean from 0.48 to 0.60. Further optimization by incorporating the wild-type T65S mutation in the chromophore improved the quantum yield to 0.87. This variant, mCerulean3, is 20% brighter and shows greatly reduced fluorescence photoswitching behavior compared to the recently described mTurquoise fluorescent protein in vitro and in living cells. The fluorescence lifetime of mCerulean3 also fits to a single exponential time constant, making mCerulean3 a suitable choice for fluorescence lifetime microscopy experiments. Furthermore, inclusion of mCerulean3 in a fusion protein with mVenus produced FRET ratios with less variance than mTurquoise-containing fusions in living cells. Thus, mCerulean3 is a bright, photostable cyan fluorescent protein which possesses several characteristics that are highly desirable for FRET experiments.

Glucagon-like peptide 1 stimulates post-translational activation of glucokinase in pancreatic beta cells.

Glucagon-like peptide 1 (GLP-1) potentiates glucose-stimulated insulin secretion from pancreatic ß cells, yet does not directly stimulate secretion. The mechanisms underlying this phenomenon are incompletely understood. Here, we report that GLP-1 augments glucose-dependent rises in NAD(P)H autofluorescence in both ßTC3 insulinoma cells and islets in a manner consistent with post-translational activation of glucokinase (GCK). GLP-1 treatment increased GCK activity and enhanced GCK S-nitrosylation in ßTC3 cells. A 2-fold increase in S-nitrosylated GCK was also observed in mouse islets. Furthermore, GLP-1 activated a FRET-based GCK reporter in living cells. Activation of this reporter was sensitive to inhibition of nitric-oxide synthase (NOS), and incorporating the S-nitrosylation-blocking V367M mutation into this sensor prevented activation by GLP-1. GLP-1 potentiation of the glucose-dependent increase in islet NAD(P)H autofluorescence was also sensitive to a NOS inhibitor, whereas NOS inhibition did not affect the response to glucose alone. Expression of the GCK(V367M) mutant also blocked GLP-1 potentiation of the NAD(P)H response to glucose in ßTC3 cells, but did not significantly affect metabolism of glucose in the absence of GLP-1. Co-expression of WT or mutant GCK proteins with a sensor for insulin secretory granule fusion also revealed that blockade of post-translational GCK S-nitrosylation diminished the effects of GLP-1 on granule exocytosis by ∼40% in ßTC3 cells. These results suggest that post-translational activation of GCK is an important mechanism for mediating the insulinotropic effects of GLP-1.

Naturally occurring glucokinase mutations are associated with defects in posttranslational S-nitrosylation.

Posttranslational activation of glucokinase (GCK) through S-nitrosylation has been recently observed in the insulin-secreting pancreatic beta-cell; however, the function of this molecular mechanism in regulating the physiology of insulin secretion is not well understood. To more fully understand the function of posttranslational regulation of GCK, we examined two naturally occurring GCK mutations that map to residues proximal to the S-nitrosylated cysteine and cause mild fasting hyperglycemia (maturity-onset diabetes of the young; subtype glucokinase). The kinetics of recombinantly generated GCK-R369P and GCK-V367M were assessed in vitro. The GCK-R369P protein has greatly reduced catalytic activity (relative activity index 0.05 vs. 1.00 for wild type), whereas the GCK-V367M has near normal kinetics (relative activity index 1.26 vs. 1.00 for wild type). Quantitative imaging and biochemical assays were used to assess the effect of these mutants on the metabolic response to glucose, GCK activation, and S-nitrosylation of GCK in betaTC3 insulinoma cells. Expression of either mutant in betaTC3 cells did not affect the metabolic response to 5 mM glucose. However, expression of either mutant blocked the effects of insulin on glucose-stimulated nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate reduction, suggesting defects in posttranslational regulation of GCK. Each of these mutations blocked GCK activation, and prevented posttranslational cysteine S-nitrosylation. Our findings link defects in hormone-regulated GCK S-nitrosylation to hyperglycemia and support a role for posttranslational regulation of GCK S-nitrosylation as a vital regulatory mechanism for glucose-stimulated insulin secretion.

Quantitative analysis of protein-lipid interactions using tryptophan fluorescence.

The fluorescent properties of the amino acid tryptophan make it a useful tool for fluorometric assays. Because tryptophan fluorescence is remarkably sensitive to the polarity of the environment, it can be used to determine the affinity of tryptophan-containing peptides for phospholipid vesicles of varying compositions. Here, we describe a method for using tryptophan fluorescence to determine the binding affinities of peptides derived from the proteins Raf-1 and KSR-1 to small unilamellar vesicles containing phosphatidic acid. The method can be extrapolated to measure the binding of other tryptophan-containing peptides or proteins to lipid vesicles.

TIG3: a regulator of type I transglutaminase activity in epidermis.

Keratinocytes undergo a process of terminal cell differentiation that results in the construction of a multilayered epithelium designed to produce a structure that functions to protect the body from dehydration, abrasion and infection. These protective properties are due to the production of a crosslinked layer of protein called the cornified envelope. Type I transglutaminase (TG1), an enzyme that catalyzes the formation of epsilon-(gamma-glutamyl)lysine bonds, is the key protein responsible for generation of the crosslinks. The mechanisms that lead to activation of transglutaminase during terminal differentiation are not well understood. We have identified a protein that interacts with TG1 and regulates its activity. This protein, tazarotene-induced gene 3 (TIG3), is expressed in the differentiated layers of the epidermis and its expression is associated with transglutaminase activation and cornified envelope formation. We describe a novel mechanism whereby TIG3 regulates TG1 activity.

Role of phosphatidic acid in the coupling of the ERK cascade.

The production of phosphatidic acid plays a crucial role in the activation of the ERK cascade. This role was linked to the binding of phosphatidate to a specific polybasic site within the kinase domain of Raf-1. Here we show that phosphatidate promotes ERK phosphorylation in intact cells but does not activate Raf in vitro. The kinase suppressor of Ras (KSR) contains a sequence homologous to the phosphatidate binding site of Raf-1. Direct binding of phosphatidate to synthetic peptides derived from the sequences of the binding domains of Raf-1 and KSR was demonstrated by spectroscopic techniques. The specificity of these interactions was confirmed using synthetic lipids and mutated peptides in which the core of the phosphatidic acid binding domain was disrupted. Insulin and exogenous dioleoyl phosphatidate induced a rapid translocation of a mouse KSR1-EGFP construct to the plasma membrane of HIRcB cells. Mutation of two arginines located in the core of the putative phosphatidate binding site abolished dioleoyl phosphatidate- and insulin-induced translocation of KSR1. Overexpression of the mutant KSR1 in HIRcB cells inhibited insulin-dependent MEK and ERK phosphorylation. The addition of dioleoyl phosphatidate or insulin increased the co-localization of KSR1 and H-Ras and promoted the formation of plasma membrane patches enriched in both proteins and phosphatidic acid. These results, in conjunction with our previous work, suggest the formation of phosphatidate-enriched membrane microdomains that contain all components of the ERK cascade. We propose that these domains act as molecular scaffolds in the coupling of signaling events.

Activation of PKCdelta and p38delta MAPK during okadaic acid dependent keratinocyte apoptosis.

There is substantial interest in identifying agents that differentially activate keratinocyte differentiation versus apoptosis. Okadaic acid (OA) is a tumor promoter in mouse skin that also stimulates apoptosis of murine keratinocytes. OA also enhances human keratinocyte differentiation; however, the impact of OA treatment on apoptosis in these cells has not been examined. We show that OA promotes normal human keratinocyte apoptosis as evidenced by increased accumulation of cells having sub-G1/S DNA content, decreased mitochondrial integrity, increased annexin V binding, increased cytoplasmic cytochrome c level, and increased procaspase 3 and PARP cleavage. Cyclin A, cyclin D1, cdk2, cdk4, p53 and p21 levels are reduced. These changes are associated with release of the PKCdelta catalytic domain and increased phosphorylation of PKCdelta-T(505)-responses consistent with PKCdelta activation. In contrast, phosphorylation of PKCdelta-Y(311) is not increased. The apoptotic response is enhanced in OA treated cells in the presence of p38delta, a PKCdelta target. OA treatment selectively activated p38delta, and OA-dependent apoptosis is not inhibited by treatment with the p38alpha/beta inhibitor, SB203580. These findings are consistent with the idea that the response is mediated by p38delta. Our data indicate that OA is an agent that regulates both keratinocyte differentiation and apoptosis, and that this regulation is mediated via activation of a PKCdelta/p38delta signaling cascade.