Src specific activity in non-invasive (PZ-HVP-7, RWPE1), androgen-independent (CWR22Rv1), and invasive (DU145, PC3) cell lines.

<p>Src kinase specific activity was calculated by dividing Src activity (Fig. 2) by total Src protein content (Fig. 3). Src specific activity is significantly higher in aggressive than in non-cancer cell lines (P<0.0001). Error bars are SEM.</p

Src expression levels in non-invasive (PZ-HPV-7, RWPE1), androgen-independent (CWR22Rv1), and invasive (DU145, PC3) cell lines.

<p>(a) Prostate cell lysates were probed for total Src, pY416 Src and pY527 Src, where α-tubulin was used as the loading control. (b) Intensities of each band from the western blots were measured and normalized to the corresponding α-tubulin control, and then compared to cell line RWPE1 (RWPE1 as 1). Comparison between non-cancer (PZ-HPV-7, RPWE1) and aggressive cancer cell lines (CWR22Rv1, DU145, PC3) showed significant lowered levels of Src expression in the latter (p<0.001). Data are representative of at least three independent experiments and are mean with SEM.</p

Src-catalyzed phosphorylation rates, Src protein content, and Src phosphorylation status in the RWPE1-derived cell lines.

<p>Increasing invasive ability is plotted along the x-axis. (a) Grey bars are phosphorylation rates of peptide 1 by whole cell lysates (normalized by total protein content). White bars are phosphorylation rates by cell lysates due to Src kinase alone after subtracting non-Src background phosphorylation of 1. (b) Total Src content as determined by western blot analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048867#pone.0048867.s005" target="_blank">Fig. S5</a>) (c) Src kinase specific activity as assessed by measured Src activity (Fig. 6A) divided total Src protein content (Fig. 6B). (d) pY416 levels were derived from the band intensities in the western blots (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048867#pone.0048867.s004" target="_blank">Fig. S4</a>), normalized to the corresponding α-tubulin control, and then compared to cell line RWPE1 (RWPE1 as 1). Data are representative of at least three independent experiments and are shown as mean with SEM.</p

Assessment of Src kinase catalytic activity.

<p>(a) CE-LIF separation and visualization of the Src peptide substrate <b>1</b> and its chemically synthesized phosphorylated counterpart <b>2</b>. (b) Src kinase-catalyzed phosphorylation of peptide <b>1</b> as a function of time as assessed by CE-LIF.</p

Src kinase activity in non-invasive (PZ-HPV-7, RWPE1), invasive (DU145, PC3), and androgen-independent (CWR22Rv1) cell lines.

<p>Grey bars are phosphorylation rates of peptide 1 by whole cell lysates (normalized by total protein extract amount). White bars are phosphorylation rates by cell lysates due to Src kinase alone after subtracting non-Src background phosphorylation of 1. Comparison between non-cancer (PZ-HPV-7, RPWE1) and aggressive cancer cell lines (CWR22Rv1, DU145, PC3) showed significant lowered levels of Src kinase activity associated with the later (p<0.001). All experiments were performed at least in triplicate. Error bars are SEM.</p

Rational Design of a Dephosphorylation-Resistant Reporter Enables Single-Cell Measurement of Tyrosine Kinase Activity

Although peptide-based reporters of protein tyrosine kinase (PTK) activity have been used to study PTK enzymology <i>in vitro</i>, the application of these reporters to intracellular conditions is compromised by their dephosphorylation, preventing PTK activity measurements. Nonproteinogenic amino acids may be utilized to rationally design selective peptidic ligands by accessing greater chemical and structural diversity than is available using the native amino acids. We describe a peptidic reporter that, upon phosphorylation by the epidermal growth factor receptor (EGFR), is resistant to dephosphorylation both <i>in vitro</i> and <i>in cellulo</i>. The reporter contains a conformationally constrained phosphorylatable moiety (7-(<i>S</i>)-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) in the place of l-tyrosine and is efficiently phosphorylated in A431 epidermoid carcinoma cells. Dephosphorylation of the reporter occurs 3 orders of magnitude more slowly compared with that of the conventional tyrosine-containing reporter

Multicolor Monitoring of the Proteasome’s Catalytic Signature

The proteasome, a validated anticancer target, participates in an array of biochemical activities, which range from the proteolysis of defective proteins to antigen presentation. We report the preparation of biochemically and photophysically distinct green, red, and far-red real-time sensors designed to simultaneously monitor the proteasome’s chymotrypsin-, trypsin-, and caspase-like activities, respectively. These sensors were employed to assess the effect of simultaneous multiple active site catalysis on the kinetic properties of the individual subunits. Furthermore, we have found that the catalytic signature of the proteasome varies depending on the source, cell type, and disease state. Trypsin-like activity is more pronounced in yeast than in mammals, whereas chymotrypsin-like activity is the only activity detectable in B-cells (unlike other mammalian cells). Furthermore, chymotrypsin-like activity is more prominent in transformed B cells relative to their counterparts from healthy donors

Measurement of Protein Kinase B Activity in Single Primary Human Pancreatic Cancer Cells

An optimized peptide substrate was used to measure protein kinase B (PKB) activity in single cells. The peptide substrate was introduced into single cells, and capillary electrophoresis was used to separate and quantify nonphosphorylated and phosphorylated peptide. The system was validated in three model pancreatic cancer cell lines before being applied to primary cells from human pancreatic adenocarcinomas propagated in nude mice. As measured by phosphorylation of peptide substrate, each tumor cell line exhibited statistically different median levels of PKB activity (65%, 21%, and 4% phosphorylation in PANC-1 (human pancreatic carcinoma), CFPAC-1 (human metastatic ductal pancreatic adenocarcinoma), and HPAF-II cells (human pancreatic adenocarcinoma), respectively) with CFPAC-1 cells demonstrating two populations of cells or bimodal behavior in PKB activation levels. The primary cells exhibited highly variable PKB activity at the single cell level, with some cells displaying little to no activity and others possessing very high levels of activity. This system also enabled simultaneous characterization of peptidase action in single cells by measuring the amount of cleaved peptide substrate in each cell. The tumor cell lines displayed degradation rates statistically similar to one another (0.02, 0.06, and 0.1 zmol pg^–1 s^–1, for PANC-1, CFPAC-1, and HPAF-II cells, respectively) while the degradation rate in primary cells was 10-fold slower. The peptide cleavage sites also varied between tissue-cultured and primary cells, with 5- and 8-residue fragments formed in tumor cell lines and only the 8-residue fragment formed in primary cells. These results demonstrate the ability of chemical cytometry to identify important differences in enzymatic behavior between primary cells and tissue-cultured cell lines

Fluorophore Assisted Photolysis of Thiolato-Cob(III)alamins

Cobalamins are known to react with thiols to yield stable β-axial Co^III–S bonded thiolato-cobalamin complexes. However, in stark contrast to the Co–C bond in alkylcobalamins, the photolability of the Co–S bond in thiolato-cobalamins remains undetermined. We have investigated the photolysis of <i>N</i>-acetylcysteinyl cob­(III)­alamin at several wavelengths within the ultraviolet and visible spectrum. To aid in photolysis, we show that attaching fluorophore “antennae” to the cobalamin scaffold can improve photolytic efficiency by up to an order of magnitude. Additionally, electron paramagnetic resonance confirms previous conjectures that the photolysis of thiolato-cobalamins at wavelengths as long as 546 nm produces thiyl radicals

B₁₂-Mediated, Long Wavelength Photopolymerization of Hydrogels

Medical hydrogel applications have expanded rapidly over the past decade. Implantation in patients by noninvasive injection is preferred, but this requires hydrogel solidification from a low viscosity solution to occur in vivo via an applied stimuli. Transdermal photo-cross-linking of acrylated biopolymers with photoinitiators and lights offers a mild, spatiotemporally controlled solidification trigger. However, the current short wavelength initiators limit curing depth and efficacy because they do not absorb within the optical window of tissue (600–900 nm). As a solution to the current wavelength limitations, we report the development of a red light responsive initiator capable of polymerizing a range of acrylated monomers. Photoactivation occurs within a range of skin type models containing high biochromophore concentrations