10 research outputs found
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<sup>–1</sup> s<sup>–1</sup>, 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<sup>III</sup>–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 cobÂ(III)Âalamin 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
B<sub>12</sub>-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