Stereospecific Interactions of Short-Chain 3-Deoxy-PIs
A R T I C L E S
Biolabs). The construction of pET28a PTEN has been described
previously;29Escherichia coli BL21 (DE3) cells were used as the
host for protein expression. PTEN protein was purified from the
bacterial extracts using a Qiagen Ni2+-nitrilotriacetic acid agarose
column according to the manufacturer protocol. Fractions of pure
PTEN protein (as judged using SDS-PAGE) were combined and
dialyzed against 100 mM Tris-HCl (pH 8.0). Protein concentration
was determined by Lowry assay.30
atmosphere at 95% humidity. Cells were cultured in the presence
or absence of the short-chain diC8PI compounds at the indicated
concentration. Cells were also incubated with 10 µM LY294002
and/or 20 nM wortmannin. At the appropriate time, 2-5 × 105
cells were collected by centrifugation at 400g for 8 min, washed
in FACS buffer [1× phosphate-buffered saline (PBS) containing
0.5% BSA and 0.01% sodium azide], and resuspended in FACS
buffer containing 5 µg/mL propidium iodide. Samples were
incubated on ice for 10 min and then analyzed by flow cytometry
using a BD FACSCanto flow cytometer with BD FACS Diva
software (BD Biosciences, San Jose, CA).
Preparation of Cell Extracts and Western Blot Analysis. Cells
were washed twice in PBS, incubated for 20 min at ∼108 cells/mL
in RIPA buffer containing 1 mM sodium orthovanadate, 1 mM
phenylmethylsulfonyl fluoride protein inhibitor cocktail, 1 mM
okadaic acid, 1 mM sodium fluoride, and 10 mM ꢀ-glycerophos-
phate, and subjected to three freeze/thaw cycles in dry ice. Lysates
were centrifuged at 14000g for 15 min to remove insoluble material,
and then protein (20 µg/lane) was separated by polyacrylamide-SDS
gel electrophoresis and transferred to a PVDF membrane. The
membrane was blocked for 1 h in TBS-T [20 mM Tris (pH 7.6),
137 mM NaCl, and 0.05% Tween-20] containing 5% nonfat dry
milk and then incubated at 4 °C overnight in TBS-T with 1 µg/mL
primary antibody (Ab). The membrane was washed several times
in TBS-T, incubated for 60 min with a 1:2500 dilution of anti-
rabbit IgG-coupled horseradish peroxidase Ab, and developed using
enhanced chemiluminescence. Bands for the different phosphory-
lated proteins were quantified by densitometric analysis using the
band corresponding to an extract from cells incubated with L-3-
deoxy-diC8PI as a control, since this compound had no effect on
the U937 cells.
PI3K Assay. PI3K (2 µg) was added to a reaction mixture (400
µL) containing 50 mM Tris-HCl (pH 7.5), 0.1 mg/mL bovine serum
albumin (BSA) (Sigma), 50 mM NaCl, 5 mM MgCl2, 2 mM ATP,
1 mM D-diC8PI and 0-3 mM 3-deoxy-diC8PI analogue. The
mixture was incubated at 22 °C for 3 h, and then the reaction was
stopped by addition of 10 mM EDTA. Phosphorylation of PI and
PI analogues was monitored by 31P NMR spectroscopy using the
phosphodiester resonance (δP 0.23 at pH 7.5) as the standard for
integration of the PI3K phosphomonoester product. The acquisition
conditions followed those previously used for PLC assays.31,32 The
product yield was quantified by comparing the phosphomonoester
resonance for D-diC8PI(3)P to the phosphodiester peak in the
1
absence of H decoupling.
PLC Assay. PLC activities toward diC8PI and the 3-deoxy-
diC8PI analogues were measured using 31P NMR spectroscopy after
incubation for fixed times, as described for other PI-specific PLC
enzymes.31,33 The assay buffer for PLCδ1 contained 50 mM Tris-
HCl, 2 mM substrate PI, and 0.5 mM CaCl2 (pH 7.5); the incubation
temperature was 28 °C. For the recombinant L. monocytogenes PLC,
the assay buffer was 50 mM Tris-HCl containing 0.5 mg/mL BSA
(pH 7.0), and the incubation temperature was 25 °C. Enzyme
(6.9-27.6 µg of PLCδ1 or 0.04-176 µg of L. monocytogenes PLC)
was added to each 200 µL sample containing 2 mM substrate, and
the incubation time was chosen to give less than 20% PI cleavage.
The reaction was stopped by addition of 200 µL of CHCl3 to the
sample. The cyclo-inositol-1,2-phosphate (cIP) and inositol-1-
phosphate [I(1)P] contents in the aqueous phase were quantified
from the 31P NMR spectrum using added glucose-6-phosphate as
an internal standard.
PTEN Assay. Phosphatase assays were carried out in 50 µL
assay buffer [100 mM Tris-HCl (pH 8.0), 2 mM EDTA, and 10
mM dithiothreitol (DTT)]. The phosphatase reaction was initiated
by adding ∼25 µg of purified PTEN (10 µM) and quenched by
adding malachite green reagent containing 1 M HCl after a 20 min
incubation at 37 °C.34 Comparison of observed A660 changes with
those for standard Pi samples was used to calculate the reaction
rate. In most of the assays, 0.5 mM D-diC8PI(3)P was used as the
substrate, and various concentrations (0.05-2 mM) of synthetic
short-chain PIs were added to test their effect on phosphatase
activity. For estimation of the Km value for D-diC8PI(3)P, assays
were carried out in 100 µL aliquots containing six different
concentrations (0.05-1.6 mM) of substrate. Most of the assays were
done at least in duplicate.
Results
Characterization of diC8PI Compounds. Three target en-
zymes that might be inhibited by (PI3K and PTEN) or degrade
(PLC) 3-deoxy-PI molecules are soluble enzymes whose natural
substrates are membrane constituents. However, in detailed
kinetic studies with synthetic short-chain phospholipid sub-
strates, these types of enzymes typically display a preference
for molecules aggregated into a micelle as opposed to monomers
in solution, a phenomenon termed “interfacial activation”. Both
PLC and PI3K exhibit this enhanced activity with interfacial
substrates/inhibitors.35,36 Since the short-chain 3-deoxy-diC8PI
molecules, unlike the previously examined synthetic 3-deoxy-
PI molecules with longer acyl chains,7,14 can exist as both
monomers and micelles in solution, we require information on
their physical states, in particular, values of the CMC (the
concentration above which micelles form) and rough estimates
of the sizes of the micelles they form. This information is also
critical for determining the distribution of the 3-deoxy-diC8PI
species in cells at concentrations where they may cause cell
death. All of the diC8PI derivatives used in this work have CMC
values between 0.4 and 0.7 mM in 100 mM Tris HCl (pH 8),
as measured by 31P line-width changes as a function of lipid
concentration (Table 1).
U937 Cell Growth and Incubation with PI Analogues. The
U937 human leukemic monocyte lymphoma cell line was obtained
from the American Type Culture Collection (Manassas, VA). U937
cells were maintained in RPMI 1640 medium supplemented with
10% fetal bovine serum, penicillin (100 units/mL), streptomycin
(100 µg/mL), 2-mercaptoethanol (50 µM), and glutamine (2 mM)
at cell densities of 0.5-1 × 106 cells/mL at 37 °C in a 5% CO2
To get a sense of the sizes and dynamics of these micelles,
two of the synthetic PIs, L-3-deoxy-diC8PI and L-3,5-dideoxy-
diC8PI, were examined at 3 mM using high-resolution 31P field
cycling. This is a novel technique that is very sensitive to the
aggregation state of the micelles.25,26 The L-isomers of the
deoxy-diC8PIs were chosen because they had CMC values
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