A Non-radioactive Assay to Determine Isoform Activation of PLD by
Phenylephrine in CCL39 Cells
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Danielle E. Rastedt, Mark A. Wallert, and Joseph J Provost
Department of Biosciences Minnesota State University Moorhead, Moorhead MN 56563
Abstract
Methods
Phospholipase D (PLD) is an enzyme found in the cells of higher
mammals and plants. The process of PLD acting on
phosphatidylcholine (PC) to produce phospatidic acid (PA) and
choline is important in cell signaling. PA acts as a bioactive lipid
activating a number of protein kinases and other effectors and can be
further metabolized to diacylglycerol, an activator of protein kinase C
(PKC). There are two isoforms of PLD, PLD1 and PLD2. PLD1
activity is activated by the small G proteins RhoA and ARF as well as
PKC, while PLD2 is constitutively active and can be stimulated by
ARF. There is great interest in understanding which isoforms is
activated by various hormones. Therefore, several different methods
have been developed to determine its enzymatic activity. The current
method used to determine enzymatic activity is an in vivo PLD assay
using radioactive lipids. Our plan is to use fluorescent labels to
measure PLD activity in a non-radioactive assay. The three types of
fluorescent lipids used in these experiments. Two free fatty acids 4,4difluoro-5-octyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic
acid
(BODIPY C8, C5); 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (BODIPY C1, C12); and 1-myristoyl-2-[, 12[(7-nitro-2-1, 3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3phosphocholine (NBD-PC). We found that both NBD-PC and BODIPY
C1, C12 but not BODIPY C8, C5 were incorporated into the membrane
as PC. Furthermore, there is a dose and time dependent manner in
the labeling of starved CCL39 fibroblasts. We plan to show which
PLD isoforms(s) is activated by stress hormones in CCL39 cells using
this technique.
Results
Separation: Solvent system- A separation of the free fatty acid 4,4difluoro-5-methyl-4-bora-3a,4a-diaza-s-inda-cene-3-dodecanoic acid
(BODIPY C1, C12), 2-(4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-sindacene-3-dodecanoy0-1-hexadecanoyl-sn-glycero-3phosphocholine (b-BODIPY 500/510 C12-HPC), 2-(4,4-difluoro-5methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoy0-1hexadecanoyl-sn-glycero-3-phosphate, diammonium salt ((b-BODIPY
500/510 C5 –HPA), and ptd BD BODIPY were tested. 15l of 0.1g/l
or 1g/l was applied to a thin layer chromatography. Four different
solvents were tested. Solvent 1- ethyl acetate:2,2,4 trimethyl pentane:
acetic acid: water (65:10:15:50; v/v). Solvent 2- chloroform: methanol:
water: acetic acid (45:45:10:1; v/v). Solvent 3- Chloroform: methanol:
water (63:34:3; v/v). Solvent 4- chloroform: methanol: water
(45:45:10; v/v). The solvents were used to develop the TLC plate The
fluorescent lipids were detected by densitometry using UV light on the
Gel Doc 1000 (Bio-Rad).
Sensitivity of Visualization- Appropriate amounts of 2-(4,4-difluoro5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoy0-1hexadecanoyl-sn-glycero-3-phosphate, diammonium salt ((b-BODIPY
500/510 C5 –HPA)were applied to a thin layer chromatography. The
solvent used to develop the TLC plate consisted of
chloroform/methanol/water (63:34:3; v/v). The fluorescent lipids were
detected by densitometry using UV light on the Gel Doc 1000 (BioRad). A further analysis of the detection of fluorescents was achieved
by scraping the silica from the TLC plate into microfuge tube. The
silica was rinsed with 0.3mL of methanol and then vortex, centrifuged,
and incubated at room temperature for 10 minutes. 200 l was
extracted from the solution and measured for fluorescents using the
plate reader.
Cell Culture- Chinese hamster lung fibroblasts (CCL39) were
cultured in attachment culture using Dulbecco’s modified Eagle
medium (DMEM, Sigma) and 10% heat-activated fetal bovine serum
(FBS, GibocBRL) 37oC in a 5% CO2 incubator. Cells were allowed to
grow to 60% confluence in 35mm dishes. Appropriate amounts of
resuspended lipids were added to each dish, and were allowed to
incubate overnight.
Fluorescent Lipid Analysis of Free Fatty Acids- Appropriate
amounts of fluorescent lipids were dried under nitrogen in sterile test
tube along with DMEM complete media. Lipids were then
resuspended by sonication, added to 35mm dishes containing CCL39
cells at 60% percent confluence, and allowed to incubate overnight.
At the end of incubation period, cells were rinsed with ice-cold
calcium magnesium free phospho-buffered saline (CMF-PBS). Lipids
were extracted and separated with methanol/chloroform/0.1M HCl
(1:1:1;v/v). The lower phase of the solution was extracted and dried
under nitrogen and lipids were resuspended in 30 l of
chloroform/methanol (2:1; v/v) and applied to a thin layer
chromatography. The solvent used to develop the TLC plate consisted
of chloroform/methanol/water (63:34:3; v/v). The fluorescent lipids
were detected by densitometry using UV light on the Gel Doc 1000
(Bio-Rad).
Conclusions
•
NHE1 Function/Regulation
Migration/Proliferative
Signal
ab
GPCR
RTK
EGFR
PE
aq
aq g
ß
GDP
GTP
GDP
?
Ras
GTP
PLCß1
PIP2
RhoA
DAG + IP3
?
PKCn
PLD
PC
DAG
P
g
b
PTK
Raf
PKCa
ROCK
SOS
P
GRB
MEK
P
ERK
P
RSK
P
LPA
a13
GTP
b
RhoGEF
g a13
GDP
GTP
RhoA
ERM
•
Future Studies:
Further studies will be needed to finish characterizing the
fluorescent lipids (solvents, labeling, sensitivity).
Examine the ability of PLD to catalyze its reaction with
Cytoskeletal Remodeling
pHi Homeostasis
Proliferation
Migration
Invasion
BODIPY PC in cells.
Reference
K. Meier, T.Gibbs, S. Knoepp, K. Ella. 1999. Expression of
Phospholipase D Isoforms in Mammalian Cells 200-211
Foster, D.A., Xu, L. 2003. Phospholipase D in Cell
Proliferation and Cancer. Molecular Cancer Research.1:
789-800
K. Ella, G. Meier, C. Bradshaw, K. Huffman, R. Spivey, K.
Meier. 1994. A Fluorescent Assay for Agonist-Activated
Phospholipase D in Mammalian Cell Extracts. Analytical
Biochemistry. 136-142.
GM. Jenkins, MA. Frohman. 2005. Phospholipase D: A Lipid
Review. Cell Molecular Life Science
ROCK
PLD
PA
GDP
NHE1
•
•
F-Actin
Focal Adhesion
Actin Stress Fibers
Cell Shape
Gq and G13 Activation of NHE1
•
Integrins
H+
Na+
•
The solvent that provides superior separation of free fatty
acids from phosphatidylcholine (PC) and phospatidic acid
(PA) is chloroform: methanol: water (63:34:3;v/v)
The fluorescent lipids were detected by densitometry using
UV light on the Gel Doc 1000 (Bio-Rad). Phospatidic acid
(PA) was only seen visually at higher doses.
BODIPY C1, C12 4,4-difluoro-5-methyl-4-bora-3a,4a-diazas-inda-cene-3-dodecanoic acid not BODIPY C8, C5 4,4difluoro-5-octyl-4-bora-3a,4a-diaza-s-indacene-3pentanoic acid was incorporated into living cells and
converted to phosphatidylcholine ((PC).
PC
PA
ERM
NHE-1
H+
Na+
Acknowledgements
P
Support
NIH-1-R15-HL074924-01A1
NSF-MCB0080243
NSF-ILI – DUE 9750937
NSF-CCLI - DUE 0088654
NSF-RUI - MCB 0080243
NSF-MRI - DBI 0110537
Dille Fund for Excellence
MSUM Faculty Grants
MSUM Alumni Foundation
Thank you to all the
undergraduates in the lab for the
support provided