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Phosphatidic acid acts as an upstream activator of src kinase and plcg to initiate calcium release in xenopus fertilization

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Title:
Phosphatidic acid acts as an upstream activator of src kinase and plcg to initiate calcium release in xenopus fertilization
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Stafford, Jason Hugh
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English
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ix, 81 leaves : ; 28 cm

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Subjects / Keywords:
Xenopus -- Eggs ( lcsh )
Ryanodine -- Receptors ( lcsh )
Fertilization (Biology) ( lcsh )
Phosphatidic acids ( lcsh )
Fertilization (Biology) ( fast )
Phosphatidic acids ( fast )
Ryanodine -- Receptors ( fast )
Xenopus -- Eggs ( fast )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Bibliography:
Includes bibliographical references (leaves 72-81).
General Note:
Department of Integrative Biology
Statement of Responsibility:
by Jason Hugh Stafford.

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|University of Colorado Denver
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Full Text
PHOSPHATIDIC ACID ACTS AS AN UPSTREAM ACTIVATOR OF SRC
KINASE AND PLCy TO INITIATE CALCIUM RELEASE IN XENOPUS
FERTILIZATION
by
Jason Hugh Stafford
B.A., MCDB, University of Colorado at Boulder, 2001
A thesis submitted to the
University of Colorado at Denver
in partial fulfillment
of the requirements for the degree of
Master of Arts
Biology
2006


This thesis for the Master of Arts
degree by
Jason Hugh Stafford
has been approved
by


Stafford, Jason Hugh (M.A. Biology)
Phosphatidic Acid acts as an Upstream Activator of Src Kinase and PLCy to Initiate
Calcium Release in Xenopus Fertilization
Thesis directed by Professor Bradley J. Stith
ABSTRACT
In all animals that have been studied, egg activation has shown to be mediated
by a relatively short rise or sustained fluctuations in calcium ion concentration known
as calcium transients. However, the signaling pathway that causes calcium release has
yet to be fully characterized. Previous analysis of lipid signaling involved in Xenopus
fertilization led to the hypothesis that phosphatidic acid (PA) is acting as a second
messenger to increase calcium.
This thesis provides data that suggest PA is acting as an upstream activator of
Src kinase and phospholipase Cy (PLCy) to stimulate calcium release from
intracellular stores during Xenopus fertilization. First, microinjection of Fluo-4 into
albino oocytes was used to verify that treatment with exogenous PA has the ability to
cause a rise in intracellular calcium concentration in whole cells. Secondly, an
ELISA was used to determine PAs ability to stimulate Src autophosphorylation.
Similarly, Western blotting was performed to detect an increase in PLCy


phosphorylation following PA addition. Finally, IP3 assays were conducted to
determine PAs effect on IP3 levels.
Conversely, experiments were also conducted to determine whether PA-
induced Ca2+ release depends upon the activity of Src and PLC (as measured by
following IP3 production). Xenopus oocytes were incubated with various tyrosine
kinase inhibitors to .determine if Src activity was required for PA-induced calcium
release. Similarly, the PLC inhibitor U73122 and IP3-receptor antagonist 2-APB
were used to verify that PA is causing a rise in intracellular calcium by acting through
PLC to increase IP3.
The results of these experiments suggest that PA may act through Src and may
also activate PLC directly to increase intracellular calcium.
This abstract accurately represents the content of the candidates thesis. I recommend
its publication.
Signed


ACKNOWLEDGEMENT
I would like to thank Dr. Brad Stith for his support, guidance, friendship and for
providing me with so many opportunities.
I would also like to thank Dr. Timberly Roane, Dr. Gerry Audesirk, Dr. Mary Weiser-
Evans, and Dr. Cheri Jones for all their support, instruction, and friendship.
Thank you to Dr. Doug Petcoff, Ying Chang, Nancy Tseng, and Tim Silverstein for
their support and friendship.


CONTENTS
Figures......................................................................viii
Chapter
1. Introduction................................................................1
1.1 Background..................................................................1
1.2 Calcium as a Signaling Molecule............................................2
1.3 Increased Calcium Leads to Egg Activation Following Oocyte Maturation.....3
1.4 Fertilization Causes an Increase in Intracellular Calcium Concentration...6
1.5 Evidence for ProteinTyrosine Kinase Activity During Fertilization.........8
1.6 Hypothesis that PA is Acting to Increase Calcium at Fertilization.........10
1.7 Molecular Structure of Phosphatidic Acid..................................12
1.8 PA is Acting Specifically to Increase Calcium.............................13
1.9 Experimental Design.......................................................16
2. Methods....................................................................18
2.1 Xenopus laevis as Model System for Calcium Signaling......................18
2.1.1 Obtaining Xenopus Eggs...................................................19
2.1.2 Obtaining Xenopus Oocytes...............................................20
2.1.3 Fertilization of Xenopus Eggs... .7.....................................20
2.2 Measurement of PA-induced Calcium Release in Whole Cells..................21
v


2.2.1 Microinjection Procedure.........................................23
2.3 Enyzme Linked Immunosorbent Assays (ELISAs).......................25
2.3.1 PhosphoELISA Src Tyr 418 Assay...................................25
2.3.2 PhosphoELISA ERK Thr 185 / Tyr 187...............................27
2.4 Measurement of PLCy Phosphorylation via Western Blotting...........29
2.4.1 Measurement of PLCy Phosphorylation via Dot Blotting.............32
2.5 IP3 Assay......................................................... 34
2.6 Use of Fluorometer to Measure PA-induced Calcium Release in PMCs..36
3. Results.............................................................42
3.1 Measurement of PA-induced Calcium Release in Whole Cells.........42
3.2 Enzyme-Linked Immunosorbent Assays (ELISAs)......................44
3.2.1 PhosphoELISA Src Tyr 418 Assay...................................44
3.2.2 PhosphoELISA ERK Thr 185 / Tyr 187...............................49
3.2.3 PhosphoELISA Src Tyr 418 Assay Repeated..........................51
3.3 Measurement of PLCy Phosphorylation via Western Blotting.........52
3.3.1 Measurement of PLCy Phosphorylation via Dot Blotting.............54
3.4 IP3 Assay..........................................................55
3.5 Effect of Src Inhibitors on PA-induced Calcium Release.............59
3.6 Effect of PLC inhibitor U73122 and DVreceptor blocker 2-APB on PA-induced
Calcium Release........................................................63
vi


4. Discussion...............................................................64
References..................................................................72
Vll


FIGURES
Figure
Figure 1.3.1 Oocyte Maturation..................................................5
Figure 1.4.1 A Rise in Intracellular Calcium is Required for the Zygote to Undergo
Mitosis.........................................................................6
Figure 1.6.1 From left to right: Untreated Egg, Egg treated w/ 400 pM PA, Egg
treated w/ Ionophore...........................................................11
Figure 1.7.1 PLD Cleaves PC to PA and Choline..................................12
Figure 1.8.1 Model for Xenopus Fertilization...................................14
Figure 1.9.1 Experimental Design...............................................17
Figure 2.2.1 Molecular Structure of Fluo-4.....................................22
Figure 2.2.1.1 Molecular Structure of 2-APB....................................24
Figure 2.6.1 Molecular Structure of Tyrosine Kinase Inhibitors: Herbimycin A, PP2,
SU6656, Peptide A, PP2, and Tyrphostin B46................................... 38
Figure 2.6.2 Fluo-4 Calibration Curve..........................................41
Figure 3.1.1 400 pM PA Causes a Rise in Intracellular [Ca2+] in Whole Albino
Oocyes.........................................................................43
Figure 3.2.1.1 400 pM PA Appears to Increase Src Activity......................45
Figure 3.2.1.2 Src ELISA Standard Line.................................... .46
Figure 3.2.1.3 10 pg/ml Progesterone Appears to Increase Src Activity at 10
min............................................................................47
Figure 3.2.1.4 10 pg/ml Progesterone May Increase Src Activity at 30 min. and
6 hours........................................................................48
viii


Figure 3.2.2.1 MAPK ELISA Standard Line
.49
Figure 3.2.2.2 2 pM Insulin and 10 [xg/ml Progesterone both Significantly Increase
MAPK activity in Xenopus Oocytes.............................................. 50
Figure 3.2.3.1 Both Sperm & PA cause an Increase in Src Phosphorylation.........51
Figure 3.3.1 Phosphorylated PLCy................................................52
Figure 3.3.2 400 pM PA Increases PLCy Activity..................................53
Figure 3.3.1.1 Both lOmM H2O2 and 400 [xM PA Increase pTyr783 PLCy
Concentration...................................................................54
Figure 3.4.1 PA significantly increases IP3.....................................56
Figure 3.4.2 Incubation with 250 [xM B APT A-AM did not Block PA-induced IP3
Elevation.......................................................................57
Figure 3.4.3 2 [xM Herbimycin A Blocks PA-induced Elevation of IP3..............58
Figure 3.5.1 Raw Fluorescence Data Suggest 100 [xM Peptide A Inhibits PA-
induced Calcium Release.........................................................60
Figure 3.5.2 100 fxM Peptide Decreases the Total Amount of Calcium Released by
PA Treatment....................................................................61
Figure 3.5.3 Five Tyrosine Kinase Inhibitors Partially Block PA Action..........62
Figure 3.6.1 PLC activity is required for PA action.............................63
Figure 4.1 PA May Act to Increase Intracellular Calcium via Two Different
Pathways........................................................................64
IX


1. Introduction
1.1 Background
Fertilization is the process by which two haploid gametes (egg and sperm)
fuse to form a diploid zygote. The interaction between egg and sperm membranes
triggers a programmed series of cellular and biochemical events known as egg
activation. These events include the fusion of the plasma membranes of the sperm
and egg, acceleration of metabolic processes, and the cortical reaction. During the
cortical reaction, secretory vesicles beneath the plasma membrane known as cortical
granules, release degradative enzymes that cut the links between the zygote and the
vitelline envelope. Thus, the free floating egg undergoes gravitational rotation
(heavier vegetal pole rotates down) and the degradative enzymes destroy sperm
receptors to prevent the entry of additional sperm. Most importantly, activation
releases the egg from meiotic arrest allowing it to resume the cell cycle and finish
towards meiosis II. In all animals that have been studied activation has shown to be
mediated by a relatively short rise or sustained fluctuations in calcium ion
concentration known as calcium transients. In fact, calcium transients are known to
be required for mitosis and cellular responses to hormones. In addition to many other
metabolic processes, hormones raise intracellular calcium in platelet activation,
muscle contraction, insulin secretion, and antibody production (Stith, 2005).
1


1.2 Calcium as a Signaling Molecule
The resting level of free calcium in most cells is kept very low, usually about
50-100 nM (or 1 x 10'7 M). This is four orders of magnitude lower than the 1-2 mM
concentration of calcium found outside the cells. In addition, the cell is negative
inside and this attracts positively charged calcium ions. As a result, an
electrochemical gradient is generated, with the cytosol being negatively charged
relative to the extracellular fluid. This powerful gradient tends to drive the calcium
into the cytosol across both the plasma membrane and the ER membrane and this
creates a signaling pathway.
The differential is maintained by Ca2+-ATPases which are transmembrane
proteins that pump calcium either out of the cell or into the lumen of the endoplasmic
reticulum. When calcium is allowed to flow down its gradient the initial signal is
often seen to be small and localized to a discrete region of the cytoplasm. These
signals are called calcium blips, quarks, puffs, or sparks, and are localized to the area
of individual or small groups of calcium release channels in the ER. If the signal
such as sperm is strong and persistent enough, then the initial calcium blip can
propagate as a regenerative calcium wave through the cytosol, much like an action
potential in an axon (Alberts et al., 2005).
When calcium is released into the cytosol it binds calmodulin. Calmodulin is a
small (17 kDA), ubiquitous protein that does not exhibit any intrinsic catalytic
2


activity. It has a helical structure with two calcium binding sites at each end. These
binding sites exhibit positive cooperativity, meaning that when calcium binds one site
it increases the binding affinity of the adjacent site. When all four sites are occupied
by calcium the a-helical structure of the protein unwinds and the molecule bends and
wraps itself around the target molecule. This interaction induces a conformational
change that activates the target protein. Calcium and calmodulin can activate more
than a 100 different enzymes.
1.3 Increased Calcium Leads to Egg Activation Following Oocyte Maturation
In the case of egg activation calcium and calmodulin activate a kinase called
CaM Kn- CaM Kn, in turn, activates the anaphase promoting complex (APC) and
degrades a cytostatic factor (believed to be c-mos) that inhibits APC (Kaltoff, 1996).
Mos is a protein/serine kinase that is synthesized in oocytes near the end of meiosis I.
Mos is required for the increase in MPF activity during meiosis II and for the
maintenance of MPF activity during metaphase II arrest. Mos activates MEK (or
MAP kinase kinase) which, in turn, phosphorylates and activates ERK. ERK, also
known as MAP Kinase (MAPK), activates yet another protein kinase named Rsk
which inhibits the action of the APC and arrests meiosis at metaphase II. Without
Rsk inhibition, functional APC directs the proteolysis of cyclin B which results in an
exit from M phase (see Figure 1.3.1).
3


Cyclins work in conjuction with cyclin dependent kinases (CDKs), to both
initiate and terminate the succesive phases of the cell cycle. CDKs are serine-
threonine kinases that are continually present within the cell and, as their name
suggests, cyclins are expressed cyclically. The accumulation of cyclins D and E
activate CDK4 and CDK2 respectively to drive the cell through the Gi phase. The S
phase of the cell cycle is characterized by the build up of cyclin A, which also
activates CDK2. The dimerization of cyclin B and CDK 1 (or cdc 2) is called
Maturation Promoting Factor (MPF) and initiates mitosis by phosphorylating several
proteins including: 1) histone HI to promote chromosome condenstation, 2) lamin
proteins to promote nuclear envelope breakdown and 3) RNA polymerase to inhibit
transcription during mitosis. MPF causes arrest at metaphase II by phosphorylating
myosin light chains to decrease their affinity for actin, thus preventing cytokinesis
until mitosis is completed. Total cyclin B destruction is necessary for cytokinesis to
occur and cell division to be completed (see Figure 1.3.1).
4


transmembrane domain receptor that inhibits Protein Kinase A (PKA) which
normally inhibits both Mos and Polo-Like Kinase Kinase (PKK). Mos is then free to
activate MEK (MEK can be inhibited by the drug U0126) which activates MAPK.
MAPK stimulates Rsk and can feedback to activate Mos. Rsk inhibits myt leading to
MPF activation as myt normally inhibits MPF. In the second pathway, PLKK
activates Polo-Like Kinase from Xenopus (PLX) which turns on CDC25. CDC25
removes an inhibitory phosphate from MPF. At meiosis I MPF levels drop stimulates
its own degradation by activating APC. However, MPF levels begin to rise again as
Rsk activates CSF and Bubl which both inhibit APC.
5


1.4 Fertilization Causes an Increase in Intracellular Calcium Concentration
OOCYTE
MEIOSIS
INSULIN-^1^
IGF-V^ /
PROGESTERONE
.^^FERTILIZATION MITOSIS ^
sperv.-A^y
EGG ZYGOTE EMBRYO
Figure 1.4.1 A Rise in Intracellular Calcium is Required for the Zygote to
Undergo Mitosis. Hormones induce the oocytes maturation into a fertilizable egg
and the fusion sperm sperm and egg leads to an increase in [Ca2+]in.
The signaling pathway that causes calcium release, CAM Kn activation, and
subsequent cyclin B degradation has yet to be fully characterized. There are two
possible origins for the influx of calcium: the extracellular fluid or intracellular stores
(e.g. the endoplasmic reticulum). Recent findings suggest that inositol phospholipid
turnover and intracellular stores are responsible for egg activation. In this pathway,
Phospholipase C (PLC), a plasma-membrane associated enzyme, catalyzes the
cleavage of phosphatidyl inositol-4-5-bisphosphate (PI -4,5-P2 or PIP2) producing
diacylglycerol (DAG) and inositol triphosphate (IP3). It is the water-soluble IP3 that
acts as a second messenger, binding to receptors on smooth endoplasmic reticulum
that mediate calcium release. The receptors are ligand-gated calcium channels (the
6


ligand is IP3). DAG activates protein kinase C, which in turn activates a Na+/H+
pump. This pump exchanges sodium for hydrogen ions, thus raising the pH of the egg
after fertilization.
PLC has three main isoforms: p,y, and 8. The P isoform has been shown to be
activated by the G protein subfamily, Gq, and the y isoform is known to be activated
by tyrosine phosphorylation. The regulation of the 8 form is not well understood.
Initially, it was believed that PLCP was the major isoform involved in Xenopus
fertilization. It was shown that the addition of active serotonin receptors or epidermal
growth factor receptors could stimulate egg activation. These receptors were known
to activate Gq proteins. However, later it was demonstrated that inhibition of Gq
proteins did not affect the activation events induced by sperm (Runft et al., 1999).
Injection of anti-Gq antibodies into Xenopus eggs did not inhibit the rise in
intracellular calcium during fertilization.
Recent studies by S.J. Lee using sea urchin eggs, suggest that the y isoform of
PLC is responsible for egg activation (Shen et al., 1999). Microinjection of 1-5 mM
methylglyoxal bis(guanylhydrazone) (MGBG) was shown to stimulate the
phosphorylation of a synthetic protein tyrosine kinase substrate (RR-Src) and also
stimulated a transient rise in intracellular calcium. Pretreatment with the tyrosine
kinase inhibitors genistein and tyrphostin B42 blocked the MGBG-induced rise in
7


intracellular calcium. Sato and Nuccitelli showed that egg activation can be blocked
in Xenopus through the use of different protein tyrosine kinase (PTK) inhibitors such
as genistein, herbimycin A, and lavendustin A (Sato et al., 1999; Glahn et al., 1999).
There are three possible models explaining the activation of PLC by tyrosine
kinases. In the first model, the sperm receptor acts as a classic receptor tyrosine
kinase with its C terminal domain having catalytic activity. However, sequence
analysis of the sperm receptor does not find such a catalytic domain (Gilbert, 1997).
In the second model, the sperm receptor may activate a separate tyrosine kinase
through cross-linking of membrane receptors. Lastly, it is possible that PLC is
activated following fusion of the gamete membranes. In this third model, the sperm
receptor only functions in cell-cell adhesion and PLC is activated by an agent
delivered by the sperm. It is important to note that a combination these hypotheses
may be at play.
1.5 Evidence for ProteinTyrosine Kinase Activity During Fertilization
Satos research provides much data suggesting egg Src kinase as the major
PTK involved in Xenopus fertilization. Src kinase is a membrane-associated protein
that was first identified as a viral oncogene and is named after the Rous Sarcoma
Virus. Src is the prototypical member of Src family protein tyrosine kinases. The
8


family has eight members in humans: Src, Yes, Fyn, Lck, Fgr, Lyn, Hck, and Blk.
However, Satos group has used mass spectrometry and Western blotting to find that
Src alone is expressed in Xenopus eggs (Sato et al., 2004). The structure of Src
consists of a unique N terminus, a SH2 domain, a SH3 domain, and a kinase domain.
The N terminus is unique in that it contains a myristoylation signal (lipid tail) for
membrane localization. Src is activated by phosphorylation at tyrosine 416 and is
followed by autophosphorylation at tyrosine 418. However, addition of a phosphate
group to tyrosine 527, near the C-terminus can cause the enzyme to fold back on itself
by binding the SH2 domain. This obscures the kinase domain and inactivates the
enzyme.
Satos group has demonstrated that the addition of sperm to Xenopus eggs or
membrane rafts isolated from Xenopus eggs promotes Src-dependent phosphorylation
of PLCy, IP3 production, and calcium release (see Figure 1.2.2). Further, they have
shown that that calcium production can be blocked by the PLC-specific inhibitor U-
73122, the IP3 receptor antagonist heparin, and various Src inhibitors. For example,
they demonstrated that calcium release stimulated by the addition of sperm to cell-
free Xenopus extracts can be blocked with Src inhibitors PP1 and PP2 and not the
inactive derivative PP3 (Tomakov et al. 2002). In addition, both Glahn and Sato
show that microinjection of synthetic inhibitory peptides (A and Al), which bind to
Srcs SH2 domain, into whole Xenopus eggs inhibit fertilization while an inactive,
9


related peptide (A9) having a slightly different amino acid sequence does not (Glahn
et al., 1999; Sato et al., 1999). Moreover, immunodepletion of PLCy from
membrane raft preparations prevented calcium release following sperm binding (Sato
et al., 2003).
Nonetheless, Satos group does not provide any data concerning the
biochemical steps in between sperm binding and Src activation (Figure 1.2.2).
1.6 Hypothesis that PA is Acting to Increase Calcium at Fertilization
Using a novel HPLC method, Stith et al. found that a significant increase in
DAG occurred following fertilization in Xenopus (Stith et al., 1997). This increase in
DAG (62-110 pmol/cell) was determined not to be from the action of PLC as the
magnitude of the DAG increase was approximately 280-fold for that of IP3 (-170
fmol/cell). Therefore, they began to investigate the source of the measured DAG
increase. It is known that phospholipase D (PLD) can hydrolyze phosphatidylcholine
to phosphatidic acid (PA) and choline and that PA can be converted to DAG via the
action of PA phosphohydrolase (Alberts et al., 2005). Choline mass was shown to
increase by approximately 134 pmol/cell (Stith et al., 1997). These data suggested
that PLD is activated at fertilization. Previously, it had been demonstrated that PA
can activate PLC (Moolenaar et al., 1986) and this led to the hypothesis that PA is
acting as a second messenger to increase calcium during Xenopus fertilization.
10


Subsequently, Stith et al. detected an increase in both PA mass and IP3 mass
in Xenopus eggs following fertilization (unpublished results). Also, the addition of
400 pM exogenous PA was found to induce parthenogenetic activation of Xenopus
eggs. During activation, a presumptive rise in intracellular calcium causes the
migration of pigment granules towards the animal pole (see Figure 1.6.1).
Ionophores also induce parthenogenetic activation resulting in the migration of
pigment granules to the animal pole. The ionophore ionomycin increase intracellular
calcium by disrupting the membrane and allowing calcium ions to enter the cell from
the extracellular media (the ionophore A23187 causes calcium release from
intracellular stores) (Gompert et al., 2003).
Figure 1.6.1 From left to right: Untreated Egg, Egg treated w/ 400 pM PA, Egg
treated w/ Ionophore. Migration of the pigment granules in the animal pole
suggests that PA may be acting to increase intracellular calcium
11


1.7 Molecular Structure of Phosphatidic Acid
PA is an anionic membrane lipid that is derived from phosphatidylcholine
(PC). Phospholipase D (PLD) cleaves off the choline functional group on PC leaving
a bare phosphate head group to yield PA (see Figure 1.7.1). In addition to being a
possible activator of inositol phospholipid turnover, PA is known to function in
vesicle formation by facilitating bending of the plasma membrane.
§
(e:
'
NfCH3)3
Ob
(Hi
A
HiGCH Phospholipase D
(PLD)
OH
OP- oQ
;A-
H2(iCHAh2
) ?
o=c o=c
Phosphatidylcholine (PC)
/ <.
Phosphatidic Acid:(PA)
.
X(CH;i)3
Oh
I
hoch2
+ Choline
Figure 1.7.1 PLD Cleaves PC to PA and Choline. Phospholipases cleave specific
ester bonds (shown on molecule of PC). PLC cleaves PC to DAG and
phosphocholine. PLA2 cleaves PC to archidonate and a lysophospholipid and PL A]
cleaves PC to 2-acylglycerophosphocholine and a fatty acid. (Figure taken from
Alberts et al., 2005)
12


1.8 PA is Acting Specifically to Increase Calcium
This thesis provides data that suggest PA is not simply disrupting the
membrane and acting non-specifically like an ionophore to increase calcium. Rather,
the data suggest that PA may be acting as an upstream activator of Src kinase and
PLCy to stimulate calcium release from intracellular stores during Xenopus
fertilization (see Figure 1.8.1).
Although this mechanism would nicely supplement the data presented by
Satos group, there is much data suggesting PA directly activates PLC (not through
Src). Moolenaar .(Moolenaar et al., 1986) first reported PA activation of PLC (and
that PA does not simply cause calcium influx into the cell). PA [but not
phosphatidylserine (PS), phophatidylcholine (PC), lysophophatidic acid (LPA) or
phosphatidylethanolamine (PE)] stimulated the activity of PLC from Xenopus (Jacob
et al., 1993), rat (Zhou et al., 1999), bacteria (Wehbi et al., 2003), rabbit platelets
(Hashizume et al., 1992), human renal cells (Knauss et al., 1990), myocytes (Kurz et
a., 1993; Liu et al., 1999), neutrophils (Siddiqui and English, 2000), bovine
parathyroid cells (McGhee and Shoback, 1990), 3T3 cells (Murayama and Ui, 1987),
snaptosome membranes (Quian and Drewes, 1991), A431 cells (both
unphosphorylated and tyrosine phosphorylated PLC-yl; Jones and Carpenter, 1993)
and human platelets (Jackowski and Rock, 1989).). Short or long chain PA (e.g.,
13


chain length is not important) activated rabbit myocytes (LPA, DAG or PS were
ineffective; Henry et al., 1995).
| SPERM |
Figure 1.8.1 Model for Xenopus Fertilization. In this model, sperm binding
stimulates phospholipase D (PLD) to elevate PA levels in the plasma membrane of
the egg. PA then stimulates Src through a mechanism yet to be specified. Active Src
then autophosphorylates on tyrosine 418 prior to phosphorylating PLCy on tyrosine
783. Phosphorylated PLCy then cleaves PIP2 to produce IP3 and DAG. IP3 binds to
IP3 receptors on the smooth endoplasmic reticulum (where calcium ions are stored) to
initiate the calcium transients required for cell division.
14


PA (but not LPA, DAG, PE or PS) stimulated PLC isoforms (31, y and 53
(Litosch, 2000) (elevating Vm and lowering Km of PLC 53; Pawelczyk and Matecki,
1999). PA may remove an autoinhibitory domain from the active site to decrease the
Km of PLCy (no effect on Vm; Jones and Carpenter, 1993; Geng et al., 1998; Zhou et
al., 1999). In contrast, PA elevated Vra (no effect on Km) of PLCpi from cerebral
cortex (again, PA chain length was not important; Litosch, 2000; 2002; 2003).
With whole cells, 30 [xM dicapryl PA addition to neutrophils (which lack LPA
receptors) caused an increase in [Ca]jn (Siddiqui and English, 1997, 2000). PA
addition to vascular smooth muscle cells raises [Ca]in and this action is reduced by a
PLC inhibitor and a IP3 receptor blocker (Bhugra et al., 2003). In rat neonatal
cardiomyocytes, PA (but not LPA) increased IP3 mass and [Ca]m and this action was
blocked by the PLC inhibitor U73122 (inactive U73343 did not inhibit; Liu et al.,
1999). PA stimulated PLC when added to human cardiomyocytes (Dhalla et al.,
1997; but was less active in cardiomyocytes from patients with congestive heart
failure; Tappia et al., 2003). Summarizing these papers, minimal concentrations
(-50-100 [xM) of PA produce activation of PLC and elevation of intracellular
calcium.
15


1.9 Experimental Design
Subsequently, we have designed experiments that were designed to determine
whether the addition of exogenous PA could stimulate Src, PLCy, IP3 production, and
calcium production in Xenopus eggs or oocytes. First, a calcium sensitive fluor was
used to verify that treatment with exogenous PA has the ability to cause a rise in
intracellular calcium concentration in eggs. Secondly, an ELISA was used to
determine if PA treatment causes an increase in Src autophosphorylation (a measure
of Src activity). Similarly, Western blotting was performed to detect an increase in
PLCy phosphorylation following PA addition. Finally, an IP3 assay was performed to
measure if PA could elevate PLC activity and raise IP3 concentration.
Conversely, experiments were also conducted to determine whether PA-
induced Ca2+ release depends upon the activity of Src and PLC (as measured by
following IP3 production). Xenopus oocytes were incubated with various tyrosine
kinase inhibitors to determine if Src activity was required for PA-induced calcium
release. Similarly, the PLC inhibitor U73122 and DVreceptor antagonist 2-APB
were used to verify that PA is causing a rise in intracellular calcium by acting through
PLC to increase IP3 (see Figure 1.9.1). !
16


Figure 1.9.1 Experimental Design. If the tyrosine kinase inhibitors failed to block
PA-induced calcium release in the Xenopus oocytes then this would suggest that PA
does indeed activate PLC directly (and not necessarily the gamma isoform).
However, if tyrosine kinase inhibitors blocked PA action then this would corroborate
Satos assertions as to the importance of Src in Xenopus fertilization. It is important
to note however that both pathways may be present.
17


2. Methods
2.1 Xenopus laevis as Model System for Calcium Signaling
There are several advantages to using Xenopus laevis as model system for
calcium signaling. First of all, it has been used to study intracellular signaling for
over 50 years and its metabolic processes have been well studied (Nuccitelli, 1994).
Perhaps more importantly, eggs or stage VI oocytes are extremely large for a single
cell being approximately 1-1.3 mm in diameter and having a fluid volume of
approximately 500nl. Moreover, the cells divide rapidly, are available in large
quantities and are unusually robust due to a highly reinforced plasma membrane.
This property facilitates microinjection and other mechanical manipulations.
Wild type and albino Xenopus laevis were obtained from Xenopus Express
(Ft. Lauderdale, FL) and are maintained in 30 gallon plastic tanks (carpenter sinks).
Female frogs are fed 2 grams of ground beef heart every other day and the tanks are
cleaned several hours after feeding. Male frogs are fed 1.5 grams each. The frogs are
generally resistant to infection as they secrete a natural antibiotic (Nuccitelli, 1994).
10 ml of Nov Aqua water conditioner (AquaScience Research Group Inc., Kansas
City, MO) is added to each tank to remove chorine and maintain sterility.
18


2.1.1 Obtaining Xenopus Eggs
Xenopus frogs have to be injected in the lower back near lymph glands with
hormones in order to stimulate egg production. Three or four days prior to the
experiment female frogs are primed with 35-50IU pregnant mare serum gonadatropin
(PMSG) (Sigma, St. Louis, MO.). PMSG stimulates the growth of the ovarian
follicles and the corpus luteum. In order to stimulate ovulation, they were then
injected with 800 IU of human chorionic gonadotropin (HCG) the night before eggs
are collected. Eggs are harvested into 0.1M NaCl by gently rubbing the back of the
female. After the eggs are squeezed from the frogs, they are treated for 4 min. in 1%
solution of cysteine. Cysteine is an acidic amino acid that is a gentle reducing agent
and is used to remove part of the protective jelly layer that covers the eggs.
Dejellying eggs facilitates manipulation and speeds PA and other membrane
permeable agents to entry into the cells. After 4 min., the cysteine is washed away by
rinsing twice with rinse solution (0.1 M NaCl, and 0.05 M Tris-Base pH adjusted to
7.5) The eggs are then transferred to 100% Modified Barths Solution (MBS) (440
mM NaCl, 5 mM KCL, 50 mM HEPES, 4.1 mM Magnesium Sulfate Hydrate, 1.7
mM Calcium Nitrate Tetrahydrate, 2.1 mM CaC12, ph 7.5). In order to prevent
precipitation of phosphatidic acid (PA) the MBS has to be diluted to 10% prior to
addition to eggs.
19


2.1.2 Obtaining Xenopus Oocytes
In order to obtain stage VI (the final stage in oogenesis) oocytes, frogs are
primed with 35IU (but not HCG). Three or four days after PMSG injection, the frogs
are immobilized being placed in an ice water bath for at least 15 min. and then
sacrificed with a rat guillotine. The ovaries are surgically removed and placed into
room temperature 0-R2 solution (83mM NaCL, 0.5 mM CaCl2, ImM MgCl2, 10 mM
HEPES, ph adjusted to 7.5). The ovaries are washed at least three times with fresh O-
R2 to remove excess blood containing enzymes that can damage the cells. Healthy
stage VI oocytes are then manually isolated from follicular membranes using forceps
and are maintained in 0-R2.
2.1.3 Fertilization of Xenopus Eggs
In order to obtain a sperm used for fertilization, males are sacrificed and the
testes are surgically removed. After being washed and cleaned of any mesenteric fat,
both testes are macerated in 2.4 ml of 100% MBS to create a working sperm
suspension. 167 pi of this sperm suspension is added to eggs in 1 ml of 10% MBS.
The sperm become activated (begin the acrosome reaction) once added to 10% MBS
and egg activation / gravitational rotation should be observed within 5 to 7 min.
20


2.2 Measurement of PA-induced Calcium Release in Whole Cells
Fluorescent indicators are molecules that adsorb light at a certain wavelength
(excitation) and radiate light (emission) at a longer wavelength (lower energy). More
light is given off with increased calcium concentration. Fluorescent calcium
indicators are generally based on the calcium chelator BAPTA with a few modeled
after proteins. Furthermore, these indicators fall into two general categories:
ratiometric indicators and single-wavelength (SW) indicators. The intensity of
fluorescence can be affected by factors not directly related to calcium concentration
such as changes in cell thickness or loss of indicator from the cell by leakage. With
ratiometric indicators, both the calcium-free and calcium-bound forms exhibit distinct
spectra. Therefore, a ratio can be obtained between the two spectra minimizing the
effect of any artifact not related to calcium concentration (Nucitelli, 1994). Common
examples ratiometric indicators are fura-2 and indo-1.
Fluo-4, however, is a high-affinity SW indicator synthesized by directly
coupling 9-xanthone to the aromatic ring of BAPTA to produce what is known as a
fluorescein derivative (see Figure 2.2.1). It has an excitation maximum at 491 nm

and emission maximum at approximately 525 nm. Fluo-3 has a nearly identical
structure, except that the fluorines found on Fluo-4 are substituted for chlorines. The
addition of halogens to the fluorescein ensures that the phenolic OH group is always
ionized within physiological pH range by reducing the pKa of the phenolic OH group
21


from ~ 6.2 to ~ 4.5. This is necessary because the fluorescein is only weakly
fluorescent when protonated. The halogens solve maintain the fluorescein in its ionic
form, but also reduce the brightness of the fluorescence. The degree to fluorescence
quench by halogens generally follows their appearance in the periodic table
(F pH, and is 40% brighter than Fluo-3. In addition, Fluo-4 is has a greater
photostability and is better suited for use in confocal microscopy. Fluo-4 has an
excitation maximum of 491 nm and is excited much more efficiently than Fluo-3 by a
488 nm argon laser (Putney, 2000).
Figure 2.2.1 Molecular Structure of Fluo-4. The fluorines in Fluo-4 reduce the
brightness of fluorescence less than chlorines found on Fluo-3 (structure not shown).
22


2.2.1 Microinjection Procedure
Oocytes were collected from an albino frog and were microinjected with 50 nl
of 10 pM Fluo-4 (a fluorescent calcium indicator) to give a final concentration
slightly greater than 1 pM. Microinjection was performed with a PV830 Pneumatic
Picopump microinjection system obtained from World Precision Instruments (WPI)
(Sarasota, FL.). First, injection needles were made from borosilicate glass capillaries
(WPI, cat.# TW150-4) using the PUL-1 microinjection needle puller (WPI). The tips
of the needles were then broken with a pair of forceps to a diameter of 10-30 pm. Tip
diameters were measured with a micrometer calibration slide (Wild, Heerbrugg,
Switzerland). The microinjection needles were then inserted into the micropipette
holder attached to the picopump microinjection system and filled with 10 pM Fluo-4
by vacuum pressure. The volume of Fluo-4 delivered by a timed injection was then
adjusted to 50 nl by measuring a bolus of approximately 0.457 mm in diameter using
the calibration slide. The equation used for converting bolus diameter to volume was:
Equation 2.2.1.1 volume (mm3) = diameter (mm)3 (n/6)
Albino oocytes were injected in IX 0-R2 in small petri dishes. The
micromanipulator (Semicon West, Munich, Germany) was used to position the
microinjection needle so as to dimple the plasma membrane. In order to puncture
the membrane, a permanent marker was used to gently tap the back of the
micromanipulator and the Fluor-4 was injected using a timed injection (vs. a gated
23


injection). The oocytes were observed for several minutes to ensure that the puncture
wound healed and the cells did not leak cytoplasm.
After the oocytes were injected with fluor some were incubated with the IP3-
receptor antagonist 2-Aminoethoxydiphenylborate (2-APB) at a final concentration of
100 p.M for approximately 25 min. 2-APB is cell permeable and is classified as a
noncompetitive inhibitor of the IP3 receptor although the exact mechanism of its
action is unknown (Kukkonen et al., 2001).
Figure 2.2.1.1 Molecular Structure of 2-APB
Both 2-APB treated and untreated cells were examined using a fluorescent
microscope imaging system following the addition of 400 pM dicapryloyl PA (Avanti
Polar Lipids, Alabaster, AL). Maximal effective concentration was previously
determined by dose response experiments and is corroborated by Jacob et al., 1993.
Albino oocytes were used because they lack the dark pigment associated with the
animal pole which would have decreased the intensity of fluorescence. The imaging
24


system consists of: (1) a Nikon Diaphot inverted microscope and a a 75W Xenon
lamp (2) a Ludl filter wheel/shutter combination (3) a Quantex QC-200 intensified
CCD camera with high performance option (gain of greater than 45,000 candles/m2
output brightness per Lux of input illumination and detects a signal as low as 10'7 Lux
with 550 nm light).
2.3 Enyzme Linked Immunosorbent Assays (ELISAs)
2.3.1 PhosphoELISA Src Tyr 418 Assay
Several experiments were performed in order to optimize the Src [pY418]
assay for Xenopus. Commercially available Src ELISA kits are designed for use with
mammalian cells and to our knowledge this is the first report of their use in the
analysis of amphibian cells. Src [pY418] ELISA kits and ERK1/2 [pTpY185/187]
ELISA kits were obtained from Biosource International Inc. (Camarilo, CA).
In one experiment, groups of 40 wild type Xenopus oocytes were placed into
wells in a 12-well plate cell culture dish (product # 3515, Coming Inc., Coming, NY)
with each well that contained 1 ml of IX .OR-2 Control cells were untreated, but the
other cells were incubated with 400 pM dicapryloyl PA for either 5 or 10 min. In
another experiment, the cells were incubated with 10 pg/ml progesterone
(Calbiochem, La Jolla, CA) for 10, 30, 60, 90 and 120. Oocytes that were not
25


defolliculated were incubated with 10 pg/ml of progesterone for 30 min., 2 hr., 4 hr.,
and 6 hr.
In addition, groups of 40 wild type Xenopus eggs were placed into wells in a
12-well plate cell culture dish with each well that contained 1 ml of 10% MBS
(phosphatidic acid precipitates in IX MBS) and were treated with 400 pM PA for
either 15 or 90 min.
Following incubation each group was homogenized in 200 pi of ice-cold 1%
SDS lysis buffer (1% SDS, lOmM Tris-HCL ph 7.5, 150 mM NaCl, ImM EDTA,
lmM EGTA, 1 mM Na-orthovanadate, 10 mM Beta-Mercaptoethanol, 10 pg/ml
Leupeptin, and 20 pM PMSF). The protease inhibitors Leupeptin and PMSF were
added just before use, as PMSF only has a half-life of approximately 30 min. in
aqueous solution. This buffer is optimized for Src extraction from membranes and
activity and (a modification of one used by Sato et al., 2003). The cells were then
sonicated using a VirSonic 50 probe sonicator (Virtis CO. Inc., Gardiner, NY) set to
10% output for 2 minutes, incubated on ice for 5-10 minutes, and then subjected to
low centrifugation (3300g for 10 min.) using a Marathon 16km microfuge (Fisher
Scientific, Pittsburg, PA) at 4 C. The supernatant (100 pi) was then added to the
antibody coated wells provided in the ELISA kits and incubated for 2 hrs. After this
primary incubation, the wells were decanted and washed 4 times with IX wash
buffer before 100 pi of rabbit anti-Src [pY418] detection antibody was added. After
26


1 hour, the wells were decanted and washed again and 100 pi of the visualizing (anti-
rabbit IgG-HRP Working Conjugate) was added. After a 30 min. incubation and a
wash, 100 ul of stabilized Chromogen (the substrate for the HRP-conjugated
antibody) was added and after 30 minutes, the reaction was stopped by adding 100 pi
of stop solution. Absorbance was then read by a Labsystems Multiskan Ascent
photometric plate reader (Thermo Electron Corp.) at 450 nm. A standard line was
also generated using serial dilutions of reconstituted lyophilized Src [pY418] standard
derived from lysates of human platelets (provided with kit).
2.3.2 PhosphoELISA ERK Thr 185 / Tyr 187
As yields of active Src were generally low, an ERK1/2 [pTpY185/187]
ELISA kit was used to validate the use of Biosource ELISA kits to analyze Xenopus
cells. Groups of 10 oocytes were incubated with 2 pM insulin (for 10, 30, and 60
min.) and 10 pg/ml progesterone (for 2 hr. and 4 hr.). Following incubation, the cells
were homogenized in 300 pi of ice-cold 0.1% SDS lysis buffer (0.1% SDS, 1%
Triton-X 100, 0.5% Na Deoxychoate, lOmM Tris-HCL ph 7.5, lOOmM NaCl, ImM
EDTA, ImM EGTA, 10% glycerol, ImM NaF, 2mM Na3VC>4, 10 pg/ml Leupeptin,
and 20 pM PMSF). The cells were then sonicated for 2 min. at 10% output,
incubated on ice for 5-10 min., and centrifuged at 16,600 x g for 10 min. The
supernatant was then diluted 1:6.25 with standard diluent buffer (provided in the
27


kit) and 100 pi of sample was added to the antibody coated wells provided in the
ELISA kit and incubated for 2 hrs. After this primary incubation, the wells were
decanted and washed 4 times with wash buffer before 100 pi of rabbit ERK1/2
[pTpY185/187] detection antibody was added. After 1 hour, the wells were decanted
and washed again and 100 pi of the visualizing (anti-rabbit IgG-HRP Working
Conjugate) was added. After a 30 min. incubation, a wash, 100 pi of stabilized
Chromogen (the substrate for the HRP-conjugated antibody) was added, and after 30
minutes, the reaction was stopped by adding 100 pi of stop solution. Absorbance
was then read by a plate reader at 450 nm. A standard line was also generated using
serial dilutions of reconstituted lyophilized ERK1/2 [pTpY185/187] standard
(provided with kit) and was used to calculate protein concentration.
28


2.4 Measurement of PLCy Phosphorylation via Western Blotting
Groups of 15 eggs or oocytes were either untreated or treated with 400 pM
dicapryloyl PA for 2, 5, 10 or 70 min. In addition, one group was treated with
phosphatidylserine (PS) for 10 min. PS is another anionic membrane lipid that should
not activate PI turnover and therefore serves as a negative control. Following
incubation, the eggs or oocytes were homogenized in 450 pi of ice-cold lysis buffer
(0.1% SDS, 10 mM Tris-(HCl), 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1%
Triton-X, 10% glycerol, lOpg/ml Leupeptin, 10 mM PMSF, and 0.5% Na-
deoxychoate). Na-deoxychoate was added to increase the solubility of any membrane
raft-associated PLCy. The samples were then split in half (-250 pi per half) and one
half frozen at -20 C for later use. 250 pi of freon was added to the remaining half in
order to extract unwanted yolk proteins. The samples were then centrifuged at 16,600
x g for 10 min at 4C in the microfuge and the supernatant was collected. The
volume of the supernatant was carefully measured with a P-100 pipetman (VWR, San
Francisco, CA) in order to estimate total protein concentration. The P-100 pipetman
was set to deliver a volume slightly greater than that of the sample (-300 pi) and the
sample was taken up. The exact volume was then determined by dialing the volume
down until the meniscus of the sample reached the end of the pipetman tip. As a
single Xenopus oocyte or egg contains approximately 10 pg of total protein, the total
protein concentration in each sample was calculated by dividing 150 pg by the
29


volume of each sample (giving protein concentration in pg/pl which is easily
converted to mg/ml). An appropriate amount of 5X Sample Buffer (60 mM Tris-HCl
pH 7.5, 10.5% glycerol, 0.02%SDS, 0.03% Bromophenol blue, and 0.07% beta-
mercaptoethanol (was added just before use) was then added and the samples were
boiled for 15 min.
The samples were then loaded and run on NuPAGE 10% Bis-Tris (1.0 mm,
10-well) pre-cast gels (Invitrogen, Carlsbad, CA). MOPS (50mM MOPS, 50 mM
Tris, 1 mM EDTA, 0.1% SDS) was used as the running buffer and the gels were run
at a constant voltage of 200V for 50 min. The protein was then transferred to
Immobilon-P polyvinylidene difluoride (PVDF) membranes (Sigma, St. Louis MO)
at a constant voltage of 30V for over an hour. The transfer buffer used contained
10%MeOH, 25 mM Tris-(Base), and 192 mM Glycine.
Following transfer the PVDF membranes were blocked overnight with pre-
made blocking solution (LI-COR Biosciences, Lincoln, Nebraska). Membranes were
then washed with blocking solution diluted 1:1 with PBS (137 mM NaCl, 2.7 mM
KC1,10 mM Na phosphate, 2 mM K phosphate). After washing, the membranes
were incubated with primary antibody solution: 1 ug/ml Anti-PLOy Phosphospecifc
[TYR 783] (Calbiochem, La Jolla, CA) diluted 1:1000 in blocking solution + 0.1%
Tween-20. Enough solution was used to completely cover the membrane and the
membranes were left on the belly dancer (ball bearings were placed on top and mixed
30


the solution for 1 hr.). The PVDF membranes were then washed 4X, 5 min. each
with PBS + 0.1% Tween-20. Following washing, membranes were incubated for 1
hour with the secondary antibody solution: goat anti-rabbit IR dye 800 (LI-COR)
diluted 1:2000 in PBS, 0.1% Tween-20, and 0,2% SDS. The wash step was repeated
and then the Tween-20 was washed away with plain PBS. Finally, the membranes
were allowed to dry and stored in aluminum foil.
Fluorescence of the secondary antibody (goat anti-rabbit IR Dye 800) was
quantified using an Odyssey Imaging System (LI-COR). The infrared dye that is
conjugated to the secondary antibody is excited by lasers in the Odyssey System at a
780 nm and emits radiation at 800 nm. This provides a higher signal to noise ratio
than other fluorescent methods whose emission spectra lie within the visible range
because it eliminates the background fluorescence by the membrane or other
biomolecules. Also, it has equal to or higher sensitivity than chemiluminescence, but
allows direct detection eliminating the need for expensive substrates or darkrooms
(LI-COR brochure 2005).
31


2.4.1 Measurement of PLCy Phosphorylation via Dot Blotting
Groups of 15 oocytes were either left untreated to serve as a negative control
or treated with 10 mM H2O2 to serve as a positive control (H2O2 is known to promote
PLCy phophorylation; Sato et al., 1999). Experimental groups (15 oocytes) were
treated with 400 pM dicapryloyl PA for 5, 10, or 55 min. Following incubation, the
eggs or oocytes were homogenized in 450 pi of ice-cold lysis buffer (0.1% SDS, 10
mM Tris-(HCl), 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton-X, 10%
glycerol, lOpg/ml Leupeptin, 10 mM PMSF, and 0.5% Na-deoxychoate). The
samples were then split in half (-250 pi per half) and one half frozen at -20 C for
later use. 250 pi of freon was added to the remaining half in order to extract
unwanted yolk proteins. The samples were then centrifuged at 16,600 x g for 10 min
at 4C in the microfuge and the supernatant was collected. The volume of the
supernatant was carefully measured with a P-100 pipetman (see 2.4 Measurement of
PLCy Phosphorylation via Western Blotting) in order to estimate total protein
concentration.). An appropriate amount of 5X Sample Buffer (60 mM Tris-HCl pH
7.5, 10.5% glycerol, 0.02%SDS, 0.03% Bromophenol blue, and 0.07% beta-
mercaptoethanol (was added just before use) was then added and the samples were
boiled for 15 min.
5 pi of each sample was then blotted directly on the PVDF membranes. The
membranes were then blocked overnight with pre-made blocking solution (LI-COR
32


Biosciences, Lincoln, Nebraska). Membranes were then washed with blocking
solution diluted 1:1 with PBS (137 mM NaCl, 2.7 mM KC1, 10 mM Na phosphate, 2
mM K phosphate). After washing, the membranes were incubated with primary
antibody solution: 1 ug/ml Anti-PLCy Phosphospecifc [TYR 783] (Calbiochem, La
Jolla, CA) diluted 1:1000 in blocking solution + 0.1% Tween-20. Enough solution
was used to completely cover the membrane and the membranes were left on the
belly dancer (ball bearings were placed on top and mixed the solution for 1 hr.). The
PVDF membranes were then washed 4X, 5 min. each with PBS + 0.1% Tween-20.
Following washing, membranes were incubated for 1 hour with the secondary
antibody solution: goat anti-rabbit IR dye 800 (LI-COR) diluted 1:2000 in PBS, 0.1%
Tween-20, and 0.2% SDS. The wash step was repeated and then the Tween-20 was
washed away with plain PBS. Finally, the membranes were allowed to dry and stored
in aluminum foil.
Fluorescence of the secondary antibody (goat anti-rabbit IR Dye 800) was
quantified using an Odyssey Imaging System (LI-COR).
33


2.5 IP3 Assay
In one experiment, groups of 15 eggs were left untreated or incubated with
either 400 pM dicapryloyl PA or 400 pM dioctanyl phosphatidylserine (scPS)
(Avanti Polar Lipids, Alabaster, AL.) for either at various time points up to 90 min.
Groups of 15 eggs were also homogenized following fertilization.
In another experiment, groups of 15 eggs were either left untreated or
incubated with 250 pM BAPTA-AM (Calbiochem, La Jolla, CA) or 6.1% DMSO
(the carrier for BAPTA-AM) for 1 hr. Untreated cells and cells treated with BAPTA-
AM were then treated with 400 pM PA for 10 min.
In the final experiment, groups of 15 eggs were microinjected (see 2.2.1
Microinjection Procedure) herbimycin-A (see Figure 2.6.1; Calbiochem, La Jolla,
CA) at a final intracellular concentration of 2 pM. 400 pM PA was then added 30
min. after injection.
Following incubation, cells were transferred to 1.5 ml v-vials (VWR, San
Francisco, CA) and excess buffer was removed using a kimwipe. The cells were then
homogenized in 300 pi of ice-cold 25% trichloroacetic acid (TCA) solution using a
dounce homogenizer and then centrifuged at 16,600 x g for 2 minutes in the
microfuge at 4C. The supernatants were then transferred to clean v-vials using a P-
1000 pipetman and 600 pi of a 3:1 solution of freon and tri-N-octylamine was then
added in order to remove the acid. The samples were then vortexed briefly and then
34


centrifuged at 16,600 x g for 20 min. at 4C. The top (aqueous layer) was then
transferred to colorless 1.5 ml v-vials. Next, 100 pi of each sample was then
incubated with 400 pi of working receptor/tracer solution at 4C for 1 hour. This
solution is composed of a membrane preparation, derived from calf brains,
containing the IP3 receptor bound with radioactively labeled IP3 ([3H] IP3). The
experimental (cold) IP3 competes for with the labeled (hot) IP3 for fixed number
of binding sites. Following incubation, the samples were centrifuged at 16,600 x g
for 20 min. at 4C. The supernatant was removed using a P-1000 pipetman then the
residual volume was removed by a P-200 pipetman with care not to disturb the pellet.
The pellets were then solubilized by adding 100 pi of 0.15M NaOH, vortexed,
allowed to stand at room temperature for 10 min., and vortexed again. The caps of the
v-vials were then cut off and the v-vials were placed into scintillation vials. Five ml
of Budget Solve was then added directly to the v-vials allowing the scintillation
fluor to overflow into the scintillation vials. The scintillation vials were then capped,
labeled, and inverted to ensure complete mixture. The amount of hot JP3 remaining
after competition with cold IP3 in the cell extract was then quantified by using a
scintillation counter. Higher counts of radioactivity in each sample meant lower
amounts of experimental IP3 in the cell extract. A standard curve was produced using
known concentrations of IP3 and the amount of IP3 in the samples is determined by
interpolation.
35


2.6 Use of Fluorometer to Measure PA-induced Calcium Release in PMCs
Plasma membrane cortices (PMCs) were manually isolated from Xenopus
oocytes as previously described by Sadler and Mailer (1981). First, the oocytes are
transferred from 0-R2 to ice-cold Sadler Isolation Buffer (SIB) (10 mM NaCl, 10
mM HEPES, pH 7.2) in a 5 cm petri dish. The cells were then pierced at the equator
with a closed pair of watchmakers forceps at the equator and tom open by allowing
the forceps to spread. Once completely opened, the cells are flattened cytoplasm side
down and the petri dish is incubated on ice for at least 25 min. After incubation on
ice for 20 min. the membrane begins to lift away from the yolk. Using forceps, the
membranes are teased and lifted away from the underlying yolk and pigment granules
leaving behind the majority (-99%) of intracellular protein. Generally, PMCs are
10-50 nm thick and contain approximately 1 pg of total protein. Important membrane-
associated proteins, cytokeletal filaments, and organelles are retained. Therefore,
they are ideal for studying calcium signaling as Src, PLC, and calcium storage sites
all remain functional in PMCs.
Once isolated the PMCs were transferred to ice-cold Modified Sadler
Isolation Buffer (150 mM KC1,10 mM HEPES made in with Nanopure Diamond
filtered water pH 7.2). Potassium chloride was substituted for sodium chloride to
better simulate the internal conditions of the cell and nanopure water was used to
ensure the lowest possible number of calcium ions contributed by the media. Groups
36


of 3 PMCs were then transferred to microliter cuvettes (Turner Designs, Sunnyvale,
CA) and the final volume was adjusted to 200 pi.
To determine if Src activity is required for PA-induced calcium release, the
PMCs were then incubated for 1 hr. with one of several tyrosine kinase inhibitors:
Peptide A (50pM), Tyrphostin B46 (100 pM), PP2 (10 pM), SU665 (1 pM), Src
Inhibitor I (1 pM), Src Inhibitor II (1 pM), and ST638 (1 pM). The inactive
derivatives Tyrphostin A1 (100 pM) was used as a negative control (all tyrosine
kinase inhibitors/inactive derivatives were obtained from Calbiochem, La Jolla, CA)
(see Figure 2.6.1).
37


Peptide A
VAPSDSIQAEEWYFGKITRRE
Tyrphostin B46 Tyrphostin A1
o
Figure 2.6.1 Molecular Structure of Tyrosine Kinase Inhibitors: Herbimycin A,
PP2, SU6656, Peptide A, PP2, and Tyrphostin B46. The molecular structure of the
inactive derivative of Tyrphostin B46 (Tyrphostin Al) is also shown.
38


All of the Src inhibitors except for peptides A and A7 are ATP-competitive
inhibitors. Activated Src kinase transfers the y-phosphate of ATP to tyrosines
residues on target proteins. Inhibitors like tyrphostin B46 and PP2 compete with ATP
for its binding site on Src. Moreover, all of these hydrophobic compounds must be
reconstituted in organic solvents such as dimethylsulfoxide (DMSO) or ethanol,
which can be damaging to cell membranes. Peptides A and A7 on the other hand are
not ATP-competitive inhibitors and are water soluble. Peptide A is a synthetic
inhibitory peptide corresponding to residues 137-157 on Src. Its sequence overlaps
the C-terminus of the SH3 domain and the N-terminus of the SH2 domain. Peptide
A7 is shorter peptide that has a more potent inhibitory effect on Src activity with an
IC50 of 3 pM. These peptides inhibit Src activity by binding to the specific segment
in the SHI kinase domain that contains tyrosine 416.
Following incubation, 100 pi of the modified SIB was removed from the
microcuvettes and replaced with 100 pi of lpM Fluo-4 (giving a final concentration
of 500 nM). Initial calcium levels were then measured with the use of a TD-700
Laboratory Fluorometer (Turner Designs). After the initial calcium concentrations
were established for each group of PMCs 400 pM PA was added and calcium
concentrations were monitored for 10 min.
39


These preincubations were also used to determine the effect of the PLC
inhibitor U73122(100 pM), the inactive U73343(100 pM ) and the DVreceptor
antagonist 2-APB (100 pM) on PA activity.
The TD-700 is compact fluorometer having an interchangeable lamp and
optical filters that allow discrete sample measurement of various fluorescent
materials. For calcium measurement, an excitation filter was used that limits the
many wavelengths of light emitted by the lamp to 488 nm. The 488 nm light directed
at the cuvette containing the PMCs and Fluo-4 excites any fluor bound to calcium to
give off light at approximately 525 nm. The emission filter then cuts out any light
below 500 nm to ensure only light emitted from the calcium-bound Fluo-4 reaches
the light detector. The light detector is connected to digital readout that gives raw
fluorescence in fluorescence signature units (fsu). A data stream was recorded using
a Windows telnet connection. The TD-700 fluorometer was connected to the COM1
port using a 9-pin serial cable. Under the Setting option in the menu bar the
Communications option was selected to configure the COM1 port. The
Connector was set to COM1 and the Baude Rate was set to 9600. Data Bits
was set to 8, Parity was set to none, Flow Control was set to Xon/Xoff, and Stop
Bits was set to 1. The connection was then saved under TD700. Data collected
under the telnet connection was then cut and pasted into a Microsoft Excel file for
analysis.
40


In order to convert raw fluorescence into calcium concentration a calibration
curve must be generated. This was accomplished by mixing pre-made calcium
standards (World Precision Instruments, Inc., Sarasota FL) 1:1 with lpM fluo-4 and
taking readings. As calcium standards are unstable, they have to be stored at -4C and
purchased periodically to ensure accuracy. Fluorescence was recorded for the
following calcium concentrations: 5 nM, 20 nM, 50 nM, 125 nM, 250 nM, 375 nM,
500 nM, 2 pM, 5 pM, 20 pM, and 50 pM. Fluorescence at 50 pM was used to
calibrate the fluorometer at 80% saturation. Raw fluorescence was plotted against
calcium concentration and linear regression was used to generate a conversion
formula (see Figure 2.6.2):
Equation 2.6.1 [Ca2+] = (Raw fluorescence x 0.0105) 0.291
Figure 2.6.2 Fluo-4 Calibration Curve.
41


3. Results
3.1 Measurement of PA-induced Calcium Release in Whole Cells
Treatment with 400 pM PA is shown to increase the concentration
intracellular concentration of calcium in whole Xenopus oocytes. Maximal
fluorescence was observed at around 8 min. which follows the peak observed for
maximal IP3 production (see Figure 3.1.1). Furthermore, the increase in calcium was
blocked by incubation with the IP3-receptor antagonist 2-APB. If PA was increasing
intracellular calcium by simply dismpting the plasma membrane to allow Ca2+ to leak
into the cell then 2-APB should have had no effect. Moreover, treatment with the
ionophore ionomycin caused a calcium increase of greater magnitude within only 2
min. (data not shown).
42


! 4 6 8 10 12 14
TIME AFTER ADDITION (MIN)
Figure 3.1.1 400 fiM PA Causes a Rise in Intracellular [Ca2+] in Whole Albino
Oocyes. Furthermore, the IP3 receptor antagonist 2-APB is shown to block this
increase.
43


3.2 Enzyme-Linked Immunosorbent Assays (ELISAs)
3.2.1 PhosphoELISA Src Tyr 418 Assay
The exogenous addition of 400 pM PA to oocytes appears to increase
Src phosphorylation by 5 min. and Src activity may be further increased by 10 min.
The average optical density (O.D.) of the two untreated groups is 0.238 where the
average O.D. of the two groups homogenized 5 min. or 10 min. following 400 pM
PA addition is 0.319 and 0.347 respectively. While not statistically significant, these
values suggest that PA may activate Src. Unfortunately, the values were too low to
convert to actual protein concentration using the standard line generated (see Figures
3.2.1.1 and 3.2.1.2).
Progesterone has been shown to be a potent activator of Src activity in
mammalian cells (Boonyaratanakomkit et al., 2001). Therefore, Xenopus oocytes
were treated with 10 ug/ml progesterone to determine if progesterone induces an
increase in Src activity similar to that elicited by PA. The data show that
progesterone may increase Src phosphorylation by 30 min. and that Src activity may
return to basal levels by 1 hour (see Figure 3.2.1.3). When a longer time course was
examined it appeared as though Src phosphorylation/activity may increase a second
time by 6 hr. (see Figure 3.2.1.4).
44


Figure 3.2.1.1 400 pM PA Appears to Increase Src Activity. Average Src Tyr 418
phosphorylation increases at 5 min. following PA treatment and increases further at
10 min. following PA treatment.
45


b[0]D0.3651316412
b[1 ] 0.0266099819
r 2D0.9947652561
Figure 3.2.1.2 Src ELISA Standard Line.
Equation 3.2.1.2.1 O.D. = [Src pY418] X 0.0266099819 + 0.3651316412
Optical densities less than 0.365 give negative values for phosphorylated Src
concentration.
46


Figure 3.2.1.3 10 pg/ml Progesterone Appears to Increase Src Activity at 10
min. Phosphorylated Src levels appear to rapidly increase and then return to basal
levels by 120 min.
47


%
F
Figure 3.2.1.410 pg/ml Progesterone May Increase Src Activity at 30 min. and
6 hours. As noted in Figure 3.2.3 phophorylated Src levels appear to rise at 30 min.
and return to basal levels at 120 min. (2 hr.). However, when an extended time
course was examined it Src Acivity appeared to peak a second time at 6 hours. The
cells used in this experiment were not defolliculated due to progesterones ability (as
a hormone) to cross membranes.


3.2.2 PhosphoELISA ERK Thr 185 / Tyr 187
In order to determine if the low signals produced by the oocyte homogenates
were due to the incompatibility between an ELISA kit designed for mammalian cells
and our amphibian cells an ERK1/2 [pTpY185/187] ELISA was performed. 2 pM
insulin is shown to significantly increase ERK activity by 1 hr. 10 pg/ml progesterone
increases ERK activity to a similar extent at 2 and 4 hours with the highest amount of
phosphorylated ERK being observed at 4 hours (see Figure 3.2.2.2). Optical densities
were converted to protein concentrated using the standard line shown in Figure
3.2.2.I.
b[0]D-0.01 01688155
b[1]D117.5495451206
r 2D0.9932342308
Figure 3.2.2.1 MAPK ELISA Standard Line
Equation 3.2.2.1.1 [ERK pT185/pY187] = O.D. x 117.549545121 0.0101688155
49


Figure 3.2.2.2 2 pM Insulin and 10 pg/ml Progesterone both Significantly
Increase MAPK activity in Xenopus Oocytes. The time course examined for
insulin was much shorter the time course examined for progesterone as insulin is
known to activate MAPK more rapidly than progesterone. These data suggest that
protein levels in Xenopus oocytes can be analyzed with ELISA kits designed for
mammalian cells.
50


3.2.3 PhosphoELISA Src Tyr 418 Assay Repeated
Experiments conducted with the aid of Nancy Tseng and Ying Chang
demonstrate that treatment with 400 pM exogenous PA causes an increase in the
amount of phosphorylated Src kinase in Xenopus eggs greater to that induced by
fertilization (see Figure 3.2.3.1). Also, it should be noted that PA treatment raised
Src phosphorylation faster than sperm. Phosphatidylserine was used as a control and
had no effect (data not shown).
TIME (MIN AFTER SPERM
OR PA ADDITION)
Figure 3.2.3.1 Both Sperm & PA cause an Increase in Src Phosphorylation
51


3.3 Measurement of PLCy Phosphorylation via Western Blotting
The expected molecular weight of phosphorylated PLCy is 145 kDA. A band
at approximately 140 kD was observed between the SeeBlue 2+ standards myosin (A)
and phosphorylase (B) (see Figure 3.3.1).
The intensity of the bands was quantified in arbitrary fluorescence units by the
Odyssey Imaging System. Treatment with 400 pM PA appeared to increase the
amount of phosphorylated PLCy by 2 min. and increased it significantly by 10 min.
PS was used as a control and appeared to have no effect. Therefore, the data suggest
PA treatment increases PLCy activity (see Figure 3.3.2).
Figure 3.3.1 Phosphorylated PLCy. The PLCy band is located between the myosin
A and phophorylase B bands found in lane loaded with SeeBlue+2 standard
having a molecular weight of approx. 140-145 kDa.
52


Figure 3.3.2 400 p,M PA Increases PLCy Activity. PLCy phosphorylation may
increase as early as 2 min. and appears to significantly increase PLCy
phosphorylation by 10 min. PS (another anionic phopholipid) had no effect.
53


3.3.1 Measurement of PLCy Phosphorylation via Dot Blotting
Dot blots were performed in order to minimize the loss of PLCy in the pellet.
As seen in the Western Blots (see Figure 3.3.2) treatment with 400 pM PA increased
PLCy phosphorylation over time. 10 mM H2O2 was used as a positive control and is
known to increase pTyr783 PLCy to levels much higher than sperm. In these dot
blots H2O2, is shown to increase phosphorylated PLCy to levels much higher than
those induced by PA (suggesting PA and sperm have a similar effect).
O
Q
LL
o
>
(/)
LU
H
Figure 3.3.1.1 Both lOmM H2O2 and 400 pM PA Increase pTyr783 PLCy
Concentration.
54


3.4 IP3 Assay
It follows that an increase in PLC activity should correspond to an increase in
IP3 production. In fact, treatment with 400 pM dicapryloyl PA stimulated IP3
production to levels similar to those seen following fertilization (indicated by red
triangle). 600 pM PS had no effect. Treatment with ionophore does increase IP3
production, but by only half that seen with sperm or PA (Stith et al.,1993). Moreover,
addition of the calcium chelator BAPTA to the external media did not affect PA
action. This reinforces the hypothesis that PA is acting specifically through PLC to
increase calcium.
It is also important that note that PA can activate PLC without raising
intracellular calcium concentration. Incubation with 250 pM BAPTA-AM (a cell
permeable calcium chealator) did not block PA-induced IP3 elevation in Xenopus
eggs (see Figure 3.4.2).
However, microinjection of the tyrosine kinase inhibitor herbimycin A (2
(-iMfinai) did inhibit PA-induced IP3 elevation, but only by 50% (see Figure 3.4.3).
This suggests that PA may be acting through a tyrosine kinase (i.e Src) and also
through a second pathway (i.e. directly) to activate PLC and increase intracellular
concentrations of IP3.
55


Figure 3.4.1 PA significantly increases IP3. The magnitude of the PA-induced
increase in IP3 was similar to that induced by fertilization (indicated by arrow). Short
chain phosptatidylserine (scPS) had no effect.


Figure 3.4.2 Incubation with 250 fiM BAPTA-AM did not Block PA-induced IP3
Elevation. Eggs treated only with BAPTA-AM or DMSO (the carrier for BAPTA-
AM) were analyzed as controls and show that neither increase levels of IP3.
- 57


+ PA
Figure 3.4.3 2 pM Herbimycin A Blocks PA-induced Elevation of IP3. This
suggests that PA may be acting through a tyrosine kinase to increase IP3. However,
PA action was only inhibited by 50%. Asterisks denote numbers significantly greater
than EGG values; p<0.013. 2 experiments, n = 6 each group. HERB + PA is
significantly lower than PA group. . .
58


3.5 Effect of Src Inhibitors on PA-induced Calcium Release
Incubating PMCs with tyrosine kinase inhibitors did not completely block
PA-induced release of calcium. Simply looking at raw fluorescence data did not
enable determination of whether the Src inhibitors had any effect (see Figure 3.5.1).
In order to quantify examine calcium release elicited by PA treatment it was
necessary to convert the data using the conversion formula calculated from the
calibration curve (see Figure 2.6.2 and Equation 2.6.1). Also, since each
microcuvette started out with different initial concentrations of calcium, the area
under the curves was calculated using Sigma Plot to determine the total amount of
calcium released (see Figure 3.5.2). Time points following equilibration of Fluo-4
and Ca were not included in the calculation. The results of five inhibitors and the
inactive tyrphostin A1 are summarized in Figure 3.5.3. Peptide A, tyrphostin B46,
SU6656, Src Inhibitor I, and PP2 all inhibited PA-induced calcium release by an
average of nearly 40%. Meanwhile, the inactive derivative of tyrphostin B46
(tyrphostin Al) had no effect.
59


J
750
700 -I
650
600
550
3
(/>
H
LU
o
z
LU
o
CO
LU
oc
O
3
-J
LL.
^ 500
<
DC
450
400 uM PA
0 100 200 300 400 500 600
TIME (s)
Figure 3.5.1 Raw Fluorescence Data Suggest 100 pM Peptide A Inhibits PA-
induced Calcium Release. However, the total calcium amount of calcium release by
each PMC preparation needs to be calculated in order to make a accurate comparison
(see Figure 3.5.2).
60


1.5e-10 i
v>
I 1.4e-10
E
q 1.3e-10
W
w
2 1.2e-10
_i
ill
| 1.1 e-10
D
j 1.0e-10
<
O
9.0e-11
0 20 40 60 80 100 120 140
TIME (s)
400 mM PA

A = 2.17x10'
(2.17 nmoles
TOTAL CA RELEASED)
A

100 |JM Peptide A

o
> pm r a. - ^^ .. A = 1.11 x 10'9
(1.11 nmoles
TOTAL CA RELEASED)
Figure 3.5.2 100 pM Peptide Decreases the Total Amount of Calcium Released
by PA Treatment. PA-induced calcium release is decreased by approximately 49%.
61


Figure 3.5.3 Five Tyrosine Kinase Inhibitors Partially Block PA Action. Peptide
A, tyrphostin B46, SU6656, SRC inhibitor I, and PP2 all produced a similar (-50%)
inhibition of PA-induced calcium release. Tyrphostin A1 (the inactive derivative of
tyrphostin B46) had no effect.
62


3.6 Effect of PLC inhibitor U73122 and nVreceptor blocker 2-APB on PA-
induced Calcium Release
Both the PLC inhibitor U73122 and the Eyreceptor antagonist 2-APB
appeared to inhibit calcium release from PMCs treated with 400 pM PA.
Furthermore, U73343 (the inactive of U73122) had no effect. This provides further
evidence that exogenously added PA is acting specifically, through PLC to increase
intracellular calcium. It also suggests that U73122 is not as effective as 2-APB in
blocking this increase in [Ca2+].
Figure 3.6.1 PLC activity is required for PA action. The PLC inhibitor U-73122
partially blocked PA action where 2-APB completely blocked PA action
63


4. Discussion
Together, these results demonstrate that addition of PA to eggs can mimic
sperm.
Figure 4.1 PA May Act to Increase Intracellular Calcium via Two Different
Pathways. The fact that different kinase inhibitors only decreased calcium release by
40% suggests that PA may activate PLC directly to account for the remaining 60%.
First of all, it was shown that PA treatment increases calcium in whole
oocytes and that this effect could be blocked by incubation with the IP3 receptor
blocker 2-APB. The requirement of IP3 receptor function indicates that PA must be
acting a stimulus to intiate calcium release from intracellular storage sites (e.g.,
endoplasmic reticulum). Moreover, maximum fluorescence was seen at
64


approximately 8 min. where IP3 levels were seen to increase within just a few (2-3)
minutes following PA treatment. It was also shown that 250 pM BAPTA-AM added
to the external media did not affect IP3 production stimulated by PA. Therefore, one
would expect that BAPTA added to the external media would not affect PAs ability
to induce fluorescence in albino oocyctes injected with fluor. However, there are
some limitations to using this method to examine PAs effect on whole cells. The
imaging system does not have a confocal capability making determination or
localization of subtle effects such as calcium puffs impossible.
Secondly, the data suggest that PA treatment may increase tyrosine 418
phosphorylation in Src extracted from Xenopus eggs and oocytes. Src must
autophosphorylate at this tyrosine residue before it can transfer phosphates from ATP
to target proteins. Therefore, it is logical to assume that a PA induced increase in Src
phosphorylation corresponds to an increase in Src activity. One experiment indicated
elevated active Src levels at 5 min. and even higher levels at 10 min. When the data
for the 10 min. time point following treatment with 400 pM PA from all experiments
was pooled (10 total data points) the amount of phosporylated Src was found to
increase by approximately 18%. This increase was found to have borderline
significance (P<0.0513) using a two tailed, one sample t test. In addition, the
magnitude of the PA response was similar to that seen with treatment with 10 pg/ml
65


progesterone which has been shown to be a potent activator of Src activity
(Boonyaratanakomkit et al., 2001).
Regardless, the signals (O.D.s) achieved from either experiment were too low
to convert to actual protein concentrations using a standard line. Several measures
were employed in an effort to improve the signal. Src is a membrane bound protein
having a lipid tail that tightly interacts with the phospholipids that make up the
bilayer. As a result, the concentration of the detergent SDS in the lysis buffer was
raised from the suggested 0.1% to 1% and each sample was sonicated on ice in effort
to maximize the solubility of Src. In addition, overall protein concentration was
maximized by homogenizing a large group (40) of oocytes in a relatively small
volume (200 pi) of lysis buffer and adding concentrated homogenate to the antibody
coated wells rather than diluting it 1:10 with standard diluent buffer (as
recommended by the protocol provided by Biosource). A 0.1% SDS concentration
combined with 1:10 dilution would give a final concentration of 0.01% SDS added to
each well. Biosource maintains that concentrations above 0.01% SDS may cause
interference between the binding of the antibodies and the target protein (i.e. Src).
It is also possible that the low signals are not the result of low solubility, but is
the result of low expression of Src in Xenopus oocytes or eggs. It is known that both
progesterone and insulin can induce oocyte maturation via the MAP Kinsase (ERK)
signaling pathway and that MAPK expression is relatively high in Xenopus oocytes.
66


Also, MAPK is a soluble protein that does not tightly associate with cell membranes.
Therefore, we used an ERK1/2 [pTpY185/187] ELISA kit to analyze groups of
oocytes that had been treated with either 10 pg/ml progesterone or 2 pM insulin for
various time points. If we could not generate sufficient signals with the ERK ELISA
kit this might point to an incompatibility between kits designed for mammalian cells
and amphibian cells. However, this was not the case. Using groups of only 10
oocytes, 0.1% SDS lysis buffer, no sonication, and diluting the samples 1:6.25 with
diluent buffer we were able to generate signals that fit ideally within the range of a
standard line. Moreover, both progesterone and insulin stimulated a >10X increase in
MAPK activity at 4 hours and 1 hour respectively. This would imply that the use of
Src ELISA kits is appropriate in the analysis of Xenopus cells.
In fact, an experiment comparing the PA induced elevation of Src
phosphorylation to that induced by fertilization did provide desirable optical
densities. Treatment with 400 pM PA was shown to increase Src activity more
rapidly and to a greater than treatment with sperm. However, it is unknown whether
PA interacts directly with Src kinase or if this effect is mediated by other proteins.
Western Blot and Dot Blot analysis provided data that suggest tyrosine kinase
(i.e. Src) activity stimulated by exogenously added PA does cause an increase in
PLCy phosphorylation. Analysis of all the SeeBlue2+ standards it was discovered
that the band was in fact at approximately 140 kDa. Phosphorylated [Y783] PLCy
67


has an expected molecular weight of 145 kDa. Moreover, using the Odyssey infrared
imaging system the resolution of the band was greater than that reported by Sato et al
(2000). The addition of 400 pM PA was seen to increase PLCy phophorylation by 2
min., with a significant increase appearing after 10 min. This time course fits with
the model in which PA activates Src which in turn activates PLCy. PA was seen to
stimulate maximal Src autophosphorylation as early as 4 minutes, well before
maximal PLCy activation.
Also, the Dot Blots suggest that the PA stimulated increase in [pTyr 783
PLCy] is on the same order of magnitude as the increase in [pTyr 783 PLCy]
stimulated by sperm. Phosphorylated PLCy levels stimulated by either sperm (Sato et
al., 1999) or 400 pM PA are much lower that those induced by 10 mM H2O2. The
Dot Blots maximized the recovery of PLCy from the pellet, but it is also possible that
the phosphospecific PLC antibody may be binding nonspecifically to other proteins.
Where an increase in PLCy phosphorylation provided evidence for PA-
induced Src activity, an increase in IP3 production is an indicator PLC activity. 400
pM PA stimulated levels of IP3 production in Xenopus eggs nearly identical to those
seen after fertilization. This is significant as treatment with an ionophore such as
A23187 also has the ability to cause an increase in IP3 production. This is due to the
fact that the Ca2+ ions entering from the external media can bind to and activate PLC
(Gompert et al. 2003). However, addition of an ionophore only raises IP3 production
68


to about half that seen with either PA or fertilization. Moreover, it is important to
note that the addition of 600 pM PS did not cause any significant increase in IP3
production. This would suggest that the addition of anionic phospholipids at
micromolar amounts does not disrupt or damage the membrane to the extent of
allowing the passage of large ions.
In addition, it is important to note that PA has the ability to elevate levels of
IP3 without simply increasing intracellular calcium. Incubation with 250 pM
BAPTA-AM for 1 hour did not block PA-induced IP3 elevation in eggs. BAPTA-AM
is a cell permeable calcium chelator and should have kept intracellular concentrations
low. However, the exact final intracellular concentration of BAPTA-AM after thel
hour incubation was unknown.
The previous data show that exogenously added PA induces a calcium release,
increases Src phophorylation, increases PLCy phosphorylation, and stimulates IP3
production. Next, various tyrosine kinase inhibitors were employed to examine
whether the PA-induced calcium release was dependent upon Src activity. Five
different tyrosine kinase inhibitors all had the ability to partially block PA action,
while the inactive tyrphostin A1 had no effect. While ideally all the Src inhibitors
should have inactive analogs that could be used for controls, the fact that all five
blocked PA action by approximately 50% suggests that they are acting specifically
through Src inhibition. Moreover, microinjection of 2 pM herbimycin A (another
69


tyrosine kinase inhibitor) decreased PA stimulated IP3 production by approximately
50%.
Nonetheless, if Src activity were required for the calcium release caused by
PA then you would expect complete inhibition rather than just the partial inhibition
observed. This may suggest that PA not only acts through Src to increase [Ca2+]m but
also binds to and activates PLC directly; where the Src-mediated pathway is
responsible for ~ 40% of the calcium increase and the direct activation PLC by PA
contributes the remaining 60%. This type of redundancy is not unusual in metabolic
pathways. Also, as mentioned before, calcium has the ability to bind to and activate
PLC.
It is also possible that the Src inhibitors bind to and deactivate the vast
majority of Src in the PMCs, but a small amount of Src still remains active. Src
would activate a small amount of PLCy and a small amount of calcium would be
released. The small amount of calcium that is released could then initiate a positive
feedback loop to stimulate PLC. Moreover, positive feedback would not be limited to
PLC, but may also occur at the level of the IP3 receptors. IP3 receptors are sensitive
to local calcium concentration (Gompert et al., 2003). Thus, as calcium concentration
increases in the cytosol, it can induce a further release from the stores. This is known
as Ca -induced Ca -release or CICR. Further experiments are needed such
70


measuring PA-induced IP3 mass in cells or PMCs pretreated with tyrosine kinase
inhibitors.
In a similar manner, the PLC inhibitor U73122 was used to verify that PLC
activity was merely increased by PA treatment, but was also required for the observed
calcium release. However, the inactive analog U73343 did not have an effect. In
addition, the nVreceptor antagonist 2-APB demonstrated total inhibition of PA-
induced calcium release in the PMCs. This reinforces observations made in the
whole albino oocytes.
As a result, it would appear as though PA is able to act as a second messenger
to increase calcium in Xenopus fertilization. However, in order to achieve statistical
significance, many of the experiments outlined above need to be repeated. In
addition, there are several other Src inhibitors that will be examined such as peptide
A7. Perhaps more importantly, experiments will be conducted to determine if PLD
activity and PA production is required for fertilization. This will be accomplished
through microinjection of PLD morpholinos or recombinant dominant negative
Xenopus PLD lb or immunodepletion of Xenopus PLD lb from membrane rafts. If
fertilization is blocked by these methods it will provide evidence for each step in the
proposed model.
71


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