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The regulation of phospolipase C during meiotic cell division in Xenopus Laevis Oocytes

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The regulation of phospolipase C during meiotic cell division in Xenopus Laevis Oocytes
Creator:
Morrison, Thomas Edward
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English
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x, 52 leaves : ; 28 cm

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Subjects / Keywords:
Phospholipase C ( lcsh )
Cell division ( lcsh )
Xenopus laevis ( lcsh )
Progesterone ( lcsh )
Cell division ( fast )
Phospholipase C ( fast )
Progesterone ( fast )
Xenopus laevis ( fast )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Bibliography:
Includes bibliographical references (leaves 44-52).
General Note:
Department of Integrative Biology
Statement of Responsibility:
by Thomas Edward Morrison.

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|University of Colorado Denver
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Full Text
THE REGULATION OF PHOSPHOLIPASE C DURING
MEIOTIC CELL DIVISION IN XENOPUS LAEVIS OOCYTES
by
Thomas Edward Morrison
B.A., University of Colorado at Boulder, 1996
B.A., University of Colorado at Boulder, 1992
A thesis submitted to the
University of Colorado at Denver
in partial fulfillment
of the requirements for the degree of
Master of Arts
Biology
1999


This thesis for the Master of Arts
degree by
Thomas Edward Morrison
has been approved
by


Morrison, Thomas Edward (M.A., Biology)
The Regulation of Phospholipase C during Meiotic Cell Division in Xenopus
laevis oocytes
Thesis directed by Associate Professor Bradley J. Stith
ABSTRACT
Insulin, insulin-like growth factor-1 (IGF-1), and progesterone induce meiotic cell
division in the Xenopus laevis oocyte and increase inositol 1,4,5-trisphosphate
(IP3) mass. IP3 is a second messenger produced by the hydrolysis of the
membrane phospholipid phosphatidyl inositol 4,5-bisphosphate (PIP2) by the
family of phospholipase C enzymes. In order to examine the mechanism of the
stimulation of phospholipase C by progesterone we obtained a stable plasma
membrane cortex (PMC) preparation from the Xenopus oocyte. We investigated
whether progesterone acts through a G-protein, tyrosine kinase, or PI 3-kinase to
activate phospholipase C. Using the PMC preparation and G-protein activators,
we found that the stimulation of phospholipase C activity by G-proteins and
progesterone was additive. Lowered Ca++ inhibited the action of progesterone but
not G-protein activators. We then investigated whether progesterone acts through
a tyrosine kinase. Both insulin and progesterone stimulated tyrosine kinase


activity in the PMC. Insulin has been shown to act through a receptor tyrosine
kinase, but this is the first demonstration that progesterone stimulated tyrosine
kinase activity. Tyrphostin, a tyrosine kinase inhibitor, blocked both insulin and
progesterone stimulation of tyrosine kinase activity as well as phospholipase C
activation. In addition, fusion proteins containing the SH2 domain from
phospholipase C-gamma (a form of phospholipase C that is activated by tyrosine
kinases) also blocked progesterone stimulation of phospholipase C activity in the
PMC. Finally, we used the PI 3-kinase inhibitor Wortmannin to investigate
whether progesterone acts through PI 3-kinase to activate phospholipase C.
Wortmannin did not inhibit the ability of progesterone to stimulate the activity of
phospholipase C.
This abstract accurately represents the content of the candidates thesis. I
recommend its publication.
Signed
IV


DEDICATION
I dedicate this thesis to my mother for hqr unlimited devotion and never failing to
stand by me.


ACKNOWLEDGEMENT
I especially want to thank my advisor, Bradley J. Stith. His generosity and
guidance will always be remembered and appreciated. I would like to thank Ellen
J. Levy for all I have learned from her and her support. I would like to thank
Robert C. Tyler for his efforts serving on my committee. I would like to thank
Cheney Lupe, Lisa Swize, and Heather Locke for their friendship. I would also
like to thank the many other people I worked with in the lab throughout the past
two and one-half years. Finally, I would also like to thank the entire staff of the
Department of Biology for their faith in me and for a wonderful experience.


CONTENTS
Figures ............................................
Chapter
1. Introduction....................................
1.1 Xenopus laevis and the Xenopus laevis oocyte....
1.2 Progesterone induced oocyte maturation..........
1.3 Phospholipase C.................................
1.3.1 Phospholipase C-delta........................
1.3.2 Phospholipase C-beta.........................
1.3.3 Phospholipase C-gamma........................
1.4 The plasma membrane cortex preparation..........
1.5 Progesterone induced activation of Phospholipase C
2. Materials and Methods...........................
2.1 Obtaining Xenopus laevis oocytes................
2.2 The plasma membrane cortex preparation.........
2.3 Addition of hormones to whole oocytes..........
2.4 Phospholipase C activity.......................
2.5 Tyrosine kinase activity.......................
2.6 Microinjection of fusion proteins..............
vii
ix
1
3
4
6
8
8
10
13
15
17
17
17
18
18
20
20


2.7 Determination of phosphorylated amino acids in phospholipase C...21
3. Results...........................................................24
3.1 Progesterone induced meiotic cell division........................24
3.2 G-protein activators stimulate phospholipase C activity in the plasma
membrane cortex....................................................24
3.3 Progesterone stimulates tyrosine kinase activity in the plasma membrane
cortex.............................................................24
3.4 Tyrphostin, a tyrosine kinase inhibitor, inhibits hormonal stimulation of
phospholipase C activity in the plasma membrane cortex.............29
3.5 GST-SH2 fusion proteins inhibit phospholipase C activity in the PMC
and the whole oocyte...............................................29
3.6 Progesterone increases phosphorylation of phospholipase C-gamma on
serine, not tyrosine...............................................34
3.7 Wortmannin, a PI 3-kinase inhibitor, does not inhibit progesterone
induced stimulation of phospholipase C activity in the PMC and
the whole oocyte...................................................34
4. Discussion..........................................................39
References
44


FIGURES
Figure
1.1 The Cell Cycle....................................................2
1.2 Biochemical signaling pathways initiated by progesterone..........5
1.3 The Phospholipase C reaction......................................7
1.4 The Three Phospholipase C isozymes................................9
1.5 The Conventional Model for phospholipase C-beta activation.......11
1.6 The Conventional Model for phospholipase C-gamma activation......12
1.7 Regulation of phospholipase C by PI 3-Kinase.....................14
3.1 Progesterone induced meiosis.....................................25
3.2 GTP-gamma-S stimulates phospholipase C activity in the plasma
membrane cortex.................................................26
3.3 G-protein and progesterone stimulation of phospholipase C activity
in the plasma membrane cortex is additive.......................27
3.4 Progesterone stimulates tyrosine kinase activity in the plasma membrane
cortex..........................................................28
3.5 Tyrphostin, a tyrosine kinase inhibitor, inhibits hormonal stimulation of
phospholipase C activity in the plasma membrane cortex..........30
IX


3.6 Inhibition of progesterone induced phospholipase C activity in the
PMC by SH2 proteins...............................................31
3.7 Shc-SH2 domains do not inhibit progesterone induced stimulation of
phospholipase C activity..........................................32
3.8 Microinjection of SH2 proteins into whole Xenopus oocytes blocked
progesterone induced stimulation of phospholipase C activity......33
3.9 Phosphoamino acids of immunoprecipitated phospholipase C-gamma... 35
3.10 Increase in serine phosphorylation of phospholipase C-gamma
immunoprecipitated from hormone stimulated Xenopus oocytes........36
3.11 Wortmannin, a PI 3-Kinase inhibitor, does not inhibit progesterone
induced stimulation of phospholipase C activity in the plasma
membrane cortex............................................. 37
3.12 Wortmannin, a PI 3-Kinase inhibitor, does not inhibit progesterone
induced stimulation of phospholipase C activity in whole Xenopus
oocytes......................................................38
4.1 Proposed model for progesterone activation of phospholipase C during
meiotic cell division in Xenopus laevis oocytes.........................43
x


1. Introduction
Insulin, insulin-like growth factor-1 (IGF-1), or progesterone induce meiotic cell
division in the Xenopus laevis oocyte (Mailer and Koontz, 1981; Stith et al.,
1991). This process is also known as oocyte maturation or germinal vesicle
breakdown (GVBD) where breakdown refers to dissolution of the nuclear
envelope.
The Xenopus oocyte is diploid and cannot be fertilized. This cell is arrested
in early prophase near the G2/M phase border of the cell cycle (Mailer,
1990)(Figure 1.1).
Hormone addition induces oocyte maturation and the oocyte enters meiotic
M-phase. Thus, oocyte maturation consists of resumption of meiosis I, completion
of meiosis I, and entry into meiosis II. The cell becomes arrested again at
metaphase II and it is called an egg.
The egg is haploid and can be fertilized. Fertilization will release the egg
from metaphase II arrest and one of the events of fertilization is the completion of
meiosis.
1


Oocyte Maturation
. Egg arrested
Progesterone J , .
I f metaphase
Oocyte of meiosis
at
II
Figure 1.1: The Cell Cycle.
The Xenopus oocyte is arrested near the G2/M phase border. Progesterone
induces meiotic cell division and the cell becomes arrested again at metaphase II
of meiosis as an egg capable of being fertilized.
2


1.1 Xenopus laevis and the Xenopus laevis oocyte
Xenopus laevis is a clawed frog from South Africa that is widely used for research
in the areas of developmental, cell and molecular biology (Tinsley and Kobel,
1996). There are several reasons that this species is so widely used as a
laboratory animal. Oocytes or eggs can be obtained from the animals on a year-
round basis. In fact, Xenopus laevis has been used as an assay for pregnancy;
urine samples from women are injected into frogs. If the frog lays eggs, the
woman is pregnant. The frog is induced to lay eggs if the urine samples contain
the hormone chorionic gonadotropin. The processes of oocyte maturation and
fertilization closely resemble the same processes in humans. Since Xenopus
laevis have a primarily aquatic lifestyle, this allows for cleaner, cheaper, and more
convenient maintenance of a laboratory population. The animals are extremely
resistant to disease and infection and are long-lived in captivity. The large size of
Xenopus oocytes and embryos provide for many research advantages: due to their
large size, biochemical analysis can be carried out on a small number of cells.
Xenopus oocytes or eggs can be easily microinjected with mRNA, cloned DNA,
peptides, and other compounds. This has led some to describe the Xenopus
oocyte as a living test tube (Tinsley and Kobel, 1996).
3


1.2 Progesterone induced oocyte maturation
The mechanism of induction of the meiotic cell cycle by progesterone remains
unclear. Progesterone is believed to act at the cell membrane, activating signaling
pathways leading to many biochemical changes in the cell interior (Figure 1.2).
There are several lines of evidence that progesterone acts at the cell surface.
Microinjected progesterone is unable to induce meiotic cell division (Masui and
Markert, 1971; Smith and Ecker, 1971). However, progesterone linked to
polymer or bound to agarose beads is still able to induce meiotic cell division in
the oocyte (Godeau, et al., 1978, Ishikawa et al., 1977). Progesterone has also
been shown to inhibit adenylate cyclase in the oocyte membrane (Sadler and
Mailer, 1981) and stimulate phospholipase C in the oocyte membrane (Stith et al.,
1992).
About 3-4 hours after progesterone is added to oocytes, the nucleus migrates
to the cell surface and the nuclear envelope dissolves. Upon migration to the
surface, the nucleus pushes away pigment granules and causes a white spot to
start to appear. Other changes include a rapid decrease in cAMP and subsequent
decrease in cAMP-dependent protein kinase (Protein Kinase A) activity (Mailer et
al., 1979), polyadenylation of Mos mRNA (Sheets et al., 1995), an increase in
4


Progesterone
*
Phospholipase C
J
T IP3 + T DAG
J i
R?
(+)
T Ca++ T PKC
G-protein (Gj)
\<->
Adenylate Cyclase
i cAMP
i PKA
i
t Mos
I
T MPF (cdc2/cyclin B)
Figure 1.2: Biochemical signaling pathways initiated by progesterone
Progesterone acts at the cell membrane to inhibit (-) adenylate cyclase resulting
in a rapid decrease (4) in cAMP and subsequent decrease in Protein kinase A
(PKA) activity. An increase in both the activity of Mos and maturation
promoting factor (MPF) leads to GVBD in the Xenopus oocyte. Progesterone
also stimulates phospholipase C activity to produce the second messengers
inositol 1,4,5-trisphosphate (IP3) and sn 1,2-diacylglycerol (DAG). It is still not
known whether increased Ca++ or PKC activation is required for GVBD (see
question marks).
5


mos (Gebauer and Richter, 1996), and an increase in the activity of maturation
promoting factor (MPF) (Mailer, 1990). MPF is a complex of the protein cyclin
B and cdc 2, a protein serine/threonine kinase (Gautier, et al., 1988). These
events are part of the signaling pathway believed to be the major pathway leading
to oocyte maturation in Xenopus.
However, the addition of progesterone or high concentrations of insulin to the
Xenopus oocyte also stimulates phospholipase C to breakdown
phosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol 1,4,5-
trisphosphate (IP3) and sn 1,2-diacylglycerol (DAG) (Figure 1.3) (Stith et al.,
1992). IP3 binds receptors on the endoplasmic reticulum (ER) inducing the
release of internal Ca++ stores from the ER lumen. The production of EP3 and a
localized elevation of intracellular calcium may be important in the ability of
progesterone to induce meiotic cell division (Kobrinsky et al., 1995; Han and Lee,
1995; Duesbery and Masui, 1996a, 1996b).
1.3 Phospholipase C
The phospholipase C enzymes are a family of inositol phospholipid
phosphodiesterases (James and Downes, 1997, Katan, 1998; Rhee and Bae,
1997). This family of enzymes is found in bacteria, simple eukaryotes, plants,
and animals. The three major isozymes are: phospholipase C-beta,
6


The Phospholipase C Reaction
o
II
CH3-CH2(d)-C-0-CH2 O
I II
CH3-CH2(n)-C-0-CH O HO O-P-OH
III II I II
0CH2-0-P-0 C C 0_ O
I \ / \ II
O C C O-P-OH
\ I I
c c 0_
I I
OH OH
Phosphatidyl inositol 4,5-bisphosphate
(PIP2)
o
II
CH3-CH2(n>C-0-CH2
I
CH3-CH2(n)-C-0-CH
II I
O CH2-OH
sn 1,2-diacylglycerol (DAG)
\
II
O O-P-OH
II I I
O -P -o c c o_ o
11/ \ II
_0 C CO-P-OH
\ / I
cc o
Inositol 1,4,5-trisphosphate
(IP3)
Figure 1.3: The Phospholipase C Reaction.
Phospholipase C hydrolyzes the membrane phospholipid
phosphatidyl inositol 4,5-bisphosphate (PIP2) to produce the second
messengers inositol 1,4,5-trisphosphate (IP3) and sn 1,2-diacylglycerol (DAG).
7


phospholipase C-gamma, and phospholipase C-delta (Figure 1.4). All of the
isozymes share two conserved regions, the X and the Y box, which form the
catalytic domain. They also share two Ca++ binding domains in the form of an EF
hand domain and a C2 domain. All of the isozymes require low levels of Ca++ for
catalytic activity. Each of the isozymes has an N-terminal PH domain. As will be
discussed, PH domains function in protein-lipid interactions.
1.3.1 Phospholipase C-delta
Phospholipase C-delta is the smallest of the three types of mammalian
phospholipase C isozymes (MW 85,000) (Rhee, S.G., and Bae, Y.S., 1997, Katan,
M., 1998). Phospholipase C-delta may be more sensitive to stimulation by Ca++
alone than either the beta or the gamma forms, so cytoplasmic [Ca++] may
regulate the isozyme. A new class of G-proteins, termed Gh proteins, may also
activate phospholipase C-delta. The mechanisms by which phospholipase C-delta
is coupled to cell surface receptors remains unclear (Rhee, S.G., and Bae, Y.S.,
1997).
1.3.2 Phospholipase C-beta
Phospholipase C-beta contains a C-terminal tail that functions in binding the
alpha subunit of Gq heterotrimeric G-proteins (Katan, 1998). Thus, phospholipase
8


Three Types of PLC isozymes
PH EF-HAND X SH2 Y C2
PLC-7 -[

SH3
PLC-P L
PLC-6
(Rhee. S.G. and Bae, Y.S., 1997)
Figure 1.4: The Three Phospholipase C isozymes.
Phospholipase C-gamma, phospholipase C-beta, and phospholipase C-delta are
the three types of mammalian PLC isozymes. The X and the Y box form the
conserved catalytic domain. The pleckstrin homology (PH) domains function in
lipid-protein interactions. The EF Hand and C2 domain are both Ca++ binding
domains. The Src Homology (SH) domains of phospholipase C-gamma (but not
beta or delta) function in protein-protein interactions. The SH2 domains bind
phosphorylated tyrosine residues of other proteins and the SH3 domains bind
proline-rich regions of other protein.
9


C-beta is activated through cell surface receptors coupled to heterotrimeric G-
proteins (Figure 1.5) (Rhee, S.G. and Bae, Y.S., 1997, Katan, M., 1998).
Phospholipase C-beta is activated by many agonists including vasopressin, the
muscarinic path of acetylcholine, bradykinin, and angiotensin II.
1.3.3 Phospholipase C-gamma
Phospholipase C-gamma contains an N terminal and C terminal SH2 domain
(Katan, 1998, Rhee and Bae, 1997; James and Downes, 1997). SH2 domains
bind phosphorylated tyrosine residues and this leads to activation (see below).
Phospholipase C-gamma also contains an additional PH domain that functions in
protein-lipid interactions, and this domain may also play a role in activating the
enzyme. Finally, the enzyme contains an SH3 domain that specifically interacts
with proline-rich regions of other proteins (this domain does not activate the
enzyme)(Seedorf, et al., 1994). Three tyrosine residues in phospholipase C-
gamma become phosphorylated after stimulation of growth factor receptors such
as the receptors for platelet-derived growth factor (PDGF), epidermal growth
factor (EGF), fibroblast growth factor (FGF), and nerve growth factor (NGF)(Kim
et al., 1991, Kim et al., 1990; Rhee and Bae, 1997). All of these receptors are
tyrosine kinases and they phosphorylate phospholipase C-gamma on the three
tyrosines once ligand binds (Figure 1.6).
The activated receptors have
10


Ligand
Of' '^5'
GTP GDP
Figure 1.5: The Conventional Model for Phospholipase C-beta activation.
A ligand binds to the extracellular region of a G-protein-coupled receptor
(serpentine receptor) inducing a conformational change in the intracellular
region. This change results in GTP displacing bound GDP from the alpha
subunit of the receptor-associated heterotrimeric G-protein. The alpha subunit,
with bound GTP, dissociates from the beta/gamma subunits and then travels to
and activates phospholipase C-beta. PIP2 is broken down to IP3 (which releases
Ca++) and DAG (which activates PKC).
11


Growth Factor
Figure 1.6: Conventional model for Phospholipase C-gamma activation
A growth factor binds to the extracellular domain of a cell surface receptor
tyrosine kinase. The intracellular kinase domain of the receptor
autophosphorylates on tyrosine residues. Autophosphorylation creates a binding
site for the SH2 domains of phospholipase C-gamma. Phospholipase C-gamma
binds to the activated receptor, becomes phosphorylated on tyrosine residues, and
is activated. PIP2 is broken down to IP3 (which releases Ca++) and DAG (which
activates PKC).
12


phosphotyrosines that bind the SH2 regions of phospholipase C-gamma.
However, phospholipase C-gamma may also be activated by another
mechanism involving phospholipase C-gammas PH domain binding to PIP3 in
the membrane (Falasca, et al., 1998) (Figure 1.7). PEP3 is made by PI 3-kinase.
1.4 The plasma membrane cortex preparation
A unique property of the Xenopus oocyte is that a functional plasma membrane
cortex (PMC) preparation can be separated from the nucleus and 99% of cell
protein (Sadler and Mailer, 1981). The structure of the PMC consists of three
layers that prevent the preparation from falling apart. The outermost layer is the
vitelline envelope. This is an outer glycoprotein layer that surrounds the plasma
membrane of the oocyte. Just underneath is the plasma membrane: a
phospholipid bi-layer with integral and peripheral membrane proteins. The
innermost layer is the cell cortex. The cell cortex consists of a cross-linked
network of actin filaments and actin binding proteins. The cross-linking gives the
cortex the physical properties of a gel. The cortex also includes vesicles.
The PMC preparation maintains links between hormones and receptors as
well as that between receptors and immediate downstream proteins.
Thus, this unique preparation allows the study of immediate cell signaling
13


0^ Hormone
PI 3-Kinase
Figure 1.7: Regulation of Phospholipase C by PI 3-kinase
Hormone binds to the extracellular domain of a cell surface receptor tyrosine
kinase. This binding event induces the intracellular kinase domain to
autophosphorylate on tyrosine residues. Autophosphorylation creates a binding
site for the SH2 domain of the p85 subunit (regulatory subunit) of PI 3-kinase.
The pi 10 domain (catalytic) domain binds to p85 and is an activated kinase. One
substrate for PI 3-kinase is the membrane phospholipid phophatidylinositol 4,5-
bisphosphate (PIP2). PI 3-kinase phosphorylates PIP2 to produce
phosphatidylinositol 3,4,5,-trisphosphate (PIP3). The PH domain of
phospholipase C binds PEP3, thus drawing the enzyme to the membrane.
14


events at the plasma membrane. For example, Sadler and Mailer (1981) showed
that progesterone addition to the PMC inhibits the enzyme adenylate cyclase
(Figure 1.2).
1.5 Progesterone induced activation of Phospholipase C
To examine the mechanism by which progesterone stimulates phospholipase C
activity, we investigated whether progesterone acts through a G-protein, a
tyrosine kinase, or a PI 3-kinase. Using the PMC preparation and G-protein
activators we found that the stimulation of phospholipase C activity by G-proteins
and progesterone was additive. Lowered Ca++ inhibited the action of
progesterone but not G-protein activators. We then investigated the possibility
that progesterone is acting through a tyrosine kinase. Both insulin and
progesterone stimulated tyrosine kinase activity in the PMC. Insulin has been
shown to act through a receptor tyrosine kinase, but this is the first demonstration
that progesterone stimulated tyrosine kinase activity. Tyrphostin, a tyrosine
kinase inhibitor, blocked both insulin and progesterone stimulation of tyrosine
kinase activity as well as phospholipase C activation. In addition, fusion proteins
containing the SH2 domain from phospholipase C-gamma also blocked
progesterone stimulated phospholipase C activity in the PMC. Finally, we used
15


I
the PI 3-kinase inhibitor Wortmannin to investigate if progesterone was acting
through this enzyme to activate phospholipase C. Wortmannin did not inhibit the
ability of progesterone to stimulate the activity of phospholipase C.
16


2. Materials and Methods
2.1 Obtaining Xenopus laevis oocytes
Xenopus laevis females were obtained from Xenopus One (Ann Arbor, MI) or
Xenopus Express (Homosassa, FL) and maintained on a diet of ground beef heart.
Female frogs were injected with 50IU of pregnant mares serum gonadotropin
(PMSG) (Calbiochem, La Jolla, CA) and O.lmg/ml bovine serum albumin (BSA)
(Sigma, St. Louis, MO) approximately 3-4 days prior to use. This priming
reduces the time required for maturation and increases the synchrony of cell
division (Stith and Mailer, 1985). On day of use, primed female frogs were
placed on ice and sacrificed by guillotine. Ovarian tissue was removed, cleaned
of blood, and placed in oocyte Ringers solution (OR-2; 83mM NaCl, 0.5mM
CaCh, ImM MgCh, lOmM HEPES, pH 7.9) at room temperature. Stage VI (1.2-
1.3 mm diameter) oocytes were obtained after manual removal of ovarian tissue.
2.2 The plasma membrane cortex preparation
The plasma membrane cortex preparations were made from Stage VI oocytes that
were tom open in ice-cold Sadlers isolation buffer (lOmM NaCl, lOmM HEPES,
pH 7.9). The cytoplasm was washed away, the PMCs placed yolk-side down and
incubated on ice for 30-45 minutes. The PMC was lifted away from the yolk and
17


other cellular protein and washed. Groups consisted of 15 cortices in 50-100 pi
total volume. There were 4-5 groups per treatment. All treatments were done at
15C.
2.3 Addition of hormones to whole oocytes
lOpM progesterone (Sigma, St. Louis, MO) or 2pM porcine insulin (Lily,
Indianapolis, IN) was added to groups of 10-20 stage VI oocytes. The percentage
of oocytes that formed a white spot in the dark animal pole was recorded. The
formation of a white spot indicated germinal vesicle breakdown (GVBD), a major
event during meiotic cell division. Untreated oocytes were checked for
spontaneous maturation.
2.4 Phospholipase C activity
The activity of phospholipase C was measured at 15 minutes after addition of
insulin or progesterone. The PMC medium or whole oocytes were assayed for IP3
with an IP3 receptor binding assay (Inositol-1,4,5-Trisphosphate [3H]
Radioreceptor Assay Kit, NEN, Boston MA). Phospholipase C activity is thus
measured by IP3 mass.
At the appropriate time, 100 pi of 25% trichloroacetic acid (TCA) was added
to groups of 15 PMCs or 300pl of 25% TCA was added to groups of 15 whole
oocytes and the groups were homogenized. The TCA in the homogenates was
extracted with 3:1 freon (l,l,2-trichloro-l,2,2-trifluoroethane): tri-n-octylamine
18


(Sigma, St. Louis, MO). The samples were then added to a membrane prep
containing 3H-EP3 bound to IP3 receptors (purified from calf brain). After an
incubation, cold IP3 in the sample displaced bound 3H-IP3 from the EP3 receptor.
The IP3 receptor was isolated by centrifugation and the supernatant discarded. IP3
mass in the samples was determined by measuring the decrease in cpm associated
with the IP3 receptor by liquid scintillation counting.
In other experiments, GTP-gamma-S or GDP-beta-S (Calbiochem, La Jolla,
CA) was used to treat the PMC. In some experiments, tyrphostin B46
(Calbiochem, La Jolla, CA.), the inactive tyrphostin A1 (Calbiochem, La Jolla,
CA ), or Wortmannin (Calbiochem, La Jolla, CA.) was added to the PMC prior to
hormone addition. Tyrphostin B46 is an inhibitor of tyrosine kinase activity
whereas Wortmannin inhibits PI 3-kinase activity. Some experiments involved
the addition of fusion proteins containing the N and C terminal SH2 domains
from phospholipase C-gamma or the SH2 domain from She (gifts from Dr.
Nicholas Webster, University of California at San Diego) to the PMC. The fusion
protein from phospholipase C-gamma should block phospholipase C-gamma
activation by tyrosine kinases whereas the SH2 domain from She is used as a
control (does not inhibit tyrosine kinase activation of phospholipase C-gamma).
19


2.5 Tyrosine kinase activity
Tyrosine kinase activity in the PMC was measured with 0.5 mM of the peptide
substrate RR-SRC. RR-SRC (Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-
Arg-Gly) does not contain serine or threonine residues and is a good substrate for
tyrosine kinases (Pike et al., 1986; Ferry et al., 1990). At time zero, 50pCi
AT P, 5mM MgCh, RR-SRC were, along with 1 OpM progesterone or 1 pM
insulin, added to the PMC. At 15 minutes, the reaction was stopped by
transferring an aliquot to P81 paper and the papers were then placed into 300 ml
of 75mM phosphoric acid. The papers were washed three times with 75mM
phosphoric acid and radioactivity on the papers was quantified by liquid
scintillation counting. Control groups received equivalent amounts of ethanol
(the solvent for progesterone).
2.6 Microinjection of fusion proteins
Microinjection needles were made from lOpl glass micropipettes (Fisher
Scientific Company, Pittsburgh, PA) using a PUL-1 microinjection needle puller.
Using a PV380 Pneumatic Picopump (World Precision Instruments, Inc.,
Sarasota, FL) individual oocytes were pressure injected with 40-50nl of 0.52(j.g/pl
GST-SH2 fusion proteins. The oocytes were injected in the animal pole at the
border near the vegetal pole.
20


2.7 Determination of phosphorylated amino acids
in phospholipase C
Groups of 50-100 oocytes were incubated in 0.5-2.0 mCi of PO4 for 6 hours at
room temperature. The oocytes were then washed twice with fresh oocyte
Ringers solution and incubated with 2pM insulin or 10|iM progesterone for the
following time periods: 30 seconds, 1 minute, 5 minutes, 15 minutes, and 30
minutes. Oocytes were homogenized in 1 ml of ice-cold lysis buffer (20mM
HEPES, 1% w/v Triton X-100, 80mM P-glycerophosphate, 50mM sodium
fluoride, ImM phenylmethylsulfonyl fluoride, 10(!g/ml leupeptin, 10|ig/ml
aprotinin, 2mM sodium orthovanadate, 20mM EGTA, pH 7.2). The homogenates
were centrifuged for 10 minutes at 14,000XG and the supernatant was pre-cleared
with 50|il 50% Protein A-Sepharose (Sigma, St. Louis, MO.) for 1 hour at 4C.
The samples were centrifuged again and 10pl of anti-phospholipase C-gamma-1
(Upstate Biotechnology, Lake Placid, NY.) was added to the pre-cleared
supernatant. The samples were incubated with antibody for 12-14 hours at 4C.
Protein A-Sepharose (40|il of a 50% solution) was added and incubated for 1 hour
at 4C followed by centrifugation for 5 seconds at 14,000xg. The
immunoprecipitate was washed three times for 1 hour each in a low salt solution
(20mM Tris, 0.1% v/v Triton X-100, 5mM EDTA, lOOmM NaCl), a high salt
solution (20mM Tris, 0.1% Triton X-100, 5mM EDTA, 1M NaCl), and 20mM
HEPES. The samples were then centrifuged for 1 minute at room temperature
21


and the supernatant removed. To hydrolyze the peptide bonds, 50pl of 5.7N
constant boiling HC1 (Sigma, St. Louis, MO.) was added to the
immunoprecipitates, the tube was filled with nitrogen, and samples were
incubated at 110C for 1 hour. The HC1 was evaporated under nitrogen and the
samples were resolubilized in 5pl pH 1.9 buffer (50ml 88% formic acid, 156ml
acetic acid, 1794ml deionized water) and 5pl of 0. lpg/ul phosphoamino acid
standards (o-phospho-L-serine, o-phospho-L-threonine, o-phospho-L-tyrosine;
Sigma, St. Louis, MO). The samples were spotted on thin layer cellulose plates
(DC-Plastikfolien Cellulose, EM Science, Gibbstown, NJ.) and electrophoresed in
two dimensions using the Hunter Thin Layer Electrophoresis System (Model #
HTLE-7000, CBS. Scientific Company, Inc., Del Mar, CA). The samples were
electrophoresed in the first dimension for 35 minutes at 1500 volts in pH 1.9
buffer. The plates were allowed to air dry and the samples were electrophoresed
in the second dimension for 20 minutes at 1200 volts in pH 3.5 buffer (100 ml
acetic acid, 10 ml pyridine, 1890 ml deionized water). After electrophoresis was
complete, the plates were sprayed with 0.25% ninhydrin (2,2-dihydroxy-1,3-
indanedione, Sigma, St. Louis, MO) to detect phosphoamino acid standards and
baked at 65C for 10-15 minutes. The plates were then placed on film (Kodak
Biomax MS) and stored at -70C until developed. Film was developed using
Kodak GBX fixer and replenisher and Kodak GBX developer. Images were
22


scanned in (Hewlett Packard Scanjet IIcx/T) and radioactivity was quantified by
spot density (SigmaGel; Jandel Scientific).
23


3. Results
3.1 Progesterone induced meiotic cell division
lOpM progesterone induced meiotic cell division in Xenopus oocytes indicated by
formation of a white spot in the animal pole (GVBD) (Figure 3.1). 100% of the
cells matured in less than 6 hours. Untreated oocytes showed no spontaneous
maturation.
3.2 G-protein activators stimulate phospholipase C
activity in the plasma membrane cortex
The G-protein activator GTP-gamma-S was able to stimulate phospholipase C
activity in the PMC (Figure 3.2). Another G-protein activator, GDP-beta-S, also
stimulated phospholipase C activity in the PMC (Figure 3.3). Addition of this
activator together with progesterone led to an additive increase in IP3 production
(Figure 3.3).
3.3 Progesterone stimulates tyrosine kinase activity
in the plasma membrane cortex
Progesterone (5pM) and insulin (lgM) stimulated tyrosine kinase activity in the
plasma membrane cortex (Figure 3.4). Tyrphostin B46 (200pM), a tyrosine kinase
24


120
0
T--------------1------------1-------------1-------------
2 4 6 8 10
Time after progesterone addition (hrs.)
PROGESTERONE TREATED: Time (hrs.) vs. %GVBD
UNTREATED OOCYTES: Time (hrs.) vs %GVBD
Figure 3.1: Progesterone induced meiosis
10p.M progesterone was added to Xenopus oocytes. Oocytes were observed for
white spot formation (indicates GVBD). At 9 hours, oocytes were fixed in
5% TCA and dissected to confirm nuclear membrane dissolution.
25


Figure 3.2: GTP-gamma-S stimulates phospholipase C activity
in the plasma membrane cortex.
GTP-gamma-S was added to the plasma membrane cortex. Phospholipase C
activity was measured after a 15 minute incubation at 15C using an IP3 mass
assay.
26


IP (fMOL/CORTEX)
300
Figure 3.3: G-protein and progesterone stimulation of
phospholipase C activity in the plasma membrane cortex is additive
GDP-beta-S, progesterone, or both were added to the plasma membrane
cortex and phospholipase C activity was measured after 15 minute incubations.
27


Figure 3.4: Progesterone stimulates tyrosine kinase activity
in the plasma membrane cortex
Tyrosine kinase activity was measured in the plasma membrane after a
15 minute incubation with lpM insulin or 5pM progesterone. "Tyr" groups
were preincubated for 10 minutes with 200pM tyrphostin B46. The inactive
tyrphostin A1 was added to control groups. Due to variation between PMC's
obtained from different frogs, all data are represented relative to control groups.

28


inhibitor, blocked tyrosine kinase activation by either hormone.
3.4 Tyrphostin, a tyrosine kinase inhibitor, inhibits
hormonal stimulation of phospholipase C activity
in the plasma membrane cortex
Progesterone and insulin stimulated phospholipase C activity in the PMC and
tyrphostin B46 was able to block this stimulation (Figure 3.5). The inactive
tyrphostin A1 was added prior to addition of insulin or progesterone in some
groups and did not block hormonal stimulation of phospholipase C activity.
3.5 GST-SH2 fusion proteins inhibit phospholipase C
activity in the PMC and the whole oocyte
A fusion protein containing the N and C terminal SH2 domains from
phospholipase C-gamma was added to the PMC 15 minutes prior to addition of
progesterone. This fusion protein was able to inhibit progesterone induced
stimulation of phospholipase C activity (Figure 3.6). Addition of a fusion protein
containing the SH2 domain from the adapter protein SHC did not block
progesterone stimulation of phospholipase C activity (Figure 3.7). Microinjection
of the fusion protein containing the SH2 domains from phospholipase C-gamma
into whole oocytes showed a similar action as in the PMC (Figure 3.8).
29


Tyrph. A1
Tyrph. B46
Progesterone
Insulin
+
+
+
+
+
+
+
+
Figure 3.5: Tyrphostin, a tyrosine kinase inhibitor, inhibits
hormonal stimulation of phospholipase C activity in the plasma
membrane cortex
Phospholipase C activity was measured in the plasma membrane cortex
after a 15 minute incubation with lpM insulin or 5pM progesterone.
Some groups were preincubated for 10 minutes with the inactive
tyrphostin A1 or with the active tyrphostin B46.
30


CON PROG PROG +
SH2 PROTEIN
Figure 3.6: Inhibition of progesterone induced phospholipase C activity
in the PMC by SH2 proteins.
GST-SH2 fusion proteins containing the N and C terminal SH2 domains from
phospholipase C-gamma were added to the plasma membrane cortex 15
minutes prior to hormone addition at a final concentration of 20ng/|il.
Phosphlipase C activity was measured using an IP3 mass assay 15 minutes after
addition of 1 OpM progesterone.
31


CON PROG PROG +
She SH2
Figure 3.7: SH2 domains from She do not inhibit progesterone
induced stimulation of phospholipase C activity
GST-SH2 proteins containing the SH2 domain from She were added
to the plasma membrane cortex 15 minutes prior to hormone addition
at a final concentration of 20ng/jil. Phospholipase C activity was measured
using an EP3 mass assay 15 minutes after addtion of lOpM progesterone.
32


3 (fMOL/OOCYTE)
160
CON PROG PLC-gamma SH2
+ PROG
Figure 3.8: Microinjection of SH2 proteins into whole Xenopus oocytes
blocked progesterone induced stimulation of phospholipase C activity.
GST-SH2 fusion proteins containing the N and C terminal SH2 domains
from phospholipase C-gamma were microinjected to a final concentration
of 10.8ng/pl into whole Xenopus oocytes (assuming injection of 50nl into an
oocyte with an intracellular volume of 480nl; Stith and Mailer, 1984).
(groups not significantly different)
33


3.6 Progesterone increases phosphorylation of
phospholipase C-gamma on serine, not tyrosine
Phosphoamino acid analysis of phopsholipase C-gamma immunoprecipitated from
hormone treated oocytes did not show an increase in phosphotyrosine (Figure 3.9) (note
lower right dotted circle labeled P-tyr does not get darker). However, both insulin and
progesterone increased the amount of phosphoserine (Figure 3.10).
3.7 Wortmannin, a PI 3-kinase inhibitor, does not inhibit
progesterone induced stimulation of phospholipase C
activity in the PMC and the whole oocyte
Wortmannin (100pM), a PI 3-kinase inhibitor, was preincubated with the PMC
for 15 minutes or with whole oocytes for 30 minutes. If PI 3-kinase activates
phospholipase C-gamma, then Wortmannin should block progesterone stimulation
of phospholipase C-gamma. Wortmannin did not block progesterone stimulation
of phospholipase C-gamma activity in the PMC (Figure 3.11) or in the whole
oocyte (Figure 3.12).
34


Figure 3.9: Phosphoamino acids of immunoprecipitated
phospholipase C-gamma
Xenopus oocytes were incubated for 6 hours in 32PC>4. After a 15-minute
incubation in IOjiM progesterone, the oocytes were homogenized and cell lysates
immunoprecipitated with anti-phospholipase C-gamma-1 antibodies. After
hydrolysis, Hunter thin-layer electrophoresis was used to separate phosphoamino
acids. To detect 32P04, autoradiography was used. P-ser represents
phosphorylated serine residues, P-thr represents phosphorylated threonine
residues, and P-tyr represents phosphorylated tyrosine residues, which are not
seen.
35


Figure 3.10: Increase in serine phosphorylation of
phospholipase C-gamma immunoprecipited from hormone
stimulated Xenopus oocytes.
Densitometry was used to quantify Hunter thin-layer electrophoresis and analysis
of the radioactivity in the phosphoserine spot after insulin (2pM) or progesterone
(lOpM) were added to oocytes for 15 minutes. Figure represents analysis from
8 experiments.
36


Figure 3.11: Wortmannin, a PI 3-Kinase inhibitor, does not inhibit
progesterone induced stimulation of phospholipase C activity in the
plasma membrane cortex.
Plasma membrane cortices were preincubated with lOOnM wortmannin
for 15 minutes, then progesterone (10pM) was added. Phospholipase C
activity was measured after another 15 minute incubation using an EP3 mass assay.
37


CON PROG PROG+
WORT
Figure 3.12: Wortmannin, a PI 3-Kinase inhibitor, does not inhibit
progesterone induced stimulation of phospholipase C activity in whole
Xenopus oocytes.
Xenopus oocytes were preincubated in lOOnM wortmannin for 30 minutes,
then progesterone (10pM) was added. Phospholipase C activity was
measured after another 15 minute incubation using an IP3 mass assay.
38


4. Discussion
We investigated the three common paths for regulation of PLC activity during
progesterone-induced meiotic cell division in Xenopus laevis oocytes. The paths
involve G-proteins, tyrosine kinases, or PI 3-kinase. G-protein activators were
able to stimulate PLC activity in a plasma membrane cortex preparation,
indicating the presence of the beta isoform. A phospholipase C-beta isozyme has
previously been purified from the Xenopus oocyte (Filtz, et al., 1996). However,
the G-protein stimulated increase in PLC activity was additive with progesterone
stimulated activity, indicating progesterone may not be acting through a G-protein
to stimulate phospholipase C. In addition, lowering [Ca++] inhibited the action of
progesterone but not G-protein activators (data not shown).
We then investigated whether progesterone acts through a tyrosine kinase to
stimulate phospholipase C activity. Phospholipase C-gamma is activated by
tyrosine phosphorylation (Meisenhelder, et al. 1989; Schlessinger, et al., 1989;
Nishibe et al., 1989). Progesterone stimulated tyrosine kinase activity in the
plasma membrane cortex. This is the first demonstration that progesterone
stimulates a tyrosine kinase at the membrane. Further, tyrphostin B46, a tyrosine
kinase inhibitor, was able to block the progesterone stimulated increase in both
tyrosine kinase and phospholipase C activities.
39


We continued to investigate the involvement of a tyrosine kinase by using
fusion proteins containing the N and C terminal SH2 domains from phospholipase
C-gamma. Referring to figure 1.6, note that the lightly shaded portion of
phospholipase C-gamma (labeled SH2 in the figure) when added to the PMC
preparation or microinjected to a high concentration in the whole oocyte would
bind the receptor and prevent phospholipase C-gamma from binding. The SH2
proteins were able to block progesterone stimulated phospholipase C activity in
the plasma membrane cortex and we found a similar trend when the fusion protein
was microinjected into whole oocytes.
However, we were unable to find an increase in tyrosine
phosphorylation on phospholipase C-gamma using two-dimensional thin-layer
electrophoresis to analyze phosphoamino acids. This was unexpected since, once
phospholipase C-gamma is bound to the receptor through phospholipase C-
gammas SH2 domains, phospholipase C-gamma is then phosphorylated on
tyrosine. We also used a Western blot with anti-phosphotyrosine antibodies but
did not record an increase in tyrosine phosphorylation on phospholipase C-gamma
following hormonal stimulation (data not shown).
The PI 3-kinase inhibitor Wortmannin was unable to inhibit progesterone
stimulation of phospholipase C in the plasma membrane cortex and whole
oocytes. Therefore, the model presented in figure 1.7 is not pertinent to the
mechanism of action of progesterone.
40


Thus, we propose a model for progesterone stimulation of phospholipase C
that involves progesterone activation of a membrane-associated tyrosine kinase
(Figure 4.1). The activated kinase would phosphorylate an unknown
intermediate or adapter protein, and the phosphotyrosine region of this protein
would then provide a binding site for the SH2 domain of phospholipase C-gamma
resulting in activation of the enzyme. The model would explain why tyrphostin
B46 or the SH2 fusion protein would inhibit the path, but the presence of an
intermediate would explain why phospholipase C-gamma is not phosphorylated
on tyrosine. The intermediate may be a nonsoluble protein since the PMC
involves washing away most soluble proteins.
There is much recent evidence supporting the role of SH2 domains in the
activation of phospholipase C-gamma. The injection of SH2 domains from
phospholipase C-gamma has been shown to inhibit Ca++ release and other events
at fertilization in sea urchin eggs (Carroll et al., 1999) and the Ca++ release at
fertilization in starfish eggs (Carroll et al., 1997). SH2 domain binding with
phosphotyrosine-containing peptides has been shown to act as an allosteric
regulator in vitro, suggesting the SH2 domains from phospholipase C-gamma
may mediate structural rearrangements and modulate enzyme activity (Koblan et
al., 1995). This evidence supports our model involving a tyrosine-phosphorylated
intermediate.
41


The regulation of phospholipase C is still not fully described and new paths
may be discovered. For example, the phospholipid phosphatidic acid has been
shown to activate both control and tyrosine-phosphorylated PLC-gamma (Jones
and Carpenter, 1993). This suggests that tyrosine phosphorylation may not be the
only mechanism by which PLC-gamma is regulated.
As discussed earlier, the production of PIP3 in the membrane by PI 3-kinase
can activate PLC-gamma by binding to the PH domain (Falasca et al., 1998) and
more recent evidence reports the SH2 domains of phospholipase C-gamma can
also specifically interact with PIP3 (Bae, et al., 1998). However, we have not
found any evidence for a role for PI 3-kinase in progesterone action.
In conclusion, there is much recent evidence suggesting alternative
mechanisms for the regulation of PLC-gamma that do not involve direct tyrosine
phosphorylation of the lipase. Our study of the progesterone induced activation
of phospholipase C during meiotic cell division in Xenopus laevis oocytes
suggests a tyrosine kinase is involved but there may be many different
mechanisms leading to the activation of the enzyme.
42


Progesterone
PIP DAG
Release of Ca^ from ER
Figure 4.1: Proposed model for progesterone activation of phospholipase C
during meiotic cell division in Xenopus laevis oocytes.
Progesterone acts at or near the cell membrane to stimulate a membrane-
associated tyrosine kinase. The activated kinase phosphorylates an unknown
intermediate or adapter protein. The phosphotyrosine region of this protein
provides a binding site for the SH2 domains of phospholipase C-gamma resulting
in activation of the enzyme.
43


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