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The effect of microinjected ras p21 on inositol 1,4,5-trisphosphate and sn 1,2-diacylglycerol mass in Xenopus oocytes

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Title:
The effect of microinjected ras p21 on inositol 1,4,5-trisphosphate and sn 1,2-diacylglycerol mass in Xenopus oocytes
Creator:
Goalstone, Marc Lee
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Language:
English
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xi, 91 leaves : illustrations ; 29 cm

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Subjects / Keywords:
Xenopua laevis -- Experiments ( lcsh )
Oncogenes -- Experiments ( lcsh )
Cancer -- Animal models ( lcsh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Bibliography:
Includes bibliographical references.
General Note:
Submitted in partial fulillment of the requirements for the degree, Master of Arts, Biology.
General Note:
Department of Integrative Biology
Statement of Responsibility:
by Marc Lee Goalstone.

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|University of Colorado Denver
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Auraria Library
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ocm28528513
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LD1190.L45 1992m .G62 ( lcc )

Full Text
THE EFFECT OF MICROINJECTED ras p21
ON INOSITOL 1,4,5-TRISPHOSPHATE AND
sn 1,2-DIACYLGLYCEROL MASS IN
Xenopus OOCYTES
by
Marc Lee Goalstone
B.A., The Colorado College, 1974
A thesis submitted to the
Faculty of the Graduate School of the
University of Colorado at Denver
in partial fulfillment
of the requirements for the degree of
Master of Arts
Biology
1992
f


This thesis for the Master of Arts
degree by
Marc Lee Goalstone
has been approved for the
Department of
Biology
by
Date'
Corinne Campbell
r


Goalstone, Marc Lee (M.A., Biology)
The Effect of Microinjected ras p21 on Inositol
1,4,5-trisphosphate and sn 1,2-diacylglycerol
Mass in Xenopus Oocytes.
Thesis directed by Assistant Professor Bradley J.
Stith.
ABSTRACT
Ras-buffer (33 nl) or Ki-ras-Val[12] (40 ng)
was microinjected into Xenopus oocytes and IP3 or
DAG mass was quantified at time points post
injection. IP3 mass increased 34 fmol/cell at 4 h
after ras p21 injection and decreased by 17
fmol/cell at GVBDso- DAG mass showed a decrease
(from basal levels) of 51 pmol/cell at 30 min
after ras p21 injection and a 14 pmol/cell
decrease at GVBD50 from GVBD25> GVBD75 and
GVBD100 levels.
The increase of IP3 at 4 h may be associated
with the time necessary for injected ras p21 to be
post-translationally modified. Increases in IP3
increase cytosolic concentrations of calcium.
Since MPF activity is correlated to the release of
iii


oocytes from the G2/M block, phosphorylation of
its sub-component p34 may be dependent upon a
calcium-dependent protein kinase pathway.
Decreases in DAG mass may be due to the
drawing away of GAP or other ras p21-activators by
injected ras p21 from endogenous ras
membrane-bound proteins. Since ras p21 is
associated with degradative pathways producing DAG
and not synthetic pathways using DAG, inhibition of
ras p21 action would decrease the net mass of DAG.
The decrease of IP3 and DAG mass at GVBD50
was not expected. Previous literature demonstrated
that progesterone- or insulin-induced meiosis
increased the mass of both. However, the larger
changes in DAG than in IP3 suggest that DAG comes
from a different source than IP3 (e.g.,
phosphatidylcholine) since equivalent amounts of
each would be generated from hydrolysis of
phosphatidyl 4,5-bisphosphate (PIP2). Thus IP3
mass represents the breakdown of PIP2, whereas DAG
mass reflects the breakdown of other phospholipid
sources.
iv


This abstract accurately represents the content of
the candidate's thesis. I recommend its
publication.
Signed
v


DEDICATION
I wish to dedicate this thesis to those who
are living, those who may be dying and those who
have died.
To those who are living I wish to thank my
wife, Janet, for her never ending support and
love. I will be ever grateful for her kind words
and inspiration. Thank you. To my children, Sarah
and Hannah, thank you for your understanding and
love.
To those who may be dying I dedicate this
thesis to my father-in-law, Everette Dahlinger,
who is dying from a progressive form of lymphoma.
Mr. Dahlinger has given me a new understanding of
life through his eyes. Thanks, Dad.
To those who have died I dedicate this
thesis to my mother, Glendeen Goalstone, who died
of cancer on April 14, 1988. Mom, I love you.


ACKNOWLEDGEMENTS
I would like to thank Dr. Bradley Stith for
his countless hours of support and encouragement.
He has taught me the skills of being a good
scientist and the importance of patience and
inquiry. I also thank Dr. Stith for his friendship
and confidence. Thank you, Dr. Stith, for allowing
me to work in your laboratory.
I would like to thank Sally Silva for all the
hours of help, advise and most of all friendship.
Sally has been a G-d send for me and I will miss
her. I wish her well in her new profession in
medicine. Her compassion will heal many. Thank
you, Sally.
Next, I would like to thank Chris Jaynes and
Allan Kirkwood for the hours they spent with me,
teaching me the intricate skills of the lab and
putting up with my strange sense of humor. Thanks,
gentlemen.
I would like to thank Dr. Alan Brockway and
Dr. Corinne Campbell for their support and humor.
I have learned much from both of you and hope that
I too will be an inspiration to others as you have
been to me.
vii


I would also like to thank Ron Espinoza and
Dawn Roberts for their help and support. Good luck
to both of you.
This work was supported in part by a grant
awarded to Dr. Stith from the National Science
Foundation.
viii


CONTENTS
CHAPTER
1. INTRODUCTION.................................1
Background.................................1
The Xenopus Cell Model.....................6
Ras p21....................................9
Structure...............................9
Post-translational Modification...........20
Regulation of Ras p21 Activity............26
Receptor Regulation of Ras p21.......26
Intrinsic Nucleotide Exchange
Activity...............................26
Extrinsic Nucleotide Exchange
Activity...............................28
Intrinsic Control of GTPase
Activity...............................30
Extrinsic Control of GTPase
Activity...............................30
Regulation of the Cell Cycle by
Ras p21...................................31
Nuclear Events.........................31
Cytoplasmic Events.....................36
Insulin's Mechanism of Action
in Xenopus................................36
Second Messengers.........................38
Second Messenger Function..............38
ix


Ras p21 Mediates Insulin Activity.........43
Hypothesis................................44
2. MATERIALS AND METHODS.......................47
GVBD......................................47
Maintenance...............................47
Priming...................................48
Xenopus Oocytes...........................48
Injection of Ras-buffer or
Ki-ras-Val[ 12 ]..........................48
DAG and IP3 Mass Measurements.............49
DAG Assay.............................49
DAG Standard Line.....................54
IP3 Mass Assay........................55
IP3 Standard Line.....................57
Statistical Analysis......................58
3. RESULTS.....................................60
Ras p21 Induces Meiosis...................60
IP3.......................................60
IP3 Mass: 0 to 5 Hours................61
IP3 Mass at GVBD......................62
DAG.......................................62
DAG Mass: 0 to 5 Hours................62
DAG Mass at GVBD......................68
x


4. DISCUSSION..................................70
Basal Levels of IP3 and DAG............70
IP3 Increase at 4 Hours...................71
DAG Decreases at 30 Minutes
after Ras Injection.......................72
IP3 and DAG Mass During GVBD............75
Possible Sources of IP3 and DAG........75
Future Experiments........................76
APPENDIX
A. CHEMICALS, SOLUTIONS AND EQUIPMENT...........78
Chemicals.................................78
Solutions.................................80
Equipment.................................82
REFERENCES
83


CHAPTER 1
INTRODUCTION
Background
Cancer is a major leading cause of death in
the United States. Many of these cancers are due to
either the existence of oncogenes in the genomes of
these tumor forming cells or the overexpression of
the products of proto-oncogenes in normal cells.
Oncogenes are tumor producing-genes (Tabin et
al., 1982). Oncogenes comes from the Greek word,
onkos, meaning mass and the Greek word, genea,
meaning type. Oncogenes are altered forms of normal
cellular genes, called proto-oncogenes, that are
involved with normal cell proliferation.
Oncogenes are typically transformed from
proto-oncogenes through mutations in the sequence
of nucleic acids that make up chromosomes (Fasano
et al., 1984; Santos et al., 1983; Balmain et al.,
1983). Chromosomal mutations occur due to the
effect of physical or chemical mutagens on an
organism's DNA. Physical, chemical or viral
intervention can cause deletions (i.e., loss of one
1


or more nucleotides), point mutations (e.g.,
guanine is substituted for cytosine), or additions
(i.e., insertion of one or more nucleotides) to a
cell's nucleic acid sequence. Unfortunately some
cells do not recognize the altered nucleotide
sequence and consequently do not repair the
deleterious modification. Alternatively, an
oncogene is inserted in a cell's genome by a virus
Early in the twentieth century Francis Peyton
Rous was the first to note that cancer in
connective tissue of chickens was due to a
retro-virus now known as Rous Sarcoma Virus (RSV).
Protein products of RSV oncogenes were later
designated "src" (from sarcoma, or skin cancers)
proteins, because they were found abundantly in
cancers of the skin.
Viral-induced neoplasia was also noted by
Harvey (1964) and Kirsten and Meyer (1967). These
scientists have correlated the presence of murine
sarcoma retro-viruses with the transformation of
normal rodent cells to cancerous cells. A set of
oncogenes from these retro-viruses was to be known
later as rodent-associated sarcoma (ras) oncogenes
2


Scientists discovered ras oncogenes when they
began using gene transfer assays with viral-induced
animal turmors. Researchers established a
correlation between ras oncogenes and human tumors
(Santos et al., 1983). By 1982 ras genes became the
focus of attention when transforming ras oncogenes
were, in fact, identified in human tumors (Tabin et
al., 1982). Conseguently, three closely-related
transforming ras proteins (generally designated
viral-ras, or v-ras proteins) have been described
and have been found in cancerous tissue: Harvey-ras
(Ha-v-ras), Kirsten-ras (Ki-v-ras) or
neuroblastona-ras (N-v-ras) (Valencia et al.,
1991). These three ras proteins have 75-80%
sequence homology. There are other less
closely-related ras proteins that have only 30-50%
sequence homology, but are not able to to induce
turmors: ral, rap, rab and Rho.
Based on ras gene sequence analysis, there is
a high degree of conservation throughout eukaryotic
ras genes (Furmanski et al., 1985). Ras proteins
are thought to play a fundamental role in basic
cellular events, such as: cell proliferation
(Mulcahy et al., 1985); terminal differentiation
3


(Bar-Sagi et al., 1985); and human cancer (Tabin et
al., 1982; Willecke and Schafer, 1984).
Revealing the mechanism of action of ras
proteins in the regulation of cell division will
hopefully have an inpact upon our understanding and
comprehension of turmorigenesis.
A primary guestion was; is whether ras
oncogenes and oncogenic ras proteins induce
neoplasia (cell division) or are a consequence of
it? First, Bar-Sagi et al. (1986) have
demonstrated that injection of transformed ras
proteins induced the appearance of surface ruffling
and fluid-phase pinocytosis in fibroblasts.
Increased ruffling and pinocytosis is a
proliferative response associated with platelet
derived growth factor stimulation in NIH 3T3
fibroblasts. Second, Zhang et al. (1990) have
demonstrated that normal cells can be transformed
by over production of cellular ras p21 (c-ras) p21.
Zhang suggests that overexpression of c-ras p21 in
normal cells might be evoked by a deletion of a
tumor suppressor gene or existence of an oncogenic
enhancer transcriptional protein.
4


Thirdly, the ability of ras to induce
neoplasia is supported by v-ras protein induction
of meiosis in Xenopus oocytes (Birchmeier et al.,
1985; see later discussion of Xenopus system).
Fourthly, ras proteins are thought to mediate
insulin-induced meiotic division in Xenopus oocytes
(see later discussion).
Fifth, both insulin and v-ras proteins induce
similar second messengers: sn 1,2-diacylglycerol
(DAG) and inositol 1,4,5-trisphosphate (IP3). Using
labeling studies, Lacal et al. (1987) have
suggested that v-ras proteins increase DAG and, to
a lesser extent, IP3. Fleischman et al. (1986) have
shown that insulin and microinjected v-ras proteins
induced increases in levels of DAG and IP3 labeling
in NIH 3T3 cells. Insulin and insulin-like growth
factor-1 (IGF-1) induce the production of second
messenger DAG (Stith et al., 1991) and IP3 mass
(Stith et al., 1992b) in Xenopus oocytes. The
purpose of this work is to examine more accurately
whether ras increases DAG or IP3 mass in Xenopus
oocytes.
5


The Xenopus Cell Model
Xenopus laevis is a carnivorous frog that is
indigenous to the muddy rivers of South Africa.
Advantages of its use are: one, the frog is
commercially available year round (Xenopus One, Ann
Arbor, Michigan) and two, females are gravid year
round, producing five to six thousand oocytes at
one time (i.e., 5-6 g, with 1 oocyte equaling about
1 mg). Three, oocytes from Xenopus frogs are
rapidly induced to enter meiotic cell division upon
in vitro addition of insulin, IGF-1 or
progesterone. Four, injection of v-ras p21 in
oocytes can induce maturation (Birchmeier et al.,
1985). Five, Xenopus oocytes are large, 1.1-1.3 mm,
which enables micropipette injection of proteins
involved in cell signaling pathways (e.g.,
antibodies to ras p21, Furth et al., 1982; to
phospholipases, Garcia de Herreros et al., 1991; to
oncogenic ras p21 Gibbs et al., 1989; or protein
kinase C, Stith and Mailer, 1987).
Stage VI oocytes (FIG. 1) average 1.2 mm in
diameter and have dark animal and light vegetal
hemisphere. As seen in figure 2, Xenopus oocytes
exhibit a visual cue (i.e., a white spot in the
6


1.2 mm
Fig. 1. Xenopus laevis oocytes at different
stages of oogenesis. Diameter of oocyte varies
according to stage of development: stage I,
0.3 mm; stage II, 0.45 mm; stage III, 0.6 mm;
stage IV, 0.9 mm; stage V, 1.0 mm; and stage VI,
1.2 mm.
7


Fig. 2. Xenopus oocytes exhibit a visual cue
of a white spot at the animal pole as a result
of hormone- or ras p21-induced meiosis. White
appears when oocyte enters meiosis and correlates
to germinal vesicle breakdown (GVBD).
8


pigmented, animal pole) when cells enter prophase
after induction by hormone (Mailer and Koontz,
1981) or microinjection of v-ras p21 (Birchmeier et
al., 1985). The appearance of the white spot, is
due to movement of the nucleus to just below the
cell surface as it pushes away pigment granules;
this correlates with the breakdown of the nuclear
envelope (Mailer, 1990). The breakdown of the
nuclear envelope is called germinal vesicle
breakdown (GVBD) and signals the oocyte's entry
into meiosis I (Mailer, 1990). This event is also
referred to as the oocyte's release from its
G2/M-phase (G2/M) block by mitogenic induction.
Ras p21
Structure
Ras p21 is a 21,000 Dalton protein product of
the ras gene that binds guanine nucleotides. Ras
p21 contains 189 amino acids, four major regions (a
region is a contiguous sequence of amino acids in a
protein, such as residues 1 through 80) and five
structural domains (domains are groups of amino
acids that function as a unit, such as a binding
pocket for a substrate) (Barbacid, 1987). The five
9


structural domains have been designated: (a)
essential; (b) effector; (c) nucleotide binding;
(d) antigenic determinant; and (e) dispensable
(FIG. 3).
The essential domains. The essential domains
lie within residues 5-63, 77-92, 109-123, 139-165
and 186-189. These amino acid seguences in ras p21
are necessary for its function as a transducer;
that is, the activity by which ras p21 transfers
external signals to the inside of cells. Without
any of these sequences, ras p21 cannot transform
(induce cancerous growth) NIH 3T3 cells (Valencia
et al., 1991). Many ras proteins have the following
amino acid sequences in these highly conserved
regions: GXXGXE, residues 10-16; DXXGXE, residues
57-62; NKXD, residues 116-119; and EXSAX, residues
143-147 (G, glycine; X any amino acid; E, glutamic
acid; D, aspartic acid; N, asparagine; K, lysine;
S, serine; and A, alanine) (John et al., 1988).
The effector domain. The effector domain is
that sequence which contains residues 32-40 and is
associated with ras p21 interaction with a molecule
called guanosine triphosphatase-activating protein
(GAP) (Cales et al. 1988). GAP increases ras p21
10


160
140
I
I
143-147
180
| C00H
CAAX
Dispensable domains
Effector domain
Essential domotns

Y13-259 epitope
Nucleotide binding

Activating point mutotlons
Fig. 3 Primary structure of ras p21, showing functional domains
and residues at which point mutations (**) occur in mutated forms
ras proteins.


intrinsic GTP hydrolyzing activity and may be
involved in effector activity (McCormick, 1990).
The nucleotide binding domain. The nucleotide
binding domain (i.e., where GDP or GTP bind to ras
p21) is made up of four separate and essential
amino acid sequences: 12-16, 59-63, 116-119 and
143-147 (Santos and Nebreda, 1989). Although these
four sequences are structurally separated, the
tertiary folding of the ras p21 protein places
these four sequences into proximity of each other
(John et al., 1988). Deletions of these sequences
or mutations of specific residues within these
sequences can impede guanine nucleotide binding to
prevent regulation of the ras protein activity
(Willumsen et al., 1986).
Five residues within the above noted domains
are very important in binding the guanine
nucleotides: glycine 12, aspartate 57, alanine 59,
aspartate 116 and aspartate 119 (FIG. 4). Alanine
59 and aspartate 57, along with a magnesium atom,
stabilize the gamma phosphate of GTP. However, when
alanine 59 is substituted with a threonine residue
the bond between the beta and gamma phosphate of
GTP is weakened. Phosphorylation of threonine 59
12


Fig. 4 Guanine nucleotide binding pocket of
ras p21. Residue positions 57,59/ 116 and 119
(shown here) along with residue positions 12
and 143 are involved with binding guanosine
diphosphate (GDP) or guanosine triphosphate
(GTP).
13


confers constitutive "on" action to the ras
protein. Aspartates 116 and 119 bind ionicly to the
guanine base, holding it in place so as to align
the rest of the nucleotide relative to the ras
protein (John et al., 1988).
In cellular ras (c-ras) p21, position 12 is a
glycine amino acid which lies inside a glycine-rich
sequence. This glycine-rich sequence imparts a
tight loop to the ras p21 molecule in this region;
a loop that is just large enough to bind guanine
nucleotides. Point mutations at position 12 affect
this helical loop, and thus its guanine nucleotide
binding and exchange activity. Introduction of
valine at position 12 causes ras p21 to have a
greater affinity for GTP, thus producing an active
ras protein that cannot be turned off. The val 12
mutant is used in most studies because of its high
activity.
Using plasmid constructs and expression in
Escherichia coli, John et al. (1988) have examined
point mutations in ras p21 proteins at residues 12
and 59. It is apparent that mutations at either one
or both residues evokes an extreme decrease in
GTPase activity and, thus, an increase in ras p21
14


activity (TABLE I). Different point mutations
produce various GDP dissociation rates (John et
al., 1988). Table II shows the percentage of GDP
dissociation rate (high dissociation rate, high ras
p21 activity) of viral ras p21 (v-ras p21) verses
c-ras p21.
Note that GDP dissociation rates decrease with
any point mutations except for those with threonine
at position 59. A point mutation to threonine 59
leads to an autophosphorylation of this threonine
residue from GTP, leading to the release of GDP
(John et al., 1988; Shih et al., 1980).
An antigenic determinant. One antigenic
determinant, or epitope, of ras p21 is located at
residues 63-73. These amino acids are recognized by
the anti-ras p21 antibody Y13-259 (Furth et al.,
1982; Santos and Nebreda, 1989). The epitope
residues, 63-73, conformationally lie near the
nucleotide binding seguences 12-16 and 59-63. Thus,
it not surprising that ras p21 exchange of GTP for
GDP is inhibited by Y13-259. This inhibition was
also noted after the antibody was co-injected with
ras p21 into E5 clones of NFS/N1-H7 cells (Rizzo et
al., 1991) or Xenopus oocytes (Trahey and
15


TABLE I
GTPase rate constants of various ras p21
Protein GTPase
p21(Gly!2,Ala59) [c] 1c x 103(min~M 10.2
p21(Vall2,Ala59) * 2.0
p21(Argl2,Thr59) [v] 1.4
p21(Gly!2,Thr59) 2.4
p21(Vall2,Thr59) 1.8
p21(Arg!2,Ala59) 1.4
p21(Glyl2,His61) 1.9
p21(Vall2,His61) 1.7
GTpase rate constants of different mutated
v-ras p21 proteins. GTpase rate constants were deter-
mined at 370 C, 5 uM p21 and 50 uM [gamma-32p]-GTP
in a total volume of 200 ul of buffer B (64 mM Tris-
HC1 pH 7.6, 10 mM MgCl2/ 1 mM dithioerythritol and
1 mM NaN3. [c], cellular ras p21; [v], viral ras p21;
other mutants are cDNA constructs (John et al., 1988).
(*) ras p21 used in our experiments.
16


TABLE II
GDP dissociation rate constants for single and
double mutant ras p21 proteins.
Protein GDP dissociation rate constants
k x 102(min-l)
p21(Glyl2,Ala59) [c] 0.79
p21(Vall2,Ala59) * 0.23
p21(Argl2,Ala59) 0.37
p21(Glyl2,Thr59) 4.4
p21(Arg!2,Thr59) [v] 4.0
p21(Vall2,Thr59) 2.1
GDP dissociation constant rates of different
mutant v-ras p21 proteins. GDP dissociation constant
rates were determined at 37 C, 10 mM Mg2+,
2 uM p21, 10 uM [8-3H]-GDP, 0.9 mM EDTA, pH 7.6
and buffer A (buffer B without Mg2+: 64 mM Tris-
HC1 pH 7.6, 1 mM dithioerythritol and 1 mM NaN3).
[c], cellular ras p21; [v], viral ras p21? other
mutants are cDNA constructs (John et al., 1988).
(*) ras p21 used in our experiments.
17


McCormick, 1987; John et al., 1988). Therefore, the
presence of the ras p21-specific antibody Y13-259
will inhibit ras p21 activity. These results again
suggest that changes in the sequence of amino acids
or the physical environment at or near residues
12-16 and 59-63 are involved in the regulation of
ras p21 activity.
Dispensable domains. Finally, there are
domains of ras p21 that are not important or
necessary for ras p21 activity. Excision of
sequences 1-4, 64-76, 92-108, 124-138 and 166-185
do not significantly alter the intrinsic activity
of ras p21 (Santos and Nebreda, 1989).
Major regions. The five structural domains
discussed above lie inside four major regions of
the ras protein molecule. The first region,
residues 1-80, contains the amino terminal residue
and sequences that are not only highly conserved
throughout all eukaryotic ras proteins, but are
also homologous to the alpha subunit of transducer
G-proteins. This region has 8096 or greater homology
in all ras proteins (Valencia et al., 1991). As
noted above, this region contains the nucleotide
binding area, the antigenic determinant sequence
and the GAP effector domain.
18


The second region, residues 81-160, has a
70-80% homology throughout eukaryotic ras proteins
(John et al., 1988). The third region, residues
161-185, is considered the hypervariable region and
has less than 70% homology among the ras protein
family. Residues in this region are specific to
each ras protein homolog, such as rab and rap
(Santos and Nebreda, 1989).
The fourth region consists of only four amino
acids: CAAX (C, cysteine? A, any aliphatic or
uncharged amino acid; and X, any amino acid)
(Valencia et al. 1991). These four amino acids
comprise the carboxyl terminal tail of the ras
molecule where lipid tails, such as farnesyl, are
added (Hancock et al., 1989; Gutierrez et al.,
1989). This post-translational modification of ras
proteins results in a change from a cytosolic
protein to a membrane-associated molecule
(Gutierrez et al., 1989). Without these four
residues, ras proteins cannot not be isoprenylated,
cannot associate with membranes and are
non-functional. Isoprenylation (see
post-translational modification heading for more
detail) is the act by which a farnesyl group is
19


attached to the C-terminus residue (cysteine 186)
on ras p21, with possible subsequent palmitoylation
of one or more upstream cysteine residues.
Post-translational Modification
Proximity to the plasma membrane is a
requisite to ras p21 function (Willumsen et al.,
1984? Hancock et al., 1989). Since all ras proteins
are made on free, cytosolic ribosomes (i.e., those
not associated with the endoplasmic reticulum and
Golgi apparatus of the secretory pathway) ras
proteins are not originally membrane-associated.
Ras p21 membrane association is achieved by
post-translational modification where
isoprenylation of the ras protein molecule takes
place (FIG. 5). Prior to isoprenylation, ras p21
does not associate with the membrane and cannot
induce proliferative events.
Gutierrez et al. (1989) have demonstrated
through pulse chase experiments that nascent and
cloned ras proteins translocate from the cytosolic
fractions of the cell to the membrane fractions of
the cell. This process requires more than three
hours in COS-1 cells.
20


pro-p21
N-
jSH |SH
Cys
181/4
CH3
\
AAX
"Cys AAX
186
STEP I
N.

H
Cys
m-p21 181/4
(Famesyl)
_Cys-OMe
186
STEP 2
(Palmitoyl)
Plasma
Membrane
Fig. 5. Ras p21 is post-translationally modified from a cytosolic protein
(pro-p21) to a membrane-bound protein (m-p21). Ras p21 is modified
in a two step enzymatic process: (1) proteolytic cleavage of the last
three amino acids (AAX), carboxy-methylation and famesylation of
Cys 186, and (2) palmitoylation of upstream cysteine residues
(e.g., Cys 181/4).
21


Using sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) and isoelectric
focusing gel electrophoresis (IEF), Gutierrez et
al. (1989) have demonstrated that the anchored or
mature form of ras p21 (m-p21) is achieved through
two steps. Gutierrez suggests that progenitor-ras
p21 (pro-p21) has a molecular weight of 23kDa and
is proteolytically cleaved to produce a cytosolic
protein with molecular weight of 21kDa. Yet, IEF
experiments have shown that two bands exist for
cytosolic ras p21, suggesting that ras p21 is
further processed by a second step that caps the
cytosolic ras protein. This would account for two
bands on the IEF gel: one with the capping molecule
and one without. Capped c-p21 is resistant to
further proteolysis. Carboxy-methylation would
preclude further proteolytic cleavage of the ras
protein, and complete the second step of protein
modification.
Isoprenylation (i.e., farnesylation) can occur
after proteolysis by carboxypeptidase removes the
last three amino acids on the ras protein and
exposes cysteine 186 of the CAAX tail. A mutation
of cysteine 186 to serine 186 precludes the
22


isoprenylation of ras p21 and the activation of ras
p21 (Gutierrez et al., 1989). It is this step of
farnesylation that confers hydrophobicity to the
ras p21 molecule so that c-p21 becomes anchored to
the inner leaflet of the plasma membrane.
Though all membrane-associated ras proteins
are farnesylated not all are palmitoylated. After
farnesylation, palmitic acid may be bound to ras
p21 residues cysteine 184 and 181 (Hancock et al.,
1989). Farnesylation, but not palmitoylation is
essential for ras protein activity. Hancock et al.
(1989) has employed the use of complementary-DNA
(cDNA) and expression vectors to construct mutated
ras proteins whose C-terminus sequences differ from
c-ras p21. With COS-1 cells, ras p21-association
with the plasma membrane requires farnesylation,
but not palmitoylation. Hancock et al. (1989) also
have demonstrated that cysteine residues 181 and
184 (upstream of cysteine 186) are necessary for
palmitolytion, but not for ras p21 activity (TABLE
III) .
The use of a trimutant ras p21 (i.e., serine
181, serine 184, and valine 12) can induce
cancerous activity in COS-1 cells without the
23


TABLE III
Carboxy-terminus sequences of ras and ras-related
small GTP binding proteins
Group IIC
181 184 186
PDESGPGCMSCKCVLS
SDDGTQGCMGLPCVVM
DDKTPGCVKIKKCVIM
AQPDKDDPCCSACNIQ
Group IIB
K(B)-ras E G K K K K K K SKTKCVIM
rap-la T P V E K K K P K K K S C L L L
Group IIC has cysteine (C) residues 181 and 184,
that are palmitoylated and upstream of cysteine residue
186, which is farnesylated. Group IIB
has lysine residues (K) upstream of cyteine 186 that
are not palmitoylated, but because of their basic charge
they are attracted to the acidic charge of the phospho-
lipids in the plasma membrane. These ras and ras-
related proteins are polybasic proteins (Hancock
et al., 1989) .
H-ras
N-ras
K(A)-ras
rap-2
24


presence of palmitoylation (Hancock et al., 1989).
This again suggests that palmitoylation is not
necessary for ras p21 activity.
Palmitoylation may not be necessary for ras
p21 activity, but it seems to confer a greater
avidity by the ras protein for the membrane. Group
IIB (KB-ras, rap la from table III) are
farnesylated, but not palmitoylated. Increased
avidity for the membrane is accomplished by ionic
interactions between the lysine (K) residues and
the phospholipids of the plasma membrane. These
basic residues are positively charged at a
physiological pH and negatively charged phosphate
groups of the phospholipid membrane would be
strongly attracted to them.
It is important, then, to study these
post-translational modifications in eukaryotic
cells. Eukaryotic cells are capable of
post-translational modification, but recombinant
ras protein studies expressed in E. coli cannot be
studied without fault since prokaryotes do not have
the mechanism to modify a protein (i.e.,
post-translational modification). We have chosen
the Xenopus oocyte cell model since it is a cell
25


which has the ability to post-translationally
modify a protein.
Regulation of Ras p21 Activity
There are five modes of regulation of ras p21
activity: regulation by (1), a mitogen receptor;
(2), intrinsic control of nucleotide exchange
activity; (3), extrinsic control of nucleotide
exchange activity; (4), intrinsic control of GTPase
activity; and (5), extrinsic control of GTPase
activity by GAP.
Receptor Regulation of Ras p21
Yarden and Ullrich (1988) and Cantley et al.
(1991) suggest that tyrosine kinase receptors, such
as the insulin and insulin-like growth factor-1
receptors, activate ras p21. Tyrosine residues on
ras p21 might be phosphorylated directly by the
receptor kinase or an intermediate kinase (in a
kinase cascade).
Intrinsic Nucleotide Exchange Activity.
When ras p21 interacts with a mitogen-bound
receptor, ras p21 releases bound GDP, binds GTP and
activates (FIG.6).
In its unactivated state (i.e., without
26


Fig. 6. Interaction of ras p21 and other cellular proteins. Ras p21
binds GDP when inactive. When hormone (H) binds to membrane
receptor (R), ras p21 releases GDP, binds GTP* with the help of a
putative guanine nucleotide exchange factor (GeF) and becomes
activated. Intrinsic hydrolysis of ras p21-bound GTP* is augmented
by GTPase activating protein (GAP), causing ras p21 to be bound
to GDP again and thus becoming inactivated.
27


mitogen-bound receptor), ras p21 exhibits a high
affinity for GDP; a binding constant for GDP of 1 x
10ll or a K<3 of 10-H M (Kd represents
that concentration of the ligand that is required
for half maximal binding). Though GTP pools in
Xenopus oocytes are thirty times greater than GDP
pools (Trahey and McCormick, 1987; John et al.
1988), quiescent ras p21 retains a stereochemical
conformation that prefers GDP over GTP. However,
when a mitogen-bound receptor interacts with ras
p21, ras p21 affinity increases for GTP and
decreases for for GDP.
Extrinsic Nucleotide Exchange Activity.
Not unlike another family of guanine
nucleotide binding molecules, such as elongation
factor-Tu (EF-Tu) of bacteria, elongation factor-1
(EF-1) of eukaryotes and eukaryotic initiation
factor-2 (eIF-2), ras p21 guanine nucleotide
exchange is thought to be influenced by exchange
factor molecules (West et al., 1990; Wolfman and
Macara, 1990) (FIG. 6).
West et al.(1990) have demonstrated a
stimulatory exogenous exchange factor that is
localized at the plasma membrane. West et al.
28


(1990) have used purified ras p21, bovine brain
extracts, and [3h]GDP to perform in vitro assays
of GDP dissociation rates. West et al. (1990)
demonstrated that exchange rates of GTP for
[3h]GDP on ras p21 were twenty-five times greater
in the presence of membrane fractions than with
cytosolic fractions. In the presence of this
exchange molecule, ras p21 activity was stimulated.
This particular exchange molecule had a molecular
weight of 100 kDa and was designated as ras guanine
nucleotide exchange factor (rGEF).
Contrary to a membrane-associated stimulatory
exchange factor, rGEF, Wolfman and Macara (1990)
have demonstrated that a stimulatory guanine
nucleotide exchange factor was localized in the
cytosol from rat brains. Wolfman and Macara (1990)
have shown that in the absence of cytosol
dissociation rates of GDP is much slower than in
the presence of cytosolic fractions. Wolfman and
Macara designated their exchange protein as ras
guanine nucleotide releasing factor (rGRF). Though
West and colleagues and Wolfman and Macara have
found different locations for exchange proteins
from different cell types, both exchange factors
had molecular weights about 100-160 kDa.
29


Intrinsic Control of GTPase Activity.
Ras p21 can slowly hydrolyze bound GTP (Santos
and Nebreda, 1989; Macara, 1991; Downard et al.,
1990). Hydrolysis of bound GTP produces a ras-bound
GDP molecule and inorganic phosphate and
inactivates ras p21.
Extrinsic Control of GTPase Activity.
Ras p21 GTPase activity is regulated by its
association with the cytosolic molecule GTPase
activating protein (GAP) (Santos and Nebreda, 1989;
McCormick, 1990). GAP is a 125 kDa protein that can
stimulate ras p21 intrinsic GTPase activity
resulting in inhibition of ras p21 activity (Trahey
and McCormick, 1987; Gibbs et al, 1989). In spite
of known inhibition of ras p21, it is uncertain as
to whether GAP is needed with ras p21 to act as an
effector complex (Gibbs et al., 1989; Zhang et al.,
1990; Hall, 1990).
GAP binds ras p21 through phosphorylated
tyrosine residues on ras p21. The conserved
sequences on GAP, called src homology-2 (SH-2)
regions, bind phosphorylated tyrosine residues on
ras p21 (those tyrosines that presumably have been
phosphorylated by tyrosine kinase receptors) (Gibbs
30


et al., 1989). Thus, growth factor receptor
tyrosine kinases might regulate ras p21 directly
through phosphorylation of ras p21 tyrosine
residues that bind SH-2 regions of GAP.
Regulation of the Cell Cycle by Ras p21
The M phase of the cell cycle, as seen in
figure 7, is made up of two major events: nuclear
division (i.e., meiosis or mitosis) and cytoplasmic
division. Nuclear division is subdivided into
phases which include specific nuclear events.
Nuclear Events
Meiosis Meiosis is divided up into two parts,
meiosis I and meiosis II. At the end of meiosis II
the original chromosomal number is reduced by half.
Each part consists of four phases; prophase,
metaphase, anaphase and telophase. Prophase I
consists of chromosomal condensation and nuclear
envelope breakdown. Metaphase I is when
chromomosomal pairs (i.e., homologous pairs or
tetrads) align along the metaphase plate. Anaphase
I is when separation and reduction of chromosomal
matter occurs: homologous pairs separate, but not
sister chromatids. Telophase I leads directly into
31


G2/M

G0
Fig. 7. The cell cycle. Interphase includes
Gap-1 (Gl), synthesis of DNA (S) and Gap-2
(G2), but does not include meiosis or mitosis
(M). Oocytes arrest in oogenesis at the G2/M
block. Hormone- or v-ras p21-induced meiosis
releases oocyte from G2/M block and allows
oocyte to enter prophase I.
32


prophase II. Chromosomes remain condensed in
prohase II and once again align along the metaphase
plate in metaphase II. Sister chromotids separate
in anaphas II. Telophase II ends the meiotic
process, the chomosomes relax and the nuclear
envelope reappears.
Mitosis. Mitosis is another nuclear event that
can occur during M phase. During mitosis the
nuclear events pass through the four phases once.
Chromosomal number does not reduce during mitosis.
Interphase. Interphase includes all phases
except those four found in M phase. These phases
are Gap-1 (Gi), synthesis phase (S), Gap-2
(G2)and Gc
The S phase (i.e., DNA synthesis) of the cell
cycle is when chromosomes are replicated. Gap-1 is
after M phase but before S phase, while Gap-2 is
after S phase, but before the M phase. Growth of
the cell occurs in Gi, whereas replication of
cellular organelles (e.g., mitochondria and golgi)
occurs in G2
G0 is when the cell is out of the cell cycle
and is not about to divide again (e.g., brain
cells). Cells may re-enter the cycle if mitogens or
growth factors are added.
33


Restriction points. Both gap phases have
restriction points. These are time points in the
cell cycle that when passed the cell is committed
to the next step in the cycle.
The first restriction point of the cell cycle,
at the G0/Gi interface, is located near the
beginning of the Gi phase. Somatic cells are
released from their quiescent stage (G0) by a
growth factor or mitogen and enter Gi (Kaibuchi
et al., 1986). The release from the G0/Gi
arrest, or block, is characterized by the onset of
transcriptional and translational processes that
lead to DNA synthesis (or S phase; Sugimoto et al.,
1988) .
Stem cells for gamete formation, usually
arrest at the second restriction point in the cell
cycle called the G2/M block (Mailer, 1990). This
restriction point lies at the border of the G2
phase and the beginning of meiosis. Release from
this arresting point is usually controlled by the
presence of mitogens such as insulin, insulin-like
growth factor (IGF-1) or progesterone (Mailer,
1990) and is characterized by entry into meiosis.
34


Progesterone. Mailer et al. (1979) have
demonstrated that progesterone releases Xenopus
oocytes from the G2/M block by decreasing the
levels of adenosine-35'-cyclic monophosphate
(cAMP) (Mailer, 1990). Progesterone inhibits the
activity of the enzyme, adenylate cyclase (AC),
that synthesizes cAMP,. Decreases in cAMP levels,
then, induce unknown intracellular reactions that
lead to meiosis.
IGF-1. IGF-1 and insulin might induce meiosis
in Xenopus by another pathway involving ras p21,
DAG and PKC (Stith and Mailer, 1987). IGF-1 is a
7.5 kDa monomer protein, consisting of one peptide
chain of 70 amino acids and three intra-chain
disulfide bonds (Rinderknecht and Humbel, 1978).
IGF-1 exhibits 50% homology to insulin's precursor
pro-insulin (Morgan et al., 1986). IGF-l-induction
of Xenopus oocytes acts through the IGF-1 receptor
(IGF-1R) to evoke oocyte maturation (Morgan et al.,
1986; Mailer and Koontz, 1981; Stith et al., 1991).
Insulin. Insulin is a dimeric peptide.
Insulin's structure consists of one alpha and one
beta subunit. The alpha subunit contains 21 amino
acids, whereas the beta subunit contains thirty.
35


Disulfide bonds connect alpha subunit residues
cysteine 7 and cysteine 20 to beta subunit residues
cysteine 7 and cysteine 19, respectively. There are
also intrachain disulfide bonds on the alpha
subunit between cysteine 6 and cysteine 11.
Although insulin is a major regulating and anabolic
hormone (Goldfine, 1987), high concentrations can
also induce cell proliferation in Xenopus oocytes
through its association with and binding to the
IGF-1R (Mailer and Koontz, 1981). However, in
multicellular organisms with normal, low
circulating insulin levels, IGF-1 is the major
mitogen of cell proliferation (Gibbs et al., 1989).
Cytoplasmic Events
Cytokinesis is cytoplasmic division. It is
separate from nuclear division (i.e., mitosis or
meiosis) and involves cytoskeletal elements, such
as microtubules and microfilaments.
Insulin's Mechanism of Action in Xenopus
Xenopus oocytes have insulin receptors as
shown in studies of insulin binding studies (Mailer
and Koontz, 1981). Insulin acts through an insulin
or IGF-1 receptor because inhibition of insulin's
36


effects can be produced by administration of
receptor antibodies (which bind both IGF-1 and
insulin receptors). Morgan et al. (1986) have shown
that each mitogen binds to its own receptor
four-fold greater than to the other's receptor and
that anti-IGF-1 antibodies and anti-insulin
receptor antibodies inhibit binding of IFG-1 and
insulin to their respective receptors.
Insulin's dissociation rate from a high
affinity receptor (presumably the insulin receptor)
is 1.4 nM. In addition, insulin binds to a low
affinity receptor (presumably the IGF-1 receptor)
with a Kd of 50 nM. Finally, Mailer and Koontz
(1981) have suggested that insulin acts through the
IGF-1 receptor since 50 nM insulin is required to
induce meiosis. Mailer and Koontz (1981) have
demonstrated that even at high in vitro
concentrations of insulin receptor-specific
antibodies, insulin binding to Xenopus oocytes
decreases only 50% and the time course for
insulin-induced maturation in oocytes is
unaffected. Taken together, these observations
strongly suggest that insulin binds to the IGF-1R
for meiotic induction.
37


Second Messengers
Second messengers are those molecules that
act as intracellular mediators of extracellular
signals. Adenosine-35'-cyclic monophosphate
(cAMP), inositol 1,4,5-trisphosphate (IP3) and sn
1,2-diacylglycerol (DAG) are examples of second
messengers. Shortly after a mitogen interacts
with a cell surface receptor, second messengers
are produced. Since one external mitogen generates
thousands of internal second messengers,
second messengers amplify the signal (FIG. 8).
DAG can be generated by the breakdown of at
least two phospholipids; phosphatidylinositol
4,5-bisphosphate (PIP2) or phosphatidylcholine
(PC). Phosphatidylcholine is hydrolyzed to the
second messenger DAG and the by-product
phosphocholine (PhCho) by the enzyme
phosphatidylcholine-specific phospholipase
(PC-PLC) (FIG. 9). Whereas hydrolysis of
phosphatidylinositol 4,5-bisphosphate (PIP2) by
PIP2-specific phospholipase C produces both second
messengers DAG and IP3 (FIG. 10).
Second Messenger Function
IP3. Once IP3 is generated from the
38


p34 and cell division
Fig. 8. Possible mechanism of action of insulin
(H). Second messengers inositol 1,4,5-trisphosphate
(IP3) and sn 1,2-diacylglycerol (DAG) are
generated when ras p21 is activated and breaks
down phosphatidylinositol 4,5-bisphosphate (P).
DAG activates protein kinase C (PKC). Whereas
IP3 acts to release calcium into the cytosol
from calcium storage organelles.
39


Phosphatidylcholine phosphocholine
Fig. 9. Hydrolysis of phosphatidylcholine (PC) is
accomplished by the enzyme phosphatidylcholine-
specific phospholipase C (PC-PLC). DAG and phospho-
choline are products PC hydrolysis.
40


(PIP2)
(iP3)
Fig.10. Hydrolysis of phosphatidylinositol 4,5-
bisphosphate is accomplished by the enzyme
phospholipase C gamma-1. Phosphatidylinositol 4,5-
bisphosphate (PIP2) produces sn 1,2-diacylglycerol
(DAG) and inositol 1,4,5-trisphosphate (IP3).
41


hydrolysis of PIP2, IP3 diffuses from the inner
plasma membrane to receptors that are located on
the smooth endoplasmic reticulum (SER) and calcium
storage bodies called calcisomes (Delisle, 1991).
Calcium ions are released into the cytoplasm once
IP3 interacts with IP3 receptors. Iontophoresis of
0.73 fmole of IP3 into Xenopus eggs produces an
increase in intracellular calcium from 0.25 uM to
1.58 uM (Busa et al., 1985).
DAG. DAG activates protein kinase C (PKC)
both in vivo and in vitro (Ballester et al.,
1987). Phorbol esters act like DAG to stimulate
PKC and they can induce ooctyes enter meiosis
(Stith and Mailer, 1987). Microinjection of PKC
stimulates rasp21 action in Xenopus oocytes
(Kamata and Kung, 1990). V-ras p21-induced GVBD
increased from 34% to 74% when PKC was .
co-injected. PKC may activate ras p21 by
phosphorylating GAP and thus block GAP from
inactivating ras p21 or inhibited GAP by a
molecule that is phosphorylated by PKC and
therefore an unknown protein mediates PKC
regulation of GAP (Cantley et al., 1991).
42


Ras p21 Mediates Insulin Activity
Incubation of Xenopus oocytes in insulin
induces membrane ruffling (Korn et al., 1987),
phosphorylation of S6 kinase (Thomas et al., 1982)
and maturation (Mailer and Koontz, 1981).
Microinjection of transforming ras p21 into
Xenopus oocytes also effects membrane ruffling
(Korn et al., 1987), S6 kinase phosphorylation
(Kamata and Kung, 1990), and maturation
(Birchmeier et al., 1985).
As noted earlier, the monoclonal ras antibody
Y13-259 binds Ki-, Ha- and N-ras p21 (Furth et
al., 1982). Microinjection of Y13-259 into Xenopus
oocytes inhibits insulin-induced, but not
progesterone-induced maturation (Korn et al.,
1987; Deshpande and Kung, 1987; Garcia de Herreros,
1991). Microinjected Y13-259 also inhibits
insulin-induced or ras p21-induced DNA synthesis
in 3T3 fibroblasts (Furth et al., 1982; Mulcahy et
al., 1985; Yu et al., 1988; Larrodera et al.,
1990).
The idea that ras p21 is involved in cell
proliferation is the following: co-microinjection
of ras p21 and ras antibody Y13-259 into BALB/3T3
43


fibroblasts inhibits DAG synthesis (Chiarugi et
al., 1989) and co-microinjection of ras p21 and
Y13-259 inhibits insulin-induced meiosis in
Xenopus (Korn et al./ 1987; Garcia de Herreros et
al., 1991).
Insulin-induced maturation in Xenopus
produces an increase in both DAG (Stith et al.,
1991) and IP3 mass (Stith et al., 1992b). Ras
p21-induced maturation has been shown to generate
increased labeling of DAG, but not IP3 in NIH 3T3
fibroblast cells (Lacal et al., 1987; Wolfman and
Macara 1987) or BALB/3T3 fibroblasts (Chiarugi et
al., 1989). However, v-ras increases DAG in
Xenopus, but only a slight increase in IP3 levels
has been shown in ras p21-induced Xenopus (Lacal,
1990; Wolfman and Macara, 1990).
Hypothesis
Microinjected transforming ras p21 induces
Xenopus oocytes to enter meiosis (Birchmeier et
al. 1985). Ras p21 may mediates insulin- and
IGF-1- induction of meiosis in Xenopus oocytes
(Garcia de Herreros et al., 1991). Since insulin
produces changes in Xenopus DAG mass (Stith et
44


al./ 1991) and IP3 mass (Stith et al., 1992b), we
suggest that microinjection of transforming ras
p21 into Xenopus oocytes will produce similar
changes in mass of these two second messengers.
Lacal et al. (1987) have investigated the
effect of microinjection of v-ras p21 on
phosphoinositol and DAG levels in Xenopus oocytes.
Lacal microinjected [3h]-glycerol or
[3H] -myo-inositol into oocytes, incubated the
cells for five hours and then microinjected v-ras
p21. Using column fractionation, Lacal determined
that microinjection of v-ras p21 increased
inositol phosphate 80%, inositol bisphosphate 20%,
but inositol trisphosphate did not increase
signigicantly from basal levels. However, using
thin layer chromatography Lacal demonstrated that
DAG levels increased five fold within 20 min after
microinjection of v-ras p21.
We find fault with Lacal's methods for the
following reasons: one, incubation of oocytes with
[^Hj-glycerol or [^Hl-myo-inositol for 5 h is
not long enough for these molecules to reach
equilibrium (equilibrium labeling of precursors
must be demonstrated in order to measure
45


production of IP3); two, only some degradative
pathways were analyzed, no synthetic pathways were
measure; and three, all molecular species of DAG
(e.g., inactive 1,3-diacylglycerol) and
phosphoinositides (e.g., inactive inositol
1 3,5-trisphosphate) were assayed. Therefore,
Lacal failed to guantify the total mass of second
messengers, sn 1,2-diacylglycerol and inositol
1,4,5-trisphosphate, before and after
microinjection of v-ras p21.
Use of assays for DAG and IP3 can quantify
total mass levels of the active isomers of these
two second messengers by all synthetic and
degredative pathways after microinjection of v-ras
p21 into Xenopus oocytes. We will examine whether
microinjection of v-ras p21 alters the mass of DAG
and IP3 in Xenopus oocytes.
46


CHAPTER 2
MATERIALS AND METHODS
GVBD
To obtain GVBD25 (GVBD25 means that 25%
of the cells have a white spot), GVBD50f
GVBD75 and GVBD100 for DAG and IP3 mass
assays, oocytes were placed in 2ml of OR-2 (83 mM
NaCl, 0.5 mM CaCl2/ 1 mM MgCl2 and 10 mM
Hepes, at pH 7.5) in 12-well culture dish. Groups
of oocytes were observed at about 40-80X with a
dissecting scope. Insulin (2 uM) (Sigma, St.
Louis, MO) or Ki-ras-Val[12] (Oncogene Science,
Manhasset, NY) was used to induce meiosis.
Maintenance
Female Xenopus laevis frogs (Xenopus One, Ann
Arbor, Ml) were fed with a diet of 1.5 g of cubed
beef heart every other day. The water in which the
frogs lived was replaced every other day with
fresh, oxygenated and de-chlorinated water. A
twenty-four hour clock was used to set a 12 h day
and 12 h night cycle. Ambient temperature was 23 C.
47


Priming
Selected female frogs were primed four to
seven days in advance of each experiment with 35
I.U. of pregnant mare serum gonadotropin (PMSG)
(Sigma Chemical Company, St. Louis, MO). Priming
increased the synchrony of response and shortened
the time required for treated oocytes to enter
into prophase I of meiosis (Stith and Mailer,
1984).
Xenopus Oocyte
Oocytes were manually dissected from excised
ovarian fragments. Stage VI oocytes (1.2 mm or
greater) were selected for each experiment, since
they are competent to enter meiosis.
Injection of Ras-buffer or Ki-ras-Val[12]
A Pico-pump PV830 (World Precision
Instruments, Inc., Sarasota, FL) was used to
inject solutions. Control cells were injected with
ras-buffer (50 mM Tris, 1 mM MgCl2^ 1 mM
NaH2P04, 0.1 mM EDTA, 5 mM DTT and 50*
Ethylene Glycol, pH 7.9), whereas experimental
groups received Ki-ras-Val[12] (Oncogene Science,
48


Inc., Manhasset, NY) into oocytes. To deliver 40
ng of Ki-ras-Val[12], the following microinjection
standardization procedures were followed. A
droplet of 0.4 mm in diameter was injected into
the air. Using the formula, 'ft x (d)3/6, where
"d" eguals the diameter, the droplet egualed to
33.51 nl. Using a Ki-ras-Val[12] stock
concentration of 1.2 mg/ml, the 0.4 mm droplet
contained 40.21 ng of ras protein. This amount was
sufficient to induce oocyte GVBD (Pan and Cooper,
1990) (Birchmeier et al. 1985). Ras-buffer or
Ki-ras-Val[12] solution was injected into the
animal hemisphere of each oocyte just above the
the eguator (FIG. 11).
DAG and IP3 Mass Measurements
DAG Assay
DAG mass was determined by measuring the
amount of 32p_phOSphatatic acid produced when
DAG kinase (Lipiden, Westfield, NJ),
[gamma-32p]_ATP (ICN Radiochemicals, Inc.,
Irvine, CA) and DAG from oocyted extracts were
added (Kennerly et al., 1979; Preiss et al., 1986;
Wright et al., 1988; Shih et al., 1980). The
49


glass micropipette
Fig. 11. Microinjection of ras-buffer or v-ras p21
solution into animal hemisphere of Xenopus oocyte
is accomplished by using a glass micropipette and
a PicoPump PV830 (World Precision Instruments, Inc.,
Sarasota, FL). Amount of solution delivered is
dependent upon the diameter of the sphere of liguid
expressed by the pump. 40 ng (33 nl) of 1.2 mg/ml
Ki-ras-Val[12] or 33 nl of ras-buffer was injected
into each oocyte in order to determine changes in
Ip3 and DAG mass as a result of microinjection
treatments.
50


amount of 32p_p-hOSphoryiation was quantified by
scintillation counter (LKB-Wallace, Pharmacia,
Gaithersburg, MD) and DAG mass was calculated from
a standard regression line.
Dissected oocytes were isolated in groups of
ten. Ras-buffer or Ki-ras-Val[12] (40 ng) was
injected into each oocyte. Typically, there were
5-6 groups for each time point. Each group was
transferred to a 2 ml glass mortar, which
contained 1 ml of ice cold (-20 C)
chloroformrmethanol 1:2 (v/v). Oocytes were
immediately homogenized with a "B" glass pestle
and the homogenate was transferred to a
nitrogen-filled 1.6 x 100 mm glass test tube. The
mortar was washed with 2 ml more of
chloroformrmethanol 1:2 (v/v) and this volume was
added to the original homogenate in the glass test
tube. 1 M NaCl (0.7 ml) was added, the tube was
vortexed for 20 seconds and the monophase was
broken with 1 ml each of 1 M NaCl and chloroform.
The NaCl neutralized partially-charged DAG to
maximize DAG extraction in the organic phase. The
tubes were centrifuged (clinical centrifuge, 60
sec, maximum setting) and the upper aqueous layer
51


was discarded and chloroform was dried under
nitrogen.
Twenty microliters of 1.5% cardiolipin
(Avanti Polar Lipids, Inc., Alabaster, AL) in 5 mM
octyl-beta-D-glucopyranoside (Calbiochem) and 1 mM
DETAPAC (diethylenetriamine penta-acetic acid,
Sigma, St. Louis, MO) was added to the dried
oocyte extract, standard and blank test tubes. The
detergent was used to make the hydrophobic DAG
molecules miscible in the aqueous solutions and
available to DAG kinase.
Seventy microliters of the DAG kinase
solution (50 ul of [100 mM imidazole,100 mM NaCl,
25 mM MgCl2/ 2 mM EGTA pH 6.7], 10 ul of 20 mM
dithiothreitol, and 10 ul of 0.5 mg/ml DAG kinase)
was added to each test tube and vortexed. To start
the reaction, 10 ul of [gamma-32p]_ATP was added
to each tube. After a 30 min incubation at 25
C, the reaction was stopped by the addition of 3
ml of chloroformrmethanol (1:2, v/v).
DAG was then extracted as before; 1 M NaCl
(0.7 ml) was added, each sample vortexed, and 1 M
NaCl and chloroform (1 ml each) were added to
break the monophase. After centrifugation (1 min,
52


clinical centrifuge), the organic layer of each
tube was transferred to a small (10 x 75 mm) glass
test tube and dried down under nitrogen.
Each sample was re-dissolved in 100 ul of 20%
methanol in chloroform (i.e., chloroformrmethanol
4:1, v/v) and a 25 ul aliquot spotted on a Merck
50 Thin Layer Chromatographic (TLC) Plate (Merck,
EM Separations, Gibbstown, NJ). TLC plates were
developed with chloroform:acetone:methanol:acetic
acid:water (10:4:3:2:1, v/v).
Radioactive TLC plates were air dried and
placed with a sheet of XAR film (Kodak, Rochester,
NY), into a light-tight holder. Fluorescent tags
were placed on the TLC plates so that orientation
of the TLC plate on the exposure film could be
determined. After exposure at -70 C overnight,
film was developed and 32p_iabeied phosphatadic
acid spots on the plates were identified, cut out,
and placed into scintillation vials. Six
milliliters of organic scintillation fluid
(HFP-20, Research Products, Inc., Mt. Prospect,
IL) were added to each scintillation vial and
counts were determinded in a LKB scintillation
counter (LKB-Wallace, Pharmacia, Gaithersburg,
MD) .
53


DAG standard line.
In each day's experiement standard amounts of
DAG (Avanti Polar Lipids, Birmingham, AL) (0.4
nmole; 0.8 nmole; 1.04 nmole; 1.44 nmole; and 1.84
nmole) were used for a standard regression line.
One test tube, designated "blank", contained DAG
kinase only (no oocyte homogenate or standard).
The blank was used to determine DAG mass in the
DAG kinase solution (a crude enzyme preparation)
and was subtracted from sample and standard DAG
mass values.
A standard line was calculated by regression
line analysis. The regression line correlated
standard DAG mass values to [32p]_pA counts per
minute (cpm). Absolute values of DAG mass in
nanomoles were graphed on the ordinate, while
scintillated cpm for [32p]pa were graphed on the
abscissa. If a standard produced 4,007 cpm and the
blank counts were 500 cpm, the adjusted counts
were 3,507 cpm. A typical regression line was;
y = (1.14 x 10~4)x + 2.21 x 10-4, where "y"
equalled DAG in nmol and "x" equalled [32p]_pa
cpm minus blank cpm. The regression coefficient
was: R = .998 (n=9). So, 3,507 cpm was equivalent
to 0.4 nmol.
54


DAG mass was represented as "RELATIVE DAG
LEVELS"; that is, relative to the control (i.e.,
basal) DAG mass. This was done to minimize the
large difference between basal DAG levels in
oocytes from different frogs (see Results
section). For example, a 28% relative decrease in
DAG mass would equate to an absolute decrease in
DAG mass from a control level of 0.994 to 0.71
nmol/10 cells, or a decrease of 28 pmol/cell.
Although a 25 ul aliquot was taken from a 100
ul total volume of sample, cpm were not multiplied
by four since only 25 ul of total 100 ul of
standards were used.
IP3 Mass Assay
IP3 mass was measured with a receptor
(isolated from bovine brain extract) competitive
ligand binding assay (New England Nuclear,
Wilmington, DE) (Challis et al., 1988; Bredt et
al. 1989). IP3 mass in oocyte homogenates was
determined by measuring the decrease in cpm as
cold IP3 from oocytes displaced tritiated IP3
bound to IP3 receptors.
As with the DAG assay, each treatment
consisted of three to six groups, but there were
55


15 oocytes per group. At designated time points,
each group was transferred to a 1.5 ml Eppendorf
"V" vial, homogenized in 250 ul of 20%
trichloroacetic acid (TCA) (total volume of 300
ul) with a pestle and allowed to sit in ice for 15
minutes in order to maximize IP3 extraction. Each
vial was centrifuged (1 min, 15,000 x g, 5 C).
The supernatant was transferred to a new "V"
vial and 500 ul of Freon:Tri-n-ocylamine (Sigma
Chemical Co., St. Louis, MO) (3:1, v/v) was added.
Vials were vortexed for 20 seconds and centrifuged
(20 min, 15,000 x g). The organic extracted TCA as
the acid would displace tritiated IP3 from the
receptor.
A 100 ul aliquot of the total 300 ul aqueous
layer was placed in a 0.5 ml "V" vial. In addition
to oocyte extracts, other vials contained IP3
standard (New England Nuclear [NEN]) (48 nM; 9.6
nM; 4.8 nM; or 2.4 nm) or blanking solution. The
blanking solution, which contained a high
concentration (12 nM) of inositol
1,2,3,4,5,6-hexaphosphate (IP6), displaced all
[3h]IP3 from its recptors. This resulted in a
cpm value that represented background,
56


non-specific trapping of tritiated IP3 in the
receptor pellet (obtained later).
Four hundred microliters of a receptor tracer
solution ([3h]IP3 bound to its receptor) was
added, vortexed for 20 seconds and incubated for
1 h at 5 C.
Vials were centrifuged (20 min, 15,000 x g)
in order to pellet IP3-bound receptor. The
supernatant was removed and 0.15 M NaCl (50 ul)
was added to each vial and vortexed (10 min, R.T.)
in order to resuspend the pellet. Each "V" vial
was placed into a scintillation vial with 6 ml of
aqueous scintillation fluid (Budget Solve,
Research Products, Inc., Mt. Prospect, IL) and
counted in the scintillation counter.
IP3 Standard Line.
The standard line correlated the log of IP3
standards (picomoles) to a ratio denoted B/B0
(coordinate). A typical standard line was:
y = (-47.88 _+ 5.42 s.d.)logIP3 + 57.34 +_ 3.92 s.d.
(n=8) and the regression coefficient was 0.98. The
B value was the cpm of the receptor in the
presence of oocyte extract or added IP3 standard.
B0 was the maximal cpm of receptor in the
57


presence of distilled water, which did not reduce
tritiated IP3 binding. Both B and B0 were
corrected by subtracting cpm of blanking tube and
ratio, B/B0, was represented as a percentage.
For example, if an expermental group (B) had 4,100
cpm, the distilled water (B0) value was 6,000
cpm and the blank value was 200 cpm, B/B0 would
equal [(4,100 200)/(6,000 200)] x 100, which
equaled 67.24%. The amount of 67.24 was then
transformed with the standard regression line;
this was a value of (-)0.207 for log IP3. Taking
the antilog, the value of 0.621 (pmol) was
calculated for IP3 mass per 100 ul. The IP3 mass
was corrected by multiplying by a factor of three,
since only a 100 ul aliquot of the original 300 ul
oocyted extract was used, and value obtained was
for 15 oocytes. Thus, using the correction factors
of 3 and 1/15, a B/B0 value of 0.621 pmol/15
cells equated to an IP3 mass of 27.95 fmol/cell.
Statistical Analysis
Data will be presented in figures as the mean
+. standard error of the mean (s.e.), and in text
as the mean + standard deviation (s.d.).
58


Differences between experimental and control
groups were examined with two-tailed one-sample or
pooled Student's _t-test (Dyna-Stat Professional,
Dynamic Microsystems, Pittsburgh, PA).
59


CHAPTER 3
RESULTS
Ras p21 Induces Meiosis
In our hands, 40 ng of ras Ki-ras-Val[12]
induced meiosis in 90% of cells and required 7.54
_+ 1.49 h s.d. (n=7). Non-transforming ras p21 were
less effective than transforming ras p21 in their
ability to induce meiosis (GBVD50 at 15 h vs. 8
h) .
IP3
IP3 Mass: 0 to 5 hours
Using the standard line equation from the
methods section, a basal B/B0 of 77.72 _+ 10.79
s.d. (n=10) was equivalent to 75.1 fmol/cell.
Since oocytes are approximately 500 nl in free
volume (Stith and Mailer, 1984), and assuming no
localized concentration of IP3, the IP3 mass
concentration can be estimated to be 150 nM.
A significant (P <.03) increase in IP3 mass
occurred in the oocytes at 4 h after
Ki-ras-Val[12] injection. As seen in figure 12,
60


RELATIVE B/B
TIME AFTER P21 RAS-VAL12
INJECTION (HRS)
Fig. 12.-. Relative IP3 mass levels were compared at
designated time points after microinjection of 33 nl
of ras-buffer or v-ras p21 (40 ng) into Xenopus
oocytes. A relative IP3 mass of 1.0 equated to an
absolute value of 75 fmol/cell. Right side of figure
shows absolute values of IP3 mass in femtomoles per
cell. Asterisk denotes significance at (P < 0.03).
61
IP3 (IMOL/CELL)


40 ng of Ki-ras-Val[12] produced a 10% increase in
IP3 mass relative to basal values of "1.0". This
equated to an increase in absolute IP3 mass from
75.1 fmol/cell to 109.07 fmol/cell, or 33.97
fmol/cell. The estimated IP3 concentration
increased from about 150 nM to 219 nM at 4 h.
IP3 Mass at GVBD
The B/B0 ratio of "1.0" for controls was
80.88 + 10.33 s.d. with the standard line for
these experiments (y = (-41.89 _+ 5.23 s.d.)x =
54.72 +_ 3.88 s.d., n=8). This B/B0 equated to
47.49 fmol/cell.
As seen in figure 13, there was a trend for a
decrease in IP3 mass as the oocyte entered into
meiosis. There was a significant decrease in IP3
mass at GVBDso* Basal IP3 mass was 47.49
fmol/cell and 30.5 fmol/cell at GVBD50. This
difference was 17 fmol/cell or a 36% decrease of
IP3 mass.
DAG
DAG Mass; 0 to 5 hours
The range of basal DAG mass suggested two
distinct groups of oocytes (FIG. 14). The first
62


RELATIVE B/Bo
1.15
.. 24.4
1.10
1.05
1.00
*
i >
10 O 10 o
in N o
Q Q Q
CQ CQ CQ Q
> > CQ
<3 O O > O
30.5
38.0
47.5
Fig. 13- Relative IP3 mass levels were compared at
designated time points after microinjection of 33 nl
of ras-buffer or v-ras p21 (40 ng) into Xenopus
oocytes. A relative IP3 mass of 1.0 equated to an
absolute value of 47.5 fmol/cell. Right side of
figure shows absolute values of IP3 mass in femtomoles
per cell. Asterisk denotes significance at (P 0.05).
63


C0
CL
D
O
QC
o
LI.
o
CC
LU
m
0.5-75 1-1.5
BASAL DAG LEVELS (n MOL/10 CELLS)
Fig. 14 This figure demonstrates the variability
of basal DAG mass in Xenopus oocytes. The range of
DAG mass was contained in two distinct groups. The
first group varied from 0.5 nmol/10 cells to 0.75
nmol/10. Whereas the second group varied from 1.0
nmol/10 cells to 1.5 nmol/10 cells. The (0)
represents an "n" of zero.
64


group varied from 0.5 nmol/10 cells to 0.75
nmol/10 cells and the second group of oocytes
fell between 1.0 nmol/10 cells and 1.5 nmol/10
cells.
Ki-ras-Val[12] injection induced a decrease
in DAG levels (P<0.05) at 30 min (FIG. 15). At
30 min, DAG mass had decreased from a basal amount
of 0.99 nmol/10 cells (99 pmol/cell) to 0.48
nmol/10 cells (48 pmol/cell). This was a decrease
of .51 nmol/10 cell (51 pmol/cell) or a decrease
of 51.5%.
Subsequently, a slow increase of DAG mass
ensued from 30 min to 5 h after Ki-ras-Val[12]
injection. DAG mass increased to 85 pmol/cell by
4 h and reached basal values by 5 hours.
Different ras p21 proteins produced similar
similar decreases in DAG mass (FIG. 16). Even
microinjected non-transforming forms of ras p21
(e.g., Ha-ras-Gly[12], Ki-ras-Gly[12]) induced a
DAG mass decrease similar to transforming ras p21
(Ha-ras-Val[12], Ki-ras-Val[12]).
DAG mass decreased significantly (P<0.05)
from 0.5 nmol/10 cells (50 pmol/cell) at basal,
ras-buffer injected cells to 40 pmol/cell. These
values equated to a 10% decrease in DAG mass.
65


RELATIVE DAG LEVELS
0 1 2 3 4 5
TIME AFTER RAS VAL12 INJECTION (HRS)
Fig. 15 Relative DAG mass levels were compared at
disignated time points after microinjection of 40 ng
of v-ras p21 into Xenopus oocytes. A relative value
of 1.0 equated to 0.994 +_ 0.389 nmol/10 cells (s.d.)
of DAG mass (P<0.05) (n=9). Right side of figure
shows absolute values of DAG mass in picomoles per
cell.
66
DAG (pMOL/CELL)


CO
-J
_l
LU
O
O
O
c
CD
<
Q
Fig. 16. Relative potency of different transforming
and non-transforming molecular species of ras p21
that were microinjected into Xenopus oocytes.
Oocytes were injected with 33 nl of ras-buffer or
v-ras p21 (40 ng) and homogenized 2 h after injection
and assayed for DAG mass.
67


DAG Mass at GVBD
DAG mass changes during white spot appearance
was complicated (FIG. 17). Basal DAG mass was 1.31
+_ 0.48 nmol/10 cells (131 pmol/cell) and GVBD25,
GVBD75 and GVBD10O DAG mass were not
significantly greater. At 50% GVBD, the DAG mass
was 117 pmol/cells and this was significantly
(P/0.05) less than DAG mass at GVBD25,
GVBD75 and GVBDioO* This decrease at GVBD50
was similar to that found for IP3 mass at
GVBD50 Both IP3 (17.04 fmol/cell) and DAG
14 pmol/cell) decreased at GVBDsq*
68


1.4
0
<
Q
LLl
>
I
<
_l
LU
cn
1.2 -
1.0 -
0.8
f
W o in o
CVJ W o
G Q G T
G m m G
> > >
0 0 0 > 0
Fig. 17. Relative DAG mass levels were compared at
designated time points after microinjection of 33 nl
of ras-buffer or v-ras p21 (40 ng) into Xenoous
oocytes. A relative DAG mass of 1.0 equated to an
absolute value of 1.31 + 0.48 nmol/10 cells (s.d.).
69


CHAPTER 4
DISCUSSION
Basal Levels of IP3 and DAG
Basal levels of IP3 remained constant for
0-5 h at 75.1 fmol/cell. With other cells at white
spot appearance (6-9 h after injection), basal
levels were 47.49 fmol/cell. Both levels of IP3
mass were in range of those reported by Stith et
al. (1992b) (29 fmol/cell to 77 fmole/cell) and
were not significantly different.
It is important to note that IP3 mass at
basal levels for ras-buffer injected oocytes was
compared to basal levels of IP3 in untreated
cells. There was no significant (PC0.05)
difference between the two groups. Each group's
relative B/B0 level was 70 + 0.8 and 71 _+ 1.9,
respectivly.
Basal levels of DAG mass at 0-5 h were 50
pmol/cell and at white spot appearance, 131
pmol/cell. Basal levels of DAG mass at 0-5 h was
very close to basal DAG levels reported by Stith
70


et al. (1991) (46 + 12.5 pmol/cell), but DAG
levels (131 pmol/cell) at GVBD in our experiments
were higher than at 0-5 hour and Stith et al.
(1991). Though 131 pmole/cell is comparatively
high it does fall within the range of basal DAG
mass for the second group of oocytes seen in
figure 16.
IP3 Increase at 4 Hours
The increase of 1096 from relative basal
levels in IP3 mass at 4 h after Ki-ras-Val[12]
injection into oocytes can be compared with
changes induced by insulin. Stith et al. (1992b)
demonstrated insulin-induced an IP3 increase at 15
min and 2 h after addition. If ras p21 requires
about 3 h to activate (associate with membrane),
the increase at 4 h may be similar to the early 15
min IP3 increase with insulin.
Increases in MPF activity are thought to be
correlated with the release of the oocyte from the
g2/M block and entry into prophase I of meiosis
(Mailer, 1990). Since one of the two components of
MPF is a protein kinase (p34) and both components
are phosphorylated at different times in the cell
71


cycle, and since IP3 increases intracellular
levels of calcium, an increase in IP3 could effect
an increase in the activity of MPF and
calcium-dependent protein kinases. Thus, the
oocyte would enter meiosis due to increases in
calcium-induced phosphorylation of p34 protein
kinase substrates.
DAG Decreases at 30 Minutes after Ras Injection
DAG decreases by 30 min after ras injection
into oocytes. We speculate that this DAG decrease
could be due to injected, inactive Ki-ras-Val[12]
drawing an activator from endogenous ras p21.
If the degradative pathways leading to the
production of DAG are decreased, while synthetic
pathways are not affected, a decrease in DAG mass
would occur. Since ras p21 may induce breakdown of
phospholipids and produce DAG, any decrease in ras
p21 activity would decrease the production of DAG.
Microinjected, inactive ras proteins could inhibit
endogenous ras p21 activity (which produces a
constant, basal level of DAG) by competing for
ras-activating molecules. GAP may be an activator
of endogenous ras p21. If GAP molecules are pulled
72


away from membrane-bound endogenous ras proteins
by microinjected cytosolic inactive ras proteins,
a decrease in the degradative pathway of membrane
phospholipids which produce DAG would occur.
Synthetic pathways of DAG are not thought to be
affected by the activity of ras p21. Since GAP may
bind inactive non-transforming ras p21, this would
explain why microinjection of non-transforming ras
proteins have the same effect on DAG mass as do
more potent transforming ras proteins. However,
since transforming ras p21 is much more effective
in inducing GVBD, the ability to decrease DAG does
not correlate with ability to induce meiosis.
These findings are not in agreement with
Lacal et al. (1987). Briefly, Lacal noted an
immediate (2 min) increase in label turnover of
diacylglycerol after microinjection of
transforming ras p21 into Xenopus oocytes. Lacal
also reported that label turnover of
diacylglycerol as well as phosphoinositides
remained elevated above basal levels even after 12
hours after injection of ras proteins. We do not
agree with this work for the following reasons.
One, Lacal employed the use of label turnover
73


assays. Two, Lacal examined only degradative
pathways labeled by glycerol; that is, pathways
that involved the breakdown of phospholipids to
produce DAG. DAG can also be produced in synthetic
pathways, which can be determined by mass assays,
but not by label turnover assays. Third, there are
several DAG and IP3 isomers. Since Lacal did not
separate isomers, all molecular species of
diacylglycerol (e.g., 1,3 diacylglycerol) and.
phosphoinositides (e.g. inositol
1,3,5-trisphosphate) generated by the breakdown of
these radiolabeled phospholipids would be
quantified. Only the isomers sn 1,2-diacylglycerol
and inositol 1,4,5-trisphosphate function as
second messengers. PKC is only activated by sn
1,2-diacylglycerol and calcium is only released
when inositol 1,4,5-trisphosphate binds to IP3
receptors on the endoplasmic reticulum. Our mass
assays are specific for these two second
messengers. Four, Lacal preincubated oocytes with
labeled glycerol or myo-inositol for only 5 hours
before he began to inject ras p21. Five hours may
not be sufficient to label DAG and IP3 precursors
to equilibrium. Thus, determinations would only be
74


based on those phospholipids that were able to
incorporate the tritiated precursors in the 5 h
period. PC requires 15-20 h labeling time (Stith
et al. 1992b).
IP3 and DAG Mass During GVBD
Both IP3 and DAG mass decrease during white
spot appearance (i.e., GVBD). DAG mass was
significantly lower at GVBD50 than at GVBD25,
GVBD75 and GVBDioO* Decreases in IP3 and DAG
mass were not expected since previous literature
demonstrated increases in mass of both second
messengers as a result of progesterone- or
insulin-induced meiosis (Stith et al., 1991; Stith
et al., 1992b).
Possible Sources of IP3 and DAG
Changes in DAG mass at 4 h and at GVBD were
much greater than changes in IP3 mass at similar
times. DAG mass decreased by 51 pmol at 4 h,
whereas IP3 exibited an increase of only 34 fmol.
At GVBD50, DAG mass decreased by 14 pmol,
whereas at the same time, IP3 decreased by only 17
fmol. Since changes in DAG mass are much greater
75


in magnitude than IP3, this suggests that DAG mass
is primarily not from PIP2 sources. PIP2
metabolism would produce similar magnitude changes
in DAG and IP3. The DAG mass assay (pmolar
sensitivity) is less sensitive than the IP3 assay
(fmolar sensitivity). This suggests that the IP3
assay reflects PIP2 breakdown and the DAG assay
reflects the breakdown of other sources (e.g., PC,
Stith et al., 1992b).
Future Experiments
The above thesis lends itself well to future
experiments concerning the mechanism of ras p21 in
the cell cycle. The following are a few ideas in
which to further explore ras p21 action in the
cell. One, does the increase in ras p21 activity
increase the in vivo activity of PKC? We will
examine the MARCKS protein, a substrate of PKC,
and determine the amount of PKC activity by the
amount of phosphorylation of the MARCKS protein.
We will also determine in vivo PKC activity by
experiments which study the translocation of PKC
to the membrane as the active form is particulate.
By similar methods, we might examine the time of
76


ras p21 movement to the membrane and, thus, the
time of ras activation. Two, is ras p21 action
inhibited by antibodies to PIP2? We will inject
antibodies to PIP2 into Xenopus oocytes and
determine if PIP2 breakdown and ras p21 action are
inhibited. If PIP2 antibodies do not inhibit ras
p21 activity, then ras action in oocytes may not
be dependent upon IP3 or PIP2 degradation.
Finally, whether ras can significantly increase
intracellular calcium will by examined. If so, the
intracellular addition of EGTA may block ras p21
action.
77


APPENDIX A
CHEMICALS, SOLUTIONS AND EQUIPMENT
ATP
32p_ATP
CaCl2
Cardiolipin
Chloroform
DAG Kinase
DAG Standards
DETAPAC
DTT
EDTA
EGTA
Chemicals
(Calbiochem, LaJolla, CA)
(ICN Radiochemicals, Irvine,
CA)
(Baker)
(Calbiochem)
(Baker)
(Lipiden, Westfield, NJ)
(Avanti Polar Lipids,
Birmingham, AL)
(Sigma) Diethylenetriamine
penta-acetic acid
(Sigma) dithiothreitol
(Sigma)
(Sigma)
78


Ethylene Glycol (Sigma)
Freon - (Sigma) 1,1,2-trichloro-
1,2,2-tri fluoroethane
Hepes - (R.P.I.)
Imidazole - (Sigma)
IP3 Standards (New England Nuclear) kit # NEK-064
[3h]IP3 - (New England Nuclear) [3h]IP3 kit,
cat. # NEK-064)
Insulin - (Sigma)
Ki-ras-Val[12] - (Oncogene Science,
Manhasset, NY)
Methanol - (Baker)
MgCl2 - (Baker)
NaCl - (Baker)
NaH2P04 - (Mallinckrodt)
NaOH - (Baker)
Octyl-B-D-glucopyranoside - (Calbiochem)
PMSG - (Sigma)
Scintillation fluor (IP3 assay) - (R.P.I.)
Budget Solve,
Scintillation fluor (DAG assay) - (R.P.I.)
HFP-20,
TCA - (Baker) Trichloroacetic acid
Trioct - (Sigma) Tri-n-octylamine
79


Tris
(Sigma)
Solutions
[32p]ATP Stock -100 ul [32p]ATP
180 ul 1 M imidazole
18 ul 100 mM DETAPAC
1,111 ul dH20
Cardiolipin:Octyl-B-D-Glucopyranoside -
25 mg/ml, dried down under N2
of cardiolipin
253.0 mg Ocytyl-B-D-Glucopyranoside
33.7 ul 100 mM DETAPAC
3,336.0 ul dH20
DAG kinase add 1 ml dH20 to 1 mg/ml
DAG Reaction Buffer 100 mM Imidazole
100 mM NaCl
25 mM MgCl2
2 mM EGTA (pH 6.7)
DTT solution
- 990 ul dH20
10 ul 100 mM DETAPAC
3 mg DTT
Freon:Trioct
3:1 (v/v)
Insulin
(Sigma) 143 uM stock, [final] = 2 uM
NaOH
(Baker) 0.15 M stock
80


PMSG - (Sigma) Pregnant Mare Serum
Gonadotropin 116.67 U/ml
ras-buffer 50 mM Tris
1 mM MgCl2
1 mM NaH2P04
0.1 mM EDTA
5 mM DTT
50 % Ethylene Glycol
0R-2(Ringers) 83 mM NaCl
0.5 mM CaCl2
1 mM MgCl2
10 mM Hepes pH 7.5
TCA - Trichloroacetic acid 2 g in
10 ml dH20
TLC 180 ml Chloroform
72 ml Acetone
54 ml Methanol
36 ml Acetic Acid
18 ml dH20
Working Receptor/Tracer Dilute Receptor/Tracer
15-fold with Assay
Buffer (NEN)
81


Centrifuges Eguipment Clinical Microfuge
Pneumatic Pico Pump - (World Precision Instruments, Inc.) PV830 Sarasota, FL)
Scintillation counter - (LKB-Wallace, Pharmacia) Gaithersburg, MD)
TLC plates - (Merck) Kieselgel 60 TLC plates (E.M. Separations, Gibbstown, NJ)
XAR film - (Kodak, Rochester, NY))
82


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