Identification and Cloning of Xp95, a Putative Signal
Transduction Protein in Xenopus Oocytes*
Shaoli
Che
,
Heithem M.
El-Hodiri§,
Chuan-Fen
Wu§,
Mayra
Nelman-Gonzalez
,
Michael M.
Weil¶,
Laurence D.
Etkin§,
Richard B.
Clark
, and
Jian
Kuang
**
From the
Departments of Clinical
Investigation, § Molecular Genetics, and ¶ Experimental
Radiation Oncology, the University of Texas, M. D. Anderson Cancer
Center, Houston and the
Department of Pharmacology, the
University of Texas Medical School, Houston, Texas 77030
 |
ABSTRACT |
A 95-kDa protein in Xenopus oocytes,
Xp95, was shown to be phosphorylated from the first through the second
meiotic divisions during progesterone-induced oocyte maturation. Xp95
was purified and cloned. The Xp95 protein sequence exhibited homology
to mouse Rhophilin, budding yeast Bro1, and Aspergillus
PalA, all of which are implicated in signal transduction. It also
contained three conserved features including seven conserved tyrosines,
a phosphorylation consensus sequence for the Src family of tyrosine
kinases, and a proline-rich domain near the C terminus that contains
multiple SH3 domain-binding motifs. We showed the following: 1) that
both Xp95 isolated from Xenopus oocytes and a synthetic
peptide containing the Src phosphorylation consensus sequence of Xp95
were phosphorylated in vitro by Src kinase and to a lesser
extent by Fyn kinase; 2) Xp95 from Xenopus oocytes or eggs
was recognized by an anti-phosphotyrosine antibody, and the relative
abundance of tyrosine-phosphorylated Xp95 increased during oocyte
maturation; and 3) microinjection of deregulated Src mRNA into
Xenopus oocytes increased the abundance of
tyrosine-phosphorylated Xp95. These results suggest that Xp95 is an
element in a tyrosine kinase signaling pathway that may be involved in
progesterone-induced Xenopus oocyte maturation.
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INTRODUCTION |
Xenopus oocyte maturation is the process through which
fully grown Xenopus oocytes (stage VI) mature into
unfertilized eggs (1-3). Stage VI Xenopus oocytes are
naturally arrested at the G2/M boundary of the first
meiotic division. The G2/M block of stage VI oocytes is
released by the steroid hormone progesterone, which initiates a series
of events that lead to induction of the first and second meiotic
divisions. The events that are instrumental for maturation induction
include a decrease in cAMP-dependent protein kinase
activity, synthesis of the c-Mos protein, activation of multiple
signaling protein kinases, including Mos protein kinase (4, 5), Raf
protein kinase (6, 7), and
MAP1 kinase (8-10),
activation of Cdc25 phosphatase (11-13), and activation of multiple
mitotic kinases. These mitotic kinases, including mitotic Cdc2 kinase
(14, 15), MPM-2 epitope kinase (16), and polo-like kinase (17), induce
meiotic divisions through the phosphorylation of a great number of
proteins (18). The oocytes are then arrested at the second meiotic
metaphase by cytostatic factor as mature oocytes or unfertilized eggs.
Fertilization triggers release of the metaphase arrest and induces
onset of embryonic cell cycles (19).
Many of the components in the maturation induction pathway are still
unknown. For example, it is unclear how Mos and Raf protein kinases are
activated following progesterone stimulation; how activation of MAP
kinase by Raf and Mos protein kinases leads to the activation of Cdc25
phosphatase; and how the phosphorylation orchestrated by multiple
mitotic kinases induces meiotic divisions. Moreover, whereas tyrosine
phosphorylation has been implicated in progesterone-induced
Xenopus oocyte maturation (20, 21), neither the tyrosine
kinases nor their substrates have been defined. Because most of these
events involve protein phosphorylation, identification and
characterization of novel proteins that become phosphorylated during
progesterone-induced oocyte maturation may shed new light on this process.
We describe the identification, purification, and molecular cloning of
Xp95, a novel 95-kDa protein in Xenopus oocytes which undergoes phosphorylation from the first through the second meiotic divisions. The protein sequence of Xp95 suggests that it belongs to an
emerging family of signal transduction proteins, is a substrate of
tyrosine kinases, and may interact with SH3 domain-containing proteins.
In support of the possibility that Xp95 is a substrate of tyrosine
kinases, we showed that Xp95 and an Xp95 peptide were phosphorylated
in vitro by Src kinase and to a lesser extent by Fyn
kinases. In addition, Xp95 from Xenopus oocytes and eggs was recognized by an anti-phosphotyrosine antibody, and the abundance of
tyrosine-phosphorylated Xp95 increased during oocyte maturation. Furthermore, microinjection of deregulated Src message into
Xenopus oocytes increased the abundance of
tyrosine-phosphorylated Xp95. The features of Xp95 suggest that Xp95
functions in a signal transduction pathway involved in
progesterone-induced oocyte maturation.
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EXPERIMENTAL PROCEDURES |
Preparation of Xenopus Oocyte, Egg, or Embryo
Extracts--
Interphase-arrested Xenopus oocyte extract
and M phase-arrested Xenopus egg extract were prepared as
described previously (16) with the following modifications.
Xenopus oocyte and egg extraction buffer (EB) was 80 mM
-glycerophosphate, 20 mM EGTA, and 15 mM MgCl2, pH 7.3, supplemented with 20 mM NaF, 1 mM ATP, 1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, and 0.1 mg/ml each of
leupeptin, pepstatin A, and chymostatin. Both extracts were immediately
frozen at
70 °C. To thiophosphorylate proteins in egg extract, the
extract was precipitated with 40% ammonium sulfate, desalted by gel
filtration through 10DG columns (Bio-Rad) into EB supplemented with 1 mM DTT and 50 µM ATP
S, and incubated at
room temperature for 3-4 h.
To determine the timing of Xp95 gel mobility shift during
progesterone-induced Xenopus oocyte maturation, surgically
removed stage VI oocytes cultured in modified Barth's solution (22) were treated with 1 µM progesterone. At 1-h intervals,
six oocytes were collected and immediately homogenized by pipetting
with 60 µl of EB supplemented 1 mM DTT and 0.1 mg/ml each
of leupeptin, pepstatin A, and chymostatin. The homogenate was then
centrifuged at ~14,000 × g for 30 min, and the layer
between the surface lipid layer and bottom yolk pellet was collected
and immediately frozen at
70 °C.
To examine the Xp95 gel mobility shift after fertilization, egg laying
was induced by injection of 800 units of human chorionic gonadotropin
(Sigma) into the dorsal lymph sacs of adult female frogs. On the
following day, sperm was prepared from testes surgically removed from
adult male frogs and suspended in 0.1× modified Barth's solution
(23). Eggs were collected, fertilized with the sperm suspension, and
then dejellied in 2% (w/v) L-cysteine dissolved in 50 mM Tris, pH 7.9. The fertilized eggs were washed
extensively with 0.1× modified Barth's solution and maintained in the
same solution at 20 °C. Six embryos were collected and extracted at 15-min intervals as described for extraction of oocytes and eggs.
Immunoblotting and Histone H1 Kinase Assay--
The anti-Cdc25
antibody was produced against a recombinant Xenopus Cdc25
fused at its N terminus to glutathione S-transferase (GST-Cdc25). GST-Cdc25 was produced in Escherichia coli TG-1
cells harboring the expression vector GEX-KG-Xenopus cdc25
and purified by the absorption of the cell lysates with
glutathione-agarose. After separation by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the GST-Cdc25
band was excised from the gel and used to immunize a rabbit. To
affinity purify the anti-Cdc25 antibody, the antiserum was incubated at
4 °C overnight with GST-Cdc25 protein immobilized onto
nitrocellulose membrane. After the membrane was washed six times with
50 mM Tris, pH 7.5, 150 mM NaCl, and 0.05%
Tween 20 (TBST), the bound antibody was eluted with MAPS II elution
buffer (Bio-Rad), neutralized to pH 7.5, concentrated by centrifugation
through Centricon-30, and stored at
20 °C. The anti-YNK1 antibody
was produced and affinity purified as described previously (24). The
anti-Xenopus MAP kinase antibody was raised in rabbit
against a Xenopus MAP kinase peptide (25). The monoclonal
anti-phosphotyrosine antibody PY99 was purchased from Santa Cruz
Biotechnology (Santa Cruz, CA).
Immunoblotting with anti-Cdc25 antibody, anti-YNK1 antibody, or
anti-MAP kinase antibody was performed using alkaline
phosphatase-conjugated goat anti-rabbit IgG (Promega Corp., Madison,
WI) as the second antibody and nitro blue tetrazolium chloride and
5-bromo-4-chloro-3-indolyl phosphate for the color development (16).
Immunoblotting with PY99 was performed using horseradish
peroxidase-conjugated goat anti-mouse IgG as the second antibody and
ECL Western blotting detection reagents for fluorescence development
(Amersham Pharmacia Biotech). The histone H1 kinase assay was performed
as described previously (16).
Phosphatase Treatment of Xp95--
20 µl of egg extract was
diluted 1:10 with EB and absorbed with 10 0.6-cm diameter discs of
nylon membrane (maximum strength Nytran; Schleicher & Schuell) at
4 °C for 2 h with continuous rotation. After the membrane discs
were washed three times with TBST and once with 50 mM
sodium citrate, pH 5.5, they were incubated in the same citrate buffer
supplemented with 2.0 units/ml potato acid phosphatase (Boehringer
Mannheim) at 37 °C for 2 h. The membrane discs were then washed
several times with TBST and eluted with a minimal volume of SDS-PAGE
sample buffer. The eluted proteins were separated by 12.5% SDS-PAGE
and immunoblotted with the anti-Cdc25 antibody.
Isolation of Xp95 from Xenopus Oocytes, Peptide Digestion, and
Amino Acid Sequence Determination--
All chromatography was
performed at 4 °C in 0.25× EB (CB) unless otherwise noted. Ammonium
sulfate precipitation was performed by incubating samples with ammonium
sulfate (Bio-Rad) at 4 °C for 30 min followed by centrifugation at
10,000 × g for 30 min at 4 °C. To purify Xp95 for
amino acid sequencing, 20 ml of oocyte extract was precipitated with
40% ammonium sulfate and desalted into CB by gel filtration through
10DG columns (Bio-Rad). The desalted sample was loaded onto a 20-ml
Q-Sepharose fast flow column (Amersham Pharmacia Biotech), which was
subsequently washed with 60 ml of the same buffer. The pooled
flow-through from the Q-Sepharose chromatography was then loaded onto a
7-ml Affi-Gel blue column (Bio-Rad), which was subsequently washed with
25 ml of CB and eluted with 25 ml of CB containing 0.4 M
NaCl. Finally, the 0.4 M NaCl eluate from the Affi-Gel blue
chromatography was precipitated with 40% ammonium sulfate, resuspended
in 200 µl of CB, and then sedimented at 4 °C through a 3-ml
10-20% sucrose gradient in a SW50.1 rotor (Beckman, Irvine, CA) at
40,000 rpm for 13 h, from which 200-µl fractions were collected.
To obtain partial amino acid sequences of Xp95, two different
procedures were performed (26). In one procedure, proteins from
partially purified Xp95 were separated by SDS-PAGE, transblotted onto
Immobilon-P nylon membrane (Millipore Corp., Bedford, MA), and
visualized by staining with 0.1% Coomassie Blue R-250 in 1% acetic
acid, 40% methanol followed by destaining with 50% methanol. The Xp95
band was then excised and digested with CNBr in situ. The
digested peptides were then separated by Tricine/SDS-PAGE (27),
transblotted onto an Immobilon-P membrane, and visualized by Coomassie
Blue staining. The most prominent band was excised from the membrane
and sequenced in situ with an automated amino acid sequencer
(model 473A, Applied Biosystems, Foster City, CA). In the alternative
procedure, the gel-separated proteins were directly stained with 0.5%
Coomassie Blue in 10% acetic acid and 45% methanol followed by
destaining with 7.5% acetic acid and 25% methanol. The Xp95 band was
then removed from the gel and digested with Lys-C. The collected
peptides were then separated by high performance liquid chromatography
(SMART; Amersham Pharmacia Biotech) on reverse-phase column µRPC
C2-C18. Two of the purified peptides were then sequenced using the same
automated amino acid sequencer.
cDNA Cloning of Xp95--
To clone a full-length cDNA
encoding Xp95, a partial Xp95 cDNA clone was first obtained by
reverse transcription-polymerase chain reaction (PCR) of
poly(A)+ RNA isolated from stage VI Xenopus
oocytes using degenerate primers. The forward primer,
GA(C/T)GCNACNAT(A/C/T)TT(C/T)GCNAA, and the reverse primer,
(C/T)TC(T/G/A)AT(A/G)TA(G/A)TC(G/A)TG), were designed based on the Xp95
amino acid sequences DATIPAN (P1, see Refs. 2-9) and HDNYIE (P2, see
Refs. 6-11), respectively. The PCR products were cloned into
pBlueScript II (KS
) (Stratagene, La Jolla, CA), and the clones
encoding the Xp95 sequence were selected by DNA sequencing. The partial
Xp95 cDNA sequence was then used to design primers that could
amplify 5'- and 3'-ends of the full-length cDNA by rapid
amplification of cDNA ends using the Marathon cDNA amplification kit (CLONTECH, Palo Alto, CA). The
resulting products were cloned into the pCR2.1 TOPO vector with a TOPO
TA cloning kit (Invitrogen, Carlsbad, CA) and verified by DNA sequencing.
In Vitro Phosphorylation of Xp95 and Synthetic
Peptides--
Both human SRC and bovine Fyn kinases were purchased
from Upstate Biotechnology Inc. (Lake Placid, NY). The human SRC kinase was a recombinant protein expressed in SF9 insect cells by baculovirus containing the human c-SRC gene and purified from the crude
cell lysates by hydroxylapatite and affinity chromatography. The bovine cellular Fyn kinase was extracted from membrane fractions of bovine thymus and purified by sequential chromatography on DEAE-Sepharose, hydroxylapatite, and phenyl-Sepharose columns, followed by Sephacryl S-200 gel filtration. Both the Src and Fyn kinase samples were indicated to be free of other protein kinase contaminants.
Phosphorylation of the standard substrate peptide KVEKIGEGTYGVVYK
confirmed that both kinases were active and that they had comparable efficiencies.
Xp95 was partially purified from Xenopus oocyte extract as
described earlier. Two Xp95 peptides, AAKKDNDFIYHDRVPDLKDLDP
(22-mer) and ELVANLKEGTKFYNDLTDIL (20-mer), were synthesized
in the Synthetic Antigen Laboratory at The University of Texas
M. D. Anderson Cancer Center using a DuPont 250 Peptide Coupler.
To assay phosphorylation of Xp95 or Xp95 peptides by Src and Fyn
kinases, ~1 µg of Xp95 or Xp95 peptides was incubated with 20 units
of Src or Fyn kinase in 30 µl of kinase reaction buffer containing
100 mM Tris, pH 7.2, 125 mM MgCl2,
25 mM MnCl2, 2 mM EGTA, 0.25 mM sodium orthovanadate, 2 mM DTT, 0.5 mM ATP, and 10 µCi of [
-32P]ATP at room
temperature for 30 min. The reaction was stopped by addition of
SDS-PAGE sample buffer. To detect phosphorylation of Xp95, the reaction
mixtures were separated by SDS-PAGE, immunoblotted with anti-YNK1
antibody, and subjected to autoradiography. To detect phosphorylation
of Xp95 peptides, the reaction mixtures were separated by
Tricine/SDS-PAGE at a constant current of 20 mA overnight. The gel was
then stained with Coomassie Blue followed by autoradiography.
Injection of RNA into Xenopus Oocytes and Examination of Xp95
Tyrosine Phosphorylation--
Ovarian tissue was surgically removed
from adult Xenopus laevis (Xenopus Express,
Beverly Hills, FL) and anesthetized by 2.5 mg/ml 3-aminobenzoic acid
ethyl ester (Sigma). Stage VI oocytes were manually defolliculated and
maintained in modified Barth's solution. Myc-Xp95 fusion cDNA was
constructed by inserting Xp95 cDNA in frame between the
EcoRI and StuI sites in the CS2+MT vector (28,
29). cDNA containing the coding region of deregulated version of
Xenopus c-src gene has been previously described
(20). RNAs for injection were produced by in vitro
transcription of the linearized plasmids using Ambion mMESSAGE
mMACHINETM SP6 Large Scale In Vitro
Transcription kit for Capped RNAs (Austin, TX). After the transcripts
were purified by LiCl precipitation, oocytes were each microinjected
with 2-3 ng of RNA in 10-20 nl of H2O or an equivalent
amount of H2O and were maintained overnight (~12 h) in
modified Barth's solution. For the experiments that involved partial
purification of Xp95 from injected oocytes by chromatography, 300 oocytes were injected with each sample, and the injected oocytes were
extracted in an Eppendorf tube with 2 volumes of EB supplemented with
20 mM NaF, 1 mM sodium orthovanadate, 1 mM DTT, and 0.1 mg/ml protease inhibitors. The extracts
were then precipitated with 40% ammonium sulfate and desalted into 0.25× EB by gel filtration through a 3-ml G-25 column. The desalted samples were absorbed with 200 µl of HiTrap Q-Sepharose resin in an
Eppendorf tube at 4 °C for 10 min, and supernatant was collected after pelleting the resin by centrifugation. The supernatants were then
absorbed with 300 µl of Affi-Gel blue resin at 4 °C for 20 min,
and the resin was recovered. The Affi-Gel blue resins were then washed
four times with 0.25× EB and eluted with 300 µl of SDS-PAGE sample
buffer at 90 °C for 5 min. For the experiments that involved
isolation of Myc epitope tagged Xp95 from injected oocytes, 60 oocytes
were injected with each sample, and the injected oocytes were extracted
in an Eppendorf tube with 4 volumes of EB supplemented with 20 mM NaF, 1 mM sodium orthovanadate, 1 mM DTT, and 0.1 mg/ml protease inhibitors. The extracts
were then absorbed with 1 µg of anti-Myc polyclonal antibody (Santa
Cruz Biotechnology, Santa Cruz, CA) immobilized onto 10 µl of
Affi-Prep protein A beads (Bio-Rad) at 4 °C for 3 h. After the
protein A beads were pelleted down by centrifugation and washed five
times with EB including 0.5 M NaCl and 0.5% Nonidet P-40,
the immobilized proteins were eluted by SDS-PAGE sample buffer.
 |
RESULTS |
Xp95 Undergoes a Phosphorylation during Progesterone-induced Oocyte
Maturation--
In the process of studying the mechanism that
initiates Cdc25 phosphorylation during Xenopus oocyte
maturation, we generated a polyclonal antibody against recombinant
Xenopus Cdc25. The affinity purified anti-Cdc25 antibody
recognized not only the 76-kDa Cdc25 but also a more prominent
polypeptide of ~95 kDa designated Xp95 (Fig.
1A). Studies were initiated to
investigate the molecular identity and possible functions of Xp95 due
to the following observations.

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Fig. 1.
Xp95 undergoes a
phosphorylation-dependent gel mobility shift during
progesterone-induced Xenopus oocyte maturation.
A, Xenopus oocyte extract was immunoblotted with
anti-Cdc25 antibody. B, oocyte and egg extracts were
immunoblotted with anti-Cdc25 antibody, and the color development was
stopped as soon as Xp95 was readily detectable. C, egg
extract was immobilized onto nylon membrane discs and incubated either
with control buffer or potato acid phosphatase for 2 h. Proteins
on the discs were then eluted with SDS-PAGE sample buffer, separated by
SDS-PAGE, and immunoblotted with anti-Cdc25 antibody. D, egg
extracts before and after thiophosphorylation were separated by
SDS-PAGE and immunoblotted with anti-Cdc25 antibody.
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Xp95 in interphase-arrested oocyte extract migrated as a single band in
SDS-PAGE; however, in M phase-arrested egg extract it became a doublet,
as shown in Fig. 1B. Since the appearance of a slower
migrating species of Xp95 in egg extract is reminiscent of many
proteins that become phosphorylated during progesterone-induced oocyte
maturation, like c-Mos (5), c-Raf (7), p42MAPK (8, 30),
Xnf7 (31), and Cdc25 phosphatase (11, 13, 32), the following
experiments were carried out to determine whether the gel mobility
shift of Xp95 was due to protein phosphorylation. First, we examined
whether dephosphorylation could eliminate the slower migrating form of
Xp95 in egg extract. Egg extract was immobilized onto nylon
membrane discs and then incubated with either potato acid phosphatase
or control buffer. Immobilization of egg extract presumably
disabled endogenous kinases that might rephosphorylate Xp95.
Immunoblotting of the proteins eluted from the membrane with anti-Cdc25
antibody showed that the phosphatase treatment eliminated the slower
migrating species while increasing the intensity of the faster
migrating species of Xp95 (Fig. 1C). Second, we tested
whether thiophosphorylation of Xp95 with the ATP analog ATP
S, which
generates thiophosphorylated proteins resistant to phosphatase activity
(33), increased the relative abundance of the slower migrating species
of Xp95. For this, egg extract was precipitated with 40% ammonium
sulfate, desalted into a buffer that included ATP
S but not ATP, and
then incubated at room temperature for phosphatase turnover. As shown
in Fig. 1D, this treatment increased the intensity of the
slower migrating species of Xp95 and decreased the intensity of the
faster migrating species. Collectively, these results indicate that
Xp95 undergoes a phosphorylationdependent gel mobility shift
during progesterone-induced Xenopus oocyte maturation.
Timing of the Xp95 Gel Mobility Shift--
Progesterone-induced
Xenopus oocyte maturation mainly consists of
progesterone-induced signal transduction and meiotic divisions. To
ascertain which process the Xp95 phosphorylation is more likely to be
associated with, the timing of Xp95 gel mobility shift during progesterone-induced oocyte maturation was determined.
Xenopus oocytes were incubated in the presence of 1 µM progesterone for 8 h and extracted at 1-h
intervals. The protein extracts were then immunoblotted with anti-Cdc25
antibodies to reveal the Xp95 gel mobility shift, with anti-MAP kinase
antibodies to reveal the activation status of MAP kinase, and assayed
for histone H1 kinase activity to reveal the activation status of
mitotic Cdc2 kinase. It has been established that MAP kinase
activation, which is associated with a gel mobility shift of the
protein (10, 30), occurs from the first to the second meiotic divisions
(8, 34), whereas mitotic Cdc2 kinase, which accounts for
80% histone H1 kinase activity in Xenopus oocytes and eggs (35), is
transiently activated at the first meiotic division, inactivated in the
interphase between the first and second meiotic divisions, and
activated again at the second meiotic division. As shown in Fig.
2A, the Xp95 doublet appeared
at approximately the same time as MAP kinase activation and remained
for the rest of the maturation process. This was in contrast to Cdc2
kinase activity, which rose, fell, and rose again during this period.
The gel mobility shift of Xp95 from the first to the second meiotic
divisions suggests that the Xp95 phosphorylation is associated with
progesterone-induced signal transduction but not with M phase
induction.

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Fig. 2.
Timing of the Xp95 gel mobility shift.
A, stage VI Xenopus oocytes were cultured in
Barth's solution containing 1 µM progesterone at
22 °C for up to 8 h, and six oocytes were extracted at 1-h
intervals. Samples were immunoblotted with anti-Cdc25 antibody
(top panel) and anti-Xenopus MAP kinase antibody
(middle panel) and assayed for histone H1 kinase assay
(bottom panel). B, Xenopus eggs were fertilized
in vitro at time 0, and six embryos were extracted at 15-min
intervals. Samples were processed as described for A.
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The metaphase arrest of mature Xenopus oocytes or
unfertilized eggs can be released by fertilization, which triggers
dephosphorylation of many proteins phosphorylated during oocyte
maturation. To determine the phosphorylation status of Xp95 after
fertilization, Xenopus eggs were fertilized in
vitro, and the eggs were extracted at 15-min intervals to examine
Xp95 gel mobility, MAP kinase activation status, and mitotic Cdc2
kinase activity. It has been established that MAP kinase becomes
inactivated after fertilization and that most, if not all, MAP kinase
remains inactivated until midblastula transition (8). In contrast,
mitotic Cdc2 kinase becomes transiently inactivated upon fertilization
and reactivated at each entry into mitosis (36). As shown in Fig.
2B, the slower migrating species of the Xp95 was lost after
fertilization around the same time of MAP kinase inactivation and did
not reappear in the first two embryonic cell cycles. In contrast, the
activity mitotic Cdc2 kinase continued to oscillate. This result
supports the possibility that the gel mobility shift of Xp95 is
associated with progesterone-induced signal transduction but not M
phase induction.
Purification and Sequencing of Xp95--
To investigate the
molecular identity of Xp95, Xp95 was purified from oocyte extracts and
its partial amino acid sequence determined. For this, we first
fractionated oocyte extract consecutively by ammonium sulfate
precipitation, Q-Sepharose chromatography, Affi-Gel blue
chromatography, and sucrose gradient sedimentation as described under
"Experimental Procedures." As shown in Fig. 3A, when the partially
purified Xp95 was separated by SDS-PAGE, the immunostained Xp95
coincided with a distinct silver-stained band indicating that Xp95 was
now separated from the rest of the proteins by SDS-PAGE. Next, we
isolated the Xp95 band from the gel and cleaved it by either
proteolysis or cyanogen bromide treatment. Finally, three of the
internal peptides were sequenced.

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Fig. 3.
Purification and sequencing of Xp95.
A, oocyte extract was sequentially fractionated as described
under "Experimental Procedures." Forty microliters of each fraction
from the sucrose gradient fractionation were separated by SDS-PAGE and
then either immunoblotted with anti-Cdc25 antibody (top
panel) or silver-stained (bottom panel). B,
gel-purified Xp95 was cleaved and three internal peptides were
sequenced. The three Xp95 peptide sequences (Xp95-P1, -2, and -3) were
aligned with the YNK1 protein sequence. The numbers next to the YNK1
sequences indicate their positions in the YNK1 protein. C,
oocyte extract and egg extract were separated by SDS-PAGE and
immunoblotted with anti-YNK1 antibody. The arrow indicates
the position of Xp95.
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The three Xp95 peptide sequences consisted of 42, 17, and 11 amino
acids. A search of protein data bases showed that none of them
exhibited homology with Xenopus Cdc25 protein sequence; however, all exhibited a high degree of homology to
Caenorhabditis elegans YNK1 (Fig. 3B). To explore
further the homology between Xp95 and YNK1, we tested whether Xp95 was
recognized by a polyclonal antibody raised against a YNK1 recombinant
protein (24). As shown in Fig. 3C, the antibody recognized a
single prominent band of 95 kDa in oocyte extract and a doublet in egg
extract in the same region. The recognition was eliminated if the
antibody was preincubated with the recombinant YNK1 protein (data not
shown). The antibody also recognized partially purified Xp95 from
oocyte extract (data not shown). The specific recognition of Xp95 by anti-YNK1 antibody, together with the sequence homology, indicates that
Xp95 is closely related to YNK1.
Xp95 Belongs to a Family of Signal Transduction Proteins--
To
explore further the molecular identity of Xp95, a full-length cDNA
encoding Xp95 was cloned by PCR-based method and Xp95 protein sequence
deduced. As shown in Fig. 4, the cDNA
contained 3506 base pairs consisting of both coding and noncoding
regions. The coding region started with ATG in the context of a Kozak
consensus sequence (37, 38) and ended with a termination codon followed by a poly(A) tail. The open reading frame encodes a protein of 867 amino acids with a calculated molecular mass of 96 kDa, which approximates the apparent molecular mass of Xp95 on SDS-PAGE. Northern
blot analysis showed that this cDNA hybridized to a single transcript of ~4 kilobase pairs (data not shown). These results indicate that this cDNA contains the entire coding sequence and most, if not all, of the noncoding sequences of the transcript.

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Fig. 4.
Sequence of Xp95 cDNA. Nucleic acid
sequence of a full-length Xp95 cDNA and the deduced amino acid
sequence from the open reading frame are shown. The
lowercase and uppercase type in the nucleic acid
sequence indicate the noncoding and coding regions of the cDNA,
respectively. The bold type sequence near the start codon
ATG matches the Kozac consensus sequence, and the bold type
in the 3'-noncoding region indicates the poly(A) tail.
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A search of protein data bases with the Xp95 protein sequence for
homologous proteins confirmed that Xp95 is homologous to YNK1. In
addition, it revealed that Xp95 is also homologous to Aspergillus PalA (39), mouse Rhophilin (40), and budding
yeast Bro1 (41). Whereas the function of YNK1 is presently unknown, the
other three proteins have all been implicated in signal transduction (see "Discussion"), indicating that Xp95 belongs to a family of signal transduction proteins.
Pairwise sequence alignment by the BESTFIT program (Genetics Computer
Group, University of Wisconsin, Madison, WI) showed that Xp95 is
homologous to YNK1, Bro1, and PalA throughout the entire length and is
homologous to Rhophilin through part of the length, i.e.
amino acids 1-530 of Xp95 is homologous to amino acids 111-640 of
Rhophilin (data not shown). This indicates that Xp95-related proteins
fall into two subfamilies. Xp95 is 39% identical and 51% similar to
YNK1, 27% identical and 39% similar to PalA, and 21% identical and
30% similar to Bro1. Thus, in the subfamily including Xp95, Xp95 is
most closely related to YNK1.
Multiple sequence alignment of Xp95 with YNK1, Bro1, and PalA revealed
three conserved features (Fig. 5). First,
there were seven perfectly conserved tyrosines suggesting that Xp95 may
be a substrate of tyrosine kinases. Second, one of the conserved tyrosines is in a highly conserved sequence context
(KDNDFIYXXXV) which matches the phosphorylation consensus
sequence (R/K)X(2,3)(D/E)X(2,3)Y for the Src
family tyrosine kinases (42). This suggests that Src family tyrosine
kinases may phosphorylate Xp95. Third, all four proteins contain a
proline-rich region near their C terminus, which contains multiple
potential SH3 domain-binding motifs PXXP (43). This raises
the possibility that Xp95 may interact with SH3 domain-containing
proteins.

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Fig. 5.
Conserved features of Xp95. The Xp95
protein sequence was aligned with protein sequences of YNK1, Bro1, and
PalA. The asterisks indicate the identical amino acid
residues, and the dots indicate similar amino acid residues.
The double underlined regions indicate consensus tyrosine
phosphorylation sites for the Src family kinases. The underlined
sequences indicate proline-rich domains. The double
underlined sequences in the underlined regions indicate
PXXP motifs.
|
|
Xp95 Is Phosphorylated by Src and Fyn Kinases--
To investigate
the possibility that Xp95 is a substrate of Src family kinases, we
first examined whether Xp95 could be phosphorylated in vitro
by Src or Fyn kinase. Xp95 was partially purified from Xenopus oocyte extract. Purified Src and Fyn kinases, free
of contaminating kinase activities, were obtained from a commercial source and were shown to have comparable efficiencies when used to
phosphorylate the standard substrate peptide KVEKIGEGTYGVVYK (data not
shown). Xp95 alone, each kinase alone, or Xp95 plus each kinase was
incubated at room temperature for 30 min in the presence of
[
-32P]ATP. All the samples were then immunoblotted
with anti-YNK1 antibody and subjected to autoradiography. Results from
immunoblotting with anti-YNK1 antibody showed that partially purified
Xp95 contained both full-length Xp95 and a partially cleaved Xp95 at
~70 kDa and that incubation with neither Src nor Fyn kinase changed
the gel mobility of Xp95 (Fig.
6A). The autoradiography
results showed that whereas incubation of Xp95 or each kinase alone
generated no 32P labeling in the immunoreactive bands,
incubation of Xp95 with either Src or Fyn kinases generated
32P labeling in both of the immunoreactive bands, although
Src kinase generated stronger labeling than Fyn kinase (Fig.
6A). Quantitation of the radioactivity in the labeled bands
by PhosphorImager analysis showed that the difference between Src and
Fyn phosphorylation was 3.5-fold. These results indicate the following:
1) Xp95 is an in vitro substrate of both Src and Fyn
kinases; 2) phosphorylation by Src kinase is more efficient; and 3)
phosphorylation by Src or Fyn kinase is not sufficient to cause the gel
mobility shift of Xp95.

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Fig. 6.
In vitro phosphorylation of Xp95
and Xp95 peptides by Src or Fyn kinases. A, Xp95
(partially purified from oocyte extract), purified Src or Fyn kinase,
or Xp95 together with either Src or Fyn kinase were incubated in the
32P-kinase reaction buffer at room temperature for 30 min.
After samples were separated by SDS-PAGE, they were immunoblotted with
anti-YNK1 antibody (left panel) and subjected to
autoradiography (right panel). B, two Xp95
peptides, the 22-mer and 20-mer, were incubated with either Src or Fyn
kinase in the 32P-kinase reaction buffer. Samples were
separated by Tricine/SDS-PAGE and subjected to autoradiography.
|
|
Next, we examined whether Src and Fyn kinases could phosphorylate a
synthetic peptide containing the conserved Src phosphorylation consensus sequence on Xp95. For this, two Xp95 peptides were
synthesized by a peptide coupler as follows: one a 22-mer containing
the Src phosphorylation consensus sequence (AAKKDNDFIYHDRVPDLKDLDP,
amino acids 310-321) and the other a 20-mer containing a different
conserved tyrosine in a conserved context (ELVANLKEGTKFYNDLTDIL, amino
acids 665-685). The two synthetic peptides were incubated with Src and Fyn kinases in the presence of [
-32P]ATP. Similar to
the result obtained with Xp95 partially purified from oocyte extract,
the 22-mer was 32P-labeled by both Src and Fyn kinases and
the labeling was much stronger by Src kinase than by Fyn kinase (Fig.
6B). In contrast, no labeling was detected on the 20-mer by
either Src or Fyn kinase. Quantitation of the radioactivity in the
labeled bands by PhosphorImager analysis showed that the difference
between Src and Fyn phosphorylation was 5-fold. These results suggest
that Src and Fyn kinases phosphorylate Xp95 on the Src phosphorylation
consensus sequence.
Both Src and Fyn kinase transcripts are detectable in
Xenopus oocytes. To determine whether Xp95 was
tyrosine-phosphorylated in vivo, we examined whether Xp95 in
either oocyte or egg extract was recognized by PY99, a characterized
monoclonal antibody that detects phosphotyrosine residues in proteins
(44, 45). First, similar amounts of proteins from Xenopus
oocyte and egg extracts were separated by SDS-PAGE and immunoblotted
with PY99 to reveal all tyrosine-phosphorylated proteins. The same
blot was also reprobed with anti-YNK1 antibody to reveal the position
of Xp95. Both the single species of Xp95 in oocyte extract and the
doublet form of Xp95 in egg extract coincided with staining by PY99.
However, the signal was much stronger in the Xp95 doublet from egg
extract than in the single species of Xp95 from oocyte extract (Fig.
7A). Second, Xp95 was
partially purified from Xenopus egg extract and immunoblotted in parallel with anti-YNK1 antibody and PY99. The doublet
form of Xp95 in the partially purified sample again comigrated with a
doublet recognized by PY99 (Fig. 7B). Third, Myc-epitope tagged Xp95 mRNA was microinjected into Xenopus oocytes
that were subsequently incubated for 10 h in the presence or
absence of progesterone. In the absence of progesterone, the injected
oocytes remained immature, whereas in the presence of progesterone
95% of the injected oocytes matured after the 10-h incubation. The Myc-tagged Xp95 was then isolated from immature or mature oocytes by
immunoprecipitation with anti-Myc antibody and the immunocomplex immunoblotted with both a commercial anti-Myc antibody and PY99. Only
the Myc-tagged Xp95 from mature oocytes coincided with staining by PY99
(Fig. 7C). These results indicate that Xp95 is
tyrosine-phosphorylated at low levels in oocytes and that the tyrosine
phosphorylation of Xp95 increases during progesterone-induced oocyte
maturation.

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Fig. 7.
In vivo phosphorylation of Xp95 on
tyrosine. A, 100-µg proteins from Xenopus
oocyte or egg extract were immunoblotted sequentially with monoclonal
antibody PY99 that recognizes phosphotyrosine and anti-YNK1 that
recognizes Xp95. B, 20 µg of partially purified Xp95 from
Xenopus eggs was immunoblotted in parallel with anti-YNK1
antibody and PY99. C, after 300 µg of proteins from
oocytes injected with Myc-tagged Xp95 RNA were immunoabsorbed with 1 µg of anti-Myc antibody, the immunocomplex was immunoblotted with
anti-Myc antibody and PY99. Lanes immature and
mature indicate sample from oocytes incubated in the absence
and presence of progesterone.
|
|
Finally, we examined whether increasing the level of Src kinase in
oocytes affected the level of tyrosine phosphorylation on Xp95.
Messenger RNA of Xenopus deregulated Src (d-Src) was produced by in vitro transcription. The RNA or
H2O was microinjected into stage VI oocytes which were then
incubated at 18 °C overnight to allow d-Src translation.
Immunoblotting of similar amounts of proteins from H2O- or
d-Src-injected oocytes as well as unfertilized eggs with anti-YNK1
antibody showed that similar amounts of Xp95 were present in different
samples and that d-Src injection did not cause Xp95 gel mobility shift
(Fig. 8A, left
panel). Probing the same blot with PY99 showed that d-Src
injection increased the amount of PY99 staining in Xp95 although the
signal was still weaker than that in the Xp95 from eggs (Fig.
8A, right panel). Xp95 was then
partially purified from both H2O- and d-Src-injected oocytes before being immunoblotted with anti-YNK1 and PY99. Although similar amounts of Xp95 from control and d-Src-injected oocytes were
again detected by anti-YNK1 antibody, only the Xp95 band from
d-Src-injected oocytes coincided with positive staining by PY99 (Fig.
8B). Finally, oocytes were injected with either Myc-tagged Xp95 RNA alone or Myc-tagged Xp95 RNA together with d-Src RNA. The
Myc-tagged Xp95 protein isolated from each group of the injected oocytes by immunoprecipitation was immunoblotted with both anti-Myc antibody and PY99. Only the Myc-tagged Xp95 from the oocytes injected with both Myc-tagged Xp95 RNA and d-Src RNA coincided with staining by
PY99 (Fig. 8C). These results indicate that d-Src injection increased the abundance of tyrosine-phosphorylated Xp95 and further support the possibility that Xp95 is a substrate of Src family kinases
or related kinases.

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Fig. 8.
d-Src injection increased the abundance of
tyrosine-phosphorylated Xp95. A, 100 µg of proteins
from H2O-injected oocytes (Control
oocytes), unfertilized eggs (Eggs), and
d-Src-injected oocytes (d-Src oocytes) were immunoblotted
sequentially with monoclonal antibody PY99 that recognizes
phosphotyrosine and anti-YNK1 that recognizes Xp95. B, 20 µg of partially purified Xp95 from H2O- or d-Src-injected
oocytes (total protein) were immunoblotted sequentially with PY99 and
anti-YNK1 antibody. C, after 300 µg of proteins from
oocytes injected with either Myc-tagged Xp95 RNA alone (Control
oocytes) or Myc-tagged Xp95 RNA together with d-Src RNA
(d-Src oocytes) were immunoabsorbed with 1 µg of anti-Myc
antibody, the immunocomplex was immunoblotted with anti-Myc antibody,
and PY99.
|
|
 |
DISCUSSION |
A series of signaling events are initiated following treatment of
Xenopus oocytes with the steroid hormone progesterone. These events lead to meiotic divisions and finally to the arrest of oocytes
in the second meiotic metaphase. In this maturation phase of oogenesis,
protein phosphorylation is known to play a crucial role in both signal
transduction and meiotic induction. In this study, we observed that
Xp95, a 95-kDa protein in Xenopus oocytes, undergoes a
phosphorylation-dependent gel mobility shift during progesterone-induced oocyte maturation and a reverse gel mobility shift
in response to fertilization. This suggests that Xp95 may be involved
in the maturation process. Prompted by this possibility, we purified
the protein to near homogeneity by a combination of standard
chromatographic procedures and gel electrophoresis. This allowed us to
obtain amino acid sequences of three internal peptides and clone a
full-length cDNA encoding Xp95 by PCR-based techniques.
Analysis of the deduced amino acid sequence of Xp95 revealed that it is
homologous to a conserved family of signal transduction proteins. The
strongest homology was to YNK1 (51% sequence similarity), a protein of
unknown function in C. elegans (24). The next was Aspergillus PalA (37% sequence similarity), a protein that
is one of six gene products implicated in the ambient pH regulation of
gene expression (39). Xp95 was also 30% similar to mouse Rhophilin, a
protein that was identified based on its association with RhoA, a
member of the Ras family of monomeric G-proteins (40). RhoA has been
implicated in various cellular responses to lysophosphatidic acid,
tetradecanoylphorbol acetate, and certain growth factors (46). Finally,
Xp95 showed 30% similarity to Bro1, a protein found in budding yeast
that has been suggested by genetic studies to interact with or run in
parallel with the protein kinase C-MAP kinase pathway involved in the
maintenance of cell integrity (41). The homology of Xp95 to a conserved family of proteins implicated in a variety of signaling pathways suggests that Xp95 is involved in progesterone-induced signal transduction during oocyte maturation.
Alignment of the five Xp95-related proteins indicated that they fell
into two subfamilies. One subfamily included Xp95, Bro1, PalA, and
YNK1. These four proteins are not only homologous to each other
throughout the entire length but also share three interesting features
as follows: a Src kinase phosphorylation consensus sequence, six
additional conserved tyrosines, and a proline-rich C-terminal domain
containing multiple SH3 domain-binding motifs. The other subfamily,
which thus far includes only Rhophilin, is neither homologous to the
other Xp95-related proteins through the entire length nor does it
contain any of the three conserved features. However, Rhophilin
contains a RhoA-binding domain in its N terminus that is absent in all
other Xp95-related proteins. The difference in the structure of the two
subfamilies of Xp95-related proteins suggests that they transduce
signals through different mechanisms.
The three conserved features of the Xp95 subfamily suggest that these
proteins transduce signals, at least in part, through tyrosine
phosphorylation and interaction with SH3 domain-containing proteins.
Although we have not experimentally addressed the possible interaction
of Xp95 with SH3 domain-containing proteins, we have obtained
substantial evidence on tyrosine phosphorylation of Xp95. We
demonstrated that Xp95 was phosphorylated in vitro by Src
and Fyn kinases and that the phosphorylation site was likely to be in
the conserved Src phosphorylation consensus sequence. In addition, we
showed that Xp95 was tyrosine-phosphorylated at low levels in oocytes
and that the tyrosine phosphorylation of Xp95 increased during
progesterone-induced oocyte maturation. Finally, we observed that
microinjection of deregulated Src message into Xenopus
oocytes increased the abundance of Xp95 that is
tyrosine-phosphorylated. Taken together, these results strongly suggest
that Xp95 is a substrate of Src family kinases or related tyrosine
kinases which participate in progesterone-induced signal transduction
in Xenopus oocytes.
Besides Src family kinases or related kinases, an additional protein
kinase must also phosphorylate Xp95 during progesterone-induced oocyte
maturation. Xp95 undergoes a phosphorylationdependent gel mobility shift during this process. However, although the abundance of
Xp95 that is tyrosine-phosphorylated increased during
progesterone-induced oocyte maturation, neither the in vitro
nor in vivo phosphorylation of Xp95 by Src kinase caused an
obvious gel mobility shift of Xp95. This indicates that the gel
mobility shift of Xp95 is caused by a different kinase that
phosphorylates Xp95 on a different site(s). Since the Xp95 gel mobility
shift coincided with MAP kinase activation, we have explored the
possibility that this additional phosphorylation is catalyzed by MAP
kinase. However, Xp95 partially purified from Xenopus oocyte
extract was unable to be phosphorylated by active MAP kinase partially
purified from Xenopus egg extract (data not shown). Mitotic
Cdc2 kinase was also unable to phosphorylate partially purified Xp95
(data not shown). Further investigation is thus required in order to
identify the kinase responsible for the Xp95 gel mobility shift during oocyte maturation.
In summary, we report here the identification and molecular cloning of
a novel protein in Xenopus, Xp95, which undergoes
phosphorylation during progesterone-induced Xenopus oocyte
maturation. Thus far, Xp95 has three interesting features. First, it
belongs to an emerging family of signal transduction proteins. Second,
it is likely to be a novel substrate of Src family kinases or related
kinases. Third, it may have the capacity to bind certain SH3
domain-containing proteins. These features suggests that Xp95 is an
element in a signal transduction pathway involved in
progesterone-induced Xenopus oocyte maturation.
 |
ACKNOWLEDGEMENTS |
The computational facility is supported by
National Cancer Institute Grant CA-16672. We thank Drs. T. Hunt and
A. R. Nebreda for providing GST-cdc25 expression vector; Dr. James
Ferrell for providing anti-Xenopus MAP kinase antibodies;
and Dr. Richard Cook for performing amino acid sequencing of Xp95. We
thank Dr. R. Allen White, Brian Kay, and David Voehringer for help in
computerized sequence analysis. We also thank Drs. Steven Marcus, Tina
Lee, and Gordon Mill for their critiques of this manuscript and
Josephine Neicheril for administrative assistance.
 |
FOOTNOTES |
*
This work was supported by National Cancer Institute Grant
R-29 GM48457-02, National Science Foundation Grant MCB-9405699 (to
J. K.), and National Cancer Institute Training Grant CA09299 (to
H. E.-H.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF115497.
**
To whom correspondence should be addressed: Dept. of Clinical
Investigation, Box 019, the University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Tel.:
713-792-2961; Fax: 713-792-3754; E-mail:
jkuang{at}notes.mdacc.uth.tmc.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
MAP, mitogen-activated protein;
DTT, dithiothreitol;
EB, Xenopus
egg extraction buffer;
GST, glutathione S-transferase;
PAGE, polyacrylamide gel electrophoresis;
TBST, Tris-based saline/Tween 20;
PCR, polymerase chain reaction;
ATP
S, adenosine
5'-O-(thiotriphosphate);
d-Src, deregulated Src;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
 |
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