From the Ottawa Civic Hospital Loeb Research Institute, Ottawa
Civic Hospital, and the Departments of Biochemistry,
§ Medicine, and ¶ Obstetrics and Gynecology, University
of Ottawa, Ottawa K1Y 4E9, Canada
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ABSTRACT |
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Insulin receptor substrate-1 (IRS-1) is
phosphorylated on multiple tyrosine residues by ligand-activated
insulin receptors. These tyrosine phosphorylation sites serve to dock
several Src homology 2-containing signaling proteins. In addition,
IRS-1 contains a pleckstrin homology domain and a phosphotyrosine
binding domain (PTB) implicated in protein-protein and protein-lipid
interactions. In a yeast two-hybrid screening using Xenopus
IRS-1 (xIRS-1) pleckstrin homology-PTB domains as bait, we identified a
Xenopus homolog of Rho-associated kinase (xROK
) as a
potential xIRS-1-binding protein. The original clone contained the
carboxyl terminus of xROK
(xROK-C) including the putative Rho
binding domain but lacking the amino-terminal kinase domain. Further
analyses in yeast indicated that xROK-C bound to the putative PTB
domain of xIRS-1. Binding of xROK-C to xIRS-1 was confirmed in
Xenopus oocytes after microinjection of mRNA corresponding
to xROK-C. Furthermore, microinjection of xROK-C mRNA inhibited
insulin-induced mitogen-activated protein kinase activation with a
concomitant inhibition of oocyte maturation. In contrast,
microinjection of xROK-C mRNA did not inhibit mitogen-activated protein
kinase activation or oocyte maturation induced by progesterone or by
microinjection of viral Ras (v-Ras) mRNA. These results suggest that
xROK
may play a role in insulin signaling via a direct interaction
with xIRS-1.
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INTRODUCTION |
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Phosphorylation of insulin receptor substrate-1 (IRS-1)1 by ligand-activated insulin receptors serves to dock several Src homology 2 domain-containing proteins (1-4). In addition to the multiple tyrosine phosphorylation sites, IRS-1 contains an amino-terminal PH domain (5, 6) and a PTB domain (7, 8) carboxyl-terminal to the PH domain (9, 10). Studies by White and co-workers (11, 12) have shown that the PH domain is required for efficient tyrosine phosphorylation of IRS-1 by the insulin receptor, although the mechanism by which the PH domain regulates this function is not clear. The presence of a PTB domain in IRS-1 explains the earlier observations that mutations in a human insulin receptor autophosphorylation site (NPEY960 (13)) or the equivalent site in insulin-like growth factor I receptor (14) diminished its ability to phosphorylate IRS-1. Biochemical and structural studies indicate that amino acids 161-265 of rat IRS-1 include the required component of the PTB domain which binds the NPEpY motif of the insulin receptor (9, 10). However, using the yeast two-hybrid assay, Gustafson and colleagues have provided evidence that additional amino acids that are carboxyl-terminal to the PTB domain (termed the SAIN domain) are also necessary for binding to the NPEpY sequence of the insulin receptor (15, 16). IRS-1 also interacts with 14-3-3 protein, a process apparently dependent on serine phosphorylation of IRS-1 (17, 18).
We have previously isolated a Xenopus cDNA encoding an
IRS-1-like protein (termed xIRS-L) (19).
Overall, xIRS-L exhibits 65% amino acid sequence identity to mammalian
IRS-1 (1) but only 45% identity to mammalian IRS-2 (20). Sequence
identity to the more recently identified IRS-3 (21) or IRS-4 (22) is even less (not shown). Therefore xIRS-L is likely the
Xenopus homolog of IRS-1 and hereafter is referred to as
xIRS-1. The putative PH and PTB domains of xIRS-1 are highly similar to
those of rat IRS-1, with 85 and 90% amino acid sequence identity,
respectively. To identify novel proteins that may bind the highly
conserved amino-terminal protein modules (PH and PTB in particular) of
IRS-1, we conducted a yeast two-hybrid screening of a
Xenopus oocyte cDNA library. We report here that the
Xenopus homolog of RhoA-associated protein kinase (xROK)
is an xIRS-1-binding protein and that binding of a noncatalytic region
of xROK
to endogenous xIRS-1 correlates with inhibition of insulin
signaling in Xenopus oocytes.
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MATERIALS AND METHODS |
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Animal and Oocyte Manipulation-- All procedures involving live oocytes were carried out in a room maintained at 18 °C. Sexually mature, oocyte-positive Xenopus laevis were purchased from NASCO and maintained according to local animal care guidelines. The frogs were injected with pregnant mare serum gonadotropin (Sigma, 50 IU/frog) 3-10 days before oocyte retrieval. A fragment of ovary was removed surgically under hypothermia. Stage VI oocytes (23) were manually defolliculated according to Smith et al. (24). Unless otherwise stated, 10 ng (in 10 nl) of mRNA was injected per oocyte. Microinjection of oocytes was performed in oocyte incubation medium OR2 (82.5 mM NaCl, 2.5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM Na2HPO4, 5 mM HEPES, pH 7.8) lacking CaCl2. Injected oocytes were incubated in OR2 (containing CaCl2) for 6 h to overnight before the addition of hormones or a second injection.
Progesterone (final concentration 10 µM) stimulation of oocytes was carried out in OR2. To ensure that oocytes respond maximally to insulin, oocytes were incubated with insulin (5 µM) in OR2 lacking K+ ions (25, 26). Elimination of K+ ions from OR2 had no effect on oocyte viability, nor did it induce oocyte maturation by itself (data not shown). To assay for meiotic maturation, oocytes were incubated overnight with insulin or progesterone. Oocytes injected with v-Ras mRNA were incubated in OR2 for 4-8 h before being scored for maturation. Oocyte maturation, as indicated by germinal vesicle (GV) breakdown or GVBD, was determined by the appearance of a white spot at the center of the animal hemisphere and confirmed, when in doubt, by bisecting the oocytes after fixation in 5% trichloroacetic acid and observing for the presence (GVBD-negative) or absence (GVBD-positive) of a GV. Oocytes were lysed by forcing them through pipette tips in phosphate-buffered saline lysis buffer (10 mM sodium phosphate, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10 µg/ml each of leupeptin and aprotinin, 1 mM phenylmethylsulfonate, and 1 mM sodium orthovanadate; 10 µl of lysis buffer/oocyte). The homogenate was centrifuged in an Eppendorf centrifuge for 15 min at 4 °C. Under these conditions, the yolk protein (vitellogenin) was not solubilized and was discarded as a pellet. The cell lysates, which were usually clouded because of the presence of lipids, were suitable for immunoprecipitation and Western blotting.Molecular Cloning and Subcloning-- The nucleotide sequence encoding amino acids 3-500 of xIRS-1 (19) was PCR-amplified using the following primers: 5'-TAT GGA TCC CTA GCC CAC AGA C; 5'-TAT GTC GAC TGA GGA GTG AGT CC. The amplified cDNA was digested with BamHI and SalI, restriction sites incorporated into the 5'- and 3'-PCR primers (underlined), respectively. The digested PCR product was then ligated to pAS2 (CLONTECH) which had been digested with the same two enzymes. The resulting plasmid, designated pAS2-xIRS-1 (3-500), was expected to express a Gal4 fusion protein containing amino acids 3-500 of xIRS-1 (19) with an extra proline at the junction between Gal4 and xIRS-1 (3-500). The subclones used for mapping binding domains (see Fig. 2) were similarly amplified by PCR using primers containing the same two restriction sites (BamHI and SalI). For the sake of brevity, only the amino acids comprising the domains of xIRS-1 (19) are indicated here: PH, 3-142; PH/PTB, 3-325; PTB, 137-325; SAIN, 306-500; PTB/SAIN, 137-500.
We subsequently transformed the new yeast strain (expressing Gal4-xIRS-1(3-500)) with a Xenopus oocyte cDNA library (CLONTECH), which was constructed in the Gal4 transactivating domain plasmid pGAD10. Subsequent screening and other yeast procedures were according to protocols provided by CLONTECH. Screening of theYeast Two-hybrid Assays--
-Galactosidase assays were
performed according to the protocols provided by
CLONTECH. The pAS2-based plasmid and pGAD10-based plasmid were cotransformed into Y1090 (CLONTECH)
yeast strain. The transformed yeast cells were plated in SD/
Trp/
Leu
and incubated at 30 °C. Filter
-galactosidase assays using
5-bromo-4-chloro-3-indolyl
-D-galactopyranoside as a
substrate were carried out (CLONTECH) when the
colonies were 1-2 mm in size (2-3 days after transformation). For
quantitative
-galactosidase assays in solution, colonies were
individually picked and inoculated into 2 ml of SD/
Trp/
Leu. When
the 2-ml cultures were saturated, they were individually diluted to 6 ml of SD/
Trp/
Leu. The diluted cultures were incubated for a further
6-10 h (until the A600 reached 1).
-Galactosidase assays were carried out using
O-nitrophenyl-
-D-galactopyranoside as a
substrate, and the results were expressed in micromoles of O-nitrophenyl-
-D-galactopyranoside
hydrolyzed/min calculated according to the CLONTECH
protocol.
Antibodies--
An internal BamHI fragment of SF7 was
subcloned into pGEX-KT (30). The resulting plasmid encoded a
glutathione S-transferase fusion protein containing amino
acids 871-1229 of xROK. The fusion protein was expressed in and
purified from bacteria and used to immunize rabbits. After four
injections with 1-month intervals, the rabbits were terminally bled and
the antiserum was used without further processing.
Other Procedures--
Immune kinase assays using myelin basic
protein (MBP) as a substrate were carried out essentially as described
in Ref. 31 except the immunoprecipitation was carried out in
phosphate-buffered saline lysis buffer. Briefly, oocyte lysates (300 µl containing approximately 1 mg of total protein, usually
representing the amount from 20 oocytes) were incubated with 5 µl of
preimmune or immune sera for 1 h (4 °C) before a further 30-min
incubation with protein A-Sepharose. Immunoprecipitates were washed
three times with phosphate-buffered saline lysis buffer and once with kinase buffer (50 mM HEPES, pH 7.3, 10 mM
MgCl2, 2 mM MnCl2, 1 mM
dithiothreitol, 0.05% Triton X-100). The kinase reaction was carried
out at room temperature for 30 min in 30 µl of kinase buffer
containing 10 µM [-32P]ATP (10 µCi)
and 5 µg of MBP. The kinase reaction was terminated by the addition
of SDS-sample buffer. 10 µl of the above reaction was resolved by
SDS-PAGE (7.5% for autophosphorylation and 15% for phosphorylation of
MBP). The 15% gels were directly dried down for autoradiography to
reveal phosphorylation of MBP. The 7.5% gels were transferred to
nitrocellulose membrane, autoradiographed, and then subjected to
Western blotting with anti-ROK
. The presence of
32P-labeled proteins did not interfere with subsequent
detection of immunoreactive proteins because the immunoblots were
typically developed for less than 10 min, whereas the autoradiography
took at least overnight.
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RESULTS |
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Identification of xROK as an xIRS-1-binding Protein--
We
initially identified 10 positive clones in a yeast two-hybrid screening
of about 700,000 colonies using xIRS-1(3-500) as bait. After isolation
of the plasmid and retransformation into yeast, four clones still
demonstrated bait-dependent activation of the Gal4
promoter. Two of these clones (SF7 and SF107) contained similar
3.5-kilobase inserts. Upon DNA sequence analysis, the two clones were
determined to be identical and contained an open reading frame of 499 amino acids, in-frame with the amino-terminal Gal4 sequence. A search
of data bases revealed high amino acid sequence identity (90%) with
the carboxyl terminus of mammalian ROK
(31, 33, 34) (Fig.
1). SF7 contained the putative Rho binding domain and the carboxyl-terminal pleckstrin/cysteine-rich domain (PH/CRD) (31, 35) (Fig. 1A). Indeed, SF7 interacted strongly with an activated mutant of RhoA (V14-RhoA) (36) in the yeast
two-hybrid assay (Fig. 2A).
Based upon these criteria, we termed the partial cDNA xROK-C.
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xROK-C Binds Endogenous xIRS-1 and Inhibits Insulin Signaling in
Xenopus Oocytes--
We used mRNA injection in Xenopus
oocytes to confirm the xIRS-1-xROK interaction and to characterize
the function of xROK
in insulin signaling. Injection of Myc-tagged
xROK-C mRNA (Fig. 3A,
lanes 1-5) but not a control RNA (lane 6) into
Xenopus oocytes resulted in the production of a protein of
approximately 80 kDa (slightly higher than the predicted molecular mass
of 70 kDa) which was recognizable by anti-Myc antibodies (Fig.
3A, lanes 4 and 5). Furthermore,
immunoprecipitation using an anti-xIRS-1 antibody (lanes 2 and 3) but not a control antibody (anti-glutathione S-transferase lane 1) coprecipitated the
Myc-tagged xROK-C. Binding of xROK-C to endogenous xIRS-1 did not
appear to be influenced by insulin stimulation (comparing lanes
2 and 3). Reciprocal experiments were also carried out
wherein anti-Myc immunoprecipitation of xROK-C coprecipitated
endogenous xIRS-1 (Fig. 3B, lane 1). A control antibody (anti-hemagglutinin, lane 3) did not precipitate
xROK-C or xIRS-1.
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Characterization of Endogenous xROK--
An
immunoprecipitation-coupled in vitro kinase assay was
performed using either preimmune or xROK
-specific immune serum. Anti-xROK
specifically brought down a protein of >200 kDa, detected both by in vitro kinase assay (Fig.
5A) and by Western blotting (Fig. 5B). In addition, the immunoprecipitated kinase was
capable of phosphorylating MBP (Fig. 5C). We did not observe
any consistent stimulation of xROK
kinase activity, either by
xROK
autophosphorylation or phosphorylation of MBP, in oocytes
stimulated with insulin (comparing lanes 2 and
3). The size of the putative xROK
was considerably larger
than either the predicted molecular mass of the cloned xROK
(159 kDa) or the reported size of mammalian ROK
(160 kDa) (31). We have
recently pasted together all of the subclones (Fig. 1A) for
in vitro transcription/translation experiments. The
full-length xROK
produced in these experiments was indeed >200 kDa
on SDS-PAGE and was recognizable by anti-xROK
serum.2
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DISCUSSION |
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Members of the Rho subfamily of small monomeric GTP/GDP-binding
proteins (including Rho, Rac, and Cdc42) function in mediating growth
factor/cytokine-induced actin cytoskeleton reorganization (43).
Specifically, Rho (RhoA, B, and C) is involved in growth factor/cytokine-induced focal adhesion/stress fiber formation, Rac is
involved in membrane ruffling, and Cdc42 promotes filopodia (44). In
addition, these GTPases have been implicated in mediating v-Ras-induced
cell transformation (45), transcriptional activation (46, 47), and in
mediating integrin-induced MAP kinase activation (48). ROK was first
identified as a serine/threonine protein kinase capable of binding to
GTP-bound RhoA (33). The function of ROK
as an effector of Rho in
promoting actin polymerization and focal adhesion formation has been
well established (31, 35). It is unclear, however, whether ROK
may
play a role in the other functions of Rho.
ROK is a relatively large protein (160 kDa) consisting of an
amino-terminal kinase domain, a central coiled coil domain that also
includes the Rho binding domain, and a carboxyl-terminal putative PH
domain that is split by the insertion of a cysteine-rich motif (31, 37)
(Fig. 1A). In the present study, we have identified a novel
interaction between the carboxyl terminus of xROK
and the xIRS-1 PTB
domain. We have also demonstrated that this binding correlated with the
inhibition of insulin signaling in Xenopus oocytes. Two
important lines of evidence support the specificity of xROK-C-induced
inhibition of insulin signaling. First, injection of xROK-C mRNA did
not affect progesterone-induced MAP kinase activation or GVBD,
suggesting that xROK-C acted in an insulin-specific pathway. Second,
injection of xROK-C mRNA did not affect MAP kinase activation or GVBD
induced by injection of v-Ras mRNA, suggesting that xROK-C acted
upstream of cellular Ras in the insulin signaling pathway. Although it
is possible that the PH/CRD domain found at the carboxyl terminus of
ROK
may nonspecifically interfere with the function of other
PH-containing proteins, notably xIRS-1, such a possibility is quite
doubtful. Whereas there is limited sequence homology between the PH/CRD
of ROK
and the PH domains of several other proteins including
pleckstrin (31, 37), no sequence homology can be detected between the
PH/CRD of ROK
(or xROK
) and the PH domain of IRS-1 (or xIRS-1)
(not shown).
The xROK-C binding site was mapped to the PTB domain of xIRS-1. This
raises the possibility that xROK-xIRS-1 interaction may interfere
with the presumed interaction of the PTB domain of xIRS-1 with the
Xenopus insulin-like growth factor I receptor. This provides
a potential explanation for the xROK-C-mediated inhibition of insulin
signaling. Interestingly, a potential serine phosphorylation site
(Ser-270) within the PTB domain has been implicated as one of the
binding sites for 14-3-3 protein (18). Further studies are required to
determine whether it is a functional PTB domain or a smaller motif
within the PTB domain (such as in the case of 14-3-3 binding) which is
responsible for binding to xROK
. It should also be pointed out that
there is no evidence that ROK
(33, 31, 35, 37) or
xROK
3 is phosphorylated on
tyrosine residues, nor is there any sequence motif in xROK
that
resembles a known PTB-binding motif (7, 8, 49, 50).
What is the functional significance of our findings? The identification
of xROK as an xIRS-1 binding protein raises the possibility that
xIRS-1 is a physiological substrate of xROK
. In this regard, xROK
may be a negative regulator of insulin signaling because serine/threonine phosphorylation of IRS-1 has often been implicated in
negative regulation of IRS-1 tyrosine phosphorylation and insulin signaling (51, 52). Alternatively, the PTB domain may serve a dual, but
sequential, function in linking both Xenopus insulin-like growth factor I receptor and xROK
. For example, the PTB domain may
mediate a transient interaction between xIRS-1 and the receptor (NPEpY
autophosphorylation site) which results in tyrosine phosphorylation of
xIRS-1. Tyrosine-phosphorylated xIRS-1 may in turn bind xROK
via
another PTB-mediated interaction resulting in modulation of xROK
kinase activity and/or subcellular localization. Studies are under way
to examine the role of full-length xROK
in insulin signaling.
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ACKNOWLEDGEMENT |
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We thank Cathy Cummings for technical assistance. We also thank J. A. Cooper for anti-xMAP kinase serum, J. C. Bell for anti-Myc antibodies, A. Hall for V14-RhoA cDNA, D. L. Turner for the CS2+MT vector, M. Wigler for v-Ras cDNA in pSP6 plasmid, and D. A. Melton for the Xenopus oocyte cDNA library.
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FOOTNOTES |
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* This study was supported by operating grants from the National Cancer Institute of Canada and the Cancer Research Society of Canada (to X. J. L.).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) AF037073.
Scholar of the Medical Research Council of Canada. To whom
correspondence should be addressed: Ottawa Civic Hospital Loeb Research
Institute, Ottawa Civic Hospital, 1053 Carling Ave., Ottawa, K1Y 4E9,
Canada. Tel.: 613-798-5555 (ext. 7752); Fax: 613-761-5411 or 761-5365;
E-mail: johne{at}civich.ottawa.on.ca.
1 The abbreviations used are: IRS-1, insulin receptor substrate-1; PH, pleckstrin homology; PTB, phosphotyrosine binding; SAIN, Shc and IRS-1 NPXpY binding domain; ROK, Rho-associated protein kinase; GVBD, germinal vesicle breakdown; PCR, polymerase chain reaction; MT, Myc tag; MBP, myelin basic protein; PAGE, polyacrylamide gel electrophoresis; MAP kinase, mitogen-activated protein kinase; CRD, cysteine-rich domain.
2 R. Booth and X. J. Liu, unpublished observations.
3 N. Ohan and X.-J. Liu, unpublished observations.
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REFERENCES |
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