From the Department of Histology and the
§ First Department of Surgery, Tohoku University School of
Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-77, Japan and the
¶ Department of Anatomy, Yamagata University School of Medicine,
Yamagata 990-23, Japan
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ABSTRACT |
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We report herein the cloning and characterization
of a novel class II phosphoinositide 3-kinase, termed PI3K-II, from
the cDNA library of regenerating rat liver. This cDNA encodes a
protein of 1505 amino acids with a calculated molecular mass of 170,972 Da. The amino acid sequence of PI3K-II
is highly similar to those of
class II PI 3-kinases, including murine Cpk-m/p170 and human HsC2-PI3K.
It contains a C2 domain at the C terminus but no recognizable protein
motifs at its N terminus. PI3K-II
displays a restricted substrate
specificity for PtdIns and PtdIns 4-P, but not for PtdIns 4,5-P2. By epitope tag immunocytochemistry, the
immunoreactivity for PI3K-II
is localized in the juxtanuclear Golgi
region at high levels and also in the plasma and nuclear membranes at
low levels. By Northern blot analysis and in situ
hybridization histochemistry, PI3K-II
mRNA expression is
confined to the liver throughout the development with much higher
expression in adult liver than in fetal liver. In addition, its
expression increases during liver regeneration after partial
hepatectomy with maximal expression after the growth period, suggesting
that PI3K-II
may function mainly in highly differentiated hepatic
cells.
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INTRODUCTION |
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Phosphoinositide 3-kinase (PI
3-kinase)1 catalyzes the
phosphorylation of phosphoinositides such as phosphatidylinositol
(PtdIns), PtdIns 4-P, and PtdIns 4,5-P2 at the D3 position
of the inositol ring to generate PtdIns 3-P, PtdIns 3,4-P2,
and PtdIns 3,4,5-P3, respectively. Although the
intracellular level of PtdIns 3-P is largely unaltered
following extracellular stimuli, the other two phosphoinositides are
almost absent in unstimulated cells but accumulate in response to a
variety of external agonists (1, 2). Since initial identification of
the molecular structure of PI 3-kinase as a form of heterodimer,
i.e. a complex composed of an 85-kDa regulatory subunit and
a 110-kDa catalytic subunit (3), cDNA cloning analyses have
revealed multiple catalytic subunits of this kinase family which can
now be classified into three classes on the basis of their structural
characteristics and in vitro lipid substrate specificity.
The class I PI 3-kinases, which have so far been reported to be
composed of p110,
,
, and
, catalyze the phosphorylation of
all PtdIns, PtdIns 4-P, and PtdIns 4,5-P2 (4-8). The class
II PI 3-kinases catalyze the phosphorylation of PtdIns and PtdIns 4-P,
but not PtdIns 4,5-P2, and contain a C2 domain at their C
termini. This class has so far been reported to be composed of two
species, HsC2-PI3K from human (9) and Cpk-m/p170 from mouse (10, 11),
both of which are homologous to PI3K_68D and Cpk from Drosophila (10,
12). The class III PI 3-kinase represents a human homologue of yeast PtdIns 3-kinase, Vps34, which is involved in trafficking of proteins from the Golgi to vacuoles. This class III PI 3-kinase catalyzes the
phosphorylation solely of PtdIns (13).
Earlier studies focused on the role of PI 3-kinases in growth factor-stimulated and tyrosine kinase receptor-mediated cascades (reviewed in Ref. 14), but recent studies support the involvement of this lipid kinase in inhibition of apoptosis, intracellular vesicle trafficking, and regulation of cytoskeletal functions in addition to mitogenic signaling (reviewed in Refs. 15 and 16). The liver may be an advantageous tissue to examine because of the following two facts: 1) the developing liver is relatively large, which makes it easy to obtain sufficient amounts of mRNA for molecular analysis, and 2) a recent gene knockout study has shown that the embryonic development of liver is severely retarded by mutation of hepatocyte growth factor whose effects on cellular functions mediated by the c-Met tyrosine kinase receptor includes induction of PI 3-kinase activity (17, 18). Moreover, the regenerating liver remnant after partial hepatectomy represents a typical model for active mitogenesis in the adult, and there has been evidence that PI 3-kinase activity is markedly enhanced in response to the hepatectomy (19). The present study addressed this issue and was undertaken to identify a novel liver-specific PI 3-kinase species by cDNA cloning from the cDNA library of regenerating rat liver. The molecular structure of a novel class II PI 3-kinase and its detailed biochemical characteristics are clarified in this report.
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EXPERIMENTAL PROCEDURES |
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Reverse Transcription-Polymerase Chain Reaction-- Regenerating liver was prepared from partially hepatectomized rats 24 h after the removal of two-thirds of liver mass according to the method of Higgins and Anderson (20). Total RNA was prepared by the guanidine method, and poly(A)+ RNA was isolated by chromatography on an oligo(dT)-cellulose column. First strand cDNA was prepared using First-Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech). Oligonucleotide primers corresponding to two regions of highly conserved amino acid sequences in PI kinase domains, (V/T)GDD(C/L)RQ (5'-CGGAATTCCGG(A/T/C)GA(T/C)GA(T/C)T(G/T)(T/C)CG(G/C)CA(G/A)GA-3') and HIDFGF (I/M) (5'-CGGAATTCAT(G/A)AA(G/A/T)CC(G/A)TC(G/A)AT(G/A)TG-3') were used for PCR amplification as sense and antisense primers. The 5' ends of both primers contained an EcoRI restriction site (underlined). PCR amplification was performed using AmpliTaq DNA polymerase as follows: 94 °C for 1 min, 42 °C for 1 min, and 72 °C for 2 min for 30 cycles. The reaction products of approximately 400 base pairs were then subcloned and sequenced.
cDNA Cloning-- Oligo(dT)-primed cDNA library from the regenerating liver of 49-day-old (P49) rat was constructed in the same way as described previously (21). Clones (approximately 2×106) derived from the cDNA library were screened by hybridization with the 400-base pair PCR products labeled with [32P]dCTP. A positive bacteriophage containing a 1.4-kb insert was isolated. The insert (pF3K4) was cloned into pBluescript, and the sequence was determined by the dideoxy chain termination method with a 377 DNA sequencer (Applied Biosystems) according to the instructions of the supplier. The missing 5' end was obtained by a rapid amplification of cDNA ends (RACE) using two gene-specific antisense primers, (5'-CGCACAGGGCCGGGTTCAGAGGAAGATG-3') and (5'-ACCGTGAAGCTCAGGTGGGAGTGGAGCC-3'), with the Marathon sense primer in PCR amplifications (Marathon cDNA Amplification Kit, CLONTECH). The missing 3' end was amplified using the gene-specific sense primer (5'-CTCGCACCATGAGAGAATCCGAGATCTG-3') and the antisense primer (5' -TGGAAGAATTCGCGGCCGCAG- 3') corresponding to the sequence of NotI (dT)18 primer which contained a NotI restriction site (Amersham Pharmacia Biotech). At least five overlapping RACE products were subcloned and completely sequenced as described above.
Expression and Preparation of a Novel Molecule and Assay for PI
3-Kinase Activity--
A NotI site was introduced by PCR at
the position of the initiation codon of a newly identified cDNA
using the primer (5'-GCGGCCGCCAAAAATGGCATACAATTGGC-3'). Resulting
NotI fragments were ligated to the same site of the expression vector, pSRE (pcDL-SR 296 in Ref. 22) as modified by
Sakane et al. (23). At the same time, FLAG marker peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (Kodak Co.), was fused upstream of the
initiation codon, ATG, of the cDNA for the newly identified molecule. FLAG-tagged molecule was recovered as described below from
COS-7 cells 3 days after the transfection by the DEAE-dextran method
(24). Cells were harvested and lysed in lysis buffer (20 mM
Tris, pH 8.0, 100 mM NaCl, 1 mM
MgCl2, 1 mM CaCl2, 10% glycerol,
1% Nonidet P-40, 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 20 µg/ml
leupeptin, 50 µg/ml soybean trypsin inhibitor). The lysate was
clarified by centrifugation (16,000 × g, 15 min), and
its protein concentration was determined by the method of Lowry
et al. (25). The supernatant was incubated with anti-FLAG
antibody-agarose beads (approximately 20 µl of beads/1 mg of lysate)
for 90 min. The resulting beads were washed repeatedly and resuspended
in assay buffer (20 mM Tris, pH 7.5, 100 mM
NaCl, 3.5 mM MgCl2). A volume of 10 µl of the
beads recovered from 100 µg of the transfected cell lysates was then
used for PI kinase assay. After 10 min of incubation with lipid
substrates (200 µM each of PtdIns, PtdIns 4-P, and PtdIns
4,5-P2) mixed with an equal amount of phosphatidylserine,
the reaction was carried out for an additional 10 min in the presence
of 40 µM ATP containing 12.5 µCi of
[
-32P]ATP. Various concentrations of wortmannin or
detergents (Triton X-100, Nonidet P-40, or CHAPS) were added to the
reaction buffer. Alternatively, 3.5 mM Mn2+ was
added instead of Mg2+. Reaction products were extracted and
resolved by thin layer chromatography (TLC) using Silica Gel 60 plates
(Whatman). For determination of the phosphorylated position on the
inositol ring, reaction products were resolved using TLC with a borate
buffer system as described by Walsh et al. (26). As
reference, [
-32P]PtdIns 3-P was produced by
phosphorylating PtdIns with A431 cell lysate, which had been reported
to contain a high level of PI 3-kinase activity (27).
[
-32P]PtdIns 4-P was produced by phosphorylating
PtdIns with the immunoprecipitates of FLAG-tagged 92-kDa PtdIns
4-kinase that we isolated from rat brain (28). For separation of PtdIns
3-P, PtdIns 3,4-P2, and PtdIns 3,4,5-P3, the
products were resolved by TLC in a buffer consisting of
chloroform/acetone/methanol/acetic acid/water (80/30/26/24/14).
RNA Extraction and Northern Blot Analysis-- Poly(A)+ RNAs were purified from liver, testis, brain, heart, lung, and spleen of the P49 rat, livers of prenatal day 18 (E18) rat, and from regenerating livers of P49 rats on the 1st, 2nd, and 3rd day after partial hepatectomy (PH1, PH2, and PH3, respectively) as described above. Each of the poly(A)+ RNA samples (2 or 5 µg) was denatured with formamide and size-separated by agarose gel electrophoresis. The RNAs were transferred and fixed to a nylon membrane (Nytran, Schleicher & Schuell) and hybridized with a [32P]dCTP-labeled probe prepared from a 6-kb NotI restriction fragment of a novel cDNA. Conditions for hybridization and washing were performed as described previously (21). The intensity of each band was measured by a Bio-Image Analyzer (Fuji Co.).
In Situ Hybridization Histochemistry--
Fresh frozen blocks of
livers and brains from adult (P49) and 7-day-old (P7) rats, whole
bodies of fetal (E15 and E18) rats, and regenerating livers of P49 rats
after partial hepatectomy (PH1, PH2, and PH3) were sectioned at 30 µm
thickness on a cryostat. The sections were hybridized for
16 h at 42 °C with [35S]dATP-labeled
oligonucleotides (5'-GGCTCACTCTGTAGTGTCATGCTGAGAAGCCTAGACCCCAGCGGA-3' (nucleotides 1944-1988)). After hybridization, the sections were rinsed twice in 2× SSC, 0.1% Sarkosyl at 42 °C for 20 min, three times in 0.1× SSC, 0.1% Sarkosyl at 42 °C for 1 h, and
dehydrated in 70 and 100% ethanol. For comparison in relative
hybridization intensity, targeted tissue sections were all mounted on
one glass slide and exposed to a Hyperfilm- max (Amersham Pharmacia
Biotech) for 3 weeks.
Immunocytochemistry and Immunoblotting-- The tissues and cells were fixed with 4% paraformaldehyde, 0.01% Triton X-100 and were incubated with the anti-FLAG antibodies (Anti-FLAG M2, Kodak). Sites of antigen-antibody reaction were visualized using the avidin-biotin complex system (Vector Laboratories) with diaminobentidine as a substrate. In the immunoblotting, the lysates of overexpressed cells were collected by centrifugation (550 × g, 10 min), and the supernatant was further centrifuged at 100,000 × g for 30 min to separate soluble and particulate fractions. The proteins of both fractions were subjected to SDS-7.5% polyacrylamide gel electrophoresis and then transferred to a polyvinylidine difluoride membrane. The membrane was incubated for 2 h at room temperature with anti-FLAG antibodies and treated with peroxidase-conjugated anti-mouse IgG antibodies for 1 h.
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RESULTS |
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The deduced amino acid sequences of the composite cDNA of the
novel molecule are presented in Fig. 1.
The putative initiation codon was preceded by in-frame stop codon at
nucleotide 57. The predicted open reading frame encoded a protein of
1505 amino acids with a calculated molecular mass of 170,972 Da. When
the sequences of the extreme 3' and 5' ends of the composite cDNA
were used in the PCR amplification, a full-length cDNA of the same
size was amplified, and its sequence was identical to that of the
composite cDNA. The deduced amino acid sequence of the novel
molecule contained a lipid kinase unique domain, a putative catalytic
domain, and a C2 domain as found previously for murine Cpk-m/p170 and
human HsC2-PI3K (9-11). The novel molecule showed 48, 42, and 30%
identities to HsC2-PI3K, Cpk-m/p170, and p110
in the lipid kinase
unique domain and 59, 57, and 42% identities to the three individual kinases in the catalytic domain, respectively. The C2 domain of the
novel molecule was located at the C terminus, and it was 40 and 32%
identical to the same domain of HsC2-PI3K and Cpk-m/p170, respectively.
The N terminus of the novel molecule did not contain any recognizable
protein motifs (Fig. 2).
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In Northern blot analysis of several tissues of P49 rat, a distinct hybridization band of 7 kb was detected only in the liver. A smaller transcript of approximately 2.5 kb was detected at a low level in the heart and testis, which was considered to be a result of alternative splicing. No significant hybridization band was discerned in the brain, lung, spleen, or kidney (Fig. 3A). The expression was detectable, though weakly, in E18 liver, and the expression level of P49 liver was approximately 10-fold higher than that of E18 liver (Fig. 3B). During liver regeneration after partial hepatectomy, the mRNA expression showed a pattern of time-dependent gradual increase, and it increased markedly on the 3rd day after hepatectomy (PH3) (Fig. 3C). The expression level of PH3 was superficially 3-fold higher and 1.6-fold higher than that of non-operated control (PH0), even after normalizing it to GAPDH mRNA expression, although a previous study has shown a slight increase in GAPDH mRNA during liver regeneration after hepatectomy (29), which was also confirmed by us.
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By in situ hybridization histochemistry of the whole body of embryos on E15 and E18 and of liver and brain on P7 and P49, the expression signals for this novel molecule were detected only in the liver, and no significant signals were detected in any other tissues of embryos and the postnatal brain (Fig. 4).
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Detection of the lipid kinase activity of the novel molecule was performed using immunoprecipitates from lysates of COS-7 cells transfected with the corresponding epitope-tagged cDNA. A single band at an approximate size of 190 kDa was visualized by silver staining on SDS-polyacrylamide gel (Fig. 5A). A single immunoreactive band of the same size was detected at almost equal intensities in both soluble and particulate fractions (Fig. 5B). In an assay for lipid kinase activity using PtdIns as a substrate, the selective production of [32P]PtdIns 3-P, but not [32P]PtdIns 4-P, was clearly revealed, compared with immunoprecipitates from lysates of cells transfected with cDNAs for 92-kDa PtdIns 4-kinase (28) as well as lysates of A431 cells, which are known to have high intrinsic activities for both PtdIns 3-kinase and PtdIns 4-kinase (27) (Fig. 5C). When PtdIns 4-P and PtdIns 4,5-P2 were used as substrates, the formation of [32P]PtdIns 3,4-P2 was detected, whereas no formation of [32P]PtdIns 3,4,5-P3 was discerned (Fig. 5D). The kinase activity was inhibited by wortmannin with an IC50 of 12 nM, whereas the PI 3-kinase activity of A431 cell lysates was inhibited with an IC50 of 5 nM under the same conditions. The kinase activity was also inhibited almost completely by addition of 0.5% Triton X-100, Nonidet P-40 or CHAPS, or in the presence of 3.5 mM Mn2+ instead of Mg2+ (data not shown).
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When COS-7 cells were transfected with the epitope (FLAG)-tagged cDNA for the novel molecule and immunostained for the FLAG-tag, immunoreactive cells accounted for about 20% of the total cell populations and appeared randomly dispersed in each culture dish. The immunoreactive products were densely aggregated in juxtaposition to the cell nuclei. In addition, the contours of the cell nuclei and cell boundary appeared clearly delineated (Fig. 6). When the transfection was made with a cDNA without the tag, no immunoreactivity was detected (data not shown).
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DISCUSSION |
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Judging from the presence of a C2 domain in its molecular
structure as well as its substrate specificity for PtdIns and PtdIns 4-P in vitro and inhibition of its kinase activity by
wortmannin and Mn2+, it is clear that this novel molecule
represents the third species of class II PI 3-kinase in vertebrates.
But it is the first vertebrate species whose biochemical
characteristics and localization in tissues and cells have been
revealed in detail (9-11). The novel molecule is thus termed as
PI3K-II, and we propose to name the previous molecules, murine
cpk-m/p170 and human HsC2-PI3K, as PI3K-II
and -II
, respectively.
Among several short cDNA clones reported in a recent study of novel
PI 3-kinases, a partial amino acid sequence of one of their clones is
found to be a presumed human homologue of PI3K-II
(30).
The appearance of PI3K-II in both supernatant and particulate
fractions, together with the absence of any transmembrane domains in
its deduced amino acid sequence, suggests that translocation of
PI3K-II
occurs between the two intracellular components although the
molecular mechanism of the translocation remains to be determined. The
present immunohistochemical analysis of COS-7 cells overexpressed with
PI3K-II
shows that the immunoreactivity for PI3K-II
is localized
in the juxtanuclear Golgi region with the highest intensity and,
furthermore, in the nuclear and plasma membranes at low levels. Whether
or not the localization of the immunoreactivity in the juxtanuclear
Golgi region by light microscopy represents the localization of this
molecule in the Golgi apparatus itself or in both the Golgi and
adjacent endoplasmic reticulum requires further double immunocytochemical analysis using specific antisera against the two
organelles. It will also be necessary to use a specific antibody against PI3K-II
to examine whether the present immunohistochemical finding represents the real localization or reflects the artificially high production of PI3K-II
protein in such cells.
The present study reveals that the gene expression of PI3K-II is
confined to the liver at both prenatal and postnatal stages, with the
latter at a much higher level. In addition, its expression increases
during liver regeneration after partial hepatectomy in a
time-dependent manner. Haber et. al. has
recently defined three chronological patterns of expression for various
genes in the regenerating liver after hepatectomy. The first pattern
parallels the major growth period of the liver that ends at 60-72 h
after hepatectomy. The second has two peaks coincident with the first and second G1 phases of the two hepatic cell cycles. In the
third pattern, the expression level reaches the maximum at 72 h,
which is maintained for a substantial length thereafter (31). The induction pattern of PI3K-II
mRNA after partial hepatectomy is similar to the third pattern. These results may suggest that PI3K-II
is not involved in the major mitogenic signaling pathway but is involved in other pathways responsible for some yet undefined liver-specific matured functions, in contrast to the expectation mentioned in the introduction. C/EBP
(CCAAT/enhancer binding protein
) represents one of the molecules exhibiting the third pattern of
the gene expression in the regenerating liver and is expressed late in
gestation and highly in the nongrowing normal adult liver (32, 33).
C/EBP
has also been shown to activate the transcription of several
tissue-specific genes such as insulin-responsive glucose transporter 4 (GLUT4) and phosphoenolpyruvate carboxykinase (PEPCK) in a coordinated
fashion (34, 35). The functional relation of PI3K-II
to C/EBP
in
the liver remains to be evaluated. With the regard to the possible
functional significance of PI3K-II
in matured cells, it should be
noted that the axonal crush was shown in our recent study to induce the
enhanced gene expression of a class I PI 3-kinase in hypoglossal
motoneurones which represent the non-mitotic cell population (36). We
have also shown in crushed/axotomized motoneurones the enhanced gene
expression of a serine/threonine protein kinase, Akt/PKB, which is
activated by PtdIns 3,4-P2, but not by PtdIns
3,4,5-P3. PtdIns 3,4-P2 is a direct product of
the class II PI 3-kinase from PtdIns 4-P although it is also a product
of the hydrolysis at the D-5 position of PtdIns
3,4,5-P3 which is produced by class-I PI 3-kinase (37). To
understand the physiological role of PI3K-II
in non-mitotic signaling in hepatic cells, further studies will be needed such as a
search for agonists that may induce enhanced activation of PI3K-II
and for proteins that may modulate the action of this molecule.
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ACKNOWLEDGEMENT |
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We thank Prof. J. A. Glomset (Univ. Washington, Seattle, WA) for helpful advice and suggestions.
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FOOTNOTES |
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* This study was supported by Grants 09044248, 09470001, and 09259203 from the Ministry of Education, Science and Culture of Japan (to H. K.) and by grants from Asaoka Eye Clinic Foundation, Hamamatsu, and JCR Pharmaceutical Co., Ashiya, Japan.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) AB009636.
To whom correspondence should be addressed: Dept. of
Histology, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-77, Japan. Tel.: 81-22-717-8033; Fax:
81-22-717-8035; E-mail: hkondo{at}mail.cc.tohoku.ac.jp.
1
The abbreviations used are: PI,
phosphoinositide; PtdIns, phosphatidylinositol; PCR, polymerase chain
reaction; RACE, rapid amplification of cDNA ends; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; TLC,
thin layer chromatography, P7, 7-day-old; P49, 49-day-old; E15,
prenatal day 15; E18, prenatal day 18; C/EBP, CCAAT/enhancer binding
protein
; kb, kilo base(s); PH1, PH2, and PH3, 1, 2, and 3 days
after partial hepatectomy, respectively.
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REFERENCES |
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