(Received for publication, November 27, 1996)
From the Third Department of Internal Medicine, Phosphatidylinositol 3-kinase (PI 3-kinase) is
stimulated by association with a variety of tyrosine kinase receptors
and intracellular tyrosine-phosphorylated substrates. We isolated a
cDNA that encodes a 50-kDa regulatory subunit of PI 3-kinase with
an expression cloning method using 32P-labeled
insulin receptor substrate-1 (IRS-1). This 50-kDa protein contains two
SH2 domains and an inter-SH2 domain of p85 A variety of growth factors and hormones mediate their cellular
effects via interactions with cell surface receptors that possess
protein kinase activity (1, 2). The interaction of most of these
ligands with their receptors induces tyrosine kinase activation and
autophosphorylation of the receptor, resulting in physical association
of these receptors with several cytoplasmic substrates having SH2
domains. Phosphatidylinositol 3-kinase (PI 3-kinase)1 has been identified through its
ability to associate with cellular protein kinases, including numerous
growth factor receptors and oncogene products (3, 4). This lipid kinase
phosphorylates phosphatidylinositol at the D-3 position of the inositol
ring in response to stimulation with a variety of growth factors and hormones (5). Although the role of this lipid product in cellular regulation remains unclear, recent reports suggest that the activation of PI 3-kinase leads to the activation of c-Akt, Rac, PKC- PI-3 kinase is composed of a catalytic 110-kDa protein (p110)
associated with a regulatory subunit (17-19). The regulatory subunit
contains two proline-rich motifs, two Src homology-2 (SH2) domains, and
a domain responsible for the binding with p110 between the two SH2
domains (3, 4). Many activated receptors with tyrosine kinase activity
interact with the SH2 domain in the regulatory subunit through
phosphorylated YXXM motifs in the receptors themselves (20),
resulting in the activation or recruitment of PI 3-kinase (21). To
date, four regulatory subunits of PI 3-kinase have been identified, two
85-kDa proteins (p85 In total, five regulatory subunits for PI 3-kinase have been identified
in mammalian cells to date, including two 85-kDa proteins, two 55-kDa
proteins, and one 50-kDa protein. In this study, we demonstrated the
tissue distributions and different roles in PI 3-kinase activation, via
insulin stimulation, of these subunits. Our data suggest that these
five regulatory subunits may have different roles in the various
responses induced by the numerous growth factors, hormones, and
oncogene products with which they interact.
The recombinant human IRS-1 protein
was prepared as described previously (24). The insulin receptor was
partially purified from human placenta on wheat germ agglutinin-agarose
as described previously (25). The 32P-IRS-1 probe was
prepared by incubating IRS-1 with activated insulin receptor in the
presence of Mn2+ and [ Northern blotting was performed using a
commercially available sheet made by Clontech (Palo Alto, CA).
Nucleotides A specific antibody against p50 The cDNAs encoding the full-length amino acid sequences
of p85 Tissues
from male Wistar rats (6-8 weeks old) were homogenized in ice-cold
lysis buffer (1/10 w/v) containing 50 mM Hepes (pH 7.5),
137 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 2 mM EDTA, 1% Nonidet P-40, 10% glycerol, 2 mg/ml aprotinin, and 34 mg/ml
phenylmethylsulfonyl fluoride. Insoluble material was removed by
centrifugation at 14,000 × g for 60 min and incubated
for 2 h at 4 °C with the antibody against a whole molecule of
p85 Twenty
rats were killed by cervical dislocation, and the brains were removed
and separated into olfactory bulbs, cerebral cortex (frontal), cerebral
cortex (temporal), cerebral cortex (occipital), superior colliculus,
inferior colliculus, caudate putamen, thalamus, hippocampus,
cerebellum, pons and medulla oblongata. Total RNA was isolated from
each part of the brain with Isogen (Nippon Gene, Japan).
[ An antisense RNA probe was prepared by in
vitro transcription from the fragment of p50 Male Wistar rats, 4 weeks of age (supplied by the Animal Breeding
Facility, Gunma University), were anesthetized with an intraperitoneal injection of sodium pentobarbital and perfused through the aorta with
an isotonic sodium chloride solution to remove blood, followed by 4%
paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Brains were then removed, washed with phosphate-buffered saline, pH 7.4, dehydrated through graded alcohols, and embedded in paraplast wax.
Serial sections (10 µM thick) were cut, mounted on
poly-L-lysine-coated slides, and stored at room temperature
until use.
After de-waxing and rehydration, tissue sections were digested with 5 µg/ml proteinase K at room temperature for 20 min. They were then
fixed in 0.4% paraformaldehyde at 4 °C for 10 min and incubated at
50 °C overnight with a hybridization solution containing 1 µg/ml
DIG-labeled cRNA, 50% formamide, 10 mM Tris-HCl, pH 7.5, 300 mM NaCl, 1 mM EDTA, 0.25% SDS, 1 × Denhardt's solution, 200 µg/ml yeast tRNA, and 10% dextran sulfate.
Following hybridization, the sections were washed in 2 × SSC,
50% formamide at 58 °C for 30 min, incubated in 1 µg/ml RNase A
solution at 37 °C for 30 min, and washed once in 2 × SSC and
twice in 0.2 × SSC at 50 °C for 20 min each time. The sections
were then incubated in a 1:500 diluted solution of polyclonal sheep
anti-DIG Fab antibody conjugated with alkaline phosphatase, before
washing and detection of the label with nitro blue tetrazolium chloride
and 5-bromo-4-chloro-3-indolylphosphate. Color development was carried
out at room temperature for 14 h, and the sections were then
washed in TE buffer (10 mM Tris-HCl, 1 mM EDTA,
pH 7.5). To inhibit further color reaction, the slides were mounted in
a mixture containing 24% polyvinyl alcohol, 12% glycerol, and 59 mM Tris-HCl, pH 7.5.
The
insoluble materials prepared from rat liver and brain (30 ml, 20 and 8 mg/ml, respectively) were directly loaded onto a DEAE-Sepharose Fast
Flow column (200 ml). The column was then washed with buffer B,
consisting of 20 mM Tris-HCl, pH 7.4, 1 mM
EDTA, 1 mM dithiothreitol, 1 µM leupeptin,
0.5 mM PMSF, and 10% glycerol. Once the absorbance eluted
at 280 nm had returned to base line, the column was eluted with a
gradient of KCl up to 0.5 M. Aliquots of the individual
fractions were assayed for PI 3-kinase activity. The pooled fractions
from the peaks (three fractions from liver chromatography, fractions A,
B, and C, and one fraction from brain chromatography, fraction D) were
concentrated 10-fold with Centriprep 10 (Amicon, MA) and subjected to
immunoblotting analysis. Concentrated fractions A and B (1 ml) were
also applied to the gel filtration column (Sephacryl S300 column,
Pharmacia Biotech Inc.), previously equilibrated with buffer B, and
eluted fractions (1 ml) were collected and assayed for PI 3-kinase
activity. Molecular weight standards were run under the same conditions as samples.
Eluted fractions from the chromatography
column or the lysates from the cells were incubated with the
appropriate antisera for 2 h at 4 °C. Protein A-Sepharose beads
were used to precipitate the immune complexes. The presence of PI-3
kinase activity in immunocomplexes was determined as described
previously (24).
All concentrated
fractions from the peaks were immunoprecipitated with the appropriate
antibodies and pelleted using protein A-Sepharose beads. The beads were
washed three times with lysis buffer A. The beads were then incubated
at 25 °C for 30 min with 20 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, and 0.1 µM
wortmannin and washed again three times with lysis buffer A. Wortmannin-labeled immunoprecipitates were separated from the beads by
boiling in Laemmli buffer. The beads were removed by centrifugation,
and the supernatants were subjected to SDS-PAGE. The wortmannin was detected by enhanced chemiluminescence using anti-wortmannin antibody, generously provided by Dr. Yuzuru Matsuda (Kyowa Hakko Co., Japan), and
horseradish peroxidase-labeled anti-rat IgG (Amersham Corp.).
PC12 and HepG2 cells were grown in Dulbecco's
modified Eagle's medium with 10% fetal bovine serum. CHO cells were
transfected with the expression vector pCAGGS containing the
full-length human insulin receptor or human IRS-1 cDNA. CHO cells
stably overexpressing human insulin receptor (CHO/IR) or human IRS-1
(CHO/IRS-1) were obtained after selection with G-418 (400 µg/ml) and
grown in Ham's F12 medium with 10% fetal bovine serum. Prior to
insulin treatment, the medium was replaced with serum-free medium
containing 0.1% bovine serum albumin, and incubation was continued for
18 h. Various concentrations of insulin were added to the medium,
and the cells were incubated for 5 min at 37 °C. After insulin
treatment, the medium was removed, and cells were collected with lysis
buffer C, consisting of 50 mM Hepes, pH 7.5, 137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 2 mM
Na3VO4, 10 mM sodium pyrophosphate,
10 mM NaF, 2 mM EDTA, 1% Nonidet P-40, 10%
glycerol, 2 µg/ml aprotinin, 5 µg/ml leupeptin, 0.5 µM pepstatin A, and 34 µg/ml PMSF. Lysates were
centrifuged at 10,000 × g for 10 min. The resulting
supernatants were assayed for PI 3-kinase activity.
The cassette cosmid for
constructing recombinant adenovirus, pAdex1wt, was the generous gift of
Dr. Izumi Saito (Institute of Medical Science, University of Tokyo).
The cDNAs encoding the full-length amino acid sequences of p85 HepG2 cells were
infected with an adenovirus expressing the full-length cDNA of
p85 A novel form of the regulatory subunit of PI 3-kinase was isolated
from a rat liver cDNA expression library. A rat liver cDNA expression library was screened with 32P-labeled
recombinant IRS-1, and 47 positive independent clones were isolated
after three or four rounds of screening. These clones included
cDNAs containing complete coding regions of p85
The five cDNAs coding for
p85 The levels of p50
Although the role of PI 3-kinase in brain and neural cells remains
unclear, all five known isoforms are abundantly expressed in brain
tissue (Fig. 2B) (22). We further investigated their distributions in various portions of the rat brain using an RNase protection assay. As shown in Fig. 3E, p50
To determine the expression of p85
As shown by the immunoblot using the specific antibody against p85 Kurosu et al. (29) reported the presence of a
46-/100-kDa heterodimer form of a PI 3-kinase, in rat liver, which was
isolated in the flow-through fraction of a DEAE-Sepharose column. In
addition, this 46-kDa protein was reported to readily be recognized by
the antibody against the whole p85 As shown in Fig. 6A, the p50
The role of insulin in the induction of
the PI 3-kinase activities associated with the various regulatory
subunits was evaluated using PC12 and HepG2 cells. PC12 cells express
all five regulatory subunit isoforms, but HepG2 cells express only
p85
The cDNA
construct for each isoform having the HA tag at its COOH terminus was
prepared, and the adenoviruses for the transient expression of these
isoforms were produced. HepG2 cells and CHO cells, expressing insulin
receptors (CHO/IR) or IRS-1(CHO/IRS-1), were infected with these
adenoviruses to achieve similar protein expression levels, as assessed
by the immunoblot using anti-HA antibody (data not shown). In HepG2
cells, the insulin stimulation induced a marked increase (25.2-fold) in
PI 3-kinase activity associated with p50
To elucidate the mechanisms accounting for the variability in the
extent of PI 3-kinase activation associated with the various regulatory
proteins, we investigated the amount of IRS-1 bound to the expressed
regulatory subunits in response to insulin. Before and after insulin
stimulation, the cells were lysed and immunoprecipitated with the
anti-HA antibody. The expressed regulatory subunit proteins of the
indicated molecular mass were observed (Fig. 9B), and
[35S]methionine-labeled IRS-1 proteins (approximately 180 kDa) associated with each of the five regulatory subunits were
measured, and the ratios of bound IRS-1/the amount of regulatory
subunit expressed were calculated for each of the isoforms (Fig.
9C). p50 Similar results were obtained in experiments using CHO/IR cells or
CHO/IRS-1 cells (Fig. 10, A and
B). The p50
In a study utilizing the expression cloning method with
32P-labeled IRS-1, we previously isolated the four
regulatory subunit isoforms for PI 3-kinase from a cDNA library
prepared from rat brain (22). In this study, we screened a cDNA
expression library prepared from rat liver by the same method and
isolated cDNA encoding a novel protein, p50 One of the two 55-kDa proteins and the 50-kDa protein were revealed to
share the two SH2 domains and an inter-SH2 domain with p85 Northern blotting using the cDNA probe coding for the N-SH2 domain
of p85 As shown in our previous report (22) and in this study, all regulatory
subunit isoforms for PI 3-kinase are abundantly expressed in brain
tissue. Although there is one report describing the important role of
PI 3-kinase in neuronal differentiation (14), no study has demonstrated
a PI 3-kinase function in neuronal cells after differentiation. The
yeast homolog of PI 3-kinase, VPS34, is required for trafficking of
proteins from the Golgi apparatus to vacuoles (33, 34). In addition,
the activation of PI 3-kinase has been implicated in histamine release
(35). Thus, PI 3-kinase may play a major role in the secretion of
various neurotransmitters. The data on the distribution of each
regulatory isoform in rat brain differed among the isoforms, possibly
offering clues as to the mechanisms regulating neurotransmitter
secretion.
There has been only one report describing a regulatory subunit with a
molecular size of approximately 50 kDa, which was recognized by the
antibody against the entire p85 It is now clear that at least four different types of growth
factor-regulated PI 3-kinase exist, including mammalian homologs of
Saccharomyces cerevisiae VPS34 (33), a G-protein-activated form termed p110 We also investigated the levels of the PI 3-kinase activities
associated with each of the five isoforms. First, the activation of
endogenous PI 3-kinase by insulin was measured using the appropriate specific antibodies in PC12 cells and HepG2 cells. In addition, these
five regulatory subunits were expressed in HepG2 cells, CHO/IR cells,
and CHO/IRS-1 cells, and the extent of activation of their associated
PI 3-kinases was compared. The results of a series of PI 3-kinase
assays can be summarized as follows; p50 In summary, there are five regulatory subunit isoforms of PI 3-kinase
which can be classified into three groups, an 85-kDa protein, a 55-kDa
protein, and a 50-kDa protein. Each isoform has a different tissue
distribution and was shown to exhibit a different level of activation,
of the associated PI 3-kinase, in response to insulin stimulation.
Given the idea that PI 3-kinase is involved in a series of systems, it
is conceivable that PI 3-kinase plays a variety of roles in response to
various stimuli. Further study is required to ascertain which isoform
corresponds to which biological phenomenon.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) D78486[GenBank]. We thank Dr. I. Saito and Y. Kanegae for
helpful advice and generous gifts of the recombinant Adex1CAlacZ, the
expression cosmid cassette, and the parental adenovirus DNA-terminal
protein complex. We also thank Dr. Yuzumi Matsuda, for the generous
gift of wortmannin antibody.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgment
REFERENCES
, but the SH3 and
bcr homology domains of p85
were replaced by a unique 6-amino acid sequence. Thus, this protein appears to be generated by
alternative splicing of the p85
gene product. We suggest that this
protein be called p50
. Northern blotting using a specific DNA probe
corresponding to p50
revealed 6.0- and 2.8-kb bands in hepatic,
brain, and renal tissues. The expression of p50
protein and its
associated PI 3-kinase were detected in lysates prepared from the
liver, brain, and muscle using a specific antibody against p50
.
Taken together, these observations indicate that the p85
gene
actually generates three protein products of 85, 55, and 50 kDa. The
distributions of the three proteins (p85
, p55
, and p50
), in
various rat tissues and also in various brain compartments, were found
to be different. Interestingly, p50
forms a heterodimer with p110
that can as well as cannot be labeled with wortmannin, whereas p85
and p55
associate only with p110 that can be wortmannin-labeled. Furthermore, p50
exhibits a markedly higher capacity for activation of associated PI 3-kinase via insulin stimulation and has a higher affinity for tyrosine-phosphorylated IRS-1 than the other isoforms. Considering the high level of p50
expression in the liver and its
marked responsiveness to insulin, p50
appears to play an important
role in the activation of hepatic PI 3-kinase. Each of the three
isoforms has a different function and may have specific roles in
various tissues.
isoform, and p70 S6 kinase (6-9). As a result, PI 3-kinase has been suggested to play essential roles in the regulation of various cellular activities, including proliferation (10, 11), differentiation (12),
membrane ruffling (13), prevention of apoptosis (14), and
insulin-stimulated glucose transport (10, 15, 16).
, p85
) and two 55-kDa proteins
(p55
/p85/AS53, p55
/p55PIK) (22-24). The two recently
cloned 55-kDa regulatory subunits, p55
and p55
, are unique
because the SH3 and bcr homology domains found in p85s are
replaced by a unique 34-amino acid residue NH2 terminus. In
this study, we screened a rat liver cDNA library using a
32P-labeled human IRS-1 protein and obtained a cDNA
that encodes a novel 50-kDa regulatory subunit for PI 3-kinase.
Sequence analysis of the cDNA revealed that this protein consists
of a unique 6- amino acid sequence at its NH2 terminus, as
well as two SH2 domains and an inter-SH2 domain of p85
. Neither the
SH3 and bcr homology domains, of the p85 regulatory subunit,
nor the unique 34-amino acid residue, of the p55 regulatory subunit,
were found in this 50-kDa protein. These sequence data indicate that
this 50-kDa protein is generated by alternative splicing of the p85
gene product. We suggest that this protein be called p50
.
Expression Screening of Rat Liver cDNA Library with Human
32P-IRS-1 Protein
-32P]ATP (24). An
oligo(dT)-primed rat liver cDNA library was prepared in UNI-ZAP XR
(Stratagene) according to the manufacturer's instructions. Sixty 15-cm
plates representing 3,000,000 independent plaques were plated and
incubated for 7 h at 37 °C. Then the plates were overlaid with
nitrocellulose filters that had been impregnated with 10 mM
isopropyl-
-D-thiogalactopyranoside and incubated for 8 h at 37 °C. The hybridization of the filters with
32P-IRS-1 probe and washing were performed as described
previously (24). The cDNA inserts in pBluescript were prepared by
in vivo excision according to the manufacturer's
instructions (Stratagene). The nucleotide sequences were determined
using an ABI automatic sequencer.
170-18 of p50
were labeled with
[
-32P]dCTP and used as probes. The filter was
hybridized and washed according to the manufacturer's instructions
(Clontech). Autoradiography was performed at
80 °C for 24-48
h.
(
p50
)
was prepared by immunizing rabbits with a 10-amino acid synthetic
peptide identical to the unique NH2-terminal region of
p50
(MHNLQTLPPK, amino acid residues 1-10). An anti-p85
specific
antibody (
p85
SH3) was prepared by immunizing rabbits
with a synthetic peptide identical to the unique SH3 region of p85
(YPFRRERPEDLELLPGDLLVVSRVALQALGVA, amino acid residues 13-44). These
peptides were coupled to keyhole limpet hemocyanin and inoculated into
rabbits. These anti sera were affinity purified with Affi-Gel 10 covalently coupled to the peptides (26). The anti-p85
specific
antibody (
p85
SH3), the anti-p55
specific antibody
(
p55
), and the anti-p55
specific antibody (
p55
) were
prepared as described previously (22). The antibody against the whole
p85
molecule (
p85PAN-UBI) was purchased from UBI.
, p85
, p55
, p55
, and p50
, as well as the HA tag
amino acid sequence (YPYDVPDYA) at each COOH terminus were subcloned
into pBacPAK9, and the baculoviruses were prepared according to the manufacturer's instructions (Clontech). The Sf9 cells, infected with a
baculovirus containing one of the five isoforms, were cultured for
48 h and then lysed in Laemmli buffer. The samples were subjected to SDS-PAGE, and immunoblotting was performed using the antibody against the HA tag, or specific antibodies, as described previously (26).
, p55
, and
p50
) of PI 3-Kinase Expressed in Various Rat Tissues
(
p85PAN-UBI) covalently coupled to protein
A-Sepharose beads purchased from UBI. The beads were washed three times
in lysis buffer A, consisting of 10 mM Tris-HCl (pH 7.8),
1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, and
0.5 mM PMSF, and separated from the beads by boiling in
Laemmli buffer. The beads were removed by centrifugation, and the
supernatants were subjected to SDS-PAGE, and immunoblotting was
performed as described previously (26). To detect the PI 3-kinase
activities, the immunoprecipitations were performed using the
appropriate antibodies, and the PI 3-kinase activities in each
immunoprecipitate were measured.
-32P]UTP-labeled RNA probes were prepared using the
nucleotides 1-350 of p85
, 1-340 of p85
, 96-354 of p55
,
66-102 of p55
, and
170-18 of p50
as templates. An RNase
protection assay was performed using an RPA IITM kit (Ambion, TX)
according to the manufacturer's instructions.
cRNA Probes and in Situ Hybridization
Histochemistry
cDNA that had
been used for Northern blot analysis, using T7 RNA polymerase, in the
presence of 350 mM digoxigenin (DIG)-linked UTP in a
20-µl reaction mixture, according to the manufacturer's instructions
(Boehringer Mannheim). A sense probe was similarly obtained with T3 RNA
polymerase.
,
p85
, p55
, p55
, and p50
, as well as the HA tag amino acid
sequence (YPYDVPDYA) at each COOH terminus, were ligated into the
SwaI sites of pAdex1wt. Recombinant adenoviruses were
obtained as described previously (27). HepG2, CHO/IR, and CHO/IRS-1
cells were infected with these viruses for 1 h and then grown for
48 h. The expression of each protein was confirmed by
immnoblotting wih anti-HA antibody.
, p85
, p55
, p55
, or p50
, p85
, p55
, p55
, or p50
and grown for 48 h.
Prior to insulin treatment, the medium was replaced with
methionine-free RPMI medium, containing 0.5% bovine serum albumin and
0.1 mCi/ml Tran35S-label (ICN) and incubated for 18 h.
Then, 10
6 M insulin was added to the medium,
and cells were incubated for 5 min at 37 °C. After insulin
treatment, the medium was removed, and the cells were collected with
lysis buffer C. Lysates were centrifuged at 10,000 × g
for 10 min. The resulting supernatants were immunoprecipitated with the
anti-HA antibody and then pelleted with protein G-Sepharose beads.
Immunoprecipitates were separated from the beads by boiling in Laemmli
buffer. The beads were removed by centrifugation, and the supernatants
were electrophoresed on 7.5% SDS-polyacrylamide gels. The gels were
dried and subjected to autoradiography.
and p85
, the
nucleotide sequences of which had previously been determined (22). In
addition, we obtained three independent cDNAs containing the
nucleotide sequence coding the NH2-terminal SH2 domain of p85
and a previously undocumented 190-nucleotide sequence at its
5
-upstream side. These cDNAs contained an open reading frame of
1275 nucleotides, and the deduced amino acid sequence is shown in Fig.
1A. The presence of this mRNA in rat
liver was confirmed by reverse transcription-polymerase chain reaction
using the 5
-primer in the newly identified nucleotide sequence and the
3
-primer in the COOH-terminal sequence of p85
(data not shown). The
deduced amino acid sequence contains 6 unique amino acids at the
NH2-terminal head of the proline-rich domain and two SH2
domains, which are identical to those of p85
. Thus, this mRNA,
as well as p55
mRNA, appears to be transcribed via alternative
splicing of the p85
gene product. We designated this protein p50
.
The splicing site of these two variants is assumed to be the same,
although the part of the p55
cDNA that is identical to that of
p85
is longer than that of p50
by two amino acids.
Fig. 1.
A, an alignment of the amino acid
sequences of p85, p50
, p55
, p55
, and p85
. The amino acid
residues for each protein, with the addition of gaps (
) to optimize
the alignment, are numbered to the right of each sequence.
Two SH2 domains and the bcr and SH3 homology domains are
boxed. The nucleotide sequence of p50
has been submitted
to the GenBankTM/EMBL Data Bank with accession number
D78486[GenBank]. B, schematic comparison of the three groups of PI
3-kinase regulatory subunits. p85
and its two variants, the
and
isoforms, were structurally divided into three groups.
C, immunoblotting of p85
, p85
, p55
, p55
, and
p50
expressed in Sf9 cells. The Sf9 cells infected with
baculoviruses containing one of each of the five isoforms were cultured
for 48 h and lysed in Laemmli buffer. The cell lysates were
subjected to SDS-PAGE, and immunoblotting was performed with anti-HA
antibody (panel a) and the protein specific antibody
indicated above each panel (panels b-f). Lane 1, control Sf9 cells; lanes 2-6, Sf9 cells expressing p85
,
p85
, p55
, p55
, and p50
, respectively.
[View Larger Version of this Image (44K GIF file)]
, p85
, p55
, p55
, and p50
, with the HA tag at their
COOH termini, were subcloned into the expression vector, and
baculoviruses recombined with these cDNA were prepared. Sf9 cells
were infected with these baculoviruses, and cell lysates were
immunoblotted with anti-HA antibody (Fig. 1C, panel a) or
specific antibodies against each isoform (Fig. 1C, panels
b-f). Bands corresponding to p50
were observed using either
the anti-HA antibody or the anti-p50
specific antibody (
p50
),
with an electric mobility of approximately 50 kDa (Fig. 1C, panel
f, lane 6). The results shown in Fig. 1C, panels b-f, indicate that none of the specific antibodies recognize other regulatory subunit isoforms. We were thus able to measure the PI
3-kinase activity associated with each of the regulatory subunit isoforms expressed endogenously in tissues or cell lines.
mRNA Is Most Abundant in Liver, but Is Also Abundant in
Brain and Kidney
mRNA expression in
various rat tissues are shown in Fig. 2A.
Northern blotting with a 5
-unique 188-nucleotide sequence located in
the 5
-untranslated region and a coding region for the
NH2-terminal 6 amino acid sequence in the p50
cDNA,
neither of which is included in the p85
cDNA nucleotide
sequence, revealed two mRNA species of 6.0 and 2.8 kb. The p50
mRNA was most abundant in liver but was also abundant in the brain
and kidney. Northern blotting using the cDNA probe coding for the
N-terminal SH2 (N-SH2) domain of p85
revealed four bands (7.7, 6.0, 4.2, and 2.8 kb), as previously reported (22) (Fig. 2B).
Among them, the 6.0- and 2.8-kb bands matched those of p50
, whereas
the 7.7- and 4.2-kb bands matched those of the SH3 p85
domain (22).
As we reported previously, a minor portion of the 4.2-kb band and a
considerable portion of the 2.8-kb band in the brain correspond to
p55
mRNAs (22). The quantities of p85
mRNA and p50
mRNA in liver appeared to be similar, judging from the Northern
blotting data.
Fig. 2.
Northern blotting of p85 and p50
mRNAs in various rat tissues. Rat multiple tissue Northern
blot was obtained from Clontech and used for the detection of mRNA.
32P-Labeled cDNA probes encoding nucleotides
170-18
of p50
, corresponding to the 5
-noncoding region and 6 unique amino
acids of p50
(A) and nucleotides 1011-2175 of p85
(B), were hybridized and washed according to the
manufacturer's instructions (Clontech).
[View Larger Version of this Image (47K GIF file)]
mRNA was abundant in the cerebral cortex (temporal and occipital),
putamen, and cerebellum. Although minor differences were observed, the
distributions of p85
and p55
were similar to that of p50
(Fig.
3, B and D). p85
mRNA was also abundant in
the superior colliculus and brainstem (pons and medulla oblongata) but
was detected in every part of the brain (Fig. 3A). On the
other hand, the expression of p55
mRNA was particularly
prominent in the cerebellum, while being barely detectable in other
parts of the brain (Fig. 3C). As to the cerebellum, in
situ hybridization histochemistry revealed p50
transcripts to
be most abundant in the cytoplasm of Purkinje cells (Fig.
4A). As reported previously (28) the PI
3-kinase and IGF-1 receptor immunoreactivities were detected in almost all Purkinje neurons in the cerebellar cortex, so we assume that p50
plays a specific role in these highly specialized cells.
Fig. 3.
Distributions of p85 (A),
p85
(B), p55
(C), p55
(D),
and p50
(E) mRNAs in the rat CNS. Rat brains
were removed and separated into olfactory bulbs, cerebral cortex
(frontal), cerebral cortex (temporal), cerebral cortex (occipital),
superior colliculus, inferior colliculus, caudate putamen, thalamus,
hippocampus, cerebellum, pons, and medulla oblongata. Total RNA was
isolated, and RNase protection assays were performed using an RPA IITM
kit (Ambion, TX) according to the manufacturer's instructions.
Upper panels are the results of autoradiography. The
radioactivities of each lane were counted with a Molecular Imager
(Bio-Rad) and are displayed in the lower panels. These
experiments were repeated three times and yielded similar results.
Lane 1, olfactory bulb; lane 2, cerebral cortex
(frontal); lane 3, cerebral cortex (temporal); lane
4, cerebral cortex (occipital); lane 5, superior
colliculus; lane 6, inferior colliculus; lane 7, caudate putamen; lane 8, thalamus; lane 9, hippocampus; lane 10, cerebellum; lane 11, pons; lane 12, medulla oblongata.
[View Larger Version of this Image (32K GIF file)]
Fig. 4.
In situ hybridization histochemistry of
p50 in rat cerebellum. Distribution of p50
mRNA in
longitudinal wax sections (10 µm) of the cerebellar lobule of rat
brain by in situ hybridization histochemistry. A,
Purkinje cells (arrows) show an intense signal with the
DIG-labeled antisense cRNA probe. B, a control section hybridized with the sense cRNA probe exhibits no signal.
Bar, 100 µm.
[View Larger Version of this Image (126K GIF file)]
,
p55
, and p50
) and Their Associated PI 3-Kinase
Activities
and its two
splice variants in different rat tissues at the protein level, lysates
from various rat tissues were immunoprecipitated with protein A-agarose beads covalently coupled to
p85PAN-UBI. This antiserum,
which is raised against the entire region and the nSH2 region of
p85
, recognizes not only p85
but also p55
and p50
. The
immunoblot obtained with
p85PAN-UBI revealed the 85-kDa
band and a broad 50-55-kDa band (Fig. 5A). The 85-kDa protein was abundantly expressed in every tissue examined, and the 50-55-kDa band was prominent in the brain, liver, and kidney
but faint in fat and muscle. By taking into consideration that the
antibody used recognizes the entire p85
molecule, the p55
and
p50
molecules, which have neither the SH3 nor the bcr homology domain, would be less effectively detected than p85
in the
immunoblot using
p85PAN-UBI. Thus, the relative amounts
of p55
and p50
proteins, as compared with the amount of p85
,
are assumed to be larger than those suggested by the data in Fig.
5A.
Fig. 5.
Immunoblotting of the protein
immunoprecipitated with p85PAN-UBI and PI-3 kinase
activities in various rat tissues. Rat tissues were homogenized
and solubilized in lysis buffer. Supernatants were collected after the
centrifugation and incubated with beads coupled to the antibody against
the entire p85
molecule. The beads were washed three times and
resuspended in Laemmli buffer. The eluate from the beads was
electrophoresed and immunoblotted with
p85PAN-UBI
(A) or with
p85
SH3,
p55
, or
p50
(B). The 50-55-kDa band, present in all lanes in B, corresponds to the heavy chain of IgG. Various rat
tissues were solubilized, and the supernatants obtained by
centrifugation were incubated with control antibody,
p85
SH3,
p55
, or
p50
. The PI-3 kinase
activities in these immunoprecipitates were measured as described under
"Experimental Procedures" (C).
[View Larger Version of this Image (60K GIF file)]
(
p85
SH3) (Fig. 5B), the expression of
p85
protein is ubiquitous. The second isoform, p55
, is expressed
abundantly in the brain but only faintly in muscle. On the other hand,
the third isoform, p50
, is expressed most abundantly in liver and in
relative abundance in the brain and kidney as shown in the immunoblot
using the specific antibody against p50
(
p50
) (Fig.
5B). These results regarding protein expression levels
appear to be consistent with Northern blotting results. Fig.
5C shows the PI 3-kinase activities associated with p85
,
p55
, and p50
proteins. p85
-associated PI 3-kinase is
ubiquitously detected, whereas p55
-associated PI 3-kinase is
detected mainly in brain and muscle, and p50
-associated PI 3-kinase
is detected in liver and brain, as well as in muscle tissues.
Binds Two Types of Catalytic Subunits of PI
3-Kinase
molecule. To determine whether this 46-kDa protein is identical to p50
, we performed the same chromatographic procedure using DEAE-Sepharose. The soluble fractions prepared from rat liver were applied to a DEAE-Sepharose column, and
fractionation was performed according to the reported procedures (29).
The obtained fractions were immunoprecipitated with the specific
antibodies against p85
or p50
, and the PI 3-kinase activities in
these immunoprecipitates were measured.
-associated PI
3-kinase was detected in two fractions. One was the flow-through
fraction, eluted at 0 mM KCl (fraction A), and the other
fraction eluted at approximately 0.1 M KCl (fraction B). On
the other hand, the PI 3-kinase activities associated with p85
proteins were observed not in the flow-through fractions but in the
fractions eluted at approximately 0.2 M KCl (fraction C),
which is in agreement with the results of Kurosu et al.
(29). The apparent molecular masses of the p50
-associated PI
3-kinases in both fractions (fractions A and B) were determined to be
160-180 kDa based on the gel filtration results (Fig. 6B). These fractions (fractions A, B, and C) were then collected and immunoprecipitated with
p85PAN-UBI. Western blotting
with
p85PAN-UBI allowed isolation of the p85
and
p50
proteins with DEAE chromatography (Fig. 6C). With
respect to the difference between the two p50
-associated PI 3-kinase
fractions, A and B, we speculate that p50
may bind to the different
catalytic subunits. To ascertain the different characteristics of the
catalytic subunits associated with p50
in the two fractions, we
attempted wortmannin labeling, followed by treatment with
anti-wortmannin antibody for detection of the catalytic subunit. The
catalytic subunit associated with p50
in fraction B was detected by
this procedure as a band of 110 kDa (Fig. 6D, lane
4). In contrast, the catalytic subunit associated with the p50
in fraction A was not detectable with the same procedure (Fig.
6D, lane 2), despite fraction A containing an amount of p50
protein similar to that of fraction B. In contrast to the case
of p50
, the PI 3-kinase activities associated with p85
and p55
were detected only in the eluted fractions containing approximately 0.2 M (fraction C) (Fig. 6A) and 0.15 M
of KCl (fraction D) (Fig. 7A), respectively,
but never in the flow-through fractions. Their associated catalytic
subunits were easily detected by the wortmannin labeling and the
following immunoblot using wortmannin antibody, as a band of 110 kDa
(Fig. 6D, lane 6, and Fig. 7C, lane 2,
respectively).
Fig. 6.
Fractionation of rat liver cytosolic PI
3-kinase activities on a DEAE-Sepharose and a gel filtration column and
detection of p110 by wortmannin labeling. A, insoluble
materials from the rat liver (30 ml, 20 mg/ml) were directly loaded
onto a DEAE-Sepharose column. The column was then washed with buffer B,
consisting of 20 mM Tris-HCl, pH 7.4, 1 mM
EDTA, 1 mM dithiothreitol, 1 µM leupeptin, 0.5 mM PMSF, and 10% glycerol. Once the 280 nm absorbance
of the eluate had returned to base line, protein was eluted (absorbance was monitored at 280 nm (· · ·)) with a gradient of KCl.
Aliquots of the individual fractions were immunoprecipitated with
p50
or
p85
SH3 and then assayed for PI 3-kinase
activity.
,
p50
;
,
p85
SH3. B,
the pooled fractions from the peaks (fractions A and B) were concentrated and applied to a gel filtration column. Eluted fractions (1 ml) were collected and immunoprecipitated with
p50
and assayed for PI 3-kinase activity.
, fraction A;
, fraction B. C, each fraction was also immunoprecipitated with
p85PAN-UBI and then subjected to SDS-PAGE.
Immunoblotting analysis was performed with
p85PAN-UBI.
Lanes 1-3, fractions A-C, respectively.
D, all fractions were immunoprecipitated with control
antibody (lanes 1, 3, and 5) and
p85PAN-UBI (lanes 2, 4, and 6) and
pelleted using protein A-Sepharose beads. The beads were washed and
incubated at 25 °C for 30 min with 20 mM Tris-HCl, pH
7.4, 0.5 mM EDTA, 0.5 mM EGTA, and 0.1 µM wortmannin. Wortmannin-labeled immunoprecipitates were
subjected to SDS-PAGE. The wortmannin labeling was detected by enhanced
chemiluminescence using anti-wortmannin antibody and horseradish
peroxidase-labeled anti-rat IgG. Lane 1, fraction A
immunoprecipitated by control antibody; lane 2, fraction A
immunoprecipitated by
p85PAN-UBI, lane 3,
fraction B immunoprecipitated by control antibody; lane 4,
fraction B immunoprecipitated by
p85PAN-UBI; lane
5, fraction C immunoprecipitated by control antibody; lane 6, fraction C immunoprecipitated by
p85PAN-UBI.
[View Larger Version of this Image (30K GIF file)]
Fig. 7.
Fractionation of rat brain cytosolic PI
3-kinase activities on a DEAE-Sepharose column and detection of p110 by
wortmannin labeling. A, insoluble materials from the rat
liver (30 ml, 8 mg/ml) were directly loaded onto a DEAE-Sepharose
column. The column was then washed with buffer B. Protein was eluted
(absorbance was monitored at 280 nm (· · ·)) with a gradient of
KCl. Aliquots of the individual fractions were immunoprecipitated with
p50
,
p55
, or
p85
SH3 and then assayed for
PI 3-kinase activity.
,
p50
;
,
p55
;
,
p85
SH3. B, fraction D was also
immunoprecipitated with
p85PAN-UBI and then subjected to
SDS-PAGE. Immunoblotting analysis was performed with
p85PAN-UBI. C, fraction D was
immunoprecipitated with control antibody (lane 1) and
p55
(lane 2) and labeled with 0.1 µM
wortmannin. Wortmannin-labeled immunoprecipitates were subjected to
SDS-PAGE. The wortmannin labeling was detected by enhanced
chemiluminescence.
[View Larger Version of this Image (21K GIF file)]
in the Insulin-induced Activation of
Associated PI 3-Kinase
, p85
, and p50
(data not shown). In PC12 cells, insulin
caused increases in the PI 3-kinase activities of the
p85
,
p55
, and
p50
immunoprecipitates up to 1.9-, 1.9-, and
3.3-fold, respectively, whereas the
p85
and
p55
immunoprecipitates showed no significant increases in PI 3-kinase
activity (Fig. 8A). In HepG2 cells, p85
and p50
responded to insulin stimulation with increases of 2.7- and
4.5-fold, respectively, whereas no significant change was observed for
p85
(Fig. 8B). In both cell lines, the degree of PI
3-kinase activation was revealed to be highest for the
p50
-associated PI 3-kinase. However, the possibility that the
different responses are due to specific antibodies, bound to the
different portions of these regulatory subunits, cannot be
excluded.
Fig. 8.
Insulin responsiveness of endogenous
regulatory subunit for PI 3-kinase. PC12 (A) and HepG2
cells (B) were grown in Dulbecco's modified Eagle's
medium. The indicated concentrations of insulin were added to the
medium, and incubation was continued for 5 min at 37 °C. After
insulin treatment, the cells were collected with lysis buffer C. The
resulting supernatants were immunoprecipitated with
p85
SH3 (
),
p85
SH3 (
),
p55
(
),
p55
(
), and
p50
(
) and assayed for
PI 3-kinase activity.
[View Larger Version of this Image (18K GIF file)]
proteins, whereas
relatively small increases (2-5-fold) were observed with p85
,
p55
, and p55
, and the increase in p85
-associated PI 3-kinase
activity was very small (Fig. 9A).
Fig. 9.
A, insulin responsiveness of
overexpressed regulatory subunits for PI 3-kinase in HepG2 cells. HepG2
cells were infected with viruses expressing the full-length amino acid
sequences of p85 (
), p85
(
), p55
(
), p55
(
), or
p50
(
), as well as the HA tag amino acid sequence at each COOH
terminus, for 1 h, and then grown for 48 h. The indicated
concentrations of insulin were added to the medium, and the cells were
incubated for 5 min at 37 °C. After insulin treatment, the cells
were collected with lysis buffer C. The resulting supernatants were
immunoprecipitated with anti-HA antibody and assayed for PI 3-kinase
activity. B, [35S]methionine labeling of HepG2
cells expressing p85
, p85
, p55
, p55
, or p50
. HepG2 cells
were infected with adenoviruses expressing the full-length cDNAs of
p85
, p85
, p55
, p55
, and p50
and then grown for 48 h. After metabolic labeling with [35S]methionine,
10
6 M insulin was added to the medium and the
cells were further incubated for 5 min at 37 °C. Then, all lysates
were centrifuged, and the resulting supernatants were
immunoprecipitated with control antibody or the anti-HA antibody and
pelleted with protein G-Sepharose beads. The immunoprecipitates were
subjected to SDS-PAGE analysis. The gels were dried and subjected to
autoradiography. These experiments were conducted three times each and
yielded similar results. C, ratios of %IRS-1
proteins/expressed regulatory proteins were calculated.
[View Larger Version of this Image (25K GIF file)]
proteins apparently associated with larger
amounts of phosphorylated IRS-1 protein, as compared with other
isoforms. In contrast, p55
associated with the smallest amount of
IRS-1, in response to insulin stimulation, among the five isoforms. The
IRS-1 protein was also measured by immunoblotting using the antibody
against IRS-1, and no significant difference was observed between the
metabolic labeling method and immunoblotting data (data not shown).
These data indicate that p50
shows the most efficient IRS-1 binding
in response to insulin. This observation may explain the high capacity
of this protein to induce PI 3-kinase activity, as compared with other regulatory proteins, in response to insulin stimulation.
-associated PI 3-kinase activity was revealed
to be elevated by 27.0-fold and by 29.0-fold in CHO/IR cells and
CHO/IRS-1 cells, respectively, whereas the PI 3-kinase activities
associated with p85
, p85
, p55
, and p55
were elevated by
only 2.5-, 0.3-, 2.0-, and 6.5-fold in CHO/IR cells and by 2.6-, 0.2-, 2.3-, and 2.8-fold in CHO/IRS-1 cells, respectively.
Fig. 10.
Insulin responsiveness of overexpressed
regulatory subunit for PI 3-kinase in CHO/IR (A) and
CHO/IRS-1 cells (B). CHO/IR and CHO/IRS-1 cells were
infected with various viruses for 1 h and grown for 48 h.
Then 106 M insulin was added to the medium,
and the cells were incubated for 5 min at 37 °C. After insulin
treatment, the cell lysates were centrifuged at 10,000 × g for 10 min. The resulting supernatants were
immunoprecipitated with anti-HA antibody and assayed for PI 3-kinase
activity.
[View Larger Version of this Image (26K GIF file)]
, which appears to be
an alternative splicing form of the p85
gene product. Thus, there
are five known regulatory subunit isoforms in mammals, including two
85-kDa proteins, two 55-kDa proteins, and one 50-kDa protein, as
demonstrated by the results of overexpression experiments using Sf9
cells (Fig. 1C). In fact, the immunoblot using the antibody
that recognizes the entire p85
molecule revealed marked expression
of 50-55-kDa proteins in various tissues (Fig. 5A). It
appears that these 50-55-kDa proteins have, to date, been regarded as
degradation products of p85
or p85
and have thus attracted little
attention. In this study, we prepared the isoform-specific antibody, as
illustrated in Fig. 1C, and demonstrated that the 50-55-kDa
bands observed by immunoblotting with this antibody that recognizes the
entire p85
molecule correspond to two 55-kDa proteins (p55
and
p55
) (22-24) and one 50-kDa protein (p50
).
and are
thus considered to be alternative splicing products from the p85
gene. Thus, the p85
gene produces three different isoforms with
molecular sizes of 85 (p85
), 55 (p55
), and 50 kDa (p50
). These
isoforms share the same two SH2 domains and an inter-SH2 domain but
contain different NH2-terminal sequences. The most
well-known isoform, p85
, contains SH3 and bcr homology domains in its NH2 terminus. The second
type isoform,
p55
, contains a unique 34-amino acid sequence, which shows
considerable similarity to the corresponding region of p55
. The
third
type isoform, p50
, contains only the unique 6-amino acid
sequence in its NH2-terminal portion. These sequence data
suggest that p50
may be the most primitive regulatory subunit form
and that p55
and p85
may have more functions than p50
,
mediated via the 34-amino acid portion, as well as the respective SH3
and bcr homology domains. In fact, the SH3 domain of p85
was shown to interact with dynamin, a GTP-binding
microtubule-associated protein (30), and also with microtubules (31) or
/
-tubulin (32). The role of the 34-amino acid sequence in the
NH2 termini of the two 55-kDa regulatory subunits remains
unknown, although the highly conserved sequence shared by p55
and
p55
may suggests a specific function of the 34-amino acid portion.
p50
contains a unique sequence of only 6 amino acids, apparently too
short to associate with other molecules. This would presumably limit
the functional capacity of this protein.
revealed four bands (7.7, 6.0, 4.2, and 2.8 kb), as
previously reported (22) (Fig. 2B). Among these bands, those of 6.0- and 2.8-kb bands matched those of p50
, whereas the 7.7- and
4.2-kb bands matched those of the SH3 p85
domain. As we reported previously, a minor portion of the 4.2-kb band and a considerable portion of the 2.8-kb band in brain correspond to p55
mRNA. It should be noted that these three regulatory isoforms are not specific to the rat and are also present in the mouse, hamster, and human, based
on the results of immunoblotting using specific antibodies (data not
shown).
molecule (29). In that study, the
46-kDa regulatory subunit formed a heterodimer with a 100-kDa catalytic
subunit in rat liver, and this heterodimer could be separated in the
flow-through fraction of a DEAE-Sepharose column. Moreover, the
46-/100-kDa PI 3-kinase was activated by G
. We suspected this
46-kDa regulatory subunit to be identical to p50
and performed the
same chromatographic procedure on a rat liver lysate using a
DEAE-Sepharose column. We found p50
to be the only major protein
detected in the flow-through fraction that associates with PI 3-kinase
and is recognized by the antibody against the entire p85
molecule.
To determine whether the molecular size of the catalytic subunit in the
flow-through fraction is 100 kDa, wortmannin labeling followed by
immunoblotting using the antibody against wortmannin was performed. The
gel chromatographic results suggest that the actual size of the
p50
-containing PI 3-kinase in the flow-through fraction, based on
our calculation, is approximately 160 kDa. Thus, we speculate that
p50
in the flow-through fraction associates with the catalytic
subunit that has a molecular size of approximately 110 kDa. However,
immunoblotting using the antibody against wortmannin failed to detect
the catalytic subunit associated with p50
in the flow-through
fraction. The other fraction containing p50
-associated PI 3-kinase
was eluted at a KCl concentration of 0.1 M. The catalytic
subunit in this fraction was detected by wortmannin labeling, followed
by immunoblotting with the antibody against wortmannin, and the
molecular size was determined to be approximately 110 kDa. Thus, there
is an apparent difference between the two catalytic subunits associated
with p50
in the flow-through fraction and that associated with
p50
in the 0.1 M KCl fraction. In contrast to p50
,
p55
or p85
having PI 3-kinase was detected only in the 0.15 M KCl or 0.2 M KCl fraction from the
DEAE-Sepharose column, respectively, and the molecular sizes of their
associated catalytic subunits were determined to be 110 kDa by
wortmannin labeling and immunoblotting with the antibody against
wortmannin. The binding motif of the p85
regulatory subunit with the
p110 catalytic subunit was reported to reside in the inter-SH2 domain
(18), and this portion of the protein was completely conserved among
p85
, p55
, and p50
. Thus, it appears quite unlikely that p50
associates with a different catalytic subunit to which p85
and
p55
cannot bind. We speculate that the difference between the two
catalytic subunits bound to p50
might be due to a modification of
the catalytic subunit, such as serine phosphorylation, although further
study is needed to clarify this issue. Taking these observations
together, we cannot rule out the possibility that p50
is identical
to the reported 46-kDa protein.
(36), and the recently cloned p170 (37). In the
case of insulin signaling, the activation of PI 3-kinase is thought to
be particularly important. Insulin stimulation induces glucose
transporter translocation to the plasma membrane in muscle and fat
cells, resulting in an increase in glucose uptake (15, 16). In
addition, insulin leads to an increase in hepatic glycogen synthesis,
recently reported to occur via the activation of c-Akt (38). These
important functions of insulin have been shown to be blocked by
specific inhibitors of PI 3-kinase, such as wortmannin (39) and
LY294002 (40), as well as by the overexpression or microinjection of
the dominant negative mutant p85
(41, 42). Furthermore,
overexpression of wild-type or constitutively active p110 induces
glucose transporter translocation to the plasma membrane, irrespective
of insulin stimulation, in 3T3-L1 cells (43, 44). These findings
strongly suggest the importance, in various insulin actions, of
activation of the SH2 domain-containing PI 3-kinase.
shows a much higher level
of activation of its associated PI 3-kinase, in response to insulin
stimulation, than the other regulatory subunits. The p85
, p55
,
and p55
subunits exhibited only moderate responsiveness to insulin.
The activation of PI 3-kinase associated with p85
was confirmed to
be low, in agreement with the results of a previous report (45). In an
effort to elucidate the molecular mechanism underlying the marked
insulin-induced PI 3-kinase activation, we demonstrated that p50
exhibits the highest affinity for phosphorylated IRS-1 in response to
insulin in vivo. Although the reason for this high affinity
of p50
for IRS-1, despite p50
, p55
, and p85
sharing the
same two SH2 domains, is unknown, we speculate that the
NH2-terminal domains of p85
and p55
form complexes with other molecules resulting in an inability to bind IRS-1 as efficiently as p50
. Considering both the high level of p50
expression in the liver and its marked responsiveness to insulin,
p50
may play a more critical role than the other isoforms in hepatic
insulin-induced activation of PI 3-kinase. p55
-associated PI
3-kinase was shown to be activated by insulin with a low affinity for
IRS-1, whereas p85
did not respond significantly to insulin despite
its association with IRS-1. Further study is needed to clarify these
issues.
*
This work was supported by Grant-in-Aid 08671136 for
Scientific Research (to T. A.) from the Ministry of Education, Science and Culture of 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.
To whom correspondence should be addressed. Tel.:
81-3-3815-5411 (ext. 3133); Fax: 81-3-5803-1874.
1
The abbreviations used are: PI 3-kinase,
phosphatidylinositol 3-kinase; IRS-1, insulin receptor substrate-1; kb,
kilobase pair(s); PAGE, polyacrylamide gel electrophoresis; PMSF,
phenylmethylsulfonyl fluoride; HA, hemagglutinin; IR, insulin
receptors; CHO, Chinese hamster ovary; DIG, digoxigenin; N-SH2,
N-terminal SH2.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.