(Received for publication, November 7, 1995)
From the
Phosphatidylinositol 3-kinase, which is composed of a 110-kDa
catalytic subunit and a regulatory subunit, plays important roles in
various cellular signaling mechanisms. We screened a rat brain cDNA
expression library with P-labeled human IRS-1 protein and
cloned cDNAs that were very likely to be generated by alternative
splicing of p85
gene products. These cDNAs were demonstrated to
encode a 55-kDa protein (p55
) containing two SH2 domains and an
inter-SH2 domain of p85
but neither a bcr domain nor a SH3
homology domain. Interestingly, p55
contains a unique 34-amino
acid sequence at its NH
terminus, which is not included in
the p85
amino acid sequence. This 34-amino acid portion was
revealed to be comparable with p55PIK (p55
) in length, with a high
homology between the two, suggesting that these NH
-terminal
domains of p55
and p55
may have a specific role that p85 does
not. The expression of p55
mRNA is most abundant in the brain, but
expression is ubiquitous in most rat tissues. Furthermore, it should be
noted that the expression of p85
mRNA in muscle is almost
undetectably low by Northern blotting with a cDNA probe coding for the
p85
SH3 domain, while the expression of p55
can be readily
detected. These results suggest that p55
may play an unique
regulatory role for phosphatidylinositol 3-kinase in brain and muscle.
Phosphatidylinositol 3-kinase (PI 3-kinase) ()(1, 2) has been implicated in the
regulation of various cellular activities, including proliferation (3, 4) , differentiation(5) , membrane
ruffling(6) , and prevention of apoptosis(7) . In
addition, PI 3-kinase activation is required for insulin-stimulated
glucose transport and insulin-dependent p70S6K activation(3) .
PI 3-kinase is a heterodimeric enzyme consisting of a regulatory
subunit (1, 2, 8, 9) and a 110-kDa
catalytic subunit (p110
,
). Recently, a novel 110-kDa
catalytic subunit (p110
), which is stimulated via G
and
G
, was cloned(10) . For the former type of PI
3-kinase, three regulatory subunit isoforms for PI 3-kinase have been
identified. Among them, p55PIK is a unique protein since the SH3 domain
and bcr homology domains found in p85
are replaced in p55PIK by a
unique 34-residue NH
terminus (11) .
In this
study, we isolated a novel alternatively spliced cDNA from the p85
gene by expression screening from a rat brain cDNA library using a
P-labeled human IRS-1 protein. This cDNA was demonstrated
to encode a 55-kDa protein, which was designated p55
, because it
is partly identical to p85
. In addition, we suggest changing the
name of p55PIK to p55
to avoid confusion between p55PIK (p55
)
and p55
. Herein, we compare the amino acid sequences of four
isoforms of the regulatory subunit of rat PI 3-kinase and show their
tissue distributions. These isoforms may be activated by different
stimuli and/or at different intercellular locations.
To confirm the
specificity of these antibodies, p85, p85
, p55
, and
p55
were expressed in Sf-9 cells using the baculovirus system.
These cDNAs coding the full amino acid sequence and the HA tag amino
acid sequence (YPYDVPDYA) at each C terminus were subcloned into
pBacPAK9 transfer vector, and the baculoviruses were prepared according
to the manufacturer's instructions (Clontech). The Sf-9 cells
infected with baculoviruses containing each of the four isoforms were
cultured for 48 h and lysed in Laemmli buffer. The samples were
subjected to SDS-PAGE, and immunoblotting was performed as described
previously (14) .
A human IRS-1 gene was successfully cloned from a human genomic library, and the complete nucleotide sequence of its coding region was determined. In comparison with the sequence reported by Araki et al.(12) , two nucleotides were revealed to be different in our IRS-1 nucleotide sequence (C to G at 2166 bp and A to G at 3432 bp). The C to G change at 2166 bp caused a change in the amino acid sequence (C to W at 382). As these differences were thought to be due to polymorphism, we prepared recombinant IRS-1 protein using a baculovirus containing this IRS-1 DNA.
A rat brain cDNA expression
library was screened with P-labeled recombinant IRS-1, and
81 positive independent clones were isolated after three or four rounds
of screening. They included cDNAs containing complete coding regions of
p85
, p85
, and p55
, of which nucleotide sequences were
determined. In addition, we obtained three independent cDNAs containing
the nucleotide sequence coding for the NH
-terminal SH2
domain of p85
and previously undocumented 166-nucleotide sequence
at its 5`-upstream side. These cDNAs contained an open reading frame of
1362 nucleotides, and the deduced amino acid sequence is shown in Fig. 1. The presence of this mRNA in rat brain was confirmed by
reverse transcription PCR using the 5`-primer in the newly identified
nucleotide sequence and the 3`-primer in the nSH2 domain or in the cSH2
domain found in p85
cDNA (data not shown). We designated this
putative protein p55
on the basis of its molecular weight.
p55
contains two SH2 domains and an inter-SH2 domain, which are
identical to those of p85
. Thus, p55
mRNA appears to be
transcribed by alternative splicing from the p85
gene. The SH3
domain and bcr homology domain found in p85
are replaced in
p55
by a unique 34-residue NH
terminus followed by a
conserved proline-rich motif (PPALPPKPPKP). Interestingly, this
34-residue region of p55
is comparable in length to the
corresponding NH
-terminal portion of p55
(11) ,
and 16 of the 34 amino acids are identical in the two peptides. These
conserved sequences suggest that their unique NH
-terminal
portion may have a specific functional role, which p85 does not.
Further study is needed to resolve this issue.
Figure 1:
Alignment of amino
acid sequences of p85, p55
, p55
, and p85
. The cDNAs
coding for the complete peptides of p85
, p55
, p55
, and
p85
were isolated by screening a rat brain expression cDNA library
with
P-labeled IRS-1 protein probe. The nucleotide
sequences were determined with an ABI automatic sequencer. 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, the bcr homology, the proline-rich, and the
SH3 domains are boxed.
The levels of
expression of p85, p55
, p55
, and p85
mRNAs in
various rat tissues were investigated, and the results are shown in Fig. 2. Northern blotting with a 5`-unique 159-nucleotide
sequence located in the 5`-untranslated region and a coding region for
the NH
-terminal 25-amino acid sequence in the NH
terminus of p55
, neither of which is included in the
p85
cDNA nucleotide sequence, revealed three mRNA species of 6.0,
4.2, and 2.8 kb in the brain (Fig. 2B). Among them, the
4.2-kb band was also detected in all of other tissues examined.
Northern blotting with nucleotides coding for the p85
SH3 domain
revealed two mRNA species of 7.7 and 4.2 kb (Fig. 2A).
In addition, the cDNA probe coding for the p85
/p55
nSH2
domains was also used for Northern blotting, and four mRNA species of
7.7, 6.0, 4.2, and 2.8 kb were observed (Fig. 2C). The
4.2-kb band was detected on all Northern blots, and the intensities of
this band were compared in various tissues. The amount of the 4.2-kb
mRNA detected with Northern blotting using a cDNA probe coding for the
p85
/p55
nSH2 domain is thought to be the sum of the amounts
of the p55
and p85
mRNAs. The intensity of the 4.2-kb band
among various tissues observed in blotting utilizing a cDNA probe
coding for the p85
/p55
nSH2 domain was revealed to be similar
to that obtained with a cDNA probe coding for the p85
SH3 domain
and differed significantly from that obtained with the p55
5`-unique cDNA probe. This result may suggest that p85
mRNA is
expressed more abundantly than p55
mRNA in most tissues, with the
apparent exceptions of brain and skeletal muscle. However, it should be
noted that in skeletal muscle the p85
mRNA expression level is
almost undetectably low, while p55
mRNA can be readily detected.
In muscle, the activation of PI 3-kinase is presumed to be involved in
insulin-stimulated glucose uptake through the translocation of GLUT4 to
the plasma membrane(3) . Therefore, it might be possible that
p55
plays a more important role than p85
in the stimulation
of glucose uptake by skeletal muscle.
Figure 2:
Northern blotting of p85, p55
,
p55
, and p85
mRNAs in various rat tissues. Rat multiple
tissue Northern blot was obtained from Clontech and used for the
detection of mRNA.
P-Labeled cDNA probes encoding
nucleotides 1-663 of p85
(panel A), 1-159 of
p55
(panel B), 1011-2175 of p85
(same as
nucleotides 201-1365 of p55
) (panel C), 96-1381 of
p55
(panel D), and nucleotides 1-2170 of p85
(panel E) were hybridized and washed according to the
manufacturer's instruction (Clontech). The intensity of the
4.2-kb bands was measured by using a BAS2000
(Fuji).
In brain, both p55 and
p55
mRNAs are expressed abundantly, as are those of p85
,
suggesting that these regulatory subunits having neither bcr homology
nor SH3 domains may have a function(s) different from that of p85. PI
3-kinase appears to be important, first, in that its activation is
essential for neurite elongation of rat PC-12 cells, and in addition,
VPS34, a yeast PI 3-kinase homologue, was shown to be involved in
vacuolar protein sorting(15) . Thus, PI 3-kinase may play an
essential role in the secretion of neurotransmitters via regulation of
vesicle sorting in the brain. Taken together, one or more of these four
regulatory subunits might be essential for neuronal differentiation,
while the others may be involved in the secretion of neurotransmitters.
Unlike p85 and p55
, p55
and p85
genes generate
only one mRNA species each, of 5.8 and 3.4 kb, respectively (Fig. 2, D and E), suggesting that no other
mRNAs are generated by alternative splicing of p55
and p85
gene products.
In order to detect the endogeneous p85,
p55
, and p55
proteins in rat tissues, we prepared specific
antibodies against each of the three. These antibodies did not
recognize different isoforms of regulatory subunits, produced in the
Sf-9 cell experiment using the baculovirus expression system (Fig. 3). As shown in Fig. 3A, by immunoblotting
using the anti-HA antibody (12CA5), the electrophoretic mobility of
p55
is essentially the same as that of p55
. The rat brain
lysates immunoabsorbed by the beads covalently coupled with
p85
, which recognize all p85
, p55
,
p55
, and p85
expressed in Sf-9 cells because of the highly
conserved amino acid sequence of these peptides (data not shown), were
subjected to SDS-PAGE and immunoblotted with control antibody,
p85
,
p85
,
p55
,
and
p55
(Fig. 3E). The immunoblot obtained
with
p85
revealed the two bands of 85 and 55
kDa, while that obtained with
p85
showed only
the 85-kDa band. In contrast,
p55
and
p55
both
showed the 55-kDa band. These results indicate the expression of these
isoforms in brain.
Figure 3:
A-D, immunoblotting of p85,
p85
, p55
, and p55
expressed in Sf-9 cells. To confirm
the specificities of the antibodies, p85
, p85
, p55
, and
p55
were expressed in Sf-9 cells using a baculovirus system. The
cDNAs coding for the full amino acid sequences and the HA-tag amino
acid sequence at the C termini were subcloned into pBacPAK9 transfer
vector (Clontech), and the baculoviruses were prepared according to the
manufacturer's instructions. The Sf-9 cells infected with
baculoviruses containing one of each of the four isoforms were cultured
for 48 h and lysed in Laemmli buffer. The samples were subjected to
SDS-PAGE, and immunoblotting was performed using anti-HA antibody (panel A),
p85
(panel B),
p55
(panel C), or
p55
(panel D),
as described previously(14) . Lane 1, control Sf-9
cells; lanes 2-5, Sf-9 cells expressing p55
,
p55
, p85
, and p85
, respectively. E,
immunoblotting of p85
, p55
, and p55
. Rat brain was
homogenized and solubilized in the lysis buffer. Supernatants were
collected after centrifugation and incubated with beads coupled with
antibody against the whole p85
molecule (Upstate Biotechnology
Inc.). The beads were washed three times and resuspended in Laemmli
buffer. The eluants from the beads were electrophoresed and
immunoblotted with control antibody,
p85
,
p85
,
p55
, or
p55
. F, PI 3-kinase activities in immunoprecipitates obtained with
control antibody,
p85
,
p85
, or
p55
. Rat brain was
solubilized and the supernatants, obtained by centrifugation, were
incubated with control antibody (lane 1),
p85
(lane 2),
p85
(lane 3),
or
p55
(lane 4). The PI 3-kinase activities of these
immunoprecipitates were measured as described under ``Materials
and Methods.''
Finally, to determine whether or not p55 is
associated with PI 3-kinase activity, as in the case of p85
, we
immunoprecipitated the rat brain soluble fraction with each control
antibody,
p85
,
p85
, or
p55
. PI 3-kinase activities in these immunoprecipitates were
measured (Fig. 3F). The immunoprecipitates obtained
with
p85
,
p85
, or
p55
were demonstrated to contain significant PI 3-kinase
activity, as compared with the control antibody immunoprecipitate. This
result strongly suggests that p55
also exists as a heterodimer
with a p110 catalytic subunit and that it functions as a regulatory
subunit of PI 3-kinase.
In this study, we showed that there are at
least four isoforms of the regulatory subunit for PI 3-kinase. All of
the four isoforms contain two SH2 domains and the binding site for
association with the p110 catalytic subunit, suggesting that these
regulatory subunits of PI 3-kinase interact with phosphotyrosine
residues on the receptors or receptor substrates through one or both of
their SH2 domains, resulting in activation of the p110 catalytic
subunit. However, SH3 and bcr homology domains found in p85 or
-
are replaced in p55
or -
by unique 34-residue NH
termini. Although the functional roles of SH3 domain have not
been understood yet, the association between SH3 domain and
proline-rich segments in various signaling proteins (i.e. dynamin(16) , paxillin(17) , hSOS1 (18) ,
p85 subunit of PI 3-kinase(19) ) is reported. Thus, the
differences in the NH
-terminal region observed among the
regulatory subunit isoforms may contribute to differences in
subcellular distributions and/or to varying degrees of PI 3-kinase
activation in response to various growth factor receptors and oncogenic
products.
In summary, we have identified a novel alternatively
spliced regulatory subunit, which may have important functions in brain
and muscle. Our future studies will focus on the variety of possible
functions mediated by differences in the NHterminal
portions of the regulatory subunits of PI 3-kinase.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s) D64045 [GenBank](rat p85), D64046 [GenBank](rat p85
), D64047 [GenBank](rat p55
), and D64048 [GenBank](rat p55
).