(Received for publication, March 6, 1997, and in revised form, April 24, 1997)
From the Department of Biochemistry, University
College London, Gower Street, London WC1E 6BT, United Kingdom, the
¶ Department of Clinical Biochemistry, University of Cambridge,
Cambridge, CB2 2QR United Kingdom, and the
Department of
Clinical Physiology, Karolinska Hospital, Stockholm, 10401 Sweden
The role of phosphoinositide 3-kinase (PI
3-kinase) in insulin signaling was evaluated in human skeletal muscle.
Insulin stimulated both antiphosphotyrosine-precipitable PI 3-kinase
activity and 3-O-methylglucose transport in isolated
skeletal muscle (both 2-3-fold). Insulin stimulation of
3-O-methylglucose transport was inhibited by the PI
3-kinase inhibitor LY294002 (IC50 = 2.5 µM).
The PI 3-kinase adapter subunits were purified from muscle lysates
using phosphopeptide beads based on the Tyr-751 region of the
platelet-derived growth factor receptor. Immunoblotting of the material
adsorbed onto the phosphopeptide beads revealed the presence of p85
,
p85
, p55PIK/p55
, and p50 adapter subunit isoforms. In
addition, p85
-NSH2 antibodies recognized four adapter subunit
variants of 54, 53, 48, and 46 kDa, the latter corresponding to the p50
splice variant. Serial immunoprecipitations demonstrated that these
four proteins were associated with a large proportion of the total PI
3-kinase activity immunoprecipitated by p85
-NSH2 domain antibodies.
Antibodies to p85
, p55PIK/p55
, and the p50 adapter
subunit also immunoprecipitated PI 3-kinase activity from human muscle
lysates. A large proportion of the total cellular pool of the 53-kDa
variant, p50, and p55PIK was present in antiphosphotyrosine
immunoprecipitates from unstimulated muscle, whereas these
immunoprecipitates contained only a very small proportion of the
cellular pool of p85
, p85
, and the 48-kDa variant. Insulin
greatly increased the levels of the 48-kDa variant in
antiphosphotyrosine immunoprecipitates and caused smaller -fold increases in the levels of p85
, p85
, and the 53-kDa variant. The
levels of p50 and p55PIK were not significantly changed.
These properties indicate mechanisms by which specificity is achieved
in the PI 3-kinase signaling system.
Insulin binding to its cell-surface receptor activates a range of intracellular signaling cascades that ultimately result in the regulation of a number of important metabolic events within the cell (1, 2). A large body of evidence indicates that PI 3-kinase1 activity is necessary for a wide range of cellular effects elicited by insulin (3-11). Specifically, several recent studies indicate that constitutive activation of PI 3-kinase is sufficient to induce stimulation of glucose uptake in adipocytes (12-14). However, many growth factors that stimulate PI 3-kinase do not stimulate glucose metabolism, suggesting that different signaling inputs have specific mechanisms for utilizing the PI 3-kinase signaling system to generate downstream responses (reviewed in Ref. 15).
Insulin and receptor tyrosine kinase-linked growth factors activate
class 1 PI 3-kinases, which consist of a 110-kDa catalytic subunit
linked to an adapter subunit (15). Three isoforms of the catalytic
subunit have been identified: p110, p110
, and p110
(16-18).
Of these, only p110
and p110
are present in insulin-responsive tissues. Two widely expressed
85-kDa forms of the adapter subunit were originally characterized: p85
and p85
. These share a high degree of sequence and structural homology, with each containing two
SH2 domains, two proline-rich domains (P1 and P2), an SH3 domain, and a
Bcr homology domain (19). Recently, several truncated isoforms of the
PI 3-kinase adapter subunit have been identified including a 55-kDa
variant of p85, termed p55
or p55PIK (20, 21), which is
encoded by a gene separate from p85
or p85
. Two splice variants
of the p85
gene have also been reported. These are a 53-55-kDa form
termed AS53 (22) or p55
(20) and a form encoding a protein with a
predicted molecular mass of 50 kDa (23, 24). PI 3-kinase purified from
liver has also been reported to associate with a 46-kDa protein that
cross-reacts with p85 antibodies (25). All of these truncated adapter
subunit variants contain two SH2 domains and the P2 proline-rich
domains, but lack the SH3 domain, the Bcr homology domain, and the P1
proline-rich domain of full-length p85.
The main mechanism by which insulin regulates PI 3-kinase activity is by tyrosine phosphorylation of specific YMXM motifs on the intracellular signaling intermediates IRS-1 and IRS-2, which enables the recruitment of PI 3-kinase via the SH2 domain on the PI 3-kinase adapter subunit (26). This increases cellular PI 3-kinase activity by a combination of increasing the catalytic activity of p110 (27, 28) and locating the p110 catalytic subunit in proximity to its substrate at the membrane.
It is not clear whether the different isoforms of the adapter subunit play independent or overlapping roles in intracellular signaling pathways. However, the possibility exists that specificity could be introduced into the PI 3-kinase system by differential recruitment of the individual adapter subunits in response to activation of different receptor tyrosine kinases. Such interactions would provide a mechanism by which multiple signaling inputs could use PI 3-kinase in different ways to obtain specific signaling outcomes.
In this study, we have investigated the PI 3-kinase signaling system in human skeletal muscle using a unique muscle strip preparation (29). The data obtained provide strong evidence that PI 3-kinase plays a crucial role in insulin signaling to glucose transport in human skeletal muscle. Furthermore, we have focused on the role of individual variants of the PI 3-kinase adapter subunit in insulin signaling. We have identified seven variants of the PI 3-kinase adapter subunit present in human skeletal muscle, and we demonstrate that these are differentially recruited into phosphotyrosine complexes in response to insulin stimulation. This study provides evidence for mechanisms by which specificity can be obtained in the PI 3-kinase signaling system.
125I-Protein A, Na125I,
[32P]ATP, ECL reagents, and Rainbow protein molecular
mass standards were from Amersham International (Buckinghamshire, United Kingdom). Phosphatidylinositol was from Lipid Products (Redhill,
United Kingdom). All other chemicals were from Sigma. Monoclonal PY20
antiphosphotyrosine antibody was from Transduction Laboratories. Rabbit
polyclonal antisera were raised against glutathione S-transferase fusion proteins corresponding to the SH3
domain (p85-SH3) or the N-SH2 (p85
-NSH2) domain of human p85
as described previously (30). Monoclonal antibodies recognizing
epitopes in the SH3 domain (U13) and in the N-SH2 domain (U2) of p85
and another specifically recognizing p85
were kindly supplied by Dr.
I. Gout and Prof. M. Waterfield (Ludwig Institute, London) (31). A
previously described polyclonal antiserum specific to p55PIK was provided by Drs. S. Pons and M. White (Joslin
Diabetes Center, Boston) (21). Antibodies to the p55
adapter subunit
and the p50 splice variant of p85
were supplied by Dr. T. Asano
(24). A peptide corresponding to amino acids 738-755 of the
platelet-derived growth factor receptor (GGYMDMSKDESVDYVPML) was
phosphorylated at the tyrosine equivalent to residue 751 and
cross-linked to Actigel (provided by Dr. B. Van Haesebroeck, Ludwig
Institute, London).
Muscle specimens were obtained from the vastus lateralis portion of the quadriceps femoris muscle from 19 healthy male subjects (age: 28.4 ± 0.7 years; weight: 76.6 ± 2.1 kg; height: 181.3 ± 1.9 cm; body mass index: 23.4 ± 0.4 kg/m2; fasting glucose: 4.9 ± 0.2 mM; and serum insulin: 6.9 ± 0.4 microunits/ml). None of the subjects were taking any medication. Following an overnight fast, the subjects reported to the laboratory at 8:30 a.m. A local anesthetic (prilocain hydrochloride, 10 mg/ml) was administered subcutaneously 15 cm above the proximal border of the patella, and a 4-cm incision was made. Two muscle specimens (250 mg each) were excised for in vitro incubation, and smaller muscle strips were prepared as described (29, 32).
Stimulation of Muscle and Determination of 3-O-Methylglucose TransportAfter preparation, the smaller muscle strips (~20 mg) were incubated at 35 °C for 10 min in a recovery solution containing oxygenated Krebs-Henseleit buffer supplemented with HEPES, 38 mM mannitol, 2 mM pyruvate, and 0.1% bovine serum albumin. The muscle specimens were subsequently incubated with inhibitors and insulin as indicated in the figure legends. For measurements of 3-O-methylglucose transport, the buffer contained 5 mM 3-O-[3H]methylglucose and 35 mM [14C]mannitol. For glucose transport experiments, the incubations were terminated by snap-freezing the muscle in liquid nitrogen. Thereafter, the muscle samples were rapidly homogenized in ice-cold buffer containing 100 mM NaF and 10 mM EDTA. Cell debris was removed by centrifugation at 5000 × g for 1 min. 3-O-Methylglucose transport was calculated as described previously (33). For immunoprecipitation experiments, the muscle samples were homogenized on ice in 50 mM Tris-Cl (pH 7.4), 1% Triton X-100, 10 mM EDTA, 1 mM NaF, 1 mM NaVO3, 2 mM aprotinin, 2 mM leupeptin, and 2 mM phenylmethylsulfonyl fluoride. Homogenates were precleared by centrifugation at 5000 × g for 10 min prior to immunoprecipitation.
Antibody Purification and Iodination10 µl of 100 mM Tris (pH 8.0) and 25 µg of swollen protein A-agarose
beads were added to 100 µl of p85-NSH2 antiserum. The solution was
mixed at 4 °C for 4 h, and the beads were collected. Beads were
washed with 100 mM Tris, and the antibody was eluted with
100 mM glycine (pH 3.0) for 5 min at room temperature. The glycine buffer was collected and neutralized with 10 µl of 1 M Tris (pH 8.0). An aliquot of purified antibody (25 µl)
was iodinated by sequential addition of 5 µl of
NaH2PO4/Na2HPO4 buffer
(0.5 M, pH 7.4) and 0.5 µCi of Na125I
followed by 10 µl of chloramine T (0.5 mg/ml). The reaction was
terminated 2 min later by the addition of 300 µl of 25 mM Tris (pH 7.4) containing 10 mM MgCl2 and 1%
bovine serum albumin. Nonincorporated 125I was separated
from iodinated antibody on a Bio-Rad Econo-Pac 10DG disposable
desalting column, and the peak iodinated antibody fraction was used for
Western blotting.
3T3-L1 fibroblasts were cultured and differentiated into adipocytes as described previously (30). Cells were used 7-11 days after differentiation, and 2-[3H]deoxyglucose uptake was determined as described previously (30).
Assessment of PI 3-Kinase ActivityPI 3-kinase activity was
determined essentially as described previously (34). Incorporation of
label from [-32P]ATP into phosphatidylinositol was
determined, and the lipid product was resolved by thin-layer
chromatography and quantitated on a Fuji BAS2000 phosphoimager.
Statistical differences between treatments were analyzed using Student's paired t test.
The ability of insulin to stimulate PI 3-kinase activity
in human skeletal muscle was initially characterized. Muscle was stimulated with insulin at 1000 microunits/ml, a concentration that
maximally stimulates glucose transport (data not shown). Insulin
stimulation increased PI 3-kinase activity associated with
phosphotyrosine-containing proteins 3.3-fold compared with basal
levels, with maximal stimulation being achieved 20 min after the
addition of insulin (Fig. 1).
Insulin-stimulated 3-O-Methylglucose Transport Is Blocked by LY294002
Insulin increased 3-O-methylglucose transport
in muscle strips 2.22-fold (basal: 0.60 ± 0.07 µmol × ml1 × h
1; insulin: 1.33 ± 0.06 µmol × ml
1 × h
1, n = 10 strips). The insulin-induced increase in
3-O-methylglucose transport was inhibited by LY294002 in a
dose-dependent manner (IC50 = 2.5 µM) (Fig. 2). The inhibition of insulin
action on muscle glucose transport by LY294002 very closely matched the effect obtained with this compound on insulin-stimulated glucose transport in 3T3-L1 adipocytes (Fig. 2).
A Number of PI 3-Kinase Adapter Subunit Variants Are Expressed in Human Muscle
Immunoblotting studies of whole muscle homogenate
using a polyclonal antibody to the SH3 domain of p85 consistently
revealed two immunoreactive bands at 85 and 87 kDa (Fig.
3A). Subsequent immunoblotting with
isoform-specific monoclonal antibodies revealed that the faster
migrating of these (lower band) represented p85
and the slower one
(upper band) represented p85
(Fig. 3B). The polyclonal
antibody raised against the N-SH2 domain revealed a prominent
immunoreactive protein at 85 kDa corresponding to p85
, but did not
recognize p85
. However, this antibody also recognized immunoreactive
proteins at 54, 53, 48, and 46 kDa in muscle homogenates. Whereas the
relative intensity of the bands varied slightly in samples from
different individuals, the 48-kDa protein was consistently the most
intense (Fig. 3A). Identical immunoreactive proteins were
recognized by antiserum raised against the same N-SH2 fusion protein in
a separate rabbit, but these proteins were not recognized by preimmune
serum from either of these rabbits (data not shown). More important,
these same immunoreactive proteins were recognized by the monoclonal
antibodies recognizing an epitope in the N-SH2 domain (Fig.
3B), but were not recognized by monoclonal or polyclonal antibodies recognizing epitopes in the SH3 domain of p85
(Fig. 3B). The lower molecular mass bands are unlikely to
represent a proteolytic product of p85
as the intensity of the bands
was not dependent on the inclusion of protease inhibitors in the
homogenization mixture and did not change with time of storage.
Furthermore, we have previously demonstrated that the p85
-NSH2
antiserum did not recognize any comparable lower molecular mass bands
in homogenates of 3T3-L1 adipocytes or 3T3-L1 fibroblasts (Ref. 30; see
also Fig. 6). The fact that the lower molecular mass bands correspond closely to those of recently reported splice variants of p85
(20-25) suggests that they are in fact variants of the PI 3-kinase adapter subunit.
Identification of PI 3-Kinase Adapter Subunit Immunoreactive Bands in Fractions Purified Using Phosphopeptide Beads
To confirm that
the lower molecular mass proteins recognized by the p85-NSH2
antibody were variants of p85, an affinity purification step was
introduced to remove any potential nonspecific reactions. In this
approach, human muscle homogenates were precipitated with phosphopeptide beads based on the amino acids surrounding Tyr-751 (p85
SH2 domain-binding region) of the platelet-derived growth factor
receptor. Such phosphopeptide beads have previously been shown to have
a high affinity for PI 3-kinase adapter subunits (31, 35). The
specificity and selectivity of this approach are demonstrated by the
fact that the p50 and p55PIK antibodies used in this study
recognize a number of bands in human muscle homogenates, whereas in the
eluate from the phosphopeptide beads, this is reduced to a single band.
The protein recognized by the p55PIK antiserum in the
phosphopeptide bead precipitates is 58 kDa, and the protein recognized
by the p50 antiserum is 46 kDa (Fig. 4), which
correspond closely with the previously reported molecular masses of
these variants in other species (21, 24). The p85
antibody
recognizes a single 87-kDa protein in muscle homogenates, and this
protein is also present in phosphopeptide beads (Fig. 4C).
Western blotting of the phosphopeptide bead eluates with the
p85
-NSH2 antibody revealed protein bands at 85, 54, 53, 48, and 46 kDa, and these were of equal intensity to the corresponding bands seen
with this antibody in muscle homogenate (Fig. 4A). Several
other components in the homogenate that were slightly immunoreactive
with the p85
-NSH2 antibody were not present in the precipitates and
were thus confirmed as nonspecific reactions.
Association of Adapter Subunit Variants with PI 3-Kinase Activity
PI 3-kinase activity was present in PI 3-kinase adapter
subunit antibody immunoprecipitates, confirming that the antibodies were recognizing functional PI 3-kinase adapter subunits. The greatest
amount of PI 3-kinase activity was found associated with antibodies
specific to p50, with lesser amounts associated with p85 and still
lower levels associated with p85
and p55PIK (Fig.
5A).
To investigate whether the lower molecular mass protein bands
recognized by the p85-NSH2 antibody were associated with PI 3-kinase
activity, serial immunoprecipitations followed by PI 3-kinase activity
assays were performed. Total p85
-associated PI 3-kinase activity was
immunoprecipitated from muscle homogenates using the p85
-NSH2
antibody (Fig. 5B). However, immunoprecipitation of an
equivalent amount of muscle homogenate with the p85
-SH3 antibody,
which recognizes both full-length p85
and p85
but not truncated
variants, precipitated a much smaller amount of PI 3-kinase activity.
This was not due to inefficient immunoprecipitation as both the
p85
-SH3 and p85
-NSH2 antibodies were equally efficient, each
being able to precipitate >85% of the full-length p85
protein (data not shown). Furthermore, subsequent immunoprecipitation with the
same antibody in the supernatant from the initial p85
-SH3 immunoprecipitation showed that no further PI 3-kinase activity could
be immunoprecipitated (data not shown). The p85
-NSH2 antibody, which
recognizes truncated adapter subunit forms, was then used to
immunoprecipitate PI 3-kinase activity remaining in the supernatant of
the p85
-SH3 immunoprecipitates. The amount of PI 3-kinase in these
immunoprecipitates was
75% of the amount of PI 3-kinase activity
directly immunoprecipitated by the p85
-NSH2 antibody from an
equivalent amount of homogenate (Fig. 5). This indicates that truncated
but functional forms of the PI 3-kinase adapter subunit are present in
human skeletal muscle.
Overall, the combination of the immunoblotting data, the phosphopeptide
bead experiments, and the PI 3-kinase assays in sequential immunoprecipitations provides strong evidence that the 54-, 53-, 48-, and 46-kDa proteins recognized by the p85-NSH2 antibody in fact
represent forms of the PI 3-kinase adapter subunit containing SH2
domains, but not the SH3 domain or Bcr homology domain.
The regulation of the multiple, structurally distinct
PI 3-kinase adapter subunit isoforms present in muscle had not been previously investigated. Therefore, the ability of insulin to induce
the recruitment of the PI 3-kinase adapter subunit variants into
antiphosphotyrosine immunoprecipitates was determined (Fig. 6). Direct blotting with iodinated p85-NSH2 antibody
was performed on antiphosphotyrosine immunoprecipitates to avoid the
problems associated with secondary antibodies recognizing IgG bands.
Use of the p85
-NSH2 antiserum also has the advantage of allowing direct comparison of the relative effects of insulin on several forms
of the PI 3-kinase adapter subunit. The p85
-NSH2 antiserum revealed
four immunoreactive bands in the antiphosphotyrosine immunoprecipitates
at 85, 53, 48, and 46 kDa. The four proteins recognized in the
antiphosphotyrosine immunoprecipitates correspond to adapter
subunit-specific bands seen in p85
-NSH2 immunoblots of
phosphopeptide bead eluates (Fig. 4). In these experiments, the 53-kDa
protein was the most prominent immunoreactive band in
antiphosphotyrosine immunoprecipitates from unstimulated muscle (Fig.
6A). The 85- and 46-kDa proteins were also readily
detectable, but the 48-kDa protein was almost undetectable in
antiphosphotyrosine immunoprecipitates from unstimulated muscle (Fig.
6A). The levels of the 46-kDa band, representing the p50
splice variant of p85, did not change significantly in
antiphosphotyrosine immunoprecipitates following insulin stimulation
(Fig. 6B). The most pronounced effect of insulin on
recruitment of adapter subunit variants into antiphosphotyrosine immunoprecipitates was on levels of the 48-kDa variant (19.5-fold stimulation; p < 0.01). Insulin caused smaller -fold
increases over basal levels of p85
(3.6-fold; p < 0.02) and the 53-kDa variant (1.7-fold; p < 0.02).
This smaller -fold increase was largely due to the presence of larger
amounts of p85
and particularly of the 53-kDa variant in
antiphosphotyrosine immunoprecipitates from unstimulated muscle.
However, when viewed in terms of absolute insulin-induced increases in
the levels of adapter subunit variants in antiphosphotyrosine
immunoprecipitates, it is seen that the increases in the amounts of
p85
and the 53- and 48-kDa forms were all very similar (Fig.
6B). In these studies, the effects of insulin on the 54-kDa
protein were not established as whereas it was recognized in direct
blots of muscle homogenates with iodinated p85
-NSH2 antibody, it was
below the level of detection in direct blots of antiphosphotyrosine
immunoprecipitates from basal and insulin-stimulated muscle (Fig.
6A). The reason for this is not clear.
As p85 and p55PIK are not recognized by the p85
-NSH2
antiserum, separate immunoblots were performed using antibodies
specific to these isoforms to determine whether they associate with
tyrosine-phosphorylated proteins in muscle in the unstimulated or
insulin-stimulated state. The p55PIK antiserum recognized a
single protein band in antiphosphotyrosine immunoprecipitates that was
identical in molecular mass to the band recognized by this antiserum in
the phosphopeptide bead eluates (i.e. 58 kDa) (Fig.
6C). The p55PIK band was present in the
antiphosphotyrosine immunoprecipitates from unstimulated muscle, and
the level was not significantly increased by insulin (Fig.
6D). The monoclonal antibody specific to p85
recognized
an 87-kDa band in antiphosphotyrosine immunoprecipitates from both
unstimulated and stimulated muscle (Fig. 6C). Insulin stimulation caused a 2.2-fold increase in the amount of p85
in antiphosphotyrosine immunoprecipitates (Fig. 6D).
Antiphosphotyrosine blotting of the phosphopeptide bead eluates was
performed to determine whether tyrosine phosphorylation of the PI
3-kinase adapter subunits contributed to their association with
antiphosphotyrosine immunoprecipitates. This failed to show any
immunoreactive bands in phosphopeptide bead eluates despite the fact
that the PY20 antibody clearly recognized a number of tyrosine-phosphorylated bands in the insulin-stimulated human muscle
homogenate (Fig. 7).
Human skeletal muscle is a major insulin target tissue in the body as it is the major site of insulin-stimulated glucose disposal in vivo (36). However, the mechanisms by which insulin stimulates glucose uptake into muscle are poorly understood. The study provides evidence that PI 3-kinase activity is essential for insulin-stimulated glucose transport in human skeletal muscle. This is based on the finding that the PI 3-kinase inhibitor LY294002 inhibits insulin-stimulated 3-O-methylglucose transport in human skeletal muscle and in 3T3-L1 adipocytes with a similar potency. Furthermore, the potency of this inhibition correlates well with the potency with which this compound inhibits the activity of purified PI 3-kinase (37). Wortmannin, an alternative PI 3-kinase inhibitor, has previously been used to study the role of PI 3-kinase in insulin-stimulated glucose transport in rodent muscle (11, 38-40). However, the concentration of wortmannin required to completely inhibit insulin-stimulated glucose transport in these studies was an order of magnitude higher than required in adipocytes and cell culture models (3, 5-7, 41). At these concentrations there is a possibility that wortmannin is inhibiting other signaling molecules such as PI 4-kinase (42) and phospholipase A2 (43). Therefore, the LY294002 data in this study strengthen the case for PI 3-kinase activity being essential for insulin stimulation of glucose transport in human skeletal muscle.
This study provides the first detailed assessment of the expression of
PI 3-kinase adapter subunits and their regulation by insulin in human
skeletal muscle. This is important as the effects of growth factors on
PI 3-kinase are mediated through the PI 3-kinase adapter subunits,
which regulate the location and specific activity of the PI 3-kinase
catalytic subunit (15). Adipocytes express only the p85 PI 3-kinase
adapter subunit (30, 44), whereas this study demonstrates that human
skeletal muscle expresses both p85
and p85
. Additionally, we
demonstrate that human skeletal muscle contains five truncated forms of
the PI 3-kinase adapter subunit. These are p55PIK and four
variants recognized by the p85
-NSH2 antibodies. The possibility that
one of the proteins recognized by the p85
-NSH2 antibodies is
p55PIK/p55
can be excluded as the p55PIK
antiserum only recognized a single band of 58 kDa in both
phosphopeptide beads (Fig. 4) and antiphosphotyrosine
immunoprecipitates (Fig. 6). This also indicates that the p85
-NSH2
antiserum is highly selective for the p85
SH2 domain as it does not
recognize p85
or p55PIK/p55
despite the fact that the
N-SH2 domains of p55PIK/p55
, p85
, and p85
are
>90% identical at the amino acid level. Therefore, the most likely
explanation for the 54-, 53-, 48-, and 46-kDa bands is that they all
contain an N-SH2 domain identical to that of p85
and are thus likely
to be p85
splice variants. Both p50 (23, 24) and p55
/AS53 (20,
22) are splice variants of p85
containing identical N-SH2 domains
and would therefore be expected to cross-react with the p85
-NSH2
antisera with a similar affinity for p85
itself. Indeed, the 46-kDa
band recognized by the p85
-NSH2 antibody in phosphopeptide bead
eluates cross-reacts with an antibody specific to the p50 splice
variant of p85
. The 53- and 48-kDa bands are likely to represent the
two described forms of the AS53 splice variant of p85
as antibodies
raised against the unique regions of AS53 recognize bands in Western blots of rat tissue (22) that have very similar characteristics to the
53- and 48-kDa bands seen in this study. The identity of the 54-kDa
band cannot be explained by any known PI 3-kinase adapter subunit
isoforms, although the molecular mass slightly greater than 53 kDa
suggests that it could represent the variant of AS53 that has an
8-amino acid splice insert in the inter-SH2 domain (22). However,
suitable reagents to assess this were not available.
Very few studies have been conducted to directly compare the function
of different PI 3-kinase adapter subunits. While p85 and p85
are
coded by separate genes, they are structurally very similar, containing
two SH2 domains, two proline-rich domains, a single SH3 domain, and a
Bcr/racGAP homology domain (19). Despite these similarities, previous
studies have suggested that differences may exist in the mechanism by
which these two isoforms control the catalytic activity of the complex.
For example, in some cell types, PI 3-kinase complexes containing
p85
appear to be less responsive to insulin than those containing
p85
(44). Furthermore, p85
is phosphorylated by p110 at serine
residues (45), whereas this does not occur in p85
(31). Little was previously known about the mechanisms by which the different truncated adapter subunit isoforms are regulated. Insulin stimulation of rat
muscle has been reported to cause a small increase in IRS-1 association
of the AS53 splice variant of p85
(22). Furthermore, coexpression of
p110, p55PIK, the insulin receptor
-subunit, and IRS-1
in Sf9 insect cells has been reported to cause an increase in the
specific activity of PI 3-kinase associated with p55PIK,
suggesting that insulin could potentially regulate this isoform (21).
This study compared the ability of insulin to induce recruitment of
multiple adapter subunit isoforms into protein complexes containing
tyrosine-phosphorylated proteins. The results provide evidence that
there are important differences in the mechanisms by which the PI
3-kinase adapter subunit isoforms are regulated by insulin. We
identified several major differences in the pattern of association of
the p85 variants with tyrosine-phosphorylated protein complexes. First,
there was a great variation in the proportion of the total pool of each
p85 variant that associated with phosphotyrosine-containing proteins in
unstimulated muscle. The levels of the 48-kDa variant associated with
antiphosphotyrosine immunoprecipitates in unstimulated muscle were
almost undetectable. Full-length p85 was readily detectable in the
same immunoprecipitates, although it still represented <5% of the
total pool of full-length p85
. In contrast, a much higher proportion
of the total of the p55PIK isoform and the 53-kDa and p50
variants was present in antiphosphotyrosine immunoprecipitates from
unstimulated muscle. The other area where major differences existed was
in the ability of insulin to increase the levels of different adapter
subunit variants in antiphosphotyrosine immunoprecipitates. Insulin
caused no significant increase at all in the levels of the 46-kDa band
or the p55PIK variant in antiphosphotyrosine
immunoprecipitates. However, insulin was able to induce the recruitment
of full-length p85
, p85
, and the 53- and 48-kDa variants into
phosphotyrosine-containing protein complexes. This indicates that these
four isoforms are the ones that contribute to PI
3-kinase-dependent insulin signaling pathways in human
skeletal muscle. Insulin induced a similar increase in the absolute
amount of full-length p85
and the 53- and 48-kDa forms in
phosphotyrosine-containing protein complexes, indicating that these may
each make similar contributions to the magnitude of the insulin
stimulation of PI 3-kinase activity. However, it was striking that the
ratio of the amount of adapter subunit present in these complexes in
the basal and insulin-stimulated states varied greatly between the p85
variants. The biggest difference was in the case of the 48-kDa variant,
where insulin increased the levels in antiphosphotyrosine
immunoprecipitates >19-fold compared with basal levels, whereas the
smallest -fold increase was for the 53-kDa form, where levels in
antiphosphotyrosine immunoprecipitates increased only 1.7-fold. If, for
example, the different adapter subunits are involved in different
signaling complexes and hence different downstream responses, it is
possible to see that this offers a mechanism by which insulin could use
PI 3-kinase-dependent signals to modulate different PI
3-kinase-dependent downstream responses to different
extents.
The mechanism by which these adapter subunit variants are
differentially recruited into complexes with tyrosine-phosphorylated proteins is not clear. The fact that all the adapter subunit variants have a minimal core containing the p110-binding region and the two SH2
domains suggests that all have a similar capability to bind to
tyrosine-phosphorylated proteins containing Tyr(P)-Xaa-Xaa-Met motifs.
However, our results demonstrate that this is not the case in reality;
thus, it appears likely that the N-terminal additions to the minimal
adapter subunit core contain elements that modulate the adapter
subunit's interaction with the tyrosine-phosphorylated proteins.
Consistent with this is the observation that structurally similar forms
of the adapter subunit behave in a similar manner. For example, p85
and p85
both contain an SH3 domain and a Bcr domain. A small amount
of the total pool of each of these isoforms is found in
antiphosphotyrosine immunoprecipitates from unstimulated muscle, and
insulin causes a significant increase in the levels of each of these in
antiphosphotyrosine immunoprecipitates. The 53-kDa variant and
p55PIK/p55
are the variants found in the greatest
amounts in antiphosphotyrosine immunoprecipitates from unstimulated
muscle, and both p55PIK/p55
and p55
/AS53 share
significant homology in the N-terminal region as both have a novel
32-amino acid sequence in place of the SH3 and Bcr domains. These
results suggest that as yet unidentified structural elements in the
N-terminal 32-amino acid extension dictate that
p55PIK/p55
and p55
/AS53 are regulated in a different
manner from full-length p85 and the 48-kDa form.
There are a number of potential mechanisms by which the multiple
adapter subunit isoforms could introduce specificity into the PI
3-kinase signaling system. The most obvious is that this allows for a
range of independently regulatable links between receptor tyrosine
kinases and the PI 3-kinase system, allowing different growth factors
to regulate PI 3-kinase in different ways. The recruitment of
individual adapter subunit isoforms will result in a parallel
recruitment of the associated catalytic subunit into the complex,
causing a net increase in production of phosphatidylinositol 3,4,5-trisphosphate. We provide evidence that such a mechanism is
functionally important as the adapter subunit variants are differentially recruited in response to insulin. The rate of
phosphatidylinositol 3,4,5-trisphosphate production can also be
regulated by the adapter subunit's ability to modulate the catalytic
activity of PI 3-kinase via the adapter subunit following its
interaction with Tyr(P)-Xaa-Xaa-Met motifs. It is currently not clear
whether all the variants of the adapter subunit are able to equally
transduce this signal to p110 to alter catalytic activity. A further
layer of specificity could be introduced into the system if the
subcellular location of different adapter subunit isoforms was
differentially regulated by different signaling inputs. We have
previously shown that insulin and platelet-derived growth factor
recruit PI 3-kinase activity to different intracellular locations in
3T3-L1 adipocytes (30). The fact that the adapter subunit isoforms
differ in the domain structure outside the minimal core consisting of
the p110-binding region and the two SH2 domains also may provide a
mechanism by which signalling specificity can be achieved. These
differences mean that in addition to the SH2 domain-directed
interaction, the different adapter subunit isoforms could interact with
different signaling complexes as a result of these differences in the
N-terminal sequences. For example, the Bcr domain in full-length p85
and p85
is potentially able to interact with Rac GTPase (46),
whereas the SH3 domain may interact with molecules involved in
downstream responses including Src (47),
-actinin (48), and dynamin (49). However, the physiological relevance of such interactions has
never been fully investigated.
In summary, the results of this study provide evidence that PI 3-kinase activity is necessary for insulin stimulation of facilitated glucose transport in human skeletal muscle. We find that seven PI 3-kinase adapter subunit isoforms exist in this tissue and that these are differentially regulated by insulin. This identifies a mechanism by which insulin could utilize specific PI 3-kinase adapter subunit isoforms to regulate particular downstream responses and thus introduce specificity into the PI 3-kinase signaling system.
We thank Prof. M. Waterfield, Dr. S. Pons, Dr. M. White, Dr. T. Asano, and Dr. Brian Holloway (Zeneca Pharmaceuticals) for providing reagents.