From the Boston University School of Medicine, Boston, Massachusetts 02118
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Glut4-containing vesicles immunoadsorbed from
primary rat adipocytes possess endogenous protein kinase activity and
phosphorylation substrates. Phosphorylation of several vesicle proteins
including Glut4 itself is rapidly activated by insulin. Wortmannin
blocks the effect of insulin when added to cells in vivo
prior to insulin administration. By means of MonoQ chromatography and
Western blot analysis, vesicle-associated protein kinase is identified
as Akt-2, a lipid-binding protein kinase involved in insulin signaling. Akt-2 is found to be recruited to Glut4-containing vesicles in response
to insulin.
The regulation of postprandial blood glucose levels by insulin is
achieved mainly by increased glucose transport into skeletal and
cardiac muscle and fat (1, 2). These are the only tissues that express
a specific isoform of the glucose transporter, Glut4, which mediates
the hormonal effect (for recent reviews see Refs. 3-8). It has been
shown that in adipocytes under normal conditions, Glut4 is localized in
an intracellular microsomal compartment, "Glut4-containing
vesicles," which are translocated to the plasma membrane in response
to insulin. Because total glucose uptake into insulin-sensitive tissues
is, in general, proportional to the amount of Glut4 molecules at the
cell surface, this translocation process is usually considered as the
major mechanism of insulin action on glucose transport.
The protein composition of Glut4-containing vesicles is now rather well
characterized. Besides Glut4, they include a novel insulin-regulated
aminopeptidase (IRAP), the IGFII/Man
6-phosphate1 receptor, the
transferrin receptor, and a recently cloned protein, sortilin (reviewed
in Refs. 9 and 10; see also Refs. 11 and 12). These proteins, which
have extracellular functional domains, represent major constituents of
Glut4-containing vesicles as shown by silver and Coomassie staining
(13, 14). In addition to these major "cargo" proteins,
Glut4-vesicles are enriched with peripheral and integral membrane
proteins that are thought to be involved in membrane trafficking and
fusion, such as vesicle-associated membrane protein-2 (VAMP2),
cellubrevin, secretory
carrier-associated membrane
proteins (SCAMPs), low molecular mass GTP-binding proteins, phosphatidylinositol kinases, and several others (reviewed in Refs. 9
and 10). Although the biochemical mechanism of translocation of
Glut4-containing vesicles to the cell surface is still unknown, there
is evidence suggesting that these vesicles represent a subcompartment of the endosomal system in insulin-sensitive cells in which recycling is inhibited under basal conditions, i.e. in the
absence of hormone (14). Insulin administration to cells may release
this trafficking block by, for example, removal of an endogenous
inhibitor from Glut4-containing vesicles, or by disassociating these
vesicles from an intracellular anchor, thus leading to their default
fusion with the plasma membrane. Indirect support for this hypothesis derives from recent data showing that the introduction of the cytoplasmic portion of several vesicular proteins, such as Glut4 (15)
or IRAP (16), causes Glut4 translocation to the plasma membrane,
presumably as a result of competing with the endogenous proteins for
the putative inhibitor or anchoring protein. After the trafficking
block is released, Glut4-vesicles fuse with the plasma membrane, most
likely, via a v-SNARE/t-SNARE-mediated process (reviewed in Ref.
8).
A major question in the field that still remains to be resolved is the
signal transduction pathway that couples activated insulin receptor
with the Glut4-containing compartment (vesicles) and triggers its
recruitment to the plasma membrane. At present, we know only about the
upstream part of this pathway: insulin receptor, insulin receptor
substrates, PI 3-kinase, PDK1, and Akt/PK B (17). The downstream
signaling components, proximal to Glut4-vesicles, remain unknown.
We show here that Glut4-containing vesicles immunoisolated from rat
adipocytes possess a tightly associated protein kinase activity and
several phosphorylation substrates. The vesicle-associated protein
kinase has been identified as Akt-2 by MonoQ chromatography and Western
blot analysis. Phosphorylation of vesicle component proteins as well as
artificial substrates by the vesicle-associated protein kinase is
rapidly increased by insulin in a wortmannin-sensitive fashion. These
data may provide a missing link in the insulin signal transduction
pathway by directly coupling Glut4-containing vesicles to the
previously established enzymatic cascade.
Antibodies--
In the present study, we used the monoclonal
anti-GLUT4 antibody 1F8 (18), the anti-SCAMPs antibody 3F8 (19), and
the anti-Akt-2 sheep antibody (Upstate Biotechnology, Lake Placid, NY).
Isolation and Fractionation of Rat Adipocytes--
Adipocytes
were isolated from the epididymal fat pads of male Sprague-Dawley rats
(200-250 g) by collagenase digestion (20) and transferred to KRP
buffer (12.5 mM HEPES, 120 mM NaCl, 6 mM KCl, 1.2 mM MgSO4, 1 mM CaCl2, 0.6 mM
Na2HPO4, 0.4 mM
NaH2PO4, 2.5 mM
D-glucose, 2% bovine serum albumin, pH 7.4). Insulin was administered to cells (where indicated) to a final concentration of 10 nM. After that, KCN was added to cells to final
concentration of 2 mM for 5 min, cells were washed three or
four times with HES buffer cooled to 14-16 °C (20 mM
HEPES, 250 mM sucrose, 1 mM EDTA, 5 mM benzamidine, 1 mM phenylmethanesulfonyl
fluoride, 1 µM pepstatin, 1 µM aprotinin, 1 µM leupeptin, pH 7.4), homogenized with a Potter-Elvehjem
Teflon pestle, and subcellular fractions were prepared as described
previously (21). In some experiments, phosphatase inhibitors (a mixture
of 25 mM Na4P2O7, 50 mM NaF, 5 mM Na3VO4)
were added to homogenization buffer with no significant effect.
Isolated membrane fractions were resuspended in PBS, which contained
all of the protease inhibitors listed above.
Immunoadsorption of GLUT4-containing Vesicles--
Protein
A-purified 1F8 antibody, as well as nonspecific mouse IgG (Sigma), were
each coupled to acrylic beads (Reacti-gel GF 2000, Pierce) at a
concentration 0.4 and 0.6 mg of antibody/ml of resin, respectively,
according to the manufacturer's instructions. Before usage, the beads
were saturated with 2% bovine serum albumin in PBS for at least 1 h and washed with PBS. The light microsomes (LMs) from rat adipocytes
were incubated separately with each of the specific and nonspecific
antibody-coupled beads overnight at 4 °C. The beads were washed
twice with PBS and twice with protein kinase buffer (10 mM
Tris, 10 mM KCl, 100 mM NaCl, 5 mM
MgCl2, pH 7.8), and the adsorbed material was used for
phosphorylation experiments as described in the following paragraph.
Phosphorylation of Glut4-containing
Vesicles--
[ Anion Exchange Chromatography--
To separate proteins by anion
exchange chromatography, membrane samples were solubilized in 1%
Triton X-100 for at least 2 h at 4 °C, centrifuged, and applied
to a 1-ml Amersham Pharmacia Biotech MonoQ column equilibrated with 20 mM Tris, 50 mM NaCl, 0.1% Triton X-100, pH
8.0. Elution was carried out with a linear gradient of NaCl (final
concentration, 0.5 M; total volume of the gradient, 30 ml)
at a flow rate of 0.5 ml/min. Thirty 1-ml fractions were collected and
analyzed for the total protein content and protein kinase activity.
Concentration of NaCl in the gradient fractions was re-evaluated with
the help of a digital conductivity meter (VWR). No Triton X-100 was
present in the samples or in the buffers upon fractionation of
cytosolic fractions.
Gel Electrophoresis and Immunoblotting--
Proteins were
separated in SDS-polyacrylamide gels according to Laemmli (22), but
without reducing agents, and were transferred to Immobilon-P membrane
(Millipore) in 25 mM Tris, 192 mM glycine. Following transfer, the membrane was blocked with 10% nonfat dry milk
in PBS for 2 h at 37 °C. Proteins were visualized with specific antibodies, horseradish peroxidase-conjugated secondary antibodies (Sigma), and an enhanced chemiluminescent substrate kit (NEN Life Science Products). Autoradiograms were normally exposed overnight in a
storage phosphor screen cassette and quantitated in a PhosphorImager (Molecular Dynamics).
Protein Content--
Protein content was determined with a BCA
kit (Pierce) according to manufacturer's instructions.
Glut4-containing Vesicles Contain an Insulin-stimulated Protein
Kinase Activity and Phosphorylation Substrates--
Glut4-containing
vesicles were immunoadsorbed from intracellular membranes of rat
adipose cells treated and not treated with insulin, and
[
Along with protein kinase(s), Glut4-containing vesicles may also
contain an endogenous phosphatase activity, which may alter the results
of the in vitro phosphorylation. To check this possibility, Glut4-vesicles were immunoadsorbed from insulin-treated and untreated cells, phosphorylated in vitro as described above,
thoroughly washed of radioactive ATP, and incubated under the same
conditions for another hour. This additional incubation in the absence
of ATP does not change the total pattern of phosphorylation or specific incorporation of radioactive phosphate into individual proteins (not shown).
In the next experiments, we explored the substrate specificity of the
vesicle-associated protein kinase. As is shown in Fig. 2, myelin basic protein (MBP) was
phosphorylated to a greater extent than other substrates analyzed. A
small amount incorporation of radioactive phosphate into total fraction
of histones (Sigma) was also detected. On the other hand, neither
casein nor a synthetic peptide corresponding to the phosphorylation
site on the regulatory p85 subunit of PI 3-kinase (25) was
phosphorylated by a vesicle-associated protein kinase (Fig. 2),
although it could be phosphorylated by immunoprecipitated PI
3-kinase.2 This, together
with other evidence (see Fig. 5), suggests that PI 3-kinase which, in a
recent study, was found to be associated with Glut4-containing vesicles
in an insulin-dependent manner (26), does not phosphorylate
their component proteins in vitro. It was also found that
the vesicle-associated protein kinase is fairly specific for ATP and
cannot use GTP, taken at an equal concentration (data not shown).
As an additional control for phosphatase activity, in vitro
phosphorylated MBP was incubated with Glut4-containing vesicles immunoadsorbed from insulin-treated and not treated cells. No dephosphorylation of 32P-labeled MBP was detected under
these conditions (not shown).
Because cytosolic proteins can, theoretically, be nonspecifically
associated with the immunoadsorbed material and dissociate with an
increase in the ionic strength of the washing buffer, it seemed
essential to determine how tightly the endogenous protein kinase is
associated with Glut4-containing vesicles. Fig.
3 shows that extensive wash of
immunoadsorbed Glut4-containing vesicles with a high ionic strength
buffer cannot remove endogenous protein kinase activity from this
compartment and does not change the pattern of phosphorylated
proteins.
As is shown in Figs. 1-3, some increase in the activity of the
vesicle-associated protein kinase always takes place after adipocytes are stimulated by insulin for 15 min. This time point was chosen because the effect of insulin on Glut4 recruitment to the plasma membrane reaches the maximum at about this time (for recent study see
Ref. 27). However, activation of the vesicle-associated protein kinase
may precede vesicle translocation. Thus, we immunoadsorbed Glut4-containing vesicles from adipocytes treated with insulin for 2, 4, 8, and 16 min and determined the level of protein kinase activity in
this material. Fig. 4A shows
that phosphorylation of the vesicle proteins by the endogenous protein
kinase is rapidly increased by insulin and then gradually declines
after 2 min of insulin stimulation.
In this and other (Figs. 1, 3, and 5) experiments, however, the effect
of insulin on phosphorylation of Glut4-containing vesicles in
vitro may and should depend on the phosphorylation status of the
substrate proteins in living cells, which is hard if not impossible to
measure. In other words, if insulin causes dephosphorylation of
Glut4-containing vesicles in vivo, we may see an increase in their phosphorylation in vitro even if the level of the
vesicle-associated protein kinase activity stays the same. To
distinguish between these two possibilities, we measured the activity
of this protein kinase toward an exogenous substrate, MBP (Fig.
4B). Because the pattern of MBP phosphorylation mirrors the
incorporation of radioactive phosphate into component proteins of
Glut4-vesicles (Fig. 4A) and given the lack of the
detectable phosphatase in this preparation, we conclude that protein
kinase activity associated with Glut4-vesicles is indeed stimulated by
insulin with the maximum at 2 min after insulin administration. Thus,
in all following experiments, the effect of insulin on phosphorylation
of Glut4-vesicles was measured after 2 min of insulin treatment.
To determine whether vesicle-associated protein kinase may participate
in the transduction of the hormonal signal from the cell surface to its
final target: Glut4-containing vesicles, we carried out experiments
with wortmannin, a specific inhibitor of PI 3-kinase and its downstream
signaling. The left panel of Fig.
5 demonstrates that the addition of
wortmannin (5 µM) to the in vitro protein
kinase assay has no effect on phosphorylation of vesicle proteins by
the endogenous protein kinase. This result provides additional evidence
that Ser/Thr kinase activity of PI 3-kinase is not responsible for
phosphorylation of Glut4-vesicles. In analogous experiments, we have
shown that neither PK I (an inhibitor of protein kinase A) nor
calphostin C (an inhibitor of protein kinase C) has any effect on the
pattern of phosphorylated proteins in Glut4-vesicles (not shown). In
contrast, wortmannin in a much lower concentration (100 nM)
was able to prevent insulin-stimulated increase in phosphorylation of
different proteins in Glut4-vesicles when added to adipose cells
in vivo, 20 min prior to insulin administration (Fig. 5,
right panel, and Table I).
Identification of the Vesicle-associated Protein Kinase Activity as
Akt-2--
Data shown in Figs. 4 and 5 suggest that the
vesicle-associated protein kinase may participate in the insulin
signaling downstream of PI 3-kinase. Moreover, in these experiments we
have detected a protein with the molecular mass of 60 kDa, the
phosphorylation of which is transiently activated by insulin in a
wortmannin-sensitive fashion. After 2 min of insulin stimulation, p60
represents one of the major phosphorylated proteins in Glut4-vesicles,
whereas after 15 min of insulin treatment its phosphorylation is hardly detectable (compare Figs. 4 and 5 with Figs. 1 and 3). The molecular mass of this protein corresponds well to that of the newly described lipid-binding Ser/Thr protein kinase, Akt. As has recently been shown,
Akt is activated by insulin in adipose and muscle cells (28-31), is
located downstream of PI 3-kinase (reviewed in Ref. 17), and may
mediate the effect of insulin on Glut4 translocation (32-35). Therefore, we decided to check whether Akt is
directly present in Glut4-containing vesicles. As is seen in Fig.
6, Glut4-vesicles include visible amounts
of Akt-2, an isoform of the enzyme that is predominant in adipocytes
(36). These data correspond well to recent results of Calera et
al. (37), who demonstrated by immunoadsorption and sucrose
gradient centrifugation that Akt-2 may interact with Glut4-containing
vesicles in an insulin- and wortmannin-sensitive fashion. Note, that
according to Western blot analysis, only a small portion (5-7%) of
the total cellular Akt-2 was recovered in the fraction of light
microsomes with the major pool of the enzyme being localized in the
cytosol. Of this membrane-associated enzyme, 3-4% was found in
Glut4-containing vesicles (Fig. 6). However, it is still an open
question as to what portion of the LM fraction represents membranes and
what portion is accounted for by ribosomes, cytoskeleton, heavy protein complexes, etc., that are also pelleted under conditions used to purify
so-called "light microsomes" (38). Thus, Fig. 6 may considerably
underrepresent the fraction of the total membrane-associated Akt-2 that
is present in Glut4-containing vesicles.
Glut4-containing vesicles from insulin-treated cells contain at least
three times more Akt-2 than vesicles from basal cells (Fig. 6). This is
consistent with our data about insulin-stimulated increase in
phosphorylation of vesicle proteins (Figs. 1-5) and suggests that this
phenomenon is likely to be explained by recruiting more active Akt-2
onto Glut4-vesicles.
To estimate to what extent Akt-2 associated with Glut4-containing
vesicles can account for the total protein kinase activity present in
this compartment, we immunoadsorbed Glut4-containing vesicles on 1F8
beads, solubilized their component proteins with 1% Triton,
fractionated this material on a MonoQ column, and determined protein
kinase activity in the chromatorgaphic fractions using MBP as a
substrate (Fig. 7, top panel).
In parallel, we fractionated Triton-solubilized total LMs from adipose
cells on the same column under the same experimental conditions and
determined the position of Akt-2 by Western blotting (Fig. 7,
bottom panel). As is seen in Fig. 7, the major peak of the
protein kinase activity associated with Glut4-containing vesicles is
eluted in fractions 5 and 6, which exactly corresponds to the position
of the membrane-associated Akt-2. Thus, we conclude that Akt-2 is
responsible for the major part of the total MBP kinase activity present
in Glut4-vesicles.
To prove that Akt-2 can phosphorylate component proteins of
Glut4-vesicles, we have purified this enzyme from the cytosol of
insulin-stimulated adipocytes by MonoQ chromatography (Fig. 8) and analyzed phosphorylation of
immunoadsorbed Glut4-vesicles in the absence and in the presence of the
Akt-2-containing chromatographic fraction. In these experiments, after
the completion of protein kinase reaction, immunobeads were eluted
first with 1% Triton X-100 and then with SDS-containing Laemmli sample
buffer to separate Glut4 from other phosphorylated proteins. Fig.
9 shows that exogenous Akt-2 considerably
facilitates incorporation of radioactive phosphate into Glut4-vesicles,
but the pattern of phosphorylated proteins in this compartment is
virtually the same in the absence and in the presence of the exogenous
kinase. In particular, the addition of cytosolic Akt-2 stimulates
phosphorylation of Glut4 itself, p110, p39 SCAMP, p25, and a doublet of
low molecular mass proteins with Mr of 18-21
kDa (compare Fig. 1 with Fig. 9). Thus, exogenous Akt-2 phosphorylates
the same protein substrates in Glut4-vesicles as the endogenous
vesicle-associated insulin-activated protein kinase. This, together
with other evidence presented above, strongly suggests that Akt-2 not
only binds to Glut4-vesicles but also is responsible for
phosphorylation of several proteins in this compartment, including
Glut4 itself.
Results presented here demonstrate that after insulin stimulation
of native rat adipocytes, Akt-2 is rapidly recruited to Glut4-containing vesicles where it can phosphorylate several component proteins of this compartment including Glut4 itself.
Because the amount of Akt-2 recruited onto Glut4-vesicles is rather
low, an important question is the stoichiometry of phosphorylation of
Glut4-vesicles by this protein kinase. Unfortunately, such calculations
are not very accurate, because it is virtually impossible to estimate
the amount of individual proteins in Glut4-vesicles. We know, however,
that when we were isolating individual proteins from these vesicles for
sequencing, we obtained from about 50 mg of LMs, 23 pmol of IRAP
(gp160), 3 pmol of the IGFII/Man 6-phosphate receptor (13), and 8 pmol
of sortilin (11). For this work, we take for immunoadsorption 150 µg
of LMs, so we are likely to obtain not more than 0.01-0.1 pmol of the
individual proteins. Usually, specific activity of ATP in our assay is
2000 cpm/pm, so if incorporation of 32P is 1:1, there
should be 20-200 cpm in a band. This is about what we detect. It must
also be considered that IRAP, the IGFII/Man 6-phosphate receptor, and
sortilin are the major proteins in these vesicles, and the amount of
SCAMPs or p25 may be lower by an order of magnitude, because we were
never even able to see these proteins by silver staining. Lastly,
phosphorylation in our assay takes place not in solution but rather on
the surface of the vesicles (see below), which theoretically should
decrease the efficiency of phosphorylation. So we believe that the
specific incorporation of radioactive phosphate into individual
component proteins of Glut4-vesicles in vitro is at least
1:1.
Phosphorylation of Glut4 has previously been detected and studied in
several laboratories (39-44). In these studies, only one phosphorylation site (Ser488) was found on the cytoplasmic
C terminus of the transporter molecule (39, 40), However, no
insulin-dependent increase in Glut4 phosphorylation was
detected in vivo (39, 40). On the other hand, it has been
shown that mutation of Ser488 as well as the following
double leucine motif (Leu489-Leu490)
significantly impairs Glut4 internalization and targeting (reviewed in
Refs. 5 and 8). Other evidence indicates that translocation of Glut4
from an intracellular pool to the plasma membrane is accompanied by an
unknown modification of the C terminus of the transporter. This causes
"unmasking" of this region of the Glut4 molecule as revealed by the
increased binding of antibodies (45, 46). It has also been suggested
that a regulatory protein may interact with the C terminus of Glut4 in
an insulin-dependent manner and thus control its
intracellular trafficking (15).
Under the conditions of phosphorylation in the experiments presented
here (intact vesicles attached to immunobeads), radioactive phosphate
can be incorporated only into cytoplasmic portions of Glut4 and other
substrates by a peripherally associated protein kinase. Ser-488 is
localized in the cytoplasmic tail of Glut4 and lies in
Arg-Xaa-Xaa-Ser-Leu motif, which represents a phosphorylation site of
Akt (47). It seems likely, therefore, that this residue may be a target
of Akt associated with Glut4-containing vesicles. It will be
interesting to check this hypothesis experimentally and to determine
whether phosphorylation of Glut4 may play a role in the unmasking of
the C terminus of the transporter and its interaction with a putative regulator.
Identification of other phosphorylation substrates in Glut4-vesicles,
such as low molecular mass proteins p25 and p18/21, may also be of
great importance for further understanding of insulin signaling. An
attractive hypothesis is that p25 represents one of the low molecular
mass GTP-binding proteins, and p18/21 may be VAMP and/or cellubrevin.
Glut4-vesicles were reported to contain Rab4 (48), which, in addition,
may be phosphorylated in response to insulin (49). Although we do not
see any significant enrichment of Rab4 in Glut4-containing
vesicles,3 p25 may still
represent some other member of the Rab family of similar
electrophoretic mobility. As it has recently been demonstrated, VAMP
can be phosphorylated in vitro by
calcium/calmodulin-dependent protein kinase II (50),
although the functional role of this phosphorylation remains unknown.
The potential biological significance of sortilin and SCAMPs
phosphorylation is also not known. We are now trying to determine whether regulated secretion of other membrane compartments in different
cells is accompanied by phosphorylation of these proteins.
So, what can be a mechanism for the recruitment of Akt-2 onto
Glut4-vesicles? As it has been shown previously, the latter have high
levels of PI 4-kinase activity (51), and PI 3-kinase is targeted to
these vesicles in response to insulin (26). Thus, after insulin
stimulation, Glut4-containing vesicles should accumulate phosphatidylinositol 3,4,5-trisphosphates and phosphatidylinositol 3,4-bisphosphates, which serve as additional binding sites for Akt
(52-54). This hypothesis is consistent with previous studies demonstrating the importance of membrane recruitment for the activation and function of this enzyme (32, 55, 56). Binding of Akt to
Glut4-vesicles may lead to an increase in phosphorylation of their
component proteins. Furthermore, it is tempting to propose that
this regulatory event may affect interaction of Glut4-vesicles with a
putative trafficking inhibitor (see the Introduction) and thus trigger
exocytosis (Fig. 10).
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-32P]ATP (50-100 µM,
300-5000 cpm/pmol) and phosphorylation substrates (as specified
separately for each experiment) were added to Glut4-containing vesicles
immunoadsorbed on the beads (see the previous paragraph) in protein
kinase buffer. To provide an efficient mixing, the volume of the liquid
phase exceeded the volume of settled beads 2-fold. This suspension was
intensively shaken for 30 min at room temperature, and immunobeads were
separated from the liquid phase. Beads were then washed twice from the
excess of radioactive ATP with protein kinase buffer and 10 mM Tris, pH 7.4, and eluted with either 1% Triton X-100 in
protein kinase buffer or Laemmli sample buffer without
2-mercaptoethanol. In the experiments when exogenous protein substrates
were added to protein kinase assay, 50-µl aliquots of the liquid
phase were applied on 2 × 2 cm squares of Whatman P81
chromatography paper, washed three times with 75 mM
phosphoric acid, and counted in a scintillation counter by Cherenkov.
Alternatively, phosphorylated proteins were electrophoresed, and dried
gels were exposed in a storage phosphor screen cassette and quantitated
in a PhosphorImager (Molecular Dynamics). In cases when the exogenous
substrates were represented by short synthetic peptides, bovine serum
albumin (final concentration, 1%) and trichloroacetic acid (final
concentration, 10%) was added to samples, which were then applied on
Whatman P81 paper and processed as described above.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-32P]ATP was added directly to the material adsorbed
on the beads as described under "Materials and Methods." Under
these conditions, radioactive phosphate is incorporated into several
proteins in Glut4-vesicles. Although the pattern of minor
phosphorylated proteins varies to some extent in different experiments,
we consistently detect phosphorylation of polypeptides with molecular
masses 110, 50, 39, and 25 kDa (Fig.
1A). Insulin stimulation of
adipocytes for 15 min results in the significant (p < 0.01) increase in the incorporation of radioactive phosphate into these
proteins 1.8 ± 0.3, 2.1 ± 0.5, 2.8 ± 0.4, and
3.2 ± 1.3-fold, correspondingly. The electrophoretic mobility of
three of the phosphorylated substrates completely matches that of the
major constituents of Glut4-vesicles, gp110, or sortilin (11), Glut4,
and the high molecular mass isoform of the SCAMP triplet, p39 (19)
(Fig. 1A). To further identify these proteins, 1F8-bound
material from insulin-stimulated cells was subsequently eluted with PBS
containing 1% Triton X-100 and, after that, with Laemmli sample
buffer. As we have shown earlier (13), under these conditions, all
vesicular proteins with the exception for Glut4 can be recovered in the
Triton eluate, whereas Glut4 is resistant to Triton elution and can be
removed from immunobeads only with SDS-containing Laemmli sample
buffer. Triton eluate from 1F8 beads was used for immunoprecipitation of SCAMPs according to the previously published protocol (19). As
expected, phosphorylated Glut4 was detected in the SDS eluate from 1F8
beads, whereas phosphorylated SCAMPs were solubilized in 1% Triton
X-100 and were immunoprecipitated with the specific antibodies (Fig.
1B). The nature of the low molecular mass phosphorylated protein (p25) remains unknown (see "Discussion"). Several other phosphorylated bands can be noticed on the autoradiogram shown in Fig.
1A, including those with the molecular masses over 200 kDa
and below 20 kDa. Electrophoretic mobilities of these proteins correspond to that of the known components of Glut4-vesicles: the
IGFII/Man 6-phosphate receptor (p230) (23) and VAMP (p18) (24). We are
currently trying to identify these proteins.
View larger version (30K):
[in a new window]
Fig. 1.
Phosphorylation of Glut4-containing vesicles
by an endogenous protein kinase in vitro. A, LMs
(0.15 mg of protein) from adipocytes untreated ( ) and treated with
insulin for 15 min (+) were immunoadsorbed with 50 µl of 1F8- or
IgG-coupled beads and phosphorylated in vitro as described
under "Materials and Methods." Material bound to the beads was then
eluted with Laemmli sample buffer, electrophoresed, transferred to
polyvinylidene difluoride membrane, and probed for individual proteins
of Glut4-containing vesicles (their positions are shown on the
left, and the positions of the molecular mass standards on
the right). After Western blot was complete, the membrane
was exposed in a PhosphorImager overnight, and the resulting
autoradiogram is shown. B, LMs (0.15 mg of protein) from
insulin-treated adipocytes were immunoadsorbed with 50 µl of
1F8-coupled beads or IgG-coupled beads (not shown) and phosphorylated
in vitro as described under "Materials and Methods."
Material bound to the beads was eluted with 1% Triton X-100 in PBS and
then with Laemmli sample buffer (lanes 1 and 2).
Triton-eluted material was immunoprecipitated with 4 µg of
anti-SCAMPs monoclonal antibody 3F8 (19) and 40 µl of anti-IgM
antibodies immobilized on agarose (Sigma) (lanes 3 and
4). Lanes 1 and 3, Western blot with
1F8 and 3F8 antibodies respectively; lanes 2 and
4, autoradiogram of phosphorylated proteins. A
representative result of nine independent experiments is shown.
View larger version (20K):
[in a new window]
Fig. 2.
Substrate specificity of the
vesicle-associated protein kinase. LMs (0.15 mg of protein) from
adipocytes untreated and treated with insulin for 15 min were
immunoadsorbed with 50 µl of 1F8-coupled beads and phosphorylated
in vitro as described under "Materials and Methods"
using the following exogenous substrates: 1, none;
2, synthetic peptide (165 µg) corresponding to
phosphorylation site (Asn600-Leu615) on p85
subunit of PI 3-kinase; 3, dephosphorylated casein (86 µg,
Sigma); 4, total histones (86 µg, Sigma); 5,
myelin basic protein (86 µg, Sigma). The figure shows the mean
values ± S.E. of three independent experiments. Absence of the
error signs on some bars indicates that the error is
virtually undetectable.
View larger version (53K):
[in a new window]
Fig. 3.
Salt resistance of the vesicle-associated
protein kinase. LMs (0.15 mg of protein) from adipocytes untreated
( ) and treated with insulin for 15 min (+) were
immunoadsorbed with 50 µl of 1F8- or IgG-coupled beads. Immunobeads
were washed twice with PBS with final salt concentration corresponding
to 0.1, 0.25, and 0.5 M NaCl and, after that, twice with
protein kinase buffer (see "Materials and Methods").
Phosphorylation assay was then carried out in the absence
(A) or in the presence (B) of 86 µg of myelin
basic protein, as described under "Materials and Methods."
A shows a representative result of three independent
experiments (insulin-treated cells only). The positions of the
molecular mass standards are shown on the right.
B shows the mean values ± S.E. of three independent
experiments. The results of nonspecific binding (not shown) were
subtracted from the experimental values.
View larger version (22K):
[in a new window]
Fig. 4.
Activation of the vesicle-associated protein
kinase by insulin; time course. Rat adipocytes were
treated with insulin for specified periods of time and fractionated by
differential centrifugation. LMs (0.15 mg of protein) were
immunoadsorbed with 50 µl of 1F8- or IgG-coupled beads and
phosphorylated in vitro in the absence (A) or in
the presence (B) of 86 µg of myelin basic protein, as
described under "Materials and Methods." A shows a
representative result of three independent experiments. The positions
of the molecular mass standards are shown on the right.
B shows the mean values ± S.E. of three independent
experiments.
View larger version (50K):
[in a new window]
Fig. 5.
Wortmannin inhibits
insulin-dependent activation of the vesicle-associated
protein kinase in vivo, but not in vitro.
Left panel, LMs (0.15 mg of protein) from adipocytes
untreated and treated with insulin for 2 min were immunoadsorbed with
50 µl of 1F8 beads and phosphorylated in vitro in the
absence or in the presence of 5 µM wortmannin.
Right panel, adipocytes were pre-incubated with 100 nM wortmannin (where indicated) for 20 min before insulin
administration for 2 min, and Glut4-vesicles were immunoadsorbed and
phosphorylated in vitro as described in the legend to the
left panel. The positions of the molecular mass standards
are shown on the right. A representative result of four
independent experiments is shown. Quantitation of the results shown in
the right panel of the figure is presented in Table I.
The effect of wortmannin on insulin-stimulated phosphorylation of
Glut4-containing vesicles by an endogenous protein kinase
View larger version (39K):
[in a new window]
Fig. 6.
Akt-2 is specifically present in
Glut4-containing vesicles from rat adipocytes. LMs (0.15 mg of
protein) from adipocytes untreated and treated with insulin for 2 min
were immunoadsorbed with 50 µl of 1F8 or IgG beads. Eluted material
along with original LMs (100 µg) and cytosol (Cyt) (100 µg) was analyzed by Western blot with anti-Akt-2 and anti-Glut4
antibodies. A representative result of three independent experiments is
shown.
View larger version (27K):
[in a new window]
Fig. 7.
Vesicle-associated protein kinase
co-fractionates with Akt-2 on a MonoQ column. Top
panel, LMs (1.4 mg of protein) from adipocytes treated with
insulin for 2 min were immunoadsorbed with 400 µl of 1F8 beads and
eluted with 1.6 ml of 1% Triton X-100 in protein kinase buffer. Eluted
material was fractionated on a MonoQ column (Amersham Pharmacia
Biotech) as described under "Materials and Methods." Protein kinase
activity was measured in aliquots (70 µl) of the gradient fractions
with MBP (10 µg) as a substrate. After phosphorylation, samples were
electrophoresed, and MBP-incorporated radioactivity was measured in a
PhosphorImager. Bottom panel, LMs (1 mg) from adipocytes
treated with insulin for 2 min were solubilized in 1% Triton X-100 and
fractionated on a MonoQ column in parallel with the material eluted
from 1F8 beads. Aliquots (120 µl) of even gradient fractions were
analyzed by Western blotting with anti-Akt-2 antibody.
Triton-solubilized LMs (40 µg) was loaded on the first lane of the
gel as a reference. A representative result of three independent
experiments is shown.
View larger version (33K):
[in a new window]
Fig. 8.
Purification of Akt-2 from cytosol on a MonoQ
column. Cytosol (5.2 mg) from adipocytes treated with insulin for
2 min was fractionated on a MonoQ column (Amersham Pharmacia Biotech)
as described under "Materials and Methods." Fractions were analyzed
for the total protein content (upper panel), and aliquots
(120 µl) of even fractions were analyzed by Western blotting with
anti-Akt-2 antibody (bottom panel). A representative result
of five independent experiments is shown.
View larger version (78K):
[in a new window]
Fig. 9.
Exogenous Akt-2 can phosphorylate component
proteins of Glut4-containing vesicles. LMs (0.2 mg of protein)
from adipocytes untreated and treated with insulin for 2 min were
immunoadsorbed with 50 µl of 1F8 beads and phosphorylated in
vitro as described under "Materials and Methods." An aliquot
(77 µl) of Akt-2-containing fraction 10 (Fig. 8) was added instead of
an equal volume of buffer to the protein kinase assay where indicated.
After phosphorylation, beads were washed of radioactive ATP and eluted
first with 1% Triton X-100 and then with Laemmli sample buffer. Eluted
material was electrophoresed, and radioactive proteins were analyzed in
a PhosphorImager. The positions of the molecular mass standards are
shown on the right. A representative result of three
independent experiments is shown.
DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
View larger version (13K):
[in a new window]
Fig. 10.
A proposed insulin signaling pathway in fat
cells. Glut4-containing vesicles possess a high basal level of PI
4-kinase activity (51). Insulin stimulation causes rapid targeting of
PI 3-kinase to this compartment (26). These two enzymes working
together produce phosphatidylinsositol phosphates that serve as docking
sites for the recruitment and activation of Akt. The latter
phosphorylates vesicle proteins, which leads to disassociation of
vesicles from an intracellular anchor and their default fusion with the
plasma membrane.
The role of Akt in activation of glucose transport in adipose cells has
recently been questioned based on the inability of the
dominant-negative Akt mutant to inhibit this process in transfected 3T3-L1 cells (57). However, in another set of experiments,
kinase-inactive Akt mutant significantly inhibited
insulin-dependent translocation of Glut4 in rat adipose cells
(35). Moreover, ceramide appears to inhibit Akt kinase activity in
parallel with insulin-stimulated Glut4 translocation (58). We believe,
therefore, that Akt does have an important role in propagation of the
insulin signal in adipose cells, and our results suggest that it may
play an important role in the downstream signaling events proximal to
Glut4-containing vesicles.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Galini Thoidis for help with several experiments, insightful discussions, and critical reading of the manuscript and to Dr. Ron Morrison for valuable comments on the manuscript. We especially thank Dr. Olga Rudick for advice and help with the analysis of the data.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant R01DK52057 from the National Institutes of Health, by Research Grant 197029 from the Juvenile Diabetes Foundation, and by a research grant from the American Diabetes Association (to K. V. K.).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: Boston University
School of Medicine, Dept. of Biochemistry, K401, 715 Albany St.,
Boston, MA 02118. Tel.: 617-638-5049; Fax: 617-638-5339; E-mail:
kandror{at}med-biochem.bu.edu.
The abbreviations used are: IGF II, insulin-like growth factor; Glut4-vesicle, Glut4-containing vesicle; PI, phosphatidylinositol; PBS, phosphate-buffered saline; LM, light microsome; MBP, myelin basic protein.
2 S. Heydrick and K. V. Kandror, unpublished observations.
3 K. V. Kandror and P. F. Pilch, unpublished observations.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|