Department of Biochemistry, School of Medical Sciences, University of
Bristol, Bristol, BS8 1TD, UK
* These authors contributed equally to this work
Author for correspondence (e-mail:
j.tavare{at}bris.ac.uk
)
Accepted 14 May 2002
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Summary |
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Key words: Insulin, GLUT4, Signalling, Protein kinase B/Akt, Adipocytes
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Introduction |
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It is well established that phosphoinositide 3-kinase (PI 3-kinase) is
pivotally involved in insulin-induced glucose uptake
(Kanai et al., 1993;
Okada et al., 1994
;
Hara et al., 1994
), although a
parallel PI 3-kinase-independent pathway, comprising CAP, Cb1, C3G and TC10,
has also been proposed to play a role
(Baumann et al., 2000
;
Chiang et al., 2001
). The
mechanism by which PI 3-kinase mediates the effect of insulin on GLUT4
translocation is controversial. Two protein kinases that lie downstream of PI
3-kinase have received considerable attention, namely protein kinase B (PKB)
and the atypical protein kinase C-family members
and
(PKC
and PKC
).
PKB is composed of an N-terminal pleckstrin homology domain (PH domain), a
protein kinase domain and a C-terminal regulatory domain containing the
phosphorylation site Ser473 (reviewed in
Vanhaesebroeck and Alessi,
2000). Binding of phosphoinositide-3,4,5-trisphosphate
[PtdIns(3,4,5)P3] to the PH domain allows recruitment of
PKB from the cytoplasm to the plasma membrane, where it is phosphorylated and
activated by PDK1 on Thr308 and, by an as yet ill-defined kinase, on Ser473.
PKB can be rendered constitutively active by substitution of Thr308 and Ser473
for aspartic acid residues, which mimic phosphorylation [PKB[DD]
(Alessi et al., 1996
)], or by
insertion of a myristoylation signal sequence from c-src at the
N-terminus [Myr-PKB (Andjelkovic et al.,
1997
)]. The latter allows constitutive membrane localisation and
thus phosphorylation on Thr308 and Ser473
(Andjelkovic et al., 1997
).
Constitutively active mutants of PKB mimic the ability of insulin to
promote glucose uptake and GLUT4 translocation in muscle and adipose cells
(Kohn et al., 1996;
Tanti et al., 1997
),
suggesting that this protein kinase may be a crucial mediator of insulin's
effect on glucose transport. Studies using a kinase-inactive PKB mutant
(Cong et al., 1997
), in which
the PDK1 and PDK2 phosphorylation sites were additionally mutated [PKB-AAA
(Wang et al., 1999
)], and
inhibitory anti-PKB antibodies (Hill et
al., 1999
) were found to block insulin-stimulated glucose uptake.
This is consistent with a role for PKB in mediating insulin-stimulated GLUT4
translocation. Furthermore, adipocytes isolated from diabetic rats or humans
exhibited a parallel defect in insulin-stimulated glucose uptake and PKB
activation and phosphorylation (Carvalho et
al., 2000a
; Carvalho et al.,
2000b
).
Equally, there is evidence against a role for PKB and this includes a
report that a dominant-negative PKB failed to block insulin-stimulated glucose
uptake in adipocytes under conditions where endogenous PKB activation was
severely compromised (Kitamura et al.,
1998). Also, we (L.M.F. and J.M.T., unpublished) (see Results) and
others (Wang et al., 1999
)
have been unable to demonstrate inhibitory effects of a cytosolic
kinase-inactive PKB on insulin-stimulated GLUT4 translocation.
Constitutively active forms of PKC and PKC
, in which the
pseudo-substrate inhibitory region was deleted, promote GLUT4 translocation
and glucose uptake in adipocytes in the absence of insulin
(Bandyopadhyay et al., 1997
;
Standaert et al., 1997
;
Bandyopadhyay et al., 1999
)
(L.M.F. and J.M.T., unpublished). Furthermore, kinase-inactive mutants of
PKC
and PKC
have been reported to block insulin-stimulated GLUT4
translocation. These data argue in favour of an additional role for the
atypical PKCs in insulin action on glucose uptake.
Growth factors have been reported to induce a translocation of PKB from the
cytosol to the plasma membrane and then to the nucleus
(Meier et al., 1997;
Goransson et al., 1998
).
Interestingly, insulin has also been reported to stimulate a recruitment of
PKB to GLUT4-containing vesicles (Calera et
al., 1998
; Kupriyanova and
Kandror, 1999
). In the current study, therefore, we have examined
the functional consequences of targeting kinase-inactive and constitutively
active mutants of PKB to GLUT4 vesicles by fusion with GLUT4 itself. Our
results suggest that PKB may indeed have an important functional role in GLUT4
vesicle translocation by acting at, or within the vicinity of, the GLUT4
vesicles themselves.
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Materials and Methods |
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Plasmids
The plasmid pIRAP-GFP comprises IRAP tagged at the C-terminus with GFP and
was generated from a partial IRAP cDNA clone kindly provided by S. Keller
(Dartmouth Medical School, Hanover, USA). An IRAP fragment (bases 66-533) was
generated by PCR using the primers 5'-TTTTAAGCTTGCGAAGATGGAGACC-3'
and 5'-TTTTGGATCCAATCGGCTGAATGAG-3'. The resulting PCR product was
cloned into the HindIII and BamHI sites of the mammalian
expression vector pcDNA3 (Invitrogen, Carlsbad, USA) to produce pcDNA3-IRAP.
GFP was subcloned at the 3' end of IRAP by first amplifying using PCR a
GFP fragment using the primers 5'-TTTTGGATCCAGTAAAGGAGAAGAAGAA-3'
and
5'-TTTTCTCGAGTTACCTCAGGTCCTCCTCCGAGATCAGCTTCTGCATTTTGTATAGTTCATC-3';
this incorporates a Myc-epitope tag at the 3'end of the GFP moiety, and
BamHI and XhoI sites at the 5' and 3' ends of
the PCR product, respectively. This PCR fragment was then subcloned into the
BamHI and XhoI sites of pcDNA3-IRAP. The GFP moiety in the
resulting plasmid essentially replaces the lumenal amino peptidase domain of
IRAP.
Plasmids (pCMV5) containing (i) wild-type PKB (PKB-WT), (ii) constitutively
active PKB[Thr308Asp/Ser473Asp] (PKB[DD]), (iii) constitutively
active myristoylated PKB (Myr-PKB), (iv) kinase-inactive PKB[Lys179Ala]
(PKB[KD]) and (v) PKB lacking the PH-domain (PKB[PH];
i.e. a deletion of amino acid residues Met1Ala117) were kindly provided
by D. Alessi (University of Dundee) and B. Hemmings (Friedrich Miescher
Institute, Basel). All the PKB sequences included an N-terminal HA-tag. A
kinase-inactive mutant of Myr-PKB (Myr-PKB[KD]) and a kinase-inactive
PKB lacking the PH domain (
PH-PKB[KD]) were constructed by
site-directed mutagenesis (thus introducing a K179A substitution) using the
QuikChangeTM site-directed mutagenesis kit (Stratagene Cloning Systems,
La Jolla, CA) and the plasmids Myr-PKB or PKB[
PH] described
above as templates, respectively.
PKB mutants were fused to the N-terminus of GLUT4 as follows.
PKB[KD]-GLUT4 and PKB[DD]-GLUT4 were constructed by PCR
amplification of fragments of pPKB[KD] and pPKB[DD],
respectively, between bases 550 and 1638 using the primers
5'-TTTTAAGCTTGACGAGATGTATCCTTACGACGTCCCCGACTACGCCAGTCTGATGGACTTCCGGTCG-3'
and 5'-TTTTGGATCCGGCCGTGCTGCTGGC-3'; an N-terminal HA-tag is thus
introduced into each construct. The resulting PCR product was cloned into the
HindIII and BamHI restriction sites of the plasmid
pGFP-GLUT4 (Dobson et al.,
1996), such that the PKB sequence replaces the GFP sequence at the
5' end of full-length GLUT4 in the plasmid pcDNAI/Neo. Thus the PKB
sequences within PKB[KD]-GLUT4 and PKB[DD]-GLUT4 lack the PH
domain.
The human FKHR sequence (bases 386-2353) was amplified by PCR from HEK293 cDNA using the primers 5'-TTTTCTCGAGATGGCCGAGGCGCCTCAGGTG-3' and 5'-TTTTGTCGACTCAGCCTGACACCCAGCTATG-3' and was cloned into pGEM-TEasy (Promega). At recombination sites were subsequently added by PCR at the 5' and 3' extremities of the FKHR cDNA and the product cloned downstream of EGFP (Clontech) in pCI-Neo using the GatewayTM Cloning System (Life Technologies).
Cell culture, adipocyte differentiation and microinjection
3T3-L1 fibroblasts were grown on coverslips, differentiated into adipocytes
and microinjected with plasmid DNA as previously described
(Oatey et al., 1997). Plasmids
were injected at 30 to 150 µg/ml, and then the cells were then incubated in
DMEM containing 10% (v/v) myoclone-plus foetal calf serum for 16-24 hours. The
cells were serum starved for 2 hours prior to any further manipulations. Where
appropriate, 100 nM insulin was added for 30 minutes prior to fixation.
Immunofluorescence analysis
Cells were fixed and permeabilised using 4% paraformaldehyde and 1% Triton
X-100 in phosphate-buffered saline (PBS), respectively. For IRAP detection,
the cells were incubated for 45 minutes with a 1:500 dilution of anti-IRAP
antiserum (a gift from S. Keller, University of Dartmouth, New Hampshire, USA)
and then for 30 minutes with TRITC-conjugated anti-rabbit IgG. All antibody
dilutions were in 3% bovine serum albumin in PBS. The presence of PKB
expression was detected using 10 µg/ml of monoclonal anti-HA antibody
(Berkley Antibody Co. Richmond, USA) followed by incubation with
TRITC-conjugated anti-mouse IgG as described previously
(Foran et al., 1999). For
transferrin-Alexa568 uptake assays, the cells were serum starved and placed in
medium containing 20 µg/ml transferrin-Alexa568 for 1 hour at 37°C and
then fixed in 4% paraformaldehyde for subsequent immunofluorescence analysis
as described above.
Confocal microscopy, image and statistical analysis
Laser-scanning confocal microscopy of fixed cells was performed with a
Leica SP2 inverted Confocal Imaging Spectrophotometer controlled with Leica
Confocal Software (Leica, Heidelberg, Germany). Visualisation of GFP was
achieved by excitation with a 488 nm laser and collection of fluorescence
using a 500-530 nm emission window. Detection of TRITC-conjugated antibody
staining was achieved by excitation with a 543 nm laser, and collection of
emitted fluorescence was achieved by using a 570-620 nm window. Adipocytes
were classified as exhibiting IRAP-GFP translocation to the plasma membrane by
the visual presence of a well defined ring of GFP fluorescence around the
plasma membrane as previously described
(Foran et al., 1999;
Thurmond and Pessin, 2000
).
The results were validated by double-blind analysis by two independent workers
and are expressed as mean±s.d. of the percentage of insulin-responsive
cells (i.e. the proportion of cells that displayed a translocated phenotype)
in three independent experiments, with each data point comprising a minimum of
50 cells. In some experiments further quantification was achieved using
Metamorph by drawing a region of interest around the `outer' and `inner' faces
of the plasma membrane and determining the total integrated fluorescence
intensities enclosed by each of these regions (Iouter and
Iinner, respectively). The amount of IRAP-GFP in the
plasma membrane was taken as
100(IouterIinner)/Iouter
as previously described by us (Foran et
al., 1999
). For quantification of FKHR translocation, the
fluorescence intensities were calculated within a region of interest drawn
around the entire cell and another inside the nuclear envelope
(Inuclear and Icellular,
respectively). The amount of FKHR in the nucleus, as a percentage of the
total, was taken as
100(Inuclear/Icellular).
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Results |
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|
Placing a 27 kDa green fluorescent protein (GFP) tag at the N-terminus of
GLUT4 appears not to perturb the distribution or trafficking of GLUT4 relative
to that expected of the native protein
(Oatey et al., 1997;
Fletcher et al., 2000
). By
contrast, placing GFP at the C-terminus of GLUT4 causes mistargeting, at least
in some cells (L.M.F. and J.M.T., unpublished). We reasoned, therefore, that
in order to target PKB mutants to GLUT4 vesicles we should place the PKB
moiety at the N-terminus of GLUT4. Furthermore, the PH domain was removed from
the PKB moiety to prevent inadvertent targeting of the contruct to the plasma
membrane.
The PKB[DD]-GLUT4 (Fig. 1E,F) and PKB[KD]-GLUT4 (Fig. 1G,H) constructs were expressed in 3T3-L1 adipocytes by microinjection, and the cells were then either fixed and stained with antibodies to the GLUT4-vesicle-resident protein, IRAP (Fig. 1E,G), or the cells were incubated prior to fixation in the presence of transferrin-Alexa568 to fluorescently label recycling endosomes to equilibrium (Fig. 1F,H).
It is well established that GLUT4 and IRAP colocalise in adipocytes, and
IRAP can be considered to be an excellent surrogate GLUT4 vesicle marker
(Kandror and Pilch, 1994;
Martin et al., 1997
). Given
that the PKB constructs were tagged to GLUT4 we could not examine their
distribution relative to native GLUT4. For this reason, we examined the
colocalisation of the targeted PKB constructs with endogenous IRAP. The
results demonstrate that, in the basal state, PKB[DD]-GLUT4 and
PKB[KD]-GLUT4 were targeted to intracellular vesicles that also
contain endogenous IRAP, as would be expected of native GLUT4 (see
Fig. 1E for
PKB[DD]-GLUT4 and Fig.
1G for PKB[KD]-GLUT4). Indeed, the colocalisation of the
PKB-GLUT4 constructs and endogenous IRAP was extremely high.
GLUT4 and IRAP co-exist within a highly specialised intracellular membrane
pool in adipocytes called `GLUT4 storage vesicles' [GSVs
(Rea and James, 1997)]. Both
proteins, however, are also found within recycling endosomes that contain
transferrin receptors and GLUT1. To examine the degree to which the PKB-tagged
GLUT4 constructs were excluded from the endosomal pool we labelled recycling
endosomes to equilibrium with transferrin-Alexa568. Both
PKB[DD]-GLUT4 and PKB[KD]-GLUT4 displayed a partial
colocalisation with transferrin receptors with significant targeting to
vesicles that exclude the fluorescent dye
(Fig. 1F,H) as exhibited by
native GLUT4. Taken together, these data demonstrate that PKB mutants can be
efficiently targeted to bona fide GLUT4/IRAP vesicles in 3T3-L1 adipocytes by
fusion at the N-terminus of GLUT4.
We next examined whether insulin was capable of inducing a translocation of
the PKB[DD]-GLUT4 and PKB[KD]-GLUT4 fusion constructs to the
plasma membrane as it does native GLUT4. The PKB[DD]-GLUT4 and
PKB[KD]-GLUT4 constructs were expressed in 3T3-L1 adipocytes, which
were then treated with or without insulin for 30 minutes. Insulin induced a
translocation of PKB[DD]-GLUT4 to the plasma membrane as we have
previously demonstrated for a GFP-tagged GLUT4
(Fig. 2A)
(Oatey et al., 1997;
Foran et al., 1999
). By
contrast, insulin appeared to be incapable of promoting a translocation of the
PKB[KD]-GLUT4 fusion protein to the plasma membrane
(Fig. 2B).
|
IRAP-GFP is a suitable surrogate marker for GLUT4 trafficking in
3T3-L1 adipocytes
We next wanted to examine the effect of the fusion proteins on
insulin-dependent translocation of endogenous GLUT4. However, as we were
already overexpressing GLUT4 in the cells and because we are unable to
distinguish native GLUT4 from the transfected PKB[DD]-GLUT4 and
PKB[KD]-GLUT4 constructs, we examined the translocation of IRAP
instead.
To visualise IRAP translocation in transfected cells, we used an IRAP-GFP
fusion protein in which the GFP moiety was placed at the C-terminus such that
the GFP effectively replaced the lumenal amino peptidase domain (see Materials
and Methods). This IRAP-GFP fusion protein almost completely colocalised with
endogenous GLUT4 (Fig. 3A) and
showed only a partial overlap in expression with transferrin-Alexa568 labelled
recycling endosomes (Fig. 3B). The construct translocates to the plasma membrane in response to insulin
(Fig. 3C) and re-internalises
efficiently after the withdrawal of the insulin signal (L.M.F. and J.M.T.,
unpublished). This makes IRAP-GFP an excellent surrogate marker for GLUT4
vesicle distribution in these studies. Indeed, it appears to be an improvement
on our previously reported GFP-GLUT4 construct
(Oatey et al., 1997), which we
find does not efficiently re-internalise upon insulin withdrawal
(Powell et al., 1999
).
|
Effects of PKB-tagged GLUT4 fusion proteins on IRAP translocation to
the plasma membrane
Cong et al. reported that a PKB[K179A] mutant has a dominant-negative
effect on insulin-stimulated GLUT4 translocation in rat adipose cells
(Cong et al., 1997). However,
we have been consistently unable to demonstrate any significant
dominant-negative effect of a PKB[KD] mutant on insulin-stimulated
IRAP-GFP translocation (Fig.
4A, Fig. 5A,B) or
on GFP-GLUT4 translocation (P. Oatey and J.M.T., unpublished) in 3T3-L1
adipocytes.
|
|
In contrast to the apparent lack of a dominant-negative effect of the PKB[KD], when this mutant was targeted to GLUT4 vesicles by fusion with GLUT4 (i.e. PKB[KD]-GLUT4), we found an unexpectedly profound inhibition of insulin-stimulated IRAP-GFP translocation to the plasma membrane (Fig. 4B) whether we quantified the response by determining the number of responding cells (Fig. 5A) or whether we determined it more quantitatively by establishing the amount of IRAP-GFP residing at the plasma membrane (Fig. 5B). This was consistent with the fact that the PKB[KD]-GLUT4 construct itself did not translocate to the plasma membrane in response to insulin (Fig. 2B). We also found that a mutant PKB possessing an N-terminal myristoylation signal sequence and a Lys179Ala mutation, which renders it kinase-deficient (Myr-PKB[KD]), also had a dominant-negative effect on insulin-stimulated IRAP-GFP translocation (Fig. 4C; Fig. 5A,B).
The PKB[KD]-GLUT4 construct differs from the PKB[KD] in two respects; (i) it is targeted to the GLUT4 vesicle, and (ii) it lacks the PH domain. To ensure that it was the targeting of PKB[KD] to GLUT4 that induced dominant-negativity, rather than the removal of the PH domain, we next examined the effect of a untargeted PKB[KD] mutant in which the PH domain was deleted. When this mutant was co-expressed with IRAP-GFP it exhibited no apparent dominant-negative effect (Fig. 5C), thus demonstrating that dominant-negativity of PKB[KD]-GLUT4 was indeed the result of targeting the PKB moiety to GLUT4 vesicles.
To determine whether the PKB[KD]-GLUT4 blocked any other effects
of insulin we examined the ability of insulin to induce the translocation of
the Forkhead transcription factor, FKHR, out of the nucleus, an effect which
is PKB-dependent and has been proposed to underlie the ability of insulin to
repress the expression of several metabolic genes in liver cells
(Guo et al., 1999;
Rena et al., 1999
). We
overexpressed a GFP-tagged FKHR in 3T3-L1 adipocytes and found that, as
expected, insulin caused a pronounced exit of FKHR out of the nucleus
(Fig. 6A,C). The ability of
insulin to promote FKHR translocation out of the nucleus was unaffected by the
presence of the PKB[KD]-GLUT4 construct
(Fig. 6B,C).
|
We next examined the effect of expressing constitutively active PKB mutants
on IRAP-GFP translocation in 3T3-L1 adipocytes. Others have previously
demonstrated that membrane-targeted constitutively active PKB mutants promote
an increased GLUT4 translocation and glucose uptake in both adipose and muscle
cells (Tanti et al., 1997;
Hajduch et al., 1998
). We have
previously demonstrated that a PKB mutant, rendered constitutively active by
virtue of replacement of both its regulatory Thr308 and Ser473 phosphorylation
sites with aspartic acid residues (PKB[DD]), also causes constitutive
translocation of GLUT4 in the absence of insulin
(Foran et al., 1999
). This was
also confirmed to be the case for IRAP-GFP
(Fig. 7A,D). Indeed, as we have
previously noted for GFP-tagged GLUT4, the PKB[DD] mutant promoted a
visible translocation of IRAP-GFP in approximately 60% of the 3T3-L1
adipocytes that co-expressed PKB[DD] and IRAP-GFP
(Fig. 7D). This effect was
indistinguishable from that of insulin alone
(Fig. 7D). The remaining 3T3-L1
adipocytes (approximately 40%) appear to be considerably less sensitive to the
actions of insulin and to the plasma-membrane-localised PKB[DD]
mutant such that IRAP-GFP cannot be visibly detected at the cell surface.
|
In contrast to the effect of PKB[DD], when PKB[DD] was targeted to GLUT4 vesicles (PKB[DD]-GLUT4; Fig. 7B,D), it had a weak insulin-like effect on IRAP-GFP translocation (translocation occurring in approximately 25% of the cells (Fig. 7D), and this effect was additive with insulin. However, we consistently found that those cells that exhibited a visible translocation of IRAP-GFP also had high levels of expression of the PKB[DD]-GLUT4 mutant (data not shown).
The constitutively active Myr-PKB had a profound effect on IRAP-GFP translocation, such that approximately 80% of the cells responded, but the additional presence of insulin had no further effect (Fig. 7C,D).
![]() |
Discussion |
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In the current study, we visualised GLUT4 vesicle translocation using IRAP,
a well established bona fide GLUT4 vesicle-resident protein, tagged with GFP
(Kandror and Pilch, 1994;
Martin et al., 1997
). The GFP
moiety of IRAP-GFP is expressed either within the vesicle lumen or on the
extracellular face of the plasma membrane after translocation although,
despite the fact that GFP fluorescence is reported to be pH sensitive
(Miesenbock et al., 1998
), we
do not find any detectable change in fluorescence intensity of the GFP moiety
during exocytosis (data not shown). As we have previously shown for GFP-tagged
GLUT4 (Oatey et al., 1997
;
Fletcher et al., 2000
),
IRAP-GFP expressed in 3T3-L1 adipocytes exhibited colocalisation with
endogenous GLUT4, partial colocalisation with transferrin-Alexa568-labelled
recycling endosomes and a translocation to the plasma membrane in response to
insulin (Fig. 3); these are all
characteristics expected of native IRAP and are also observed with the
PKB[DD]-GLUT4 construct. IRAP-GFP was, therefore, used as a surrogate
marker for the localisation for GLUT4/IRAP-containing vesicles.
It has been previously reported that a PKB[K179A] mutant has a
dominant-negative effect on insulin-stimulated GLUT4 translocation in rat
adipose cells, although the inhibition of the insulin effect was only
approximately 20% (Cong et al.,
1997). In 3T3-L1 adipocytes, we have consistently found no
significant effect using a similar PKB mutant (PKB[KD]) construct
that appears to be expressed predominantly within the cytosol
(Fig. 1B,
Fig. 4A), although small
amounts were also found in the plasma membrane. The fact that insulin
stimulated the translocation of PKB[DD]-GLUT4, but not
PKB[KD]-GLUT4, to the plasma membrane gave us our first clue that
PKB[KD]-GLUT4 may act as a dominant-negative protein capable of
blocking GLUT4/IRAP vesicle translocation.
Our further investigations demonstrated that a PKB[KD] mutant targeted to the GLUT4 vesicle (PKB[KD]-GLUT4; Fig. 4B) had a potent dominant-negative effect on IRAP-GFP translocation (Fig. 5A,B). Furthermore, a kinase-inactive PKB containing a myristoylation signal sequence (Myr-PKB[KD]), which was expressed both at the plasma membrane and within intracellular vesicles containing endogenous IRAP (Fig. 4C), was also a potent dominant-negative inhibitor of IRAP-GFP translocation (Fig. 4C, Fig. 5A,B). As both PKB[KD]-GLUT4 and Myr-PKB[KD] are targeted to intracellular vesicles containing IRAP, we concluded that PKB[KD] must be targeted to intracellular membranes to exhibit a dominant-negative effect on insulin-stimulated GLUT4/IRAP vesicle translocation.
Interestingly, the dominant-negative effect of this GLUT4 vesicle-targeted PKB[KD] mutant was specific to blocking IRAP translocation, as the insulin-dependent translocation of FKHR out of the nucleus was not inhibited by the construct (Fig. 6). As the ability of insulin to promote FKHR translocation is dependent on the activation of PKB, this strongly suggests that PKB activation, and by inference PI 3-kinase and PDK1 activation, are largely intact in cells expressing the PKB[KD]-GLUT4 construct. Thus the ability of the PKB[KD]-GLUT4 construct to inhibit IRAP translocation is likely to be the result of a localised inhibition of phosphorylation of a specific PKB substrate that resides at or close to the GLUT4 vesicle.
GLUT4 continuously recycles between the cell interior and plasma membrane
(Yang and Holman, 1993;
Holman et al., 1994
). Although
we do not detect any of the PKB[KD]-GLUT4 fusion protein in the
plasma membrane, it is possible that PKB[KD]-GLUT4 and
Myr-PKB[KD] exhibit dominant-negative effects only when they reside
in the plasma membrane. This is unlikely, however, because we find that the
PKB[KD] mutant, which is clearly visible in the plasma membrane in
some cells, is without any detectable dominant-negative effect on IRAP-GFP
translocation in those cells.
Our data may provide an explanation for the inability of some groups to
observe a dominant-negative effect of PKB mutants on GLUT4 translocation and
glucose uptake. In studies where a dominant-negative effect has been observed
(e.g. Cong et al., 1997;
Wang et al., 1999
), but not
others (Kitamura et al.,
1998
), the PKB mutants may have been expressed at a level
sufficient for the endogenous PKB associated with GLUT4 vesicles to be
inhibited. In the study of Kitamura and colleagues
(Kitamura et al., 1998
), the
dominant-negative PKB mutant (Akt-AA) potently (>80%) blocked the
stimulation of endogenous PKB. However, this mutant may have lacked a
dominant-negative effect on GLUT4 translocation because it did not block the
activation of the GLUT4-resident pool of PKB, which may make up the residual
(20%) activity that remained.
A logical conclusion from our data is that PKB plays a crucial role at, or
in the vicinity of, GLUT4 vesicles. In our study we used PKB (Akt1) in
the fusion constructs, although others have reported that PKBß (Akt2)
localises to GLUT4 vesicles. Although Akt1-/- knockout mice exhibit
normal whole body glucose tolerance and disposal
(Cho et al., 2001a
), a role
for PKB
(Akt1) in adipose tissue glucose uptake cannot be excluded as
(i) insulin-stimulated glucose uptake into adipocytes of the mice was not
measured and (ii) the defect in insulin-stimulated glucose uptake into
adipocytes (and muscle) from Akt2-/- mice was only partial
(Cho et al., 2001b
). Thus it
remains possible that PKB
and PKBß play redundant roles in
insulin-stimulated GLUT4 translocation.
Lowering the temperature of 3T3-L1 adipocytes to 23°C causes an
accumulation of GLUT4 vesicles underneath the plasma membrane in response to
insulin as a result of a proposed block in the final fusion step
(Elmendorf et al., 1999). Our
data suggest that PKB does not act at this fusion step, since we do not
observe IRAP-GFP vesicles accumulating under the plasma membrane in
insulin-treated cells expressing PKB[KD]-GLUT4. As we observe no
difference in IRAP-GFP distribution between non-injected and
PKB[KD]-GLUT4-expressing cells, the dominant-negative
PKB[KD]-GLUT4 must act on the initial mobilisation of GLUT4/IRAP
vesicles by insulin.
Dominant-negative PKB[KD]-GLUT4 may act by preventing the
phosphorylation of substrates by endogenous PKB in the vicinity of the
GLUT4/IRAP vesicle. Indeed, partially purified PKBß/Akt2 has been
reported to phosphorylate several integral components of the GLUT4 vesicle
(Kupriyanova and Kandror,
1999), although the identity and function of these substrates are
currently not known. Alternatively, the dominant-negative
PKB[KD]-GLUT4 could act by sequestering upstream activators such as
PDK1, thus preventing the activation of endogenous PKB
and PKBß.
Thus, although we have used PKB
in our constructs, it may be able to
block the activation of PKBß by this mechanism. This is considered
unlikely, however, as the PKB[KD]-GLUT4 mutant did not block the
PKB-dependent translocation of FKHR out of the nucleus, suggesting that PKB
activation is largely intact (Fig.
6). The dominant-negative PKB[KD]-GLUT4 may act by
interfering with the stimulation of atypical PKCs
/
by insulin
in the vicinity of the GLUT4 vesicles, as these are also phosphorylated and
activated by PDK1 and are reported to play a role in insulin-stimulated
glucose uptake (Bandyopadhyay et al.,
1997
; Kotani et al.,
1998
). However, again we would consider this to be unlikely as the
insulin-stimulated translocation of FKHR from the nucleus also requires PI
3-kinase and PDK1 activation (both of which are required for PKC
activation), and our data suggest that these events must also be largely
intact in cells expressing the PKB[KD]-GLUT4 construct.
A constitutively active PKB[DD] fully mimics the effect of insulin
causing a visible translocation of IRAP-GFP in approximately 60% of the cells
(Fig. 7A,D), although the
effect of PKB[DD] and insulin were not additive. By contrast,
PKB[DD] targeted to GLUT4 vesicles appears to only partially mimic
the effect of insulin on IRAP-GFP translocation
(Fig. 7B,D). Careful
examination of many confocal images revealed that the cells that exhibit a
high level of PKB[DD]-GLUT4 expression also contain significant
amounts of IRAP-GFP in the plasma membrane; at lower levels of
PKB[DD]-GLUT4 expression, both PKB[DD]-GLUT4 expression and
IRAP-GFP are exclusively intracellular. This suggests that
PKB[DD]-GLUT4 does not promote IRAP-GFP translocation unless, as a
result of overexpression, sufficient PKB[DD]-GLUT4 leaks out to the
plasma membrane from where it promotes GLUT4/IRAP vesicle translocation. This
would be consistent with the fact that PKB[DD] causes
insulin-independent GLUT4/IRAP vesicle translocation and is predominantly
expressed within the plasma membrane (Fig.
7A,D). The effect of exclusive plasma membrane localisation of
active PKB, using a K-Ras CAAX-targeting motif for example, is difficult to
assess as this mutant has been reported to exhibit dominant-negative rather
than constitutive activity (van Weeren et
al., 1998). It should also be noted that we cannot exclude the
possibility that the PKB[DD] moiety tethered to GLUT4 lacks
dominant-negative activity because it has restricted access to the putative
substrate located on the vesicle as a result of its orientation away from the
vesicle surface.
In conclusion, we propose a model in which PKB activation at, or close to, the GLUT4 vesicle is necessary but not sufficient to induce GLUT4 vesicle translocation to the plasma membrane. PKB activation must also occur in the plasma membrane to promote efficient GLUT4 vesicle translocation, as suggested by the fact that a constitutively active plasma-membrane-localised PKB (i.e. the PKB[DD] mutant) can stimulate GLUT4/IRAP translocation in the absence of insulin. Identification of the intracellular protein(s) and/or substrates with which PKB[KD]-GLUT4 interacts to prevent GLUT4 vesicle translocation will provide important clues as to how insulin stimulates GLUT4 translocation in adipose cells.
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