From the Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755 and the § Harvard Microchemistry and Proteomics Analysis Facility, Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138
Received for publication, February 12, 2003, and in revised form, March 5, 2003
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ABSTRACT |
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Insulin stimulates the rapid translocation of
intracellular glucose transporters of the GLUT4 isotype to the plasma
membrane in fat and muscle cells. The connections between known insulin signaling pathways and the protein machinery of this
membrane-trafficking process have not been fully defined. Recently, we
identified a 160-kDa protein in adipocytes, designated AS160, that is
phosphorylated by the insulin-activated kinase Akt. This protein
contains a GTPase-activating domain (GAP) for Rabs, which are small G
proteins required for membrane trafficking. In the present study we
have identified six sites of in vivo phosphorylation on
AS160. These sites lie in the motif characteristic of Akt
phosphorylation, and insulin treatment increased phosphorylation at
five of the sites. Expression of AS160 with two or more of these sites
mutated to alanine markedly inhibited insulin-stimulated GLUT4
translocation in 3T3-L1 adipocytes. Moreover, this inhibition did not
occur when the GAP function in the phosphorylation site mutant was
inactivated by a point mutation. These findings strongly indicate that
insulin-stimulated phosphorylation of AS160 is required for GLUT4
translocation and that this phosphorylation signals translocation
through inactivation of the Rab GAP function.
Insulin rapidly stimulates glucose transport into fat and muscle
cells by causing the insertion of additional glucose transporters of
the GLUT4 isotype into the plasma membrane, in a process referred to as
GLUT4 translocation. The overall process consists of generation of the
specialized vesicles containing GLUT4 from the endosomal system, the
movement of these vesicles from the perinuclear region to the plasma
membrane, and the fusion of the vesicles with the plasma membrane (1).
The steps in this process that insulin accelerates, and the complete
signaling pathways from the insulin receptor that lead to their
acceleration, have not yet been fully defined. One partial insulin
signaling pathway that has been established to be required for GLUT4
translocation is the pathway that proceeds from the receptor through
tyrosine phosphorylation of the insulin receptor substrates to
activation of phosphatidylinositol 3-kinase and generation of
phosphatidylinositol 3,4,5-trisphosphate. The latter leads to the
activation of the protein kinase Akt and also protein kinase C Recently, we reported the discovery of a new substrate for
insulin-activated Akt in 3T3-L1 adipocytes, which was designated AS160
for Akt subtrate of 160 kDa (4). The most prominent feature of AS160 is
the presence of a GTPase activating domain for a Rab. Since Rabs are
small G proteins that play critical roles in vesicle formation,
movement, and fusion (5), we investigated the role of AS160 in GLUT4
translocation. Our present results strongly indicate that
insulin-stimulated phosphorylation of AS160 is required for GLUT4
translocation and also that an active
GAP1 domain in AS160 is
required for AS160 control of GLUT4 translocation. This study thus
identifies AS160 as a new and likely key connection between the
phosphatidylinositol 3-kinase insulin signaling pathway and the vesicle
trafficking machinery in GLUT4 translocation.
Plasmids--
The plasmid encoding GLUT4 with an HA tag in an
extracellular domain and GFP fused to the carboxyl terminus was as
described in Ref. 6. The CMV-10 plasmid encoding human AS160 with a
triple FLAG tag at the amino terminus was as described in Ref. 4. Mutations in AS160 in this vector were made with the QuikChange XL
site-directed mutagenesis kit from Stratagene, and the mutations were
verified by DNA sequencing.
Antibodies--
Antibodies were purchased from the following
sources (catalog number in parentheses): mouse monoclonal anti-FLAG
(F-3165), Sigma; mouse monoclonal anti-HA (MMS-101P), Berkeley
Antibody Company; Cy3-conjugated goat anti-mouse immunoglobulin
(115-165-146), Jackson ImmunoResearch. Affinity-purified rabbit
antibody against the carboxyl terminus of mouse AS160 was as described
in Ref. 4.
Purification of AS160 and Mass Spectrometry--
AS160 was
purified from five 10-cm plates of unstimulated 3T3-L1 adipocytes and
from five plates treated with 160 nM insulin for 10 min. As
described in detail in Ref. 4, cells were lysed in SDS/dithiothreitol,
excess nonionic detergent was added, and the AS160 was isolated by
immunoprecipitation with 30 µg of antibody against the carboxyl
terminus, followed by SDS-PAGE of the immunoprecipitate. Approximately
100 ng of AS160 were obtained in each case. The AS160 was digested in
gel with trypsin. The tryptic peptides were analyzed by microcapillary
liquid chromatography MS/MS on an ion trap mass spectrometer
(Thermo-Finnigan LCQ DECA XP Plus), as described in Ref. 7.
To detect a specific site of phosphorylation, targeted ion MS/MS
was conducted for each putative phosphopeptide and the corresponding
nonphosphorylated form.
Cell Culture and Electroporation--
3T3-L1 fibroblasts from
the American Type Culture Collection were carried as fibroblasts and
differentiated as described previously (4). For transfections, cells at
day 4 of differentiation were detached with 0.25 mg/ml trypsin, 0.5 mg/ml collagenase, washed with PBS, and electroporated in a 0.5-ml
cuvette (cells from approximately one 10-cm plate) at 0.18 kV and 975 microfarads in a Bio-Rad Gene Pulser II with 75 µg HA-GLUT4-GFP and
100 µg FLAG-tagged AS160 plasmid. After electroporation the cells
were plated in four wells of a six-well plate containing glass cover
slips. After 24 h the cells were put into serum-free medium for
2 h, treated with 160 nM insulin or not for 30 min,
washed with PBS, and fixed with 4% formaldehyde in PBS for 5 min.
Approximately 20% of the cells were transfected, as assessed by the
GFP fluorescence. For each AS160 plasmid co-transfected with the
HA-GLUT4-GFP, the extent of co-transfection was assessed by
permeablizing fixed cells with 0.2% saponin, staining with anti-FLAG
followed by Cy3-conjugated anti-mouse immunoglobulin, and determining
whether cells with GFP fluorescence also exhibited Cy3 fluorescence.
Under these conditions, each cell expressing the HA-GLUT4-GFP also
expressed FLAG-tagged AS160. In some experiments transfected, unfixed
cells were solubilized in SDS sample buffer and immunoblotted for
AS160, as described in Ref. 4.
Fluorescence Microscopy and GLUT4 Translocation--
The
appearance of HA-GLUT4-GFP in the plasma membrane was detected by
reaction of its extracellular HA tag with anti-HA and Cy3-conjugated
anti-mouse immunoglobulin, according to the method described in Ref. 1.
A brief description is as follows. The fixed, nonpermeablized cells on
coverslips were reacted with 5 µg/ml anti-HA in PBS, 2% fetal calf
serum, washed, reacted with 1/800 dilution of the Cy3-conjugated
secondary antibody, washed, and mounted on slides. Fluorescence images
on the GFP and Cy3 settings were acquired on a BX 51 Olympus microscope
with a Sensicam QED CCD camera and analyzed with the IPLab software
(Scanalytics). Ref. 8 provides details of the microscopic equipment.
Each transfected cell in a field was outlined, and its average Cy3 and
GFP fluorescence intensities measured. The background intensities, measured from the same analysis of an untransfected cell in the same
field, were subtracted. To normalize for differences in the expression
of HA-GLUT4-GFP between cells, the Cy3 fluorescence intensity was then
divided by the GFP fluorescence intensity for the same cell. A typical
experiment consisted of transfecting aliquots from one preparation of
cells with HA-GLUT4-GFP plus separately the 3×FLAG-CMV-10 vector,
wild-type AS160 in this vector, and several mutants of AS160 in this
vector. Cells from each combination were treated with insulin or left
in the basal state, and the normalized Cy3 intensity was measured for
at least 50 transfected cells from each condition on coded slides to
obviate investigator bias. The -fold GLUT4 translocation was then
calculated as the ratio of the average value of the normalized Cy3
intensity for the insulin state to that for the basal state, less
1.0.
Phosphorylation Sites on AS160--
We have reported previously
that AS160 isolated from insulin-treated cells was phosphorylated on
Ser588 and Thr642 (4). To characterize
the phosphorylation of AS160 more completely, we purified AS160 from
basal and insulin-treated 3T3-L1 adipocytes by immunoprecipitation with
antibody against the carboxyl terminus, digested it with trypsin, and
searched for predicted phosphopeptides by targeted ion MS/MS. We used
the program Scansite to predict the likely sites of Akt phosphorylation
(9) and searched for the seven phosphopeptides corresponding to the
sites with the best scores. Six of these phosphopeptides, as well as
the corresponding nonphosphorylated peptides, were identified in both
the basal and insulin samples, and the sites of phosphorylation were
deduced (Fig. 1). The sequences
surrounding the six sites in mouse AS160 are: RSRCSS318V,
RRRHAS341A, RSLTSS570L, RGRLGS588M,
RRRAHT642F, and RKRTSS751T. Thus, with the
exception of Ser570, these sites are all in the
RXRXXS/T motif preferred by Akt (9). The
phosphopeptide corresponding to a seventh possible Akt site at
Ser597 was not detected, although the nonphosphorylated
peptide was detected.
To estimate the effect of insulin on the phosphorylation of each site,
we measured the total ion intensities of each phosphorylated peptide
and of the corresponding nonphosphorylated peptide in the basal and
insulin samples. For each sample, the total ion intensity of each
phosphorylated peptide and that of the corresponding nonphosphorylated
peptide were expressed as percentages of their combined total ion
intensities. The ratio of these percentages for each
phosphorylated/nonphosphorylated pair in the basal sample was then
compared with the ratio in the insulin sample, to assess the effect of
insulin on the extent of phosphorylation (7). These ratios were as
follows (listed as phosphorylation site, ratio of percentage for
phosphorylated form to that for nonphosphorylated form in the basal
sample, corresponding ratio in the insulin sample): Ser318,
27/73, 76/24; Ser341, 80/20, 96/4; Ser570,
16/84, 46/54; Ser588, 14/86, 83/17; Thr642,
27/73, 74/26; and Thr751, 10/90, 75/25. Since the factor
relating the total ion intensity of each peptide to its actual amount
in the sample varies from peptide to peptide, these values do not give
the actual molar ratios of phosphopeptide to nonphosphopeptide.
Nevertheless, the large increase in this ratio in the insulin sample
compared with that in the basal sample for the peptides encompassing
Ser318, Ser570, Ser588,
Thr642, and Thr751 demonstrates that insulin
caused a marked increase in phosphorylation at these sites.
Effect of AS160 on GLUT4 Translocation--
Insulin-stimulated
translocation of GLUT4 to the cell surface was followed by
immunofluorescence in 3T3-L1 adipocytes expressing HA-GLUT4-GFP. The
extracellularly oriented HA epitope tag on the surface of fixed
nonpermeablized cells was detected by labeling with anti-HA antibody
followed by Cy3-labeled secondary antibody. The effects of wild-type
AS160 and various mutants thereof on GLUT4 translocation were examined
by cotransfecting the adipocytes with the AS160 plasmid and the
HA-GLUT4-GFP plasmid. Fig. 2,
top, shows representative images for cells transfected with
vector, wild-type AS160, or AS160 in which four of the phosphorylation sites have been mutated to alanine (4P mutant). In the vector control,
intracellular GLUT4, given by the GFP signal, was concentrated in the
perinuclear region, and insulin treatment caused a marked increase in
the GLUT4 at the cell surface, given by the Cy3 signal. The cells
expressing wild-type AS160 appeared similar to the vector control.
However, the cells expressing the 4P mutant exhibited a pronounced
inhibition of insulin-stimulated GLUT4 appearance at the cell
surface.
As described under "Experimental Procedures," images of the type in
Fig. 2 were quantitated, and the -fold translocation of GLUT4 in
response to insulin was calculated. Fig.
3 summarizes the data from a number of
experiments. In each experiment, the vector control was included, and
the -fold translocation for it was determined. In 12 separate
experiments the values for the control ranged from 3.1 to 13.6, with
the median value at 6.7. Thus, in each experiment there was substantial
translocation, but the -fold effect varied. The variation may be due to
differences between passages of the 3T3-L1 adipocytes. To compare all
the data, we have normalized the values for insulin-stimulated GLUT4 translocation to a value of 1.0 for the vector. Fig. 3 shows that expression of wild-type AS160 had no significant effect on
translocation. However, expression of the 4P mutant markedly reduced
translocation, to 21% of value for the vector control.
Rab GAP domains contain an arginine residue that is critical for
activity, and mutation of this residue to lysine abolishes activity
(10). Alignment of the GAP domain of AS160 with other Rab GAP domains
(11) showed that this key arginine is Arg973 of AS160.
Consequently, we examined the effect of the expression of the R973K
mutant (R/K) on GLUT4 translocation. In addition, the effect of
mutations in both the phosphorylation sites and the GAP domain (4P,
R/K) was examined. The mutation in the GAP domain by itself very
slightly reduced translocation to 83% of the control value (Figs. 2
and 3). However, this mutation in the context of the phosphorylation
site mutations largely relieved the inhibition by the phosphorylation
site mutations alone (Figs. 2 and 3).
To be sure that differences in expression levels could not account for
the effects of the various mutants, we immunoblotted SDS samples of the
transfected cells for AS160, both with an antibody against the FLAG tag
(Fig. 4, upper panel) and with
an antibody against the carboxyl terminus of mouse AS160 (Fig. 4,
lower panel). Approximately equal amounts of the wild-type
and mutant forms of FLAG-tagged human AS160 were expressed. The blot
with the antibody against the carboxyl terminus also showed the
endogenous mouse AS160, which is of slightly greater mobility since it
lacks the FLAG tag. From the relative signals in the lower
panel of Fig. 4 and the ~20% transfection efficiency, we
estimate that the transfected cells express about 15 times more
FLAG-tagged human AS160 than endogeneous mouse AS160. This estimate may
be low if the antibody against the mouse AS160 carboxyl terminus does
not react as well with the human AS160, which differs from mouse AS160
by four out of 12 amino acids in the carboxyl terminus.
We also examined the effect of cumulative mutations to alanine of the
six phosphorylation sites in AS160 on GLUT4 translocation. Most of the
inhibition was achieved with the combined T588A and S642A mutations
(Fig. 5).
Our results show that AS160 undergoes a marked increase in
phosphorylation at five sites in response to insulin. Since each site
lies within the consensus sequence for Akt phosphorylation, and since
we have shown previously that Akt phosphorylates the Thr642
site (4), it seems likely that activated Akt is responsible for
phosphorylation at these sites, although it remains possible that one
or more other insulin-activated kinases, such as protein kinase C
Expression of AS160 mutated at two or more of its phosphorylation sites
markedly inhibited insulin-stimulated GLUT4 translocation, whereas
equivalent expression of wild type AS160 had no effect. This result
strongly indicates that phosphorylation of AS160 is necessary for GLUT4
translocation to occur. Expression of AS160 with combined mutations in
the phosphorylation sites and the Rab GAP domain largely reversed the
inhibition given by AS160 mutated at the phosphorylation sites alone.
This result demonstrates that the inhibitory effect of AS160 mutated at
its phosphorylation sites requires a functional Rab GAP domain. A
hypothesis that explains these findings is the following:
insulin-stimulated translocation of GLUT4 requires a Rab in its active
GTP form; in the unstimulated state this Rab is maintained in its
inactive GDP form by the GAP domain of AS160; phosphorylation of AS160
inhibits its GAP activity toward this Rab, through an effect either on
GAP function and/or on localization of the protein; as a consequence
the GTP form of the Rab increases, and the Rab-dependent
step(s) in GLUT4 translocation proceed.
If the above hypothesis is correct, it might be thought that AS160 with
an inactive GAP domain would trigger translocation in the absence of
insulin, whereas in fact expression of the GAP mutant of AS160 did not
have this effect. One possibility is that extent of expression of the
GAP mutant was not sufficient for it to block the action of the
endogenous AS160. A second possible explanation is that generation of
the GTP form of a Rab is only one of several signals required for GLUT4
translocation. There is considerable evidence that a second signaling
pathway involving activation of the Rho-type G protein TC10 and the
rearrangement of cortical actin is required for GLUT4 translocation
(3). This pathway is independent of Akt activation. In addition, there is suggestive evidence that Akt phosphorylation of the syntaxin 4-interacting protein Synip may be required for GLUT4 translocation (12, 13).
This study indicates that AS160 is a key component linking the
phosphatidylinositol 3-kinase insulin signaling pathway to the vesicle
trafficking machinery in GLUT4 translocation. In the future it will be
important to identify the Rab(s) on which AS160 acts and to determine
whether phosphorylation of AS160 directly inhibits its GAP activity.
Rab4 and Rab11 are present in the intracellular vesicles that contain
GLUT4 (reviewed in Ref. 14). Consequently these two Rabs are candidates
to be substrates for the GAP function of AS160. We are currently
attempting to determine whether AS160 exhibits GAP activity toward
either of these Rabs or toward another one of the ~60 Rabs present in
mammals (5).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
,
and there is evidence that GLUT4 translocation requires the activation
of one or both of these kinases (reviewed in Refs. 2 and 3). However,
although a substrate linking either kinase to GLUT4 translocation has
been sought for several years, hitherto none has been clearly identified.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic diagram of human AS160. AS160
contains two phosphotyrosine binding domains (PTB) and a Rab
GAP domain (4). The sites of phosphorylation and the arginine residue
critical for GAP activity are designated. The sites of phosphorylation
were identified in mouse AS160; since mouse AS160 has not yet been
completely cloned, the corresponding sites on the human protein are
given.
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Fig. 2.
Fluorescent images of total and cell-surface
HA-GLUT4-GFP in 3T3-L1 adipocytes. Cells were transfected with
HA-GLUT4-GFP and either vector, wild-type AS160 (WT), AS160
mutated to alanine at phosphorylation sites Ser318,
Ser588, Thr642, and Ser751
(4P), AS160 mutated to lysine at Arg973
(R/K), or the AS160 with the combined
phosphorylation site and GAP mutations
(4P,R/K). Basal (B) and
insulin-treated (I) cells were examined for total
HA-GLUT4-GFP (GFP) and HA-GLUT4-GFP at the cell surface
(Cy3), as described under "Experimental
Procedures."
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Fig. 3.
Effect of AS160 mutants on insulin-stimulated
translocation of HA-GLUT4-GFP to the cell surface. Translocation
in the presence of the vector (Vec), wild-type AS160, and
various mutants of AS160 (see the legend to Fig. 2) was measured and
calculated as described under "Experimental Procedures" and
"Results." The error bars show the S.D. for the number
of experiments given in parentheses. The HA-GLUT4-GFP at the cell
surface in the basal state in cells expressing the various forms of
AS160 was not significantly different from that at the cell surface in
the vector control, and thus the differences in this figure are due to
differences in insulin-stimulated translocation.
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Fig. 4.
Expression of AS160 and mutants thereof in
3T3-L1 adipocytes. SDS samples of the transfected cells described
in the legends to Figs. 2 and 3 containing equal amounts of protein
were immunoblotted for the exogenous FLAG-tagged human AS160
(hAS160) with anti-FLAG (upper panel) and for
this form plus the endogenous mouse AS160 (mAS160) with
antibody against the carboxyl terminus of mouse AS160. The 1× loads
were 5 µg (upper panel) and 35 µg (lower
panel). A repetition of this experiment with a second set of
transfected cells gave similar results.
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Fig. 5.
Effect of AS160 phosphorylation site
mutations on HA-GLUT4-GFP translocation. The translocation was
measured and calculated as described under "Experimental
Procedures" and "Results," and in the legend to Fig. 3. The AS160
was mutated to alanine at: single sites, Ser588 and
Thr642; Ser588 and Thr642
(2P); 2P sites plus Ser751 (3P); 3P
sites plus Ser318 (4P); 4P sites plus
Ser570 (5P); and 5P sites plus
Ser341 (6P).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
/
, also participates.
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ACKNOWLEDGEMENTS |
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We are deeply indebted to Renee Robinson for technical assistance with the mass spectrometry measurements, to Dr. Samuel Cushman for the HA-GLUT4-GFP plasmid, to Dr. Michael Czech and his associates for the guidance in the electroporation method, to Dr. Timothy McGraw for advice about the fluorescence microscopy, and to Dr. William Wickner for the use of the fluorescence microscope. We are especially grateful to Dr. Alexey Merz for his guidance in the fluorescence microscopy.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK25336 and DK42816.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.
These authors contributed equally to this study.
¶ Permanent address: Dept. of Chemistry, Abilene Christian University, Abilene, TX 79699.
To whom correspondence should be addressed: Dept. of
Biochemistry, Vail Bldg., Dartmouth Medical School, Hanover, NH
03755. Tel.: 603-650-1627; Fax: 603-650-1128; E-mail:
gustav.e.lienhard@dartmouth.edu.
Published, JBC Papers in Press, March 11, 2003, DOI 10.1074/jbc.C300063200
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ABBREVIATIONS |
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The abbreviations used are: GAP, GTPase-activating protein; GFP, green fluorescence protein; MS/MS, tandem mass spectrometry; PBS, phosphate-buffered saline; HA, hemagglutinin.
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