From the Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242
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ABSTRACT |
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Guanosine 5'-O-(3-thiotriphosphate)
(GTPS) treatment of permeabilized adipocytes results in GLUT4
translocation similar to that elicited by insulin treatment. However,
although the selective phosphatidylinositol 3-kinase inhibitor,
wortmannin, completely prevented insulin-stimulated GLUT4
translocation, it was without effect on GTP
S-stimulated GLUT4
translocation. In addition, insulin was an effective stimulant, whereas
GTP
S was a very weak activator of the downstream Akt
serine/threonine kinase. Consistent with an Akt-independent mechanism,
guanosine 5'-O-2-(thio)diphosphate inhibited
insulin-stimulated GLUT4 translocation without any effect on the Akt
kinase. Surprisingly, two functionally distinct tyrosine kinase
inhibitors, genistein and herbimycin A, as well as microinjection of a
monoclonal phosphotyrosine specific antibody, inhibited both GTP
S-
and insulin-stimulated GLUT4 translocation. Phosphotyrosine immunoblotting and specific immunoprecipitation demonstrated that GTP
S did not elicit tyrosine phosphorylation of insulin receptor or
insulin receptor substrate-1. In contrast to insulin, proteins in the
120-130-kDa and 55-75-kDa range were tyrosine-phosphorylated following GTP
S stimulation. Several of these proteins were
identified and include protein-tyrosine kinase 2 (also known as CAK
,
RAFTK, and CADTK), pp125 focal adhesion tyrosine kinase, pp130
Crk-associated substrate, paxillin, and Cbl. These data demonstrate
that the GTP
S-stimulated GLUT4 translocation utilizes a novel
tyrosine kinase pathway that is independent of both the
phosphatidylinositol 3-kinase and the Akt kinase.
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INTRODUCTION |
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It has been well established that the insulin stimulation of
glucose uptake primarily results from the translocation of the GLUT41 glucose transporter
isoform from intracellular storage sites to the cell surface membrane
in muscle and adipose tissues. Although the molecular mechanism and
signaling cascade(s) regulating the intracellular trafficking of
GLUT4-containing vesicles have not been completely elucidated, several
important effector molecules have recently been identified. Insulin
binding to the insulin receptor results in tyrosine autophosphorylation
of the -subunit and activation of its intrinsic tyrosine kinase (1).
Subsequently, the insulin receptor tyrosine kinase phosphorylates
several intracellular proteins on tyrosine residues, most notably
insulin receptor substrate 1 (IRS1) (1). The phosphorylation of these
substrates creates recognition sites for additional effector proteins
containing Src homology 2 (SH2) domains, thereby generating
multisubunit signaling complexes (1, 2). In particular, the tyrosine phosphorylation of IRS1 induces the association and activation of
phosphatidylinositol (PI) 3-kinase (3). The targeting and/or activation
of PI 3-kinase is well documented to be necessary for GLUT4
translocation (4, 5). Recently, it has been shown that insulin
stimulates the Akt kinase, also known as protein kinase B or RAC-PK
(6). Insulin-stimulated Akt kinase activity is dependent on PI 3-kinase
activation, and expression of a membrane-targeted and constitutively
active Akt kinase results in persistent GLUT4 translocation (7, 8).
Consistent with a role for GLUT4 trafficking, expression of a dominant
interfering Akt mutant inhibited insulin-stimulated GLUT4 translocation
(9).
In addition to insulin, various other stimuli display insulinomimetic
properties and can induce the translocation of GLUT4-containing vesicles to the plasma membrane. For example, introduction of guanosine
5'-O-(3-thiotriphosphate) (GTPS), a nonhydrolyzable GTP
analogue, into adipocytes rapidly stimulates GLUT4 translocation to a
similar extent as insulin (10, 11). In addition, GTP
S can stimulate
GLUT4 translocation in the absence of ATP, suggesting that ATP is
required at an early step(s) in the insulin-signaling pathway and that
a GTP-binding protein(s) functions at a more distal step(s) (11). The
stimulatory effect of GTP
S can also be mimicked by treatment with
AlF4
, which is characteristic of the
involvement of a heterotrimeric GTP binding protein (12, 13).
Consistent with this interpretation, adrenergic stimulation can induce
GLUT4 translocation and glucose uptake in both cardiac myocytes (14,
15) and brown adipocytes (16, 17). Furthermore, in transfected Chinese
hamster ovary cells and 3T3L1 adipocytes, activation of receptors
coupled to Gq also stimulate GLUT4 translocation (18). In
this regard, skeletal muscle appears to have two distinct pathways
mediating GLUT4 translocation. Similar to adipocytes,
insulin-stimulated glucose transport in muscle is PI 3-kinase dependent
(19-21). In contrast, muscle contraction/exercise or hypoxia
stimulates glucose transport through a PI 3-kinase-independent pathway,
which may utilize a distinct and separate pool of GLUT4 intracellular
vesicles (22). It has been suggested that this alternative pathway
leading to GLUT4 translocation may be mediated by an increase in
cytoplasmic calcium levels (23-25), consistent with the known
functional role of G proteins in stimulating increases in
intracellular calcium (26-30).
In the present study, we have examined the mechanism by which GTPS
stimulates GLUT4 translocation by comparing its signaling properties
with those of insulin. Our data demonstrate that GTP
S-stimulated GLUT4 translocation is independent of the PI 3-kinase, the Akt kinase,
and changes in intracellular calcium ion concentration. However,
GTP
S-stimulated GLUT4 translocation occurred through the activation
of a novel tyrosine kinase pathway, which does not involve the insulin
receptor or IRS1 but may require the tyrosine phosphorylation of
protein-tyrosine kinase 2 (PYK2, also known as CAK
, RAFTK, and
CADTK), pp125 focal adhesion tyrosine kinase (pp125FAK),
pp130 Crk-associated substrate (pp130Cas), paxillin, and
Cbl.
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EXPERIMENTAL PROCEDURES |
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Cell Culture and Differentiation of 3T3L1 Adipocytes-- Murine 3T3L1 preadipocytes were obtained from the American Type Tissue Culture repository and were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 25 mM glucose and 10% calf serum at 37 °C. Confluent cultures were induced to differentiate into adipocytes by incubation of the cells with DMEM containing 25 mM glucose, 10% fetal bovine serum, 1 µg/ml insulin, 1 mM dexamethasone, and 0.5 mM isobutyl-1-methylxanthine. After 4 days, the medium was changed to DMEM containing 25 mM glucose, 10% fetal bovine serum, and 1 µg/ml insulin for an additional 4 days. The medium was then changed to DMEM containing 25 mM glucose and 10% fetal bovine serum. Under these conditions, more than 95% of the cell population morphologically differentiated into adipocytes. All studies were performed on adipocytes between 8 and 12 days after initiation of differentiation (day 0). Prior to all experimental treatments, the differentiated adipocytes were serum-starved in DMEM containing 25 mM glucose and 0.1% bovine serum albumin for 2 h at 37 °C.
Streptolysin-O Permeabilization of 3T3L1 Adipocytes-- 3T3L1 adipocytes were permeabilized with streptolysin-O (SL-O) as described by Robinson et al. (11) with minor modifications. Briefly, 3T3L1 adipocytes were washed three times with intracellular (IC) buffer (140 mM potassium glutamate, 20 mM Hepes, pH 7.15, 7.5 mM MgCl2, 5 mM EGTA, 5 mM NaCl, 2 mM CaCl2) and incubated in IC buffer containing 0.8 IU/ml of SL-O (Murex Diagnostics Inc., Atlanta, GA) for 5 min at 37 °C. Under these conditions, more than 95% of the cells were permeabilized based upon incorporation of propidium iodide. Following SL-O permeabilization, the cells were washed two times with ICR buffer (IC buffer containing 1 mg/ml bovine serum albumin, 1 mM dithiothreitol and enriched with either 10 mM MgATP or an ATP-regenerating system: 40 IU/ml creatine phosphokinase, 5 mM creatine phosphate, and 1 mM ATP). Unless otherwise indicated, all experimental treatments were performed by incubating the cells in ICR buffer containing various additions for 15 min at 37 °C.
Single Cell Microinjection-- 3T3L1 adipocytes used for microinjection were grown on 60-mm tissue culture dishes. The cells were incubated in Krebs-Ringer bicarbonate Hepes buffer (pH 7.4), containing 2 mM pyruvate, 0.5% bovine serum albumin, and 2.5 mM glucose for 45 min prior to microinjection. Adipocytes were microinjected with antibodies over a 45-min period using an Eppendorf model 5171 micromanipulator and given injections of approximately 0.1 pl directly into the cell cytoplasm with an Eppendorf model 5246 transjector. Following microinjection of approximately 100-300 cells/dish, the buffer was changed to fresh DMEM containing 0.1% bovine serum albumin, and the cells were allowed to recover for 90 min at 37 °C, prior to permeabilization and treatment.
Plasma Membrane Sheet Assay-- Preparation of plasma membrane sheets from the adipocytes was performed essentially by the method of Robinson et al. (11). Briefly, cells cultured on 35- or 60-mm dishes, following the appropriate treatment as described in each figure legend, were rinsed once in ice-cold phosphate-buffered saline (PBS) and incubated with 0.5 mg/ml of poly-L-lysine in PBS for 30 s. The cells were then swollen in a hypotonic buffer (23 mM KCl, 10 mM Hepes, pH 7.5, 2 mM MgCl2, 1 mM EGTA) by three successive rinses. The swollen cells were sonicated for 3 s at power setting 4.5 with a model 550 Fisher sonic dismembrator fitted with a 5-mm microtip set 1 cm above the surface of the cell monolayer in 10 ml of sonication buffer (70 mM KCl, 30 mM Hepes, pH 7.5, 5 mM MgCl2, 3 mM EGTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride). The bound plasma membrane sheets were washed two times with sonication buffer and used for either indirect immunofluorescence or immunoblot analysis as described below.
Preparation of Total Cell Extracts and
Immunoprecipitation--
Total cell extracts were prepared from 60- or
150-mm plates of 3T3L1 adipocytes following the appropriate treatment
as described in each figure legend. Cells from each plate were washed
two times with ice-cold PBS and scraped into 1 ml of lysis buffer (50 mM HEPES, pH 7.8, 1% Triton X-100, 100 mM NaF,
10 mM Na3P2O7, 2.5 mM EDTA) containing 1.0 mM phenylmethylsulfonyl
fluoride, 2 mM Na3VO4, 1 mg/ml
aprotinin, 10 mM leupeptin, and 1 mM pepstatin A by rotation for 15 min at 4 °C. Insoluble material was separated from the soluble extract by microcentrifugation for 15 min at 4 °C.
Protein concentration was determined, and samples were either subjected
directly to SDS-polyacrylamide electrophoresis (as described below) or
immunoprecipitated for IRS1 or PYK2. Briefly, 3-5 mg of cellular
protein were immunoprecipitated with 5 µg of IRS1 polyclonal antibody
(IRS1; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or PYK2
polyclonal antibody (31) for 2 h at 4 °C. Immune complexes were
recovered by the addition of protein A-Sepharose (Amersham Pharmacia
Biotech) and subjected to SDS-polyacrylamide electrophoresis (as
described below).
Precipitation with GST Fusion Proteins--
GST and GST-CrkII
were prepared as described previously (32). Soluble GST fusion proteins
were covalently linked to agarose beads with Amino-Link (Pierce). 100 µg of fusion proteins bound to the beads were incubated for 2 h
at 4 °C with 4 mg of cell extracts isolated from control,
insulin-stimulated, or GTPS-stimulated cells. The beads were
subsequently pelleted, washed three times with washing buffer (10 mM Tris, pH 7.4, 2 mM EDTA, 150 mM
NaCl, 0.2% Triton X-100, 0.1% Nonidet P-40, 0.2 mM
phenylmethylsulfonyl fluoride, and 0.2 mM sodium vanadate),
and boiled in Laemmli sample buffer. The precipitated
proteins were analyzed by SDS-polyacrylamide gel electrophoresis and
Western blotting (as described below).
Electrophoresis and Immunoblotting-- Plasma membrane sheets were scraped into a buffer containing 250 mM sucrose, 20 mM Tris, pH 7.40, 1 mM EDTA and pelleted by centrifugation at 200,000 × g for 1 h at 4 °C. The pelleted fraction was solubilized in 100 µl of Laemmli sample buffer, and 5 µg of the total solubilized fraction was separated by SDS-polyacrylamide gel electrophoresis (10% polyacrylamide). The resolved proteins were then transferred to nitrocellulose membranes and immunoblotted with polyclonal rabbit GLUT4 antibody (IRGT; Charles River).
Whole cell lysates, GST precipitates, and immunoprecipitates were separated by SDS-polyacrylamide gel electrophoresis (7.5% polyacrylamide). The resolved proteins were then transferred to Immobilon P membrane (Millipore Corp.) and immunoblotted with a monoclonal phosphotyrosine antibody (PY20:HRPO; Transduction Laboratories), a monoclonal paxillin antibody (Transduction Laboratories), a monoclonal p130Cas antibody (Transduction Laboratories), and a polyclonal Cbl antibody (Santa Cruz Biotechnology). All immunoblots were subjected to enhanced chemiluminescence detection (Amersham Pharmacia Biotech).Immunofluorescence and Confocal Microscopy-- Sonicated plasma membrane sheets were fixed for 20 min at 4 °C in a solution containing 2% paraformaldehyde, 70 mM KCl, 30 mM HEPES, pH 7.5, 5 mM MgCl2, and 3 mM EGTA. The fixed plasma membrane sheets were quenched for 15 min at 25 °C in 100 mM glycine-PBS (pH 7.5). After three rinses in PBS, the sheets were blocked overnight at 4 °C in PBS containing 5% donkey serum (Sigma). The blocked sheets were incubated at 4 °C overnight with a 1:100 dilution of polyclonal rabbit GLUT4 antibody (IRGT; Charles River). For the microinjection studies, this incubation was done in combination with a 1:5000 dilution of polyclonal sheep anti-maltose binding protein antiserum (generously provided by Dr. Morris Birnbaum, University of Pennsylvania). The plasma membrane sheets were then washed for 30 min with PBS (six changes of PBS) and incubated overnight at 4 °C with a 1:50 dilution of lissamine-rhodamine-conjugated donkey anti-rabbit IgG (Jackson Immunoresearch, Inc.). For the microinjection studies, this incubation was done in combination with a 1:100 dilution of fluorescein isothiocyanate-conjugate donkey anti-sheep IgG (Jackson Immunoresearch Inc.). Following incubation with the secondary antibodies, the membrane sheets were washed for 30 min with PBS (six changes of PBS) and mounted for microscopic analysis with Vectashield mounting medium (Vector Laboratories Inc.). Confocal images were obtained on a Bio-Rad MRC 600 laser confocal microscope (University of Iowa Central Microscopy Research Facility).
Akt Kinase Activity Assay--
Akt protein kinase activity was
determined as described by Moule et al. (33). 3T3L1
adipocyte detergent cell extracts were prepared as described above, and
600 µg of cellular protein were immunoprecipitated with 5 µg of Akt
antiserum (kindly provided by Dr. Kelly Moule, University of Bristol)
for 2 h at 4 °C. Immune complexes were recovered by the
addition of protein A-Sepharose (Amersham Pharmacia Biotech). The
protein A-Sepharose beads were washed and resuspended in 40 µl of
assay buffer (20 mM MOPS, pH 7.0, 1 mM EDTA, 1 mM EGTA, 0.01% Brij 35, 5% glycerol) containing 0.1%
2-mercaptoethanol, 2.5 µM cAMP-dependent
protein kinase inhibitor peptide. The activity of Akt in the
immunoprecipitate was measured using histone H2B (0.5 mg/ml) as an
in vitro substrate. The reaction was initiated by the
addition of 100 µM [-32P]ATP (10 µCi)
for 20 min at 30 °C. The reaction was stopped by the addition of 2×
Laemmli sample buffer (25 µl), after which the samples were boiled
for 5 min at 100 °C and electrophoresed on a 16% SDS-polyacrylamide
gel. The gel was stained with Coomassie Blue, dried, and subjected to
autoradiography.
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RESULTS |
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GTP-S, but Not Insulin, Stimulation of GLUT4 Translocation in
3T3L1 Adipocytes Is Wortmannin-insensitive--
As previously observed
(10, 11), treatment of SL-O permeabilized 3T3L1 adipocytes with insulin
resulted in the translocation of GLUT4 to the plasma membrane as
detected by GLUT4 immunoblotting of isolated plasma membrane sheets
(Fig. 1A, lanes 1 and 2). Similarly, the addition of GTP
S to the
permeabilized cells also induced the translocation of GLUT4 (Fig.
1A, lanes 3 and 4). Since it is well
established that activation and/or appropriate intracellular targeting
of the PI 3-kinase is necessary for insulin-stimulated GLUT4
translocation, we next examined the role of PI 3-kinase in
GTP
S-stimulated GLUT4 translocation. In the control, unstimulated cells, there was a low level of GLUT4 immunofluorescence detected from
the isolated plasma membrane sheets (Fig. 1B, panel
1). Pretreatment with the selective PI 3-kinase inhibitor
wortmannin (100 nM) slightly reduced the amount of GLUT4
present in the isolated plasma membrane sheets from unstimulated cells
(Fig. 1B, panel 4). As expected, insulin
stimulated a large increase in the translocation of GLUT4 to the plasma
membrane, which was completely inhibited by pretreatment with
wortmannin (Fig. 1B, panels 2 and 5).
In contrast, although GTP
S stimulated a similar extent of GLUT4
translocation compared with insulin, pretreatment with wortmannin was
without effect (Fig. 1B, panels 3 and
6). In addition, pretreatment of the 3T3L1 adipocytes with
higher concentrations of wortmannin (1 µM) also inhibited
insulin-stimulated GLUT4 translocation but did not reduce the
translocation induced by GTP
S (data not shown).
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GTPS Is an Ineffective Activator of the Akt Protein
Kinase--
Recently, it has been reported that insulin stimulation
increases Akt protein kinase activity in a PI
3-kinase-dependent manner, and stable overexpression of a
constitutively active membrane-bound form of Akt kinase resulted in the
persistent translocation of GLUT4 (7, 8). We therefore examined the
ability of GTP
S to stimulate Akt kinase as a potential common point
of convergence between the insulin and GTP
S signaling pathways
leading to GLUT4 translocation. Activation of Akt protein kinase
activity was assessed by immunoprecipitation and analysis of in
vitro protein kinase activity (Fig.
2). Isolation of Akt from unstimulated
cells demonstrated a basal level of Akt protein kinase activity that
was reduced by pretreatment with wortmannin (Fig. 2, lanes 1 and 2). Insulin stimulation resulted in a marked activation
of Akt protein kinase activity, which was also attenuated by wortmannin
(Fig. 2, lanes 5 and 6). Treatment with GTP
S
resulted in an intermediate stimulation of Akt protein kinase activity
(Fig. 2, lane 3). Nevertheless, pretreatment with wortmannin
completely inhibited the GTP
S-stimulated activation of Akt protein
kinase activity (Fig. 2, lane 4). Since Akt kinase
activation is accompanied by dual phosphorylation on serine and
threonine residues (34), we also examined the effect of insulin and
GTP
S on the SDS-polyacrylamide gel electrophoretic mobility of Akt.
Consistent with Akt kinase activity, insulin stimulation resulted in a
marked reduction of Akt electrophoretic mobility, whereas GTP
S
stimulation had little effect (data not shown). Furthermore,
pretreatment of the cells with wortmannin completely prevented the
reduction in Akt electrophoretic mobility. Thus, together these data
demonstrate that GTP
S stimulation of GLUT4 translocation occurs in a
pathway that is independent of both PI 3-kinase and Akt kinase
activation.
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GDPS Inhibits Insulin Stimulation of GLUT4
Translocation--
The data presented above suggest that the GTP
S
stimulation of GLUT4 translocation occurs in a pathway(s) downstream to
and/or in parallel with the PI 3-kinase and Akt kinase. To further
characterize the relationship between insulin and GTP
S stimulation,
we next incubated the SL-O-permeabilized cells with 100 µM GDP
S prior to insulin treatment (Fig.
3). In the absence of insulin, GDP
S had no significant effect on GLUT4 translocation as detected by GLUT4
immunofluorescence of isolated plasma membrane sheets (Fig. 3A, panels 1 and 5). Insulin
stimulation resulted in a dose-dependent increase in the
amount of plasma membrane-associated GLUT4 protein (Fig. 3A,
panels 1-4). Pretreatment with GDP
S resulted in the inhibition of GLUT4 translocation at low (1 and 10 nM)
insulin concentrations but not at high (100 nM) insulin
concentrations (Fig. 3A, panels 5-8). Insulin
stimulation also resulted in a dose-dependent decrease in
Akt SDS-polyacrylamide gel electrophoretic mobility (Fig.
3B, lanes 1, 2, 5, and
8). However, pretreatment with GDP
S had no effect on the
insulin stimulation of the Akt gel shift and, hence, phosphorylation
and presumably protein kinase activation (Fig. 3B,
lanes 3, 6, and 9). These data further
support a model in which GTP
S stimulation of GLUT4 translocation is
mediated by a mechanism downstream of the Akt kinase.
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GTPS Stimulation of GLUT4 Translocation Is Tyrosine
Kinase-dependent--
Recently, several studies have
suggested that G protein-coupled receptors can modulate tyrosine kinase
signaling pathways. Having ruled out any potential role for PI 3-kinase
and Akt protein kinase, we next assessed whether GTP
S-stimulated
GLUT4 translocation was mediated by a tyrosine
kinase-dependent mechanism. This possibility was initially
examined using the relatively specific tyrosine kinase inhibitors
genistein and herbimycin A (Fig. 4). In
unstimulated cells, there was little GLUT4 detectable in the isolated
plasma membrane sheets, and pretreatment with either genistein or
herbimycin A had no effect (Fig. 4, panels 1, 4,
and 7). As expected, both genistein and herbimycin A were
effective inhibitors of insulin-stimulated GLUT4 translocation (Fig. 4,
panels 2, 5, and 8), as both of these agents inhibit the insulin receptor tyrosine kinase and IRS1 tyrosine phosphorylation (data not shown). However, to our surprise,
pretreatment with these tyrosine kinase inhibitors also prevented the
GTP
S-stimulated translocation of GLUT4 (Fig. 4, panels 3,
6, and 9).
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Insulin and GTPS Stimulate the Tyrosine Phosphorylation of
Discrete Proteins--
Based upon the inhibition of insulin- and
GTP
S-stimulated GLUT4 translocation by genistein, herbimycin A, and
the PY20 phosphotyrosine antibody, we next compared the tyrosine
phosphorylation of proteins induced by insulin and GTP
S treatment
(Fig. 6). Insulin stimulation resulted in
increased tyrosine phosphorylation of the insulin receptor
-subunit
and IRS1 (Fig. 6A, lanes 3 and 4). In
contrast, GTP
S stimulation had no effect on either insulin receptor
subunit or IRS1 tyrosine phosphorylation (Fig. 6A,
lanes 1 and 2). Instead, GTP
S appeared to
induce the tyrosine phosphorylation of several proteins in the
molecular mass range of 120-130 kDa and 55-75 kDa (Fig.
6A, compare lanes 1 and 2 with
lanes 3 and 4). To more carefully examine the
potential for IRS1 tyrosine phosphorylation, we also specifically
immunoprecipitated IRS1 from both insulin- and GTP
S-stimulated
adipocytes. Insulin stimulated the tyrosine phosphorylation of IRS1 in
the IRS1 immunoprecipitates (Fig. 6B, lanes 3 and
4), whereas there was no detectable IRS1 tyrosine phosphorylation following GTP
S stimulation (Fig. 6B,
lanes 1 and 2). IRS1 immunoblotting of that same
membrane demonstrated the presence of equal amounts of IRS1 protein
(Fig. 6B, lanes 5-8). The apparent increase in
molecular weight following both insulin and GTP
S stimulation most
likely reflects the serine/threonine phosphorylation of IRS1.
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GTPS Stimulates the Tyrosine Phosphorylation of PYK2,
pp130Cas, Paxillin, and Cbl--
One interesting tyrosine
kinase that has recently been implicated in the intracellular signaling
of several trimeric G protein-coupled receptors and by osmotic shock is
PYK2 (31, 36-39). Since osmotic shock, like GTP
S, stimulated GLUT4
translocation through a PI 3-kinase- and Akt-independent pathway (31),
we next examined the tyrosine phosphorylation of PYK2 by
immunoprecipitation. As previously reported, in unstimulated cells
there was no detectable PYK2 tyrosine phosphorylation, whereas osmotic
shock induced a marked tyrosine phosphorylation of PYK2 (Fig.
7A, lanes 1 and 2). In contrast, there was no detectable tyrosine
phosphorylation of PYK2 in response to insulin (Fig. 7A,
lane 3). Although permeabilization alone resulted in a
slight increase in the phosphotyrosine content of PYK2, GTP
S was an
effective activator of PYK2 tyrosine phosphorylation (Fig.
7A, lanes 4 and 5). Immunoblotting of
the same Western blot with a PYK2 antibody indicated the presence of
equal amounts of PYK2 in each lane (Fig. 7B, lanes
1-5).
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DISCUSSION |
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Over the past several years, significant progress has been made in our understanding of the insulin signal transduction pathway leading to GLUT4 vesicle trafficking. It is generally accepted that insulin stimulation results in the activation of the insulin receptor tyrosine kinase, leading to the tyrosine phosphorylation of the IRS family of intracellular docking proteins (1, 2). The tyrosine phosphorylation of IRS1 induces the association/targeting and/or activation of PI 3-kinase (3). Multiple studies have demonstrated that PI 3-kinase function is necessary for insulin-stimulated GLUT4 translocation (4, 5). Although the downstream target(s) of PI 3-kinase involved in GLUT4 translocation has not been identified, recent studies indicate that activation of PI 3-kinase results in the activation of the Akt serine/threonine-protein kinase (7, 8).
In addition to the identification of this signaling pathway of insulin,
there are several insulinomimetic agents that can also stimulate
glucose transport in adipocytes and muscle. In particular,
nonhydrolyzable GTP analogues have been reported to increase glucose
transport activity by inducing the translocation of GLUT4 (10, 11). We
therefore reasoned that examining the GTPS stimulation of glucose
transport might provide new insight by identifying common and/or
distinct pathways leading to GLUT4 translocation. In this regard, two
previous studies have reported contradictory findings for the role of
PI 3-kinase in GTP
S-stimulated glucose transport regulation. A study
by Clarke et al. (40) suggested that the ability of GTP
S
to stimulate 2-deoxy-D-glucose uptake by permeabilized
cells was partially blocked by wortmannin (1 µM). In
contrast, it was later reported by Herbst et al. (41) that
incubation of permeabilized 3T3L1 adipocytes with GTP
S (200 µM) did not increase PI 3-kinase activity over basal
level. Although the basis for this difference is not apparent, our data
demonstrate that GTP
S-stimulated GLUT4 translocation is completely
wortmannin-insensitive at concentrations sufficient to inhibit both the
receptor tyrosine kinase-activated PI 3-kinase and the
G
-responsive PI 3-kinase isoform (42).
One recently established function of PI 3-kinase is to couple receptor
tyrosine kinase activation to the Akt protein kinase, also known as
RAC-PK or protein kinase B (6, 43, 44). The Akt kinase is directly
activated by dual phosphorylation on serine and threonine residues
(34). In addition, the pleckstrin homology domain of Akt can bind
phosphatidylinositol 3,4-bisphosphate in vitro, which also
has been shown to stimulate its protein kinase activity (45-47). In
either case, insulin activates the Akt kinase in a PI 3-kinase
dependent manner. Furthermore, overexpression of a membrane-targeted
constitutively active Akt results in the persistent translocation of
GLUT4 (7, 8). Since it has been suggested that there are PI
3-kinase-independent pathways leading to Akt activation (48), it
remained formally possible that GTPS activated Akt, hence GLUT4
translocation, by a wortmannin-insensitive mechanism. However, our data
demonstrated that GTP
S is a very weak activator of Akt
phosphorylation and protein kinase activity. In addition, wortmannin
completely inhibited this small extent of Akt activation, yet had no
effect on GTP
S-stimulated GLUT4 translocation. Thus, these data
clearly establish the presence of a PI 3-kinase- and Akt-independent
pathway leading to GLUT4 translocation in 3T3L1 adipocytes.
In an alternative approach to identify the pathway(s) utilized by
GTPS, we observed that GDP
S was an effective inhibitor of
insulin-stimulated GLUT4 translocation at physiological but not at
supraphysiological (saturating) insulin concentrations. The inability
of GDP
S to prevent insulin-stimulated GLUT4 translocation at
supraphysiological insulin concentrations has been previously reported,
but the effect of GDP
S was not examined at physiological insulin
levels (49). There are several plausible reasons accounting for the
ineffectiveness of GDP
S at high insulin concentrations, including
the possibility that insulin can mediate GLUT4 translocation by more
than one signaling pathway, i.e. one pathway that is
dependent on a GTP binding protein(s) and another one that is not. The
former pathway would be the predominant signaling pathway under normal physiological stimulation, whereas the later pathway would be activated
only by high insulin concentrations. Alternatively, at high insulin
concentrations, the downstream effectors are maximally activated and
would require substantially greater concentrations of GDP
S to be
inhibited. In any case, the fact that GDP
S inhibited the
insulin-stimulated translocation of GLUT4 without any effect on Akt
kinase is consistent with the presence of at least one GTP-binding
protein functioning downstream of Akt. Together, these data provide
compelling evidence that neither PI 3-kinase nor Akt kinase are
involved in the GTP
S stimulation of GLUT4 translocation and that
GTP
S functions at a site distal to the Akt kinase.
Although it is generally thought that calcium does not play a
significant role in the insulin stimulation of GLUT4 translocation, it
has been hypothesized to be necessary for the
exercise/contraction-stimulated GLUT4 translocation in skeletal muscle
(23-25). In analogy with GTPS, the exercise/contraction-stimulated
GLUT4 translocation occurs by a wortmannin-insensitive pathway (22). In
addition, agonists of Gq-coupled receptors, receptors that
elicit increases in intracellular calcium, have also been reported to
induce GLUT4 translocation in both transfected Chinese hamster ovary
cells and 3T3L1 adipocytes (18). Thus, it seemed possible that
GTP
S-stimulated GLUT4 translocation occurred via mediation of an
increase in intracellular calcium. However, both an inhibitor of
intracellular calcium release and calcium chelators failed to prevent
the GTP
S-stimulated translocation of GLUT4 (data not shown).
Having ruled out PI 3-kinase, Akt kinase, and changes in intracellular
calcium, we next assessed whether GTPS stimulated GLUT4
translocation in a tyrosine kinase-dependent pathway. To our surprise, we observed that two tyrosine kinase inhibitors (genistein and herbimycin A) as well as microinjection of the PY20
monoclonal phosphotyrosine antibody were effective blockers of
GTP
S-stimulated GLUT4 translocation. Since GTP
S did not induce the tyrosine phosphorylation of either the insulin receptor or IRS1
(which would activate the PI 3-kinase and Akt kinase), the ability to
block the GTP
S signal with genistein, herbimycin A, and PY20
microinjection suggests the presence of a novel tyrosine kinase
pathway. Consistent with this interpretation, GTP
S stimulation resulted in the specific tyrosine phosphorylation of proteins in the
120-130-kDa and 55-75-kDa ranges.
To search for the identity of these proteins, we took advantage of the
ability of SH2 domains to bind with high affinity to tyrosine-phosphorylated proteins. Using a GST-CrkII fusion, we identified several proteins (pp125FAK,
pp130Cas, paxillin, and Cbl) that displayed a slight
increase in tyrosine phosphorylation following GTPS treatment (data
not shown). On the other hand, immunoprecipitation of PYK2, a newly
identified soluble tyrosine kinase that has been implicated in the
intracellular signaling of several trimeric G protein-coupled receptors
and by osmotic shock (31, 36-39), demonstrated that PYK2 was markedly tyrosine-phosphorylated in response to GTP
S but not to insulin. The
fact that both GTP
S and osmotic shock stimulation result in GLUT4
translocation by both a PI 3-kinase- and Akt kinase-independent pathway
provides suggestive evidence that PYK2 may be an important kinase
regulating these trafficking events.
In summary, we have observed that GTPS-stimulated GLUT4
translocation occurs by a novel tyrosine kinase pathway in 3T3L1 adipocytes. This alternative signaling pathway does not require tyrosine phosphorylation of the insulin receptor, IRS1, or the targeting and/or activation of either PI 3-kinase or Akt kinase. However, GTP
S stimulation results in the tyrosine phosphorylation of
several distinct proteins, including PYK2, pp125FAK,
pp130Cas, paxillin, and Cbl. Whether or not any of these
tyrosine-phosphorylated proteins are necessary and/or sufficient for
the GTP
S stimulation of GLUT4 translocation is an important future
issue for investigation.
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ACKNOWLEDGEMENTS |
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We are grateful to Robert Brown for excellent technical assistance, to Tom Moninger for assistance with the fluorescent microscopic analysis, and to Drs. Brian Ceresa and Ken Coker for helpful discussion of the data.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK49012, DK33823, and DK25925.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: Dept. of Physiology
and Biophysics, University of Iowa, Iowa City, IA 52242-1109. Tel.:
319-335-7823; Fax: 319-335-7330; E-mail: jeffrey-pessin{at}uiowa.edu.
1
The abbreviations used are: GLUT4, the
insulin-responsive glucose transporter isoform; PI 3-kinase,
phosphatidylinositol 3-kinase; IRS1, insulin receptor substrate-1; SH2,
Src homology 2; PYK2, protein-tyrosine kinase 2 (also known as CAK,
RAFTK, and CADTK); pp125FAK, pp125 focal adhesion tyrosine
kinase; pp130Cas, pp130 Crk-associated substrate; DMEM,
Dulbecco's modified Eagle's medium; SL-O, streptolysin-O; PBS,
phosphate-buffered saline; GTP
S, guanosine
5'-O-(3-thiotriphosphate); GDP
S, guanosine
5'-O-2-(thio)diphosphate; IC, intracellular; MOPS,
4-morpholinepropanesulfonic acid; MBP, maltose-binding protein; GST,
glutathione S-transferase.
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REFERENCES |
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