From the
Diabetes Section, Laboratory of Clinical
Investigation and the
Vascular Studies Unit,
Laboratory of Cardiovascular Science, NIA, National Institutes of Health,
Baltimore, Maryland 21224
Received for publication, January 29, 2003 , and in revised form, April 14, 2003.
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ABSTRACT |
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INTRODUCTION |
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The submembranous actin microfilament network links various signaling
proteins (e.g. phosphatidylinositol (PI) 3-kinase and Rho family of
proteins) that play an important role in membrane trafficking, cellular
integrity, and homeostasis
(24).
Incidentally, insulin is known to induce rapid dynamic reorganization of the
actin cytoskeleton to generate the forces necessary for plasma membrane
ruffling formation and a host of other cellular processes, including proper
insertion of insulin-regulatable glucose transporter 4 in the cell surface
(5). The stabilization of actin
network at the periphery of the cytoplasm and its attachment to cellular
membranes are orchestrated by actin-binding proteins. The filamin family of
actin-binding proteins bind to actin filaments and to a number of
macromolecules (reviewed by Stossel et al.
(6)), notably small GTPases
(7) and p21-activated kinase 1
(Pak1) (8). Filamins are
rod-shaped proteins of 280 kDa that contain an N-terminal actin-binding
domain followed by 24 repeats each of 96 amino acids. Repeat 24 contains the
dimerization domain of filamin. Significantly, most of the interactions
between filamin and its binding partners occur through the C-terminal end of
filamins, thus allowing the interwebbing of actin scaffolds with
membrane-bound proteins
(6).
Several proteins involved in signal transduction events are partitioned in lipid rafts, a process that allows proper compartmentalization and spatial/temporal organization of functionally competent signaling complexes (9). The IR segregates to glycolipid-enriched raft domains of the plasma membrane in a variety of cell types (10, 11). Of interest, inducible association of signaling proteins with lipid rafts has been shown to depend on the actin cytoskeleton through a mechanism involving raft coalescence (12, 13). Therefore, colocalization of filamin and resident raft proteins, including the scaffolding protein caveolin-1 (14), is likely to be of physiological importance in the clustering of lipid rafts and organization of multiple signaling pathways by the actin cytoskeleton. Until now, however, little is known about a role that filamins would play in the transmission of the diverse effects of insulin.
In this study, we have investigated the relative contribution of filamin A (FLNa) to the regulation of insulin signaling in human melanoma cell lines (M2 cells) that have spontaneously lost expression of FLNa, and a subline with stable expression of recombinant FLNa (A7 cells) (15). By using this cell model, we have found that FLNa expression attenuated insulin mitogenic signaling by selectively inhibiting the recruitment and tyrosine phosphorylation of Shc and subsequent activation of p42/44 MAPK. Of interest, neither early steps in insulin signaling (e.g. IR and IRS-1 tyrosine phosphorylation) nor the activation of the IRS-1/PI3-kinase/AKT pathway were affected by FLNa. MAPKs transduce a mitogenic signal by phosphorylating transcription factors such as Elk-1, which leads to regulation of critical genes (16). Our results have indicated also that FLNa binds directly to IR and that ectopic expression of a C-terminal fragment of FLNa disrupts constitutive IR-FLNa interaction in HepG2 cells, thereby inducing a marked increase in insulin-stimulated MAPK phosphorylation and Elk-1 transactivation. These results indicate that FLNa has a negative role in MAPK-mediated Elk-1 transcriptional activation in response to insulin, in part, by interacting directly with the IR.
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EXPERIMENTAL PROCEDURES |
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Plasmid ConstructioncDNA encoding the FLNa C-terminal fragment (amino acids 23572647) was amplified by PCR using full-length human filamin A cDNA (kindly provided by Dr. Yasutaka Ohta (Harvard Medical School, Boston)) as the template. The following primer pairs were used: forward primer, 5'-TAGGATCCATGGGCTATCCATATGATGTTCCAGATTATGCTCTGAACGGGGCCAAG-3'; reverse primer, 5'-CGACTAGTTCAGGGCACCACAAC-3'. The underlined nucleotides indicate the KpnI and SpeI sites in the forward and reverse primers, respectively, and the italic nucleotides indicate the HA epitope. The amplified product was digested and inserted into the KpnI/XbaI sites of pcDNA3.1 (Invitrogen). The integrity of the HA-FLNaCT construct was verified by automated sequencing.
A cDNA fragment corresponding to Arg941Ser1343
of the human IR was generated by PCR amplification to contain BamHI
and EcoRI restriction sites using pCMVHIR as the template
(18). The resulting 1206-bp
BamHI-EcoRI cDNA fragment was inserted into pGEX-4T-1 vector
(Amersham Biosciences). GST and GST-IR fusion protein were expressed in BL21,
induced by 0.5 mM
isopropylthio--D-galactopyranoside, and purified by affinity
chromatography with glutathione-Sepharose (Amersham Biosciences) according to
the manufacturer's protocols. The resulting eluates were concentrated by
ultrafiltration and stored at -70 °C. Translation and product size were
verified by analyzing an aliquot of the samples by SDS-PAGE and Colloidal blue
staining of the gel, as well as by immunoblot analysis. The integrity of the
GST-IR construct was verified by automated sequencing.
Cell TreatmentM2 and A7 cells were cultured in minimum essential medium (MEM) supplemented with 10 mM Hepes, pH 7.4, 0.25% sodium bicarbonate, 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, 8% newborn calf serum, and 2% fetal calf serum and maintained in a humidified atmosphere of 5% CO2 in air at 37 °C. Before treatment, cells were serum-starved for 18 h in MEM supplemented with 0.1% FBS, washed with phosphate-buffered saline (PBS), and then incubated in Krebs-Ringer phosphate (KRP) buffer. Cells were treated in the absence or the presence of 200 µM orthovanadate for 30 min followed by the addition of 100 nM insulin for periods up to 10 min. In some experiments, cells were stimulated with either 25 nM insulin, 20 nM EGF, 14 nM IGF-1, or 20% FBS for 15 min. These cells were washed in PBS and immersed in liquid nitrogen. The human HepG2 hepatoma cells and HEK293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) and McCoy's 5A medium, respectively, supplemented with 10% FBS, 2 mM L-glutamine, 50 units/ml penicillin, and 50 µg/ml streptomycin.
Transient Transfection AssaysHepG2 cells were cultured for 24 h until 6080% confluence was reached. Transient transfection was performed according to the manufacturer's protocol for the use of LipofectAMINE 2000. In brief, empty expression vector (pcDNA3.1) or expression plasmid encoding HA-FLNaCT was mixed with the transfection reagent and directly added to the culture plates at a ratio of 6 µg/60-mm dish. Twenty four to 48 h later, cells were used for various experiments. Expression of HA-FLNaCT was analyzed in total cell lysates by immunoblotting with anti-HA antibody (Clontech, Palo Alto, CA). Cells were serum-starved for 18 h and incubated for 30 min with 200 µM orthovanadate prior to 100 nM insulin treatment for 10 min.
Elk-1 Transactivation AssayTransactivation of Elk-1 was
examined by the PathDetect Elk-1 trans-Reporting System (Stratagene).
In brief, HepG2 cells were cotransfected with pFR-Luc, pFA-Elk-1 and 0.2 µg
of pCMV--galactosidase and either 3 µg of pcDNA3.1 or 3 µg of
HA-FLNaCT as indicated. Serum-starved cells were left untreated or
were stimulated with 100 nM insulin for 24 h. Elk-1 luciferase and
-galactosidase activities were measured using assay system kits from
Promega according to the manufacturer's instructions, and the luciferase
values were normalized to
-galactosidase.
Detergent-free Isolation of Lipid RaftsIsolation of lipid rafts was accomplished using a detergent-free sucrose gradient centrifugation method as described previously (19). In brief, cells from 150-mm culture dishes were resuspended in 1 ml of 0.5 M sodium carbonate, pH 11, supplemented with a protease inhibitor mixture (Calbiochem), and homogenized by passing cells 15 times through a 23-gauge needle and two 10-s bursts of a sonicator probe on ice. The sucrose concentration in cell extracts was adjusted to 45% (w/w) by the addition of 1.8 volume of 70% (w/w) sucrose prepared in MBS (25 mM MES, pH 6.5, 0.15 M NaCl). At the bottom of an ultracentrifuge tube, 2 ml of the extracts were placed, followed by the addition of 5 ml of 35% (w/w) and 4 ml of 5% (w/w) sucrose prepared in MBS containing 0.25 M sodium carbonate. After centrifugation at 200,000 x g for 16 h at 4 °C in a Beckman SW41 rotor, a total of 11 fractions (1 ml each) were collected from the top of each gradient and used for immunoblotting.
Immunoprecipitation and ImmunoblottingUnless otherwise indicated, cells were scraped in a lysis buffer (20 mM Hepes, pH 7.4, 137 mM NaCl, 100 mM NaF, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 0.02% NaN3, and 1 mM sodium orthovanadate) supplemented with protease inhibitor mixture (Calbiochem). After 30 min on ice, cell lysates were centrifuged (10,000 x g, 20 min, 4 °C), and the resulting clarified supernatants were collected. Equal amounts of solubilized proteins were incubated with anti-IR (1 µg of each clone), IRS-1 (5 µg), Shc (5 µg), or phosphotyrosine (5 µg) antibody at 4 °C overnight. Alternatively, to detect FLNa-IR association, cells were solubilized in TLB buffer (25 mM Tris·HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 2 mM dithiothreitol, and protease inhibitor mixture), and the clarified supernatants were incubated with IR antibodies. Then, protein A/G-agarose (Oncogene Science) beads were added, and the incubation was continued at 4 °C for 4 h. The beads were pelleted by centrifugation and washed twice in lysis buffer and twice in 50 mM Hepes, pH 7.4, supplemented with 0.1% Triton X-100 before solubilization in Laemmli sample buffer (20) supplemented with 5% 2-mercaptoethanol. In some experiments, cells were lysed directly in Laemmli sample buffer containing 5% 2-mercaptoethanol and 1 mM orthovanadate. After heating at 70 °C for 10 min, proteins were separated by SDS-PAGE and electrotransferred onto polyvinylidene difluoride membranes. Detection of individual proteins was carried out by immunoblotting with specific primary antibodies and visualized by enhanced chemiluminescence. Signals were quantitated by densitometry using the ImageQuant software (Amersham Biosciences).
Measurement of Thymidine IncorporationM2 and A7 cells were grown to 75% confluence in 6-well cluster plates. The growth medium was replaced with serum-free MEM for 18 h. Cells were stimulated with insulin at the indicated concentrations (0, 1, 10, and 100 nM) for 18 h and then pulsed with [3H]thymidine (PerkinElmer Life Sciences), 2 µCi/ml, for 2 h at 37 °C. The cells were washed three times in ice-cold PBS, and DNA was precipitated with 10% trichloroacetic acid for 30 min on ice. After a rapid wash, trichloroacetic acid-precipitable material was dissolved in 0.5 ml of 1 M NaOH and then neutralized with HCl. Levels of [3H]thymidine incorporated into DNA were measured in a scintillation counter and expressed as counts/min. Values obtained without insulin stimulation were subtracted from the corresponding values obtained after treatment.
GST Pull-down ExperimentsHepG2 cell lysates (endogenous
FLNa) or 5 µg of purified chicken gizzard FLNa (Research Diagnostics Inc.)
were either incubated with anti-FLNa antibodies for 4 h followed by
precipitation of the immune complexes with protein A/G-Sepharose beads or
incubated for 16 h with 5 µg of GST or 5 µg of GST-IR -subunit
protein preimmobilized onto glutathione-agarose (Amersham Biosciences) at 4
°C in TLB buffer. After a series of washes in TLB buffer, the bound
proteins were eluted in Laemmli sample buffer, separated by SDS-PAGE, and
immunoblotted using anti-FLNa antibody.
Fluorescence MicroscopyHepG2 cells grown on coverslips were
fixed in fresh 4% paraformaldehyde in PBS for 10 min and permeabilized in 0.1%
Triton X-100 in PBS for 10 min at room temperature. The cells were incubated
with blocking buffer (8% bovine serum albumin in PBS) for 20 min at room
temperature, washed in PBS supplemented with 0.5% bovine serum albumin and
0.05% Tween 20, and incubated with anti-IR -subunit (1:100), FLNa
(1:100), or HA (1:166) antibody for 16 h at 4 °C. After washing, cells
were stained with Alexa Fluor secondary antibody (1:1000). For
immunolocalization of F-actin, fixed cells were incubated with Alexa
Fluor-conjugated phalloidin (1:20). Nuclear counterstaining was performed by
incubating coverslips with Topro-3 in PBS for 5 min prior to mounting slides
with Vectashield (Vector Laboratories). Images were acquired using a Zeiss
LSM-410 inverted confocal microscope with a 63x oil-immersed objective
and processed by using the Metamorph software (Universal Imaging Corp.). No
fluorescent staining was observed when the primary antibody was omitted.
Statistical AnalysisQuantitative data are presented as mean ± S.E., and differences between the means were determined by analysis of variance coupled to Fisher's least significance difference for multiple mean comparisons. A p value of <0.05 was considered significant. Statistical analyses were performed with the StatView statistical software program (SAS Institute, Inc., Cary, NC).
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RESULTS |
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PI3-kinase is an important enzyme implicated in insulin signal transduction through its interaction with tyrosine-phosphorylated IRS proteins (21). It is a heterodimeric enzyme encompassing a p85 regulatory subunit and a catalytic subunit (p110). We therefore examined the cosedimentation of p85 in IRS-1 immunoprecipitates to determine whether p85 association to IRS-1 might be affected by FLNa expression. As shown in Fig. 1B, insulin was able to stimulate recruitment of PI3-kinase to IRS-1 both in vanadate-pretreated M2 and A7 cells.
Two of the major insulin signaling events initiated downstream of the IR is activation of the PI3-kinase/AKT pathway and Ras/MAPK cascade. To evaluate the involvement of FLNa expression on the regulation of these pathways, we examined the ability of insulin to activate AKT and p42/44 MAPK (also known as extracellular signal-regulated kinase, ERK 1/2) by immunoblotting cell lysates with phospho-specific antibodies (Fig. 2). The levels of phosphorylation of AKT at Ser-473, a modification required for its activation (22), were increased upon stimulation of M2 and A7 cells with insulin. In contrast to AKT, there was a marked attenuation in insulin-induced ERK phosphorylation in FLNa-expressing A7 cells (Fig. 2 upper panel, 9th versus 2nd lane). We then investigated whether the lack of FLNa plays a role in the phosphorylation of AKT and ERK in response to other stimuli. Both cell lines were equally responsive to EGF and serum; however, the increase in ERK phosphorylation levels induced by IGF-1 in M2 cells was blocked in FLNa-expressing A7 cells (Fig. 2, upper panel). Similar amounts of ERK protein were present in either cell line (Fig. 2, lower panel). Thus, expression of FLNa differentially affects ERK regulation in response to various stimuli. These results are consistent with a selective effect of FLNa in the signaling pathway used by insulin and IGF-1 to regulate p42/44 MAPK cascade.
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It is well recognized that phosphorylation of Shc by IR is necessary for activation of the Ras/MAPK pathway and mitogenesis (23). In order to investigate the ability of FLNa to modulate mitogenic responses of insulin, we incubated M2 and A7 cells with increasing concentrations of the hormone for 18 h and measured DNA synthesis. FLNa-depleted M2 cells displayed an enhanced sensitivity to insulin when compared with FLNa-expressing A7 cells (Fig. 3). Both cell lines responded to serum (10%) stimulation by increasing thymidine incorporation to the same levels. The sensitivity of insulin-dependent activation of the Shc/MAPK cascade and the subsequent mitogenic responses to FLNa expression suggest specific perturbation of the signaling pathway proximal to the IR.
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Effect of FLNa Expression on Cellular Redistribution of Signaling IntermediatesTranslocation of the Shc adaptor from the cytosol to lipid raft microdomains leads to MAPK activation (24). Moreover, the guanine nucleotide exchange factor SOS1 is prevalently cytosolic and must be brought to the cytosolic side of the plasma membrane in close juxtaposition to Ras to allow GDP/GTP exchange and Ras activation (25). To establish whether expression of FLNa could influence the ability of signaling intermediates to be redistributed upon insulin stimulation, lipid rafts were isolated by sucrose gradient centrifugation from lysates of M2 and A7 cells and immunoblotted with specific antibodies. Preliminary studies showed that both cell types contained similar levels of signaling molecules, such as Shc, SOS1, ERK, and c-Src (Fig. 4A). Light fractions, enriched in rafts, and heavy fractions, containing most soluble proteins, were first tested by immunoblot with anti-SOS1 antibodies. As shown in Fig. 4B (panels I and III), endogenous SOS1 was found only in the heavy fractions of unstimulated cells, demonstrating that SOS1 is predominantly in the cytosol. However, addition of insulin resulted in inducible localization of SOS1 in the light fractions of FLNa-depleted M2 cells but not from FLNa-expressing A7 cells (Fig. 4B, panel II versus IV). The latter cells were found to have also impaired translocation of Shc, ERK, and phosphoactive ERK to the membrane rafts following insulin treatment (Fig. 4C). Of importance, FLNa expression did not abrogate the levels of c-Src, and presumably of other raft-associated proteins, in these specialized membrane microdomains (Fig. 4C, bottom panel). These results are consistent with the hypothesis that FLNa plays an important role in insulin-dependent Shc/MAPK cascade signaling at a step proximal to the IR.
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The IR Is an FLNa-interacting ProteinFLNa interacts with a number of cell surface proteins and intracellular signaling molecules (6). We examined whether IR could form a complex with FLNa in intact cells. Our data demonstrated that IR was associated constitutively with FLNa in A7 cells but not in M2 cells that lack FLNa (Fig. 5A, right panels). Insulin treatment did not cause a significant change in the formation of the IR-FLNa complex in A7 cells. Similar results were obtained in HEK293 cells and HepG2 hepatoma whereby IR-FLNa association was detected under basal and insulin-stimulated conditions (Fig. 5A, left panels). In light of previous evidence supporting the actin-binding properties of FLNa, we sought to examine the role of cytoskeletal organization in the regulation of IR-FLNa association. To this end, HepG2 hepatoma were treated with cytochalasin D, an agent that causes depolymerization of actin (26), followed by reciprocal immunoprecipitation/immunoblotting assays as well as fluorescence microscopy. The inhibitor failed to block FLNa association to the IR, although it blocked the filamentous pattern of actin staining along the longitudinal axis of the cell (Fig. 5B, left panels). Thus, it would appear that the functional pattern of filamentous actin does not regulate IR-FLNa interaction.
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To verify independently that a population of the IR is colocalized with
FLNa, HepG2 hepatoma were stained with antibodies to FLNa and IR
-subunit. As shown in Fig.
5C, both FLNa and IR in the basal state exhibited a
prominent plasma membrane staining.
Next, to determine whether IR could be a direct FLNa-interacting partner,
lysates from HepG2 cells were incubated with a GST fusion protein containing
the cytoplasmic domain of the human IR (amino acids 9411343)
(GST-IR) and analyzed by immunoblotting for the presence of FLNa.
GST-IR
associated with FLNa, whereas GST protein alone was unable to
interact (Fig. 6, lane
6). Incubation with GST-tagged SH2SH2SH3 domain of PLC
1 did not
result in the recruitment of FLNa (data not shown). Purified chicken gizzard
FLNa was then tested for its ability to bind GST-IR
and was found also
to cosediment with recombinant IR (Fig.
6, lane 8). Taken together, these data indicate that IR
interacts with FLNa and may be physiologically relevant.
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Expression of the FLNa C-terminal Region Potentiates Insulin-mediated MAPK ActivationIn order to determine whether the C-terminal region of FLNa is required for the interaction with IR and the regulation of insulin downstream signaling, an FLNa construct comprising the C-terminal repeat regions Arg22Arg24 (amino acids 23572647) was fused to the HA epitope (HA-FLNaCT) and transiently expressed in HepG2 cells (Fig. 7A). Although ectopic expression of FLNaCT blocked the association of IR with endogenous FLNa (Fig. 7B, upper panel), it did not affect the extent of IR autophosphorylation elicited by insulin (Fig. 7B, middle panel). Shown in Fig. 7C is the quantitative data of four independent observations. We then explored the role of FLNaCT in the activation of IR-mediated downstream signaling events. HepG2 cells that were transfected with either pcDNA or FLNaCT displayed no detectable difference in basal and insulin-induced AKT phosphorylation, whereas the phosphorylation levels of ERK were significantly increased in the FLNaCT-transfected cells (Fig. 7D). Similarly, expression of FLNaCT in HEK293 cells consistently led to far greater insulin-induced phosphorylation of ERK, but not AKT, compared with the corresponding control vector-expressing cells (data not shown). It is therefore unlikely that FLNa is involved in regulating the activation of the PI3-kinase/AKT pathway by insulin.
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Engagement of the IR signaling pathway results in the activation of p42/44
MAPK (ERK) and subsequent increase in Elk-1 transcriptional activity
(27). To determine the
relative contribution of FLNaCT in the regulation of Elk-1
transcriptional activity by insulin, we used a transient transfection assay in
which HepG2 cells were cotransfected with pFA-Elk (the activation domain of
Elk-1 fused to the DNA-binding domain of Gal4), pFR-Luc (luciferase reporter
driven by Gal4 DNA-binding element repeats), pCMV--galactosidase as a
control, and either pcDNA or HA-FLNaCT. Serum-starved HepG2 cells
were treated with or without insulin, and the activity of Elk-1-mediated
luciferase activity was determined. Insulin stimulation of pcDNA-transfected
cells resulted in a 1.5-fold increase in Elk-1 transactivation
(Fig. 8; p < 0.05).
However, insulin-induced Elk-1 transactivation was potentiated 2.2-fold in
HepG2 cells that expressed HA-FLNaCT
(Fig. 8; p < 0.01),
whereas the basal level of luciferase activity was not significantly altered
(p > 0.2). Taken together, our data suggest that FLNaCT
does influence physiologically relevant MAPK-dependent effects of insulin such
as Elk-1 transcriptional activation.
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Subcellular Distribution of FLNaCT in HepG2 CellsRecent evidence points to the role of FLNa in the control of actin cytoskeleton and ruffle formation (6). We tested whether ectopic expression of FLNaCT could cause changes in cell morphology through destabilization of cell surface actin cytoskeleton. To this end, control and HA-FLNaCT-transfected HepG2 cells were stained with an antibody to HA and Alexa 568-conjugated phalloidin to detect changes in the pattern of filamentous actin. Whereas F-actin staining in cells expressing the empty vector exhibited a filamentous pattern, a marked attenuation of actin staining throughout the cell was observed upon HA-FLNCT expression (Fig. 9, D and F versus B). Of significance, expression of HA-FLNCT in unstimulated cells appeared to induce a change in cell morphology and an increase in membrane ruffling at sites where FLNCT accumulated (Fig. 9C). The pattern of HA-FLNCT staining was detected both subjacent to the plasma membrane and as a diffuse cytoplasmic distribution (Fig. 9E). Finally, overlay of anti-HA and IR staining in transfected HepG2 cells showed constitutive colocalization of FLNCT with endogenous IR at the cell membrane (Fig. 9I).
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DISCUSSION |
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In this study, we have demonstrated that human M2 cells lacking FLNa exhibit normal insulin-mediated phosphorylation of p42/44 MAPK, whereas the FLNa-expressing A7 cells are unable to elicit this phosphorylation event upon stimulation with insulin. In contrast, FLNa expression did not interfere with MAPK phosphorylation in response to EGF or serum but not IGF-1. These results would underscore the specificity of the FLNa effects toward the insulin/IGF-1 responses and support our observations indicating that FLNa expression selectively impairs insulin-dependent thymidine incorporation, while having no effect on serum-stimulated DNA synthesis. There is evidence to suggest that TNF-stimulated MAPK activation is potently inhibited in M2 cells (30), whereas heregulin efficiently stimulates MAPK in both M2 and A7 cells (8). On the other hand, the G protein-coupled extracellular calcium-sensing receptor can activate MAPK very efficiently as a result of specific interaction between FLNa and the calcium-sensing receptor (31, 32). These observations raise the interesting possibility that although FLNa is dispensable for MAPK activation in response to EGF or serum, it might be required at some regulatory step proximal to the highly homologous receptors for insulin and IGF-1, possibly in cooperation with a subset of signal transducing molecules converging to MAPK (33).
The role of Shc in insulin-induced mitogenic signaling follows a path that has been well characterized. The adaptor Shc binds via its PTB domain to the juxtamembrane region of the activated IR (34), which then leads to Shc tyrosine phosphorylation and subsequent association with pre-formed Grb2-SOS complex to the plasma membrane where Ras is known to reside (35). Because of the role of Shc in linking the activated IR to the Ras/MAPK pathway via the guanine nucleotide exchange factor SOS, elucidation of the mechanisms by which FLNa impairs Shc tyrosine phosphorylation is key for understanding the mitogenic signaling of insulin. Localization to membrane rafts is required for Shc activity (36). Indeed, addition of the membrane localization sequence for Ras leads to constitutive Shc targeting to the plasma membrane, thus resulting in ligand-independent activation of the Ras/MAPK cascade (24). This is somewhat consistent with our observation that FLNa-dependent interactions might inhibit insulin-induced Shc tyrosine phosphorylation due to impaired translocation of Shc to membrane rafts where the IR is known to reside. It is noteworthy that the ability of insulin to promote redistribution of SOS and MAPK to lipid rafts is also blocked as a consequence of FLNa expression in A7 cells. This argues well for the role of FLNa in governing insulin-dependent signaling events via regulation of protein trafficking.
FLNa is an actin-binding protein expressing a number of repeats that have
been shown to associate with receptors or signaling proteins (reviewed by
Stossel et al. (6)). For
instance, FLNa binds Smads, a group of proteins necessary for the regulation
of TGF- signaling (37).
Many proteins known to bind FLNa appear to be functionally linked to
downstream signaling components via the actin cytoskeleton. As such, FLNa is a
determinant of the submembranous cytoskeletal architecture of cells, and
consistent with this role, it has an important function in the endocytic
sorting and recycling pathways
(38,
39). The presence of filamin
has been reported to be necessary for translocation of the 5-phosphatase
SHIP-2 at the membrane ruffles and the cortical actin rim at the periphery of
the COS-7 cells after EGF stimulation
(40). Similarly, FLNa may be
required for proper cell surface expression of the D2 dopamine receptors
(41). On the other hand,
ligand-induced movement of the human androgen receptor from the cytoplasm to
the nucleus is a process that requires FLNa
(42). Unlike these
FLNa-binding proteins, the association between the IR to FLNa does not appear
to be obviously related to intact actin cytoskeletal assembly as actin
depolymerization with cytochalasin D failed to disrupt the constitutive
IR-FLNa interaction.
Immunofluorescence studies revealed that transiently expressed FLNaCT colocalizes with the IR at the surface of HepG2 cells. Whereas FLNaCT expression blocks the constitutive association between endogenous FLNa and the IR, it potentiates insulin-induced MAPK phosphorylation and Elk-1 transcriptional activity when compared with pcDNA-transfected cells treated with insulin. However, FLNaCT had no effect on insulin-induced IR or IRS-1 tyrosine phosphorylation and an increase in AKT phosphorylation elicited by insulin. It is therefore likely that overexpressed FLNaCT titrates inhibitory proteins out of endogenous signaling complexes involved in MAPK cascade activation, thereby potentiating insulin signal transduction. In light of these results, what role might FLNa play in constitutive and IR-mediated activation of MAPK? With the exception perhaps of a weak binding of MEK-1 to FLNa in two-hybrid assays (30), no component of MAPK-activating pathways other than the IR, as demonstrated here, has been shown to bind to FLNa to date. Thus, it is possible that alteration in FLNa expression might affect the signaling potential of the IR at a step proximal to the receptor, namely the tyrosine phosphorylation of a subset of protein substrates and their recruitment to appropriate location. Alternatively, a subpopulation of the IR could potentially couple to the MAPK pathway, at least through its interaction with FLNa, whereas the receptor in other subcellular locations might interact with additional scaffold proteins that enable it to modulate the IRS-1/PI3-kinase/AKT pathway.
The localization of FLNaCT to membrane ruffles in HepG2 hepatoma
may enable it to function as a scaffold upon which reorganization of membrane
actin cytoskeleton and cell morphology can take place. Activation of the small
GTPases of the Rho family and their downstream targets, Paks, produces
phenotypic changes consistent with actin cytoskeletal rearrangements. Notably,
FLNa interacts directly with Trio, a unique Rho GEF
(43), and Pak1
(8). It remains to be
determined if FLNa regulates the assembly of signaling complexes after
activation of the IR. Although insulin treatment did not appear to change the
binding of endogenous and transfected FLNa to the IR in HepG2 hepatoma and A7
cells, respectively, these experiments do not rule out a signal-dependent
posttranslational modification of FLNa. In this regard,
p56lck has been proposed to phosphorylate filamin, thus
controlling its association with cell surface receptors such as
2 integrins and actin filament cross-linking
(44). Moreover, Pak1 controls
the actin cytoskeletal and ruffle formation, in part by phosphorylating FLNa
(8). FLNa has also been found
to undergo phosphorylation in situ in response to serum,
lysophosphatidic acid, and other stimuli
(45). Thus, it is likely that
functional alterations in FLNa might occur as a consequence of phosphorylation
events. Incidentally, phosphorylation of IRS-1 on tyrosine residues allows the
recruitment of signaling-competent molecules (e.g. PI3-kinase),
whereas its phosphorylation by Ser/Thr kinases adversely affects the ability
of IRS-1 to interact with the activated IR and several signal-transducing
proteins, resulting in the development of insulin-resistant states
(46,
47). The possibility that
insulin initiates the phosphorylation of FLNa will be the subject of future
investigations. Finally, the importance of FLNa in modulating insulin signal
transduction in other insulin-sensitive cells (e.g. muscle and
adipose tissue) remains to be determined. The IR-binding properties of FLNa
may represent only one of many possible pathways leading to the control of
MAPK activation by insulin.
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FOOTNOTES |
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¶ Present address: Division of Pulmonary and Critical Care Medicine, Dept. of
Medicine, The Johns Hopkins University, Baltimore, MD 21224.
|| To whom correspondence should be addressed: Diabetes Section, NIA, National Institutes of Health, 5600 Nathan Shock Dr., Box 23, Baltimore, MD 21224-6825. Tel.: 410-558-8199; Fax: 410-558-8381; E-mail: Bernierm{at}vax.grc.nia.nih.gov.
1 The abbreviations used are: IR, insulin receptor; FLNa, filamin A; MAPK,
mitogen-activated protein kinase; IRS-1, insulin receptor substrate 1; SH2,
Src homology 2 domain; PI3-kinase, phosphatidylinositol 3-kinase; PBS,
phosphate-buffered saline; MES, 4-morpholinoethanesulfonic acid; MEM, minimum
essential medium; EGF, epidermal growth factor; FBS, fetal bovine serum;
IGF-1, insulin-like growth factor 1; GST, glutathione S-transferase;
HA, hemagglutinin; ERK, extracellular signal-regulated kinase.
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ACKNOWLEDGMENTS |
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REFERENCES |
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