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Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Diego, 9500 Gilman Dr., La Jolla, California 92093
Received 8 December 2006/ Returned for modification 31 January 2007/ Accepted 7 May 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(PKC), are activated and contribute to insulin regulation of GLUT4 translocation and glucose uptake. Akt is an important mediator of the biological functions of insulin. Krook et al. reported that insulin-stimulated Akt phosphorylation was impaired in insulin-resistant Goto-Kakizaki rats and in muscle biopsies from type 2 diabetic patients (23). Impaired activation of Akt in response to insulin has also been described in insulin-resistant human (33) and rodent (4) adipocytes, and impaired GLUT4 translocation was associated with the defective Akt phosphorylation at insulin-resistant states (4). Furthermore, in knockout animals, targeted disruption of the Akt2 (protein kinase Bß) gene causes insulin resistance and a type2 diabetes-like phenotype (5). Recently, several lines of evidence have demonstrated the importance of Akt for insulin-stimulated GLUT4 translocation. Thus, constitutively active membrane-targeted Akt induced GLUT4 translocation in the absence of insulin (21, 22), whereas expression of dominant-negative mutants and microinjection of blocking antibodies against Akt inhibited insulin-induced GLUT4 translocation (21, 42, 44). Using small interfering RNA (siRNA) to reduce the expression level of Akt, knockdown of Akt2 in adipocytes prevented insulin-induced GLUT4 translocation (17, 21, 42).
In an earlier study, we showed insulin stimulation can activate atypical PKC, which facilitates exocytosis of GLUT4 towards the plasma membrane along the microtubule system (14). We further showed that this involves Rab4 and the motor protein KIF3. However, GLUT4 proteins cannot travel along microtubules all the way to the plasma membrane. Rather, microtubule cargo interacts with the submembranous actin cytoskeletal network (45) to carry out the final stages of GLUT4 translocation. Thus, in the current study, we have focused our attention on the motor proteins which facilitate GLUT4 translocation along actin filaments (F-actin). Since insulin-induced activation of Akt is a necessary step in GLUT4 translocation, we assessed whether an actin-based motor protein might be a target of Akt. This study shows that the motor protein myosin 5a is a direct target of Akt and facilitates in the process of insulin-stimulated GLUT4 translocation along the actin network in 3T3-L1 cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) antibody, anti-myosin 5a antibody, anti-Rab4 antibody, anti-myc antibody, protein A-Sepharose, protein G-Sepharose, and horseradish peroxidase-linked anti-goat antibody were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-Akt2 antibody and anti-IRS1 antibody were from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti--actinin antibody was from Abcam (Cambridge, MA). Horseradish peroxidase-linked anti-rabbit and anti-mouse antibodies, sheep immunoglobulin G (IgG), rhodamine- and fluorescein isothiocyanate (FITC)-conjugated anti-rabbit antibody, and Cy3-conjugated secondary antibody were obtained from Jackson Immunoresearch Laboratories Inc. (West Grove, PA). Anti-GLUT4 antibody was from Biogenesis Inc. (Brentwood, NH). Anti-PKC antibody was from Transduction Laboratory (Lexington, KY). Anti-HA-11 antibody was from Covance (Princeton, NJ). Wortmannin was from Calbiochem (San Diego, CA). Dulbecco's modified Eagle medium (DMEM) and fetal calf serum were obtained from Life Technologies, Inc. (Grand Island, NY). 2-[3H]Deoxyglucose and L-[3H]glucose were from ICN (Costa Mesa, Calif.). All other reagents and chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Cell treatment and transient transfection. 3T3-L1 cells were cultured and differentiated as described previously (47). For preparation of whole-cell lysates for immunoprecipitation and immunoblotting experiments, 3T3-L1 adipocytes were starved for 16 h in DMEM containing 0.1% bovine serum albumin (BSA). The cells were stimulated with or without 0.5 or 17 nM insulin at 37°C for various periods as indicated in the figures. Differentiated 3T3-L1 adipocytes were transiently transfected by electroporation, as previously described (14). For adenovirus infection, 3T3-L1 adipocytes were transduced at a multiplicity of infection of 50 PFU/cell for 16 h with the recombinant adenovirus encoding GFP or the dominant-negative form of Akt (Akt-MAA [K179M, T308A, S473A]) as described previously (21).
Generation of mutant MGT. Myc epitope-tagged COOH-terminal globular domains of myosin 5a tail (MGT) wild type and MGT-Ser1650Ala (20a) were provided by Vladimir I. Gelfand (Northwestern University, Chicago, IL). A QuikChange kit (Stratagene, La Jolla, CA) was used for site-directed mutagenesis. An oligonucleotide in which Ser 1812 of MGT was replaced with Ala was used as the primer in the in vitro mutagenesis reaction. The sequences were confirmed by direct DNA sequencing. The resulting plasmids were denoted MGT-Ser1812Ala.
RNA interference.
The duplexes of each siRNA, targeting myosin 5a mRNA (target sequence, 5'-CGCUACAAGAAGCUCCAUA-3'; corresponding to the cDNA sequence from 2789 to 2807), Akt1 (18), Akt2 (target sequence, 5'-GAGAGGACCUUCCAUGUAG-3'; corresponding to the cDNA sequence from 469 to 487), PKC (42), and a negative control (scrambled sequence) were purchased from Dharmacon Research Inc. (Lafayette, CO). The target sequences against myosin 5a and Akt2 were chosen by a World Wide Web-based search program (www.dharmacon.com), and the absence of homology to any other gene was confirmed by a BLAST search (National Center for Biotechnology Information, National Institutes of Health). On day 8 postdifferentiation, 3T3-L1 adipocytes were electroporated with an siRNA using the Gene Pulser XCell (Bio-Rad). Electroporated cells were incubated for 48 h at 37°C prior to assays.
Immunoprecipitation and Western blotting. Coimmunoprecipitation and Western blotting experiments were performed as described previously (14). For the coimmunoprecipitation between Rab4 and myosin 5a, cells were incubated for 120 min at 4°C in phosphate-buffered saline containing 2 mM dithiobis (succinimidylpropionate) cross-linker (Pierce). Cells were lysed in a cold solubilizing buffer containing 40 mM Tris, 1 mM EGTA, 100 mM NaCl, 1 mM MgCl2, 1% Nonidet P-40, 10% glycerol, 1 mM Na3VO4, 1 mM phenymethylsulfonyl fluoride (PMSF), and 20 mM NaF, pH 7.5. The soluble fractions were immunoprecipitated with antibodies, followed by an incubation with protein A- or protein G-conjugated beads. The immunoprecipitates were boiled with Laemmli sample buffer containing 100 mM dithiothreitol and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting.
In vitro phosphorylation of MGT.
Forty-eight hours after electroporation of MGT-wild type or -Ser1650Ala into 3T3-L1 adipocytes, MGT proteins were purified using anti-c-myc agarose from Sigma Chemical Co. Akt2/PKBß (delta PH, S474D) active protein and PKC active protein were purchased from Upstate Biotechnology, Inc., and we performed in vitro phosphorylation assays following the manufacturer's procedure. Purified MGT was incubated with 600 ng of BSA, 600 ng of recombinant Akt, or 30 ng of recombinant PKC with [
-32P]ATP (20 µCi; final ATP concentration of 100 µM per assay point). After 20 min of incubation at 30°C, the reaction was terminated by addition of Laemmli sample buffer. Samples were then analyzed by SDS-PAGE. Gels were dried, and signals were detected by using a PhosphorImager (Molecular Dynamics).
RNA isolation and RT-PCR. Forty-eight hours after electroporation of siRNA, 3T3-L1 adipocytes were scraped, and total RNA was purified with an RNeasy mini-kit from QIAGEN (Valencia, CA). The reverse transcriptional PCR (RT-PCR) was performed with a myosin 5a-specific or ß-actin primer set by using a one-step RT-PCR kit from QIAGEN.
2-Deoxyglucose uptake. The procedure for evaluating glucose transport was performed as previously described (47). Glucose uptake was determined after the addition of 5 µl of substrate. 2-[3H]deoxyglucose (2-DOG) or L-[3H]glucose (0.1 µCi; final concentration, 0.01 mM) was added to provide a concentration at which cell membrane transport is rate limiting.
Microinjection and immunofluorescence staining. Microinjection was performed as previously described (15). Cells were incubated in complete medium for 48 h after microinjection and then serum starved for 5 h, followed by stimulation with insulin for 20 min. Immunostaining of GLUT4 was performed as described elsewhere (12). Cell surface GLUT4 staining was identified by immunofluorescence microscopy, and individual cells were scored as positive or negative for surface GLUT4. Approximately 300 cells per coverslip were counted by an observer blind to the experimental conditions. For myosin 5a immunofluorescence, we fixed and permeabilized 3T3-L1 adipocytes. The cells were then stained with polyclonal anti-myosin 5a antibody (LF-18 from Sigma Chemical Co.), followed by Cy3-conjugated anti-rabbit secondary antibody. Cy3 fluorescent emission was collected on a Nikon TE300 with a 40x 1.25-numerical-aperture oil immersion objective.
Membrane-targeting kinetics of HA-GLUT4-eGFP. Various siRNAs or MGT-wild type along with the HA-GLUT4-eGFP expression vector were electroporated together for scoring of HA-GLUT4-eGFP translocation. All images were collected on a Nikon TE300 with a 40x 1.25-numerical-aperture oil immersion objective. Simple PCI software (C-imaging Systems, Sewickley, PA) was used for image processing and quantification. In transfected cells, the intensities of the GFP and Cy3 signals were quantified, and background GFP and Cy3 fluorescence emissions were subtracted. The Cy3 fluorescence intensity for each HA-GLUT4-eGFP-expressing cell (reflecting cell surface HA-GLUT4-eGFP) was divided by the GFP fluorescence intensity (reflecting the total cellular HA-GLUT4-eGFP level) to determine the fraction of tagged GLUT4 transporter at the membrane. Translocation following insulin stimulation was expressed as a percentage of the maximum response. Fluorescence quantification was performed as previously described (30, 40).
Transferrin receptor trafficking assay. The transferrin receptor trafficking assay was a modified version of the method previously published (40). Briefly, control or myosin 5a siRNA along with the human transferrin receptor (TR) expression vector were electroporated together into 3T3-L1 adipocytes for assessment of TR translocation. The endogenous mouse TR was quenched by incubating cells for 18 h with a rat monoclonal antibody to the extracellular domain of the mouse TR but not the human TR (40). The starved cells were incubated in DMEM containing 10 µg/ml Cy3-transferrin for 2 h and stimulated with or without 170 nM insulin. The cells were fixed with 3.7% formaldehyde, and TR on the plasma membrane was stained with a monoclonal antibody directed against the extracellular domain of the human TR (Molecular Probes Inc., Eugene, OR) and detected with a FITC-anti-mouse secondary antibody. The total amount of TR expressed was determined by uptake from the medium of Cy3-labeled human transferrin (Sigma Chemical Co.; Cy3 labeling kit from Molecular Probes, Inc.). Images were taken, and total Cy3 and FITC fluorescence intensities per cell were calculated by dividing the total intensity by the area of the cell measured in pixels. To correct for background fluorescence, the same measurements were made for cells that did not express human TR. The background fluorescence intensity (for both the Cy3 and FITC fluorescence) was subtracted from the experimental data. The FITC/Cy3 ratio was calculated for each cell and averaged over 20 cells for each experiment.
Cosedimentation assay of myosin 5a-actin binding.
A cosedimentation assay (29) was performed by using the nonmuscle actin binding protein Biochem kit according to the manufacturer's instructions (Cytoskeleton, Denver, CO). Briefly, starved 3T3-L1 adipocytes were stimulated with or without 17 nM insulin at 37°C for 20 min. Cells were lysed in a cold actin lysis buffer containing 20 mM Tris, 140 mM NaCl, 50 mM KCl, 2 mM MgCl2, 1% Nonidet P-40, 1 mM Na3VO4, 1 mM PMSF, 2 mM dithiothreitol, and 50 mM NaF, pH 7.5. The soluble lysate was sedimented at 150,000 x g for 60 min at 20°C, and pellets were resuspended. The test protein, -actinin, or BSA was incubated with or without polymerized F-actin at room temperature for 30 min. The mixtures were pelleted by centrifugation at 150,000 x g for 1.5 h. The pellets were separated and analyzed by SDS-PAGE, and proteins were detected by immunoblotting with anti--actinin or anti-myosin 5a antibody or Coomassie brilliant blue (CBB) staining.
Isolation of GLUT4 vesicles. 3T3-L1 adipocytes were treated with or without 17 nM insulin for 15 min. Each cell was homogenized in buffer (25 mM HEPES, pH 7.4, 1 mM EDTA, 255 mM sucrose) supplemented with 0.1 mM Na3VO4, 50 mM NaF, and 1 mM PMSF, and the homogenate was centrifuged at 16,000 x g for 20 min. The supernatant was used for the adsorption of vesicles. Aliquots were incubated with antibody against GLUT4, followed by an incubation with protein G-conjugated beads. The immunoprecipitates were boiled with Laemmli sample buffer.
Subcellular fractionation. An abbreviated differential centrifugation procedure was used to obtain cytosol and plasma membrane fractions as previously described (6). 3T3-L1 adipocytes were homogenized in HES buffer (20 mM HEPES, pH 7.4, 1 mM EDTA, 250 mM sucrose) supplemented with 0.1 mM Na3VO4 and 1 mM PMSF. The homogenate was centrifuged at 19,000 x g for 20 min, and the resulting supernatant was centrifuged at 180,000 x g for 75 min at 4°C, which yielded the cytosol fraction as a supernatant. The pellet obtained on the initial spin was layered onto 1.12 M sucrose in HES buffer, followed by centrifugation at 100,000 x g for 60 min. This yielded a white fluffy band at the interface (plasma membrane fraction). The plasma membrane fraction was resuspended in HES and then pelleted at 40,000 x g for 20 min. All fractions were resuspended in HES to a final protein concentration of 1 to 2 mg/ml.
Statistics. The values are expressed as means ± standard errors. Scheffe's multiple comparison test was used to determine the significance of any differences among more than three groups. A P value of less than 0.05 was considered significant.
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RESULTS |
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and [-32P]ATP. As seen in Fig. 1G, MGT-WT phosphorylation was substantially increased by active recombinant Akt2, but not by active PKC or control buffer. Furthermore, active Akt2 did not phosphorylate MGT-Ser1650Ala (Fig. 1G), consisted with the results in Fig. 1E and F. These results indicate that myosin 5a is a direct substrate of activated Akt and that the relevant insulin-induced phosphorylation occurs at Ser1650. Knockdown of myosin 5a by RNAi inhibits insulin-stimulated glucose transport. The data in Fig. 1 suggest that myosin 5a may play a role in insulin-stimulated glucose transport. To assess this, we measured 2-DOG uptake in 3T3-L1 adipocytes with and without myosin 5a depletion by RNA interference (RNAi). Several siRNAs against myosin 5a sequences were tested, and Fig. 2 shows the results for the most effective sequence obtained. At 48 h after electroporation of siMyosin 5a, we observed a 50 to 60% decrease in myosin 5a mRNA (Fig. 2A and B) and protein expression (Fig. 2C and D), compared to the levels in cells treated with scrambled siRNA. In contrast, expression levels of myosin 1c were unaffected (Fig. 2C). Myosin 5a knockdown led to a 20 to 25% decrease in insulin-stimulated glucose transport. Although this effect was modest, it should be noted that the siRNA knockdown of myosin 5a was only 50 to 60% effective. In addition, we considered the possibility that other myosin isoforms, such as myosin 5b, might compensate for the relative absence of myosin 5a (26). To assess this possibility, we used RNAi to knock down both myosin 5a and 5b, followed by measurements of 2-DOG uptake. This double knockdown led to a further decrease in insulin-stimulated 2-DOG uptake, with inhibition of glucose transport of 49 and 66% at submaximal (0.5 nM) and maximal (17 nM) insulin stimulation, respectively (Fig. 2E). We could not determine whether myosin 5b is also an Akt substrate, since antibodies which recognize this myosin isoform are not available. Furthermore, treatment of cells with siMyosin 5b or siMyosin 5c alone had no effect on glucose transport, and the combination with siMyosin 5c had no further inhibitory effect (data not shown).
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Dominant-negative Akt causes decreased phosphorylation of myosin 5a. To further explore myosin 5a phosphorylation by Akt, we examined the effect of DN-Akt (Akt-K179M/T308A/S473A). DN-Akt inhibited Akt activity in immunoprecipitations with either anti-Akt1 or -Akt2 antibody (21), indicating that DN-Akt inhibits both Akt isoforms. As shown in Fig. 4A and B, expression of DN-Akt markedly reduced insulin-stimulated myosin 5a phosphorylation, without changing myosin 5a protein levels (Fig. 4A). These data further indicate that myosin 5a is phosphorylated by Akt. Consistent with this (42), 3T3-L1 adipocytes expressing DN-Akt exhibit decreased insulin-stimulated 2-DOG uptake compared to cells transduced with control adenovirus encoding GFP (Fig. 4C).
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, knockdown blocks insulin-induced phosphorylation of myosin 5a.
To assess the specificity of Akt-mediated myosin 5a phosphorylation, we utilized siRNAs against Akt1, Akt2, and PKC. Thus, 3T3-L1 adipocytes were electroporated with these different siRNAs, and 48 h later insulin-stimulated myosin 5a phosphorylation was determined. Each individual siRNA led to marked depletion of the target protein (Fig. 5A and B), but as seen in Fig. 5A and C, only Akt2 knockdown inhibited insulin-induced myosin 5a phosphorylation. Taken together with the in vitro phosphorylation data in Fig. 1G, these results demonstrate the specificity of insulin-directed Akt2 activity towards myosin 5a, whereas the other PI 3-kinase-dependent insulin target proteins (Akt1 and PKC) did not participate in this phosphorylation event. In addition, Fig. 5D shows that knockdown of Akt2 inhibited insulin-stimulated GLUT4 translocation, whereas knockdown of Akt1 had only marginal effects. As mentioned earlier, we have previously demonstrated that depletion, or inhibition, of PKC causes decreased insulin-stimulated GLUT4 translocation (14, 42), but this mechanism relates to proximal GLUT4 movement towards the cell surface along microtubules prior to engagement of the actin cytoskeleton (14).
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-actinin, which is known to cosediment with F-actin, and this is demonstrated in Fig. 6A. After incubation of the resuspended pellets with or without F-actin, the associated proteins were pelleted and analyzed by SDS-PAGE with either anti-myosin 5a antibody immunoblotting or CBB staining. In the absence of F-actin, myosin 5a was not detected in the pellets (Fig. 6B), whereas in the presence of F-actin, insulin stimulation led to marked cosedimentation of myosin 5a with F-actin as detected by myosin 5a immunoblotting or CBB staining. As seen in Fig. 6C and D, this ability of insulin to induce myosin 5a binding to F-actin was blocked when Akt activity was inhibited by expressing dominant-negative Akt in the cells, or when Akt2 was depleted by Akt2 siRNA.
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knockdown cells (Fig. 8C), while neither Akt2 nor PKC knockdown affected the amount of myosin 5a and Rab4. These data indicate that the insulin-stimulated interaction of myosin 5a and Rab4 is downstream of Akt2.
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DISCUSSION |
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Although the functional regulation of myosin 5 is incompletely understood, two major domains are known to participate in the motor activity of myosin 5a. A series of repeat IQ motifs are located near the motor protein head domain, and these motifs bind to calmodulins and specific myosin light chains (36). The myosin 5a tail domain is important for cargo binding and cellular localization, and overexpression of this tail domain can inhibit myosin 5a activity (45). Interestingly, by searching the myosin sequence databases, we observed that the myosin 5a tail domain contains two Akt substrate sequence repeats (RXRXXpS/T) at Ser 1650 and Ser 1812. Here we used three distinct approaches to test whether myosin 5a is a direct substrate of Akt. First, we showed a modest insulin-stimulated gel mobility shift of myosin 5a (Fig. 1B). Second, we utilized a phospho-Akt substrate-specific antibody and clearly demonstrated insulin-stimulated phosphorylation of Ser 1650 in a PI 3-kinase- and Akt2-dependent manner. Third, we purified the myosin 5a C-terminal domain (MGT) and used a direct in vitro phosphorylation assay to demonstrate that Akt2 phosphorylates myosin 5a at Ser 1650. Further, insulin-induced phosphorylation of myosin 5a stimulated the actin binding activity of this protein, and this insulin effect was inhibited by expression of dominant-negative Akt, or siRNA-mediated Akt2 knockdown, as well as expression of dominant-negative mutant myosin 5a. Insulin stimulation also caused association of myosin 5a with GLUT4 vesicles and translocation of myosin 5a to the plasma membrane along with GLUT4 and IRAP. Consistent with the motor function of myosin 5a, we found that siRNA-induced depletion of myosin 5a, or expression of dominant-negative myosin 5a, inhibited insulin-stimulated glucose transport and GLUT4 translocation without affecting the early steps of insulin signaling. From these results, we conclude that insulin induces myosin 5a activation via Akt2-mediated phosphorylation of Ser 1650 in the tail domain. In turn, activated myosin 5a then functions as a motor protein facilitating anterograde translocation of GLUT4 through the actin cytoskeleton network.
Activation of insulin receptor is rapidly followed by docking of insulin receptor substrates and stimulation of PI 3-kinase. Akt and PKC are serine/threonine kinases activated downstream of PI 3-kinase, and both are mediators of major metabolic actions of insulin, such as glucose transport and GLUT4 translocation (38). However, the signaling events downstream of these two kinases remain to be fully elucidated. Recently, a new Akt substrate, Akt substrate of 160 kDa (AS160), has been identified (19). Upon insulin stimulation, phosphorylation of AS160 through Akt leads to inactivation of AS160 GAP activity, which releases retention of GLUT4, promoting translocation (7, 28). Evidence shows that Rab8A and Rab14 are targets of AS160 and may be involved in GLUT4 translocation in the perinuclear region (16). That report suggested that AS160 serves as a modulator of basal GLUT4 trafficking. More recently, Gonzalez et al. reported that inhibition of Akt impaired GLUT4 exocytosis in AS160 knockdown adipocytes, suggesting that additional Akt substrates, other than AS160, are involved in insulin regulation of GLUT4 exocytosis (8). They also showed that Akt activity was specifically required for GLUT4 exocytosis within the region 250 nm from the plasma membrane, where F-actin is located in juxtaposition to the plasma membrane. These data are fully consistent with our results showing that myosin 5a is a new Akt2 substrate involved in GLUT4 translocation along F-actin.
It has been well demonstrated in several systems that vesicular trafficking is observed along microtubule and actin cytoskeletal structures (10, 27). Indeed, in adipocytes, disruption of microtubules (11, 24, 31) or F-actin (20) resulted in marked inhibition of insulin-stimulated GLUT4 translocation and glucose uptake. Since the plus ends of microtubules do not connect directly to the plasma membrane, vesicle cargo has to be transferred from microtubules to F-actin structures in order to reach the cell surface. Although the understanding of vesicle transfer mechanisms between these two systems is limited, it has been reported that melanosomes can be transported to melanocyte dendrites by the microtubule-based motor protein KIF3 and that the subsequent movement of these vesicles, and their tethering at the cell membrane, is dependent on myosin 5 and F-actin (46). Further support for this microtubule/actin cytoskeletal "handoff" model has been provided by colocalization and binding studies that showed the direct interaction between the myosin 5a and KIF3 tail domains (13). Close cooperation between the kinesin/microtubule and myosin 5/F-actin systems has also been observed in melanophore transport (32). In our previous study, we found that GLUT4 vesicles are localized to the perinuclear Golgi region in nonstimulated cells and, after insulin stimulation, are transported in an anterograde fashion along microtubules, and this is dependent on the microtubule-based kinesin motor protein KIF3 (14). In the overall process of GLUT4 exocytosis, this would account for GLUT4 movement along microtubules towards the cell periphery after insulin stimulation. The potential transfer mechanism of GLUT4 vesicles from microtubules to the F-actin system has not been addressed, and based on the current results, we suggest that PI 3-kinase-dependent KIF3/microtubule and myosin 5a/F-actin cooperation could be a model to translocate GLUT4 vesicles from their intracellular perinuclear loci to the plasma membrane via microtubular and then F-actin structures, under the influence of insulin.
Previous reports have shown that another microtubule motor protein, KIF5B, and the actin-based motor protein, myosin 1c, are involved in insulin-induced GLUT4 translocation (2, 37). In this regard, KIF5B was wortmannin insensitive, suggesting that a PI 3-kinase-independent pathway(s) mediates insulin's effect on this protein. Additionally, myosin 1c is now thought to facilitate the fusion of exocytic GLUT4-containing vesicles with the adipocyte plasma membrane (3). Time-lapse total internal reflection microscopy studies (25) in rat primary adipocytes demonstrated that GLUT4 vesicles rapidly move along microtubules, periodically tethering to the plasma membrane in the basal state, and that insulin halted this traffic by enhancing the tethering step. It is possible that myosin 5a participates in this tethering step.
Akt and PKC are both PI 3-kinase-dependent serine/threonine kinases, and numerous studies have demonstrated that both PKC and Akt are necessary for insulin-induced glucose transport (14, 17, 21, 22, 42, 44). These findings raise the question as to how these two similar serine/threonine kinases both play important roles in the process of insulin signaling to glucose transport. The current study, coupled with our previous reports of PKC signaling (14), provide a working model to help understand these phenomena. In a previous study, we reported that insulin stimulation of PKC mediates the activation of a kinesin family motor protein, KIF3, to facilitate anterograde movement of GLUT4 along microtubule structures. In the current study, we find that insulin-induced Akt2 activation stimulates the ability of another motor protein, myosin 5a, to mediate anterograde movement of GLUT4 by the actin cytoskeleton. This suggests that sequential cooperation of PKC/KIF3 and Akt2/myosin 5a could participate in the movement of GLUT4 cargo through the microtubule system and then on through the actin cytoskeleton, thus providing an explanation for the role of both of these serine/threonine kinases in the overall process of GLUT4 translocation. Additional supporting evidence for this idea is the subcellular localization of these two kinases. Thus, PKC has been colocalized with microtubules (32), whereas Akt is colocalized with F-actin (39). Taken together, these differences in subcellular localizations of Akt and PKC, combined with the biochemical and biological data in the current and previous studies, are consistent with the working model proposed above.
The PKC/KIF3/microtubule and Akt2/myosin 5a/actin systems are biochemically and structurally distinct. Nevertheless, GLUT4 vesicles must be recognized by both systems under the influence of insulin to complete the full process of GLUT4 translocation from its initial perinuclear localization to the cell surface. Although the mechanisms for this remain incompletely understood, we have provided some evidence for a possible linker protein between GLUT4 vesicles and both KIF3 and myosin 5a motor proteins. Thus, we have previously shown that insulin stimulates Rab4 activation in 3T3-L1 adipocytes and that Rab4 may serve as an adaptor, between KIF3 and GLUT4 vesicles, in relationship to microtubule-based movement (14). In the current study, we show that, under the influence of insulin, myosin 5a binds to GLUT4-containing vesicles and translocates to the plasma membrane. In the insulin-stimulated state, Rab4 can also coassociate with myosin 5a. This Rab4 association with myosin 5a was decreased in Akt2 knockdown but not in PKC knockdown cells, suggesting that the insulin-stimulated interaction of myosin 5a and Rab4 is downstream of Akt2. Thus, Rab4 may provide a common link between GLUT4 vesicles and the microtubular and actin-based motor proteins facilitating the transfer of GLUT4 vesicles from microtubules to the F-actin system in the process of GLUT4 translocation.
In summary, these results provide evidence that the actin-based motor protein myosin 5a is a direct substrate for insulin-stimulated Akt2. In turn, Akt2-dependent phosphorylation of myosin 5a enhances its ability to interact with the actin cytoskeleton and GLUT4 vesicles, and depletion of myosin 5a inhibits glucose transport stimulation. As such, these novel results are consistent with the view that myosin 5a is a new regulator of GLUT4 translocation, providing an important and direct functional link between the insulin-directed PI 3-kinase-Akt2 signaling pathway and GLUT4 translocation. In addition, taken together with our previous findings on the connection between activation of PKC and the microtubule-based motor protein KIF3, it is suggested that PKC and Akt2 sequentially cooperate to translocate GLUT4 vesicles from the perinuclear pool to the cell surface through a sequential KIF3/microtubule and myosin 5a/F-actin mechanism.
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ACKNOWLEDGMENTS |
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This study was funded in part by National Institutes of Health grant DK 33651 (J.M.O.) and the University of California Discovery Program Project bio03-10383 (BioStar) with matching funds from Pfizer Incorporated.
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FOOTNOTES |
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Published ahead of print on 21 May 2007.
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