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Endocrinology, doi:10.1210/en.2006-1549
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Endocrinology Vol. 149, No. 1 40-49
Copyright © 2008 by The Endocrine Society

Tyrosine Phosphorylation of Munc18c Regulates Platelet-Derived Growth Factor-Stimulated Glucose Transporter 4 Translocation in 3T3L1 Adipocytes

Mitsuhiko Umahara, Shuichi Okada, Eijiro Yamada, Tsugumichi Saito, Kihachi Ohshima, Koshi Hashimoto, Masanobu Yamada, Hiroyuki Shimizu, Jeffrey E. Pessin and Masatomo Mori

Department of Medicine and Molecular Science (M.U., S.O., E.Y., T.S., K.H., M.Y., H.S., M.M.), Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan; Gunma University Health and Medical Center (K.O.), Maebashi, Gunma 371-8510, Japan; and Department of Pharmacological Sciences (J.E.P.), Stony Brook University, Stony Brook, New York 11794-8651

Address all correspondence and requests for reprints to: Shuichi Okada, Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, 3-39-15 Showa-machi, Maebashi, Gunma 371-8511, Japan. E-mail: okadash{at}showa.gunma-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Platelet-derived growth factor (PDGF) stimulation of skeletal muscle, cultured myotubes, and 3T3L1 adipocytes results in glucose transporter 4 (Glut4) translocation, albeit to a reduced level compared with insulin. To address the mechanism of PDGF action, we have determined that the Syntaxin 4 negative regulatory protein, Munc18c, undergoes PDGF-stimulated phosphorylation on tyrosine residue 521. The tyrosine phosphorylation of Munc18c on Y521 occurred concomitant with the dissociation of the Munc18c protein from Syntaxin 4 in a time frame consistent with Glut4 translocation. Moreover, expression of the wild-type Munc18c protein did not inhibit PDGF-induced Glut4 translocation, whereas expression of Y521A-Munc18c mutant was inhibitory and failed to dissociate from Syntaxin 4. In contrast, expression of either wild-type Munc18c or the Y521A-Munc18c mutant both resulted in a marked inhibition of insulin-stimulated Glut4 translocation. Together, these data demonstrate that one mechanism accounting for the PDGF induction of Glut4 translocation is the suppression of the Munc18c negative regulation of Syntaxin 4 function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ALTHOUGH physiologically insulin is the predominant anabolic hormone regulating glucose transporter 4 (Glut4) translocation, platelet-derived growth factor (PDGF), bradykinin, and endothelin have also induced glucose uptake and the redistribution of Glut4 from intracellular storage sites to the plasma membrane, albeit to a lesser extent than insulin (1, 2). These events are functionally related to general trafficking/compartmentalization mechanisms that occur during dense core granule secretion and synaptic transmission (3, 4). In the case of the Glut4 protein, vesicles containing vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptors (v-SNAREs) [vesicle-associated membrane protein (VAMP) 2] interact with the cognate plasma membrane target SNAREs (t-SNAREs) (Syntaxin 4 and 23-kDa synaptosome-associated protein). These proteins are the isoform-specific counterparts found in neural cells that comprise the core fusogenic machinery for regulated trafficking of neurotransmitter to nerve terminals (5).

Recently, it was demonstrated that noncognate SNARE proteins could form complexes in vitro, and the properties of these complexes were similar regardless of whether the SNARE proteins were closely or distantly related (6). This implies that specificity and membrane fusion may be mediated through the actions of specific SNARE regulatory proteins (7, 8, 9, 10, 11, 12). In particular, the Munc18 proteins comprise a family of Syntaxin binding proteins and are the mammalian homologs of the Saccharomyces cerevisiae Sec1 and Caenorhabditis elegans Unc18 proteins (13). They apparently have a critical role in vesicle docking, fusion, and/or exocytosis because null or temperature-sensitive mutations in yeast and/or Drosophila homologs markedly inhibit vesicle exocytosis (14, 15). To date, four mammalian Munc18 homologs have been identified: Munc18a, b, c, and c(L) (16, 17, 18, 19, 20). Munc18a and b interact with Syntaxin 1, 2, and 3. Munc18c interacts with Syntaxin 4, the Syntaxin homolog shown to be of importance for fusion of Glut4 vesicles with the plasma membrane and, to a lesser extent, Syntaxin 2 (5, 21). In neuronal cells the binding of Munc18a to Syntaxin 1A prevents Syntaxin from forming a SNARE complex with VAMP and 25-kDa synaptosome-associated protein. In 3T3L1 adipocytes, overexpression of Munc18c inhibits insulin-stimulated glucose transport and Glut4 translocation (8, 22). In Munc18c null adipocytes, insulin-stimulated Glut4 translocation is enhanced (23). Thus, Munc18c appears to play a physiologically important inhibitory role on insulin-stimulated Glut4 translocation. However, the mechanism by which insulin and other regulator hormones controls Munc18c function remains unknown. In this study we demonstrate that PDGF stimulation of Glut4 translocation occurs through the regulation of Munc18c function by phosphorylation at Y521.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
The FLAG M2 monoclonal antibody and Syntaxin 4 polyclonal antibody were obtained from Sigma-Aldrich (St. Louis, MO). Munc18c rabbit polyclonal antibody was prepared and affinity purified as previously described (7, 22). Phosphotyrosine specific antibody (PY20 and PY99) was purchased from BD Biosciences (San Jose, CA). Myc antibody was purchased from Upstate (Charlottesville, VA). Enhanced chemiluminescence plus Western Blotting Detection System was obtained from Amersham Biosciences (Piscataway, NJ). The horseradish peroxidase (HRP)-conjugated antimouse or antirabbit IgG antibodies were obtained from Pierce (Rockford, IL). Cell culture media and reagents, and collagen-coated tissue culture wares were from Invitrogen Life Technologies (Carlsbad, CA). All of other chemicals used in this study were purchased from Sigma-Aldrich.

Cell culture
3T3L1 preadipocytes were cultured in DMEM containing 25 mM glucose, 10% calf serum at 37 C with 8% CO2. Confluent cultures were induced to differentiate into adipocytes as previously described (10).

Immunoprecipitation and immunoblotting
Fully differentiated 3T3L1 adipocytes from one 10-cm dish or electroporated fully differentiated 3T3L1 adipocytes from one 10-cm dish were frozen after variously designed stimulations as described in each figure. Thereafter, scraped frozen cells were rocked for 30 min at 4 C with NP-40 lysis buffer [25 mM HEPES (pH 7.4), 0.1% NP-40, 10% glycerol, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, 137 mM sodium chloride, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 µg/ml pepstatin, and 5 µg/ml leupeptin]. Insoluble material was separated from the soluble extract by centrifugation for 30 min at 4 C, and the total protein amount in the supernatant was determined by the bioinchoninic acid (BCA) method. Approximately 3 mg protein was typically recovered. After the addition of 2.0 µg antibody to the whole cell lysates, samples (typically 2 mg lysates) were incubated for 2 h at 4 C. Then 30 µl protein A/G-agarose was added, and samples were consistently rocked for 1 h at 4 C. After the incubation, samples were extensively washed five times with the NP-40 lysis buffer. The washed samples were resuspended in sodium dodecyl sulfate sample buffer [125 mM Tris-HCl (pH 6.8), 20% (vol/vol) glycerol, 4% (wt/vol) sodium dodecyl sulfate, 100 mM dithiothreitol, 0.1% (wt/vol) bromophenol blue], and heated at 100 C for 5 min. Samples were separated by SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes. The samples were immunoblotted with monoclonal or polyclonal-specific antibody as indicated in the figures and legends. Our immunoprecipitation efficiency was usually approximately 50–70%.

Munc18c tyrosine-phosphorylation was assessed after Munc18c immunoprecipitation. Of immunoprecipitated sample, 90% was applied on SDS-PAGE, and subjected to PY20 and PY99 phosphotyrosine immunoblotting to detect tyrosine-phosphorylated Munc18c. To assess endogenous Munc18c-Syntaxin 4 interaction, after Munc18c immunoprecipitation, 90% of samples were applied on SDS-PAGE and subjected to Syntaxin 4 immunoblotting to detect endogenous Syntaxin 4 associated with Munc18c. For the estimation of immunoprecipitated Munc18c, the 10% of the immunoprecipitate was applied on different SDS-PAGE and subjected to Munc18c immunoblotting.

Transfected Munc18c tyrosine-phosphorylation was assessed after FLAG immunoprecipitation. Of immunoprecipitated sample, 90% was applied on SDS-PAGE, and subjected to PY20 and PY99 phosphotyrosine immunoblotting to detect tyrosine-phosphorylated transfected Munc18c. To assess transfected FLAG tagged Munc18c-Syntaxin 4 interaction, after FLAG immunoprecipitation, 90% of samples were applied on SDS-PAGE and subjected to Syntaxin 4 immunoblotting to detect endogenous Syntaxin 4 associated with transfected Munc18c. For the estimation of immunoprecipitated transfected Munc18c proteins, the 10% of the immunoprecipitate was applied on different SDS-PAGE and subjected to FLAG immunoblotting. The primary monoclonal and polyclonal antibodies for immunoblottings were detected with HRP-conjugated antimouse or antirabbit IgG antibodies.

Transfection of 3T3L1 adipocytes
3T3L1 adipocytes were suspended by mild trypsinization and electroporated with a total of 1000 µg plasmid under low-voltage condition (0.16 kV, 950 µF). Under these conditions, typical transfection efficacy was 50–80%, and transfected protein expression level is approximately 20 times higher than endogenous protein expression level (24). The cells were then allowed to adhere to collagen-coated tissue culture dishes for 30–48 h, and the adipocytes were then serum starved for 4 h before incubation in the absence or presence of either 5 nM PDGF or 100 nM insulin at 37 C for the various time periods indicated in each figure.

Enhanced green fluorescent protein (eGFP)-Glut4 translocation assay
Fully differentiated 3T3L1 adipocytes were transfected with 50 µg eGFP-GLUT4 reporter plasmid plus 550 µg effector plasmids by electroporation as previously described (24, 25). After electroporation, the cells were plated on collagen-coated tissue culture dishes for 30–48 h and then serum starved for 2 h before incubation in the absence or presence of either 5 nM PDGF or 100 nM insulin at 37 C for the various time period indicated in each figure. The transfected adipocytes were washed in PBS, and fixed for 10 min in PBS containing 4% paraformaldehyde and 0.2% Triton X-100. The samples were mounted on glass slides with Dako fluorescent mounting medium (Dako Corp., Carpinteria, CA) and imaged using a confocal fluorescence microscope (model MRC-1024; Bio-Rad Laboratories, Hercules, CA).

Quantification of Glut4 translocation
Quantification of transfected Glut4 translocation was determined using a colorimetric assay as previously described besides a single-cell assay with eGFP signal by confocal microscopy (24, 25, 26, 27). Briefly, 3T3L1 adipocytes were cotransfected with 400 µg eGFP-cMyc-Glut4 plus 600 µg various other cDNAs as indicated in each figure. After basal or hormonal stimulation, the cells were cooled to 4 C and incubated with a Myc antibody, followed by HRP-conjugated antimouse IgG antibody. The specific cell surface-bound HRP was then determined by incubation with the substrate, o-phenylenediamine dihydrochloride peroxidase.

The mean value of untreated pcDNA (Invitrogen) samples was initially calculated. Each value of samples was then divided by that mean value, and those numbers were used for statistical analysis. The y-axis on each bar graph represents those results.

Statistical analysis
Each band of Western blots was scanned and analyzed by Molecular Imager FX (Bio-Rad Laboratories). We set and used the same-sized rectangle box to surround each band and analyzed the intensity of each band alone by the program in Molecular Image FX. Before we calculated the band intensity, we subtracted the background intensity, which is estimated from the band-free area by using the same rectangle box. All values are expressed as mean ± SEM. Data were evaluated for statistical significance by ANOVA and t test using the InStat 2 program (GraphPad Software Inc., San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PDGF stimulates Glut4 translocation in differentiated 3T3L1 adipocytes
Although insulin stimulation results in a robust increase in glucose uptake in 3T3L1 adipocytes, it was recently reported that approximately 50% of this effect was due to Glut4 activity, whereas the remaining 50% was due to a Glut1-dependent glucose uptake (28). Therefore, to determine specifically the effect of PDGF on Glut4 translocation without contribution of Glut1, we transfected 3T3L1 adipocytes with a Myc-Glut4-eGFP construct and assessed the cell surface exposure of the Myc epitope using a quantitative colorimetric assay (24, 25, 26, 27). Consistent with previous reports (29, 30, 31, 32, 33), PDGF stimulation resulted in an approximate 2-fold increase of Glut4 translocation at 10-min stimulation compared with unstimulated cells that gradually decreased over the next 30 min (Fig. 1AGo). As expected, insulin was a more potent activator of Glut4 translocation. At 10-min stimulation, it was a 4-fold increase and resulted in an approximate 5-fold increase at 30-min stimulation (Fig. 1BGo) (***, P < 0.001 vs. 0 min; **, P < 0.01 vs. 0 min; *, P < 0.05 vs. 0 min).


Figure 1
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  		FIG. 1. PDGF and insulin stimulate Glut4 translocation in 3T3L1 differentiated adipocytes. 3T3L1-differentiated adipocytes were transfected with Myc epitope-tagged Glut4, and cells were either untreated or stimulated by either 5 nM PDGF (A) or 100 nM insulin (B) for 10, 20, or 30 min. Translocated Myc epitope-tagged Glut4 amount at plasma membrane was estimated by quantitative colorimetric assay, as described under Materials and Methods. Data are means ± SE (n = 5). ***, P < 0.001, **, P < 0.01, *, P < 0.05 vs. untreated cells (0 min). AU, Arbitrary unit.

 
PDGF stimulation results in the tyrosine phosphorylation of Munc18c and dissociation from Syntaxin 4
Inspection of the Munc18c amino acid sequence by the motif survey program (Scansite; http://scansite.mit.edu/) revealed the presence of potential PDGF receptor tyrosine phosphorylation consensus sites at tyrosine 218 (VEKKLEDYYYKIDENG) and 521(RQKPRTNYLELDRKN). Therefore, we decided to assess the ability of PDGF stimulation to induce the tyrosine phosphorylation of the endogenous Munc18c protein by immunoprecipitation with a Munc18c antibody and subsequent phosphotyrosine immunoblotting (PY20 and PY99). Under these conditions the PDGF receptor was maximally tyrosine phosphorylated after 10-min PDGF stimulation and subsequently declined over the next 30 min (Fig. 2A-iGo).


Figure 2
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  		FIG. 2. Endogenous Munc18c undergoes either PDGF- or insulin-stimulated tyrosine phosphorylation and dissociates from Syntaxin 4. 3T3L1-differentiated adipocytes were incubated in the absence of either PDGF (A–C) or insulin (D–F, lanes 1 and 5), or in the presence of either 5 nM PDGF (A–C) or 100 nM insulin (D–F) for 10 (lanes 2 and 6), 20 (lanes 3 and 7), or 30 min (lanes 4 and 8) at 37 C. Whole cell lysates were then prepared, and either total tyrosine-phosphorylated proteins were immunoprecipitated with a PY20 phosphotyrosine antibody (A and E), or total Munc18c was immunoprecipitated with a Munc18c antibody (B and D), or Syntaxin 4 was immunoprecipitated with a Syntaxin 4 antibody (C and F), as described in Materials and Methods. The immunoprecipitates were immunoblotted with the phosphotyrosine antibody (A; provided as short exposure for IP:PY20 lanes, B and D-i), Munc18c antibody (C and E; provided as long exposure for IP:PY20 lanes, F-i, A, B, and D-ii), and Syntaxin 4 antibody (C, F-ii, B, and D-iii). The band intensity estimations in immunoprecipitated samples are provided along with a graph indicating the band intensity change profile normalized to the value at 10-min stimulation. Data are means ± SE (n = 4). ***, P < 0.001, **, P < 0.01, *, P < 0.05 vs. 0 min. The PY99 phosphotyrosine antibody immunoblottings showed the essentially identical results to that observed in the PY20 phosphotyrosine antibody immunoblottings (data not shown). AU, Arbitrary unit; WCL, whole cell lysates.

 
In unstimulated adipocytes there was a relatively low level tyrosine-phosphorylated Munc18c protein. However, 10-min PDGF stimulation resulted in a maximum increased amount of tyrosine-phosphorylated Munc18c that declined but was readily detectable over a 30-min time frame (Fig. 2B-iGo). Thus, the tyrosine phosphorylation of Munc18c paralleled that of PDGF-stimulated receptor phosphorylation. Under these conditions the total cellular Munc18c protein content and extent of immunoprecipitation remained constant (Fig. 2B-iiGo). To confirm the PDGF-induced tyrosine phosphorylation of Munc18c, we also performed immunoprecipitation with a phosphotyrosine antibody and subsequent Munc18c immunoblotting and obtained similar results (Fig. 2A-iiGo). Because Munc18c is known to associate directly with Syntaxin 4, we also assessed the potential effect of PDGF stimulation on the binding interaction between Munc18c and Syntaxin 4 (Fig. 2B-iiiGo). In the basal state, immunoprecipitation of Munc18c resulted in the coimmunoprecipitation of Syntaxin 4. However, after 10-min PDGF stimulation, there was a reduction in the amount of Syntaxin 4 coimmunoprecipitated with Munc18c protein that gradually increased over the 30-min time course examined (Fig. 2B-iiiGo). The PDGF-stimulated dissociation of the Munc18c-Syntaxin 4 complex was confirmed by first immunoprecipitation of Syntaxin 4, followed by immunoblotting for Munc18c (Fig. 2CGo).

We also assessed the ability of insulin stimulation to induce the tyrosine phosphorylation of the endogenous Munc18c protein by immunoprecipitation with a Munc18c antibody and subsequent phosphotyrosine immunoblotting (PY20 and PY99) (Fig. 2DGo). In unstimulated adipocytes there was a low level tyrosine-phosphorylated Munc18c protein similar to that observed in Fig. 2B-iGo. Ten minutes of insulin stimulation resulted in an increased amount of tyrosine-phosphorylated Munc18c that gradually increased over a 30-min time frame. However, these phosphorylation extents by insulin were much weaker than those of PDGF (Fig. 2Go, B-i and D-i). Under these conditions the total cellular Munc18c and extent of immunoprecipitation remained constant (Fig. 2D-iiGo). To confirm the insulin-induced tyrosine phosphorylation of Munc18c, we also performed immunoprecipitation with a phosphotyrosine antibody and subsequent Munc18c immunoblotting with similar result (Fig. 2EGo). We also assessed the potential effect of insulin stimulation on the binding interaction between Munc18c and Syntaxin 4 (Fig. 2D-iiiGo). In the basal state, immunoprecipitation of Munc18c resulted in the coimmunoprecipitation of Syntaxin 4. However, after 10-min insulin stimulation, there was a smaller reduction in the amount of Syntaxin 4 coimmunoprecipitation with Munc18c protein than that observed for PDGF stimulation (Fig. 2Go, B-iii and D-iii). The insulin-stimulated dissociation of the Munc18c-Syntaxin 4 complex was also confirmed by first immunoprecipitation of Syntaxin 4, followed by immunoblotting for Munc18c (Fig. 2FGo) (***, P < 0.001 vs. 0 min; **, P < 0.01 vs. 0 min; *, P < 0.05 vs. 0 min). To confirm these data, we also examined the interaction of Munc18c with Syntaxin 4 in isolated plasma membrane fractions and obtained essentially identical results to that observed in Fig. 2Go, D–F (data not shown).

Tyrosine 521 is the site for PDGF-stimulated phosphorylation and is required for dissociation of the Munc18c-Syntaxin 4 complex
To examine the potential functional role of Munc18c tyrosine phosphorylation, we transfected adipocytes with a cDNA encoding for a FLAG epitope tagged wild-type Munc18c (FLAG-WT-Munc18c) protein (Fig. 3Go). In the basal state, the FLAG-WT-Munc18c was found to coimmunoprecipitate with Syntaxin 4 (Fig. 3A-iiiGo). Similar to the endogenous Munc18c protein, there was a PDGF-stimulated time-dependent dissociation of Syntaxin 4 from the expressed FLAG-WT-Munc18c. In parallel, the FLAG-WT-Munc18c protein displayed a relatively low level of tyrosine phosphorylation in the basal state but underwent a time-dependent PDGF-stimulated increase in tyrosine phosphorylation that was more robust than that of endogenous Munc18c (Fig. 3A-iGo). As a control the total amount of expressed FLAG-WT-Munc18c protein in the cell extracts and in each immunoprecipitation remained relatively unchanged (Fig. 3A-iiGo).


Figure 3
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  		FIG. 3. FLAG-tagged wild-type Munc18c undergoes either PDGF- or insulin-stimulated tyrosine phosphorylation and dissociates from Syntaxin 4. 3T3L1 adipoctyes were transfected with the wild type of FLAG-tagged Munc18c (FLAG-WT-Munc18c) cDNA as described in Materials and Methods. Forty-eight hours later, the transfected adipocytes were incubated in the absence of either PDGF (A) or insulin (B, lanes 1 and 5), or in the presence of either 5 nM PDGF (lane 3) or 100 nM insulin (lane 3) for 10 (lanes 2 and 6), 20 (lanes 3 and 7), or 30 min (lanes 4 and 8) at 37 C. Whole cell lysates were then prepared, and transfected FLAG-WT-Munc18c was immunoprecipitated with a FLAG antibody as described in Materials and Methods. The immunoprecipitates were immunoblotted with the phosphotyrosine antibody (i), FLAG antibody (ii), and Syntaxin 4 antibody (iii). The band intensity estimations in immunoprecipitated samples are provided along with a graph indicating the band intensity change profile normalized to the value at 10-min stimulation. Data are means ± SE (n = 4). ***, P < 0.001, **, P < 0.01, *, P < 0.05 vs. 0 min. The PY99 phosphotyrosine antibody immunoblottings showed the essentially identical results to that observed in the PY20 phosphotyrosine antibody immunoblottings (data not shown). AU, Arbitrary unit; WCL, whole cell lysates.

 
Next, we assessed the ability of insulin stimulation to induce the tyrosine phosphorylation of the FLAG-WT-Munc18c protein by immunoprecipitation with a FLAG antibody and subsequent phosphotyrosine immunoblotting (PY20 and PY99) (Fig. 3B-iGo). In unstimulated adipocytes there was a similar level tyrosine-phosphorylated Munc18c protein compared with PDGF stimulation. Ten minutes of insulin stimulation resulted in a slightly increased amount of tyrosine-phosphorylated Munc18c that remained unchanged over a 30-min time frame. However, the effect of insulin to induce the tyrosine phosphorylation of the expressed FLAG epitope tag Munc18c was substantially less than that observed for PDGF induced stimulation. Under these conditions the total cellular FLAG-WT-Munc18c and extent of immunoprecipitation remained constant (Fig. 3B-iiGo). In parallel, we next determined the potential effect of insulin stimulation on the binding interaction between FLAG-WT-Munc18c and endogenous Syntaxin 4 (Fig. 3B-iiiGo). In the basal state, immunoprecipitation of FLAG-WT-Munc18c resulted in the coimmunoprecipitation of Syntaxin 4. However, unexpectedly, after 10-min insulin stimulation, there was no reduction in the amount of Syntaxin 4 coimmunoprecipitation with Munc18c protein that remained constant over the 30-min time course examined (Fig. 3B-iiiGo).

Because the expressed FLAG-WT-Munc18c protein underwent a similar pattern of PDGF-stimulated tyrosine phosphorylation and dissociation from Syntaxin 4, we examined the site of PDGF-induced tyrosine phosphorylation by expression of FLAG-Munc18c mutants in which Y218 (FLAG-Y218A-Munc18c) and Y521 (FLAG-Y521A-Munc18c) were mutated to alanine residues. In the basal state, adipocytes transfected with the FLAG-Y218A-Munc18c mutant were coimmunoprecipitated with Syntaxin 4 (Fig. 4A-iiiGo). Similar to the endogenous Munc18c and FLAG-WT-Munc18c proteins, there was a PDGF-stimulated time-dependent dissociation of Syntaxin 4 from the expressed FLAG-Y218A-Munc18c protein. In parallel, the FLAG-Y218A-Munc18c protein displayed a relatively low-level tyrosine phosphorylation in the basal state, but 10-min PDGF stimulation resulted in a maximum increased amount of tyrosine-phosphorylated Munc18c that decreased but was readily detectable over a 30-min time frame (Fig. 4A-iGo). As a control the total amount of expressed FLAG-Y218A-Munc18c protein in the cell extracts and in each immunoprecipitation remained relatively unchanged (Fig. 4A-iiGo).


Figure 4
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  		FIG. 4. Tyrosine 218 is not the site for PDGF and insulin-stimulated phosphorylation, and is not required for dissociation of the Munc18c-Syntaxin 4 complex. 3T3L1 adipoctyes were transfected with the FLAG tagged Y218A Munc18c (FLAG-Y218A-Munc18c) cDNA as described in Materials and Methods. Forty-eight hours later, the transfected adipocytes were incubated in the absence of either PDGF (A) or insulin (B) (lanes 1 and 5), or in the presence of either 5 nM PDGF (A) or 100 nM insulin (B) for 10 (lanes 2 and 6), 20 (lanes 3 and 7), or 30 min (lanes 4 and 8) at 37 C. Whole cell lysates were then prepared, and transfected FLAG-Y218A-Munc18c was immunoprecipitated with a FLAG antibody as described in Materials and Methods. The immunoprecipitates were immunoblotted with the phosphotyrosine antibody (i), FLAG antibody (ii), and Syntaxin 4 antibody (iii). The band intensity estimations in immunoprecipitated samples are provided along with a graph indicating the band intensity change profile normalized to the value at 10-min stimulation. Data are means ± SE (n = 4). ***, P < 0.001, **, P < 0.01, *, P < 0.05 vs. 0 min. The PY99 phosphotyrosine antibody immunoblottings showed the essentially identical results to that observed in the PY20 phosphotyrosine antibody immunoblottings (data not shown). AU, Arbitrary unit; WCL, whole cell lysates.

 
We also assessed the ability of insulin stimulation to induce the tyrosine phosphorylation of the FLAG-Y218A-Munc18c protein by immunoprecipitation with a FLAG antibody and subsequent phosphotyrosine immunoblotting (PY20 and PY99) (Fig. 4B-iGo). In unstimulated adipocytes there was a much lower level tyrosine-phosphorylated Munc18c protein compared with PDGF stimulation. Ten minutes of insulin stimulation resulted in a slightly increased amount of tyrosine-phosphorylated Munc18c that remained unchanged over a 30-min time frame, but the phosphorylation was much lower extent compared with PDGF stimulation. Under these conditions the total cellular FLAG-Y218A-Munc18c and extent of immunoprecipitation remained constant (Fig. 4B-iiGo). We also assessed the potential effect of insulin stimulation on the binding interaction between FLAG-Y218A-Munc18c and Syntaxin 4 (Fig. 4B-iiiGo). In the basal state, immunoprecipitation of FLAG-Y218A-Munc18c resulted in the coimmunoprecipitation of Syntaxin 4. However, unexpectedly, after 10-min insulin stimulation, there was no apparent reduction in the amount of Syntaxin 4 coimmunoprecipitated with FLAG-Y218A-Munc18c protein that was remained over the 30-min time course examined (Fig. 4B-iiiGo).

On the other hand, the expressed FLAG-Y521A-Munc18c was coimmunoprecipitated with Syntaxin 4 but was completely remained refractory to PDFG-stimulated dissociation from Syntaxin 4 (Fig. 5A-iiiGo). Moreover, there was no detectable basal or PDGF-stimulated tyrosine phosphorylation of the FLAG-Y521A-Munc18c mutant (Fig. 5A-iGo). Under these conditions the total amount of immunoprecipitated FLAG-Y521A-Munc18c protein also remained unchanged (Fig. 5A-iiGo).


Figure 5
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  		FIG. 5. Tyrosine 521 is the site for PDGF and insulin-stimulated phosphorylation, and is required for dissociation of the Munc18c-Syntaxin 4 complex. 3T3L1 adipoctyes were transfected with the FLAG tagged Y521A Munc18c (FLAG-Y521A-Munc18c) cDNA as described in Materials and Methods. Forty-eight hours later, the transfected adipocytes were incubated in the absence of either PDGF (A) or insulin (B) (lanes 1 and 5), or in the presence of either 5 nM PDGF (A) or 100 nM insulin (B) for 10 (lanes 2 and 6), 20 (lanes 3 and 7), or 30 min (lanes 4 and 8) at 37 C. Whole cell lysates were then prepared, and transfected FLAG-Y521A-Munc18c was immunoprecipitated with a FLAG antibody as described in Materials and Methods. The immunoprecipitates were immunoblotted with the phosphotyrosine antibody (i), FLAG antibody (ii), and Syntaxin 4 antibody (iii). The band intensity estimations in immunoprecipitated samples are provided along with a graph indicating the band intensity change profile normalized to the value at 10-min stimulation. Data are means ± SE (n = 4). The PY99 phosphotyrosine antibody immunoblottings showed the essentially identical results to that observed in the PY20 phosphotyrosine antibody immunoblottings (data not shown). AU, Arbitrary unit; WCL, whole cell lysates.

 
We also assessed the ability of insulin stimulation to induce the tyrosine phosphorylation of the FLAG-Y521A-Munc18c protein by immunoprecipitation with a FLAG antibody and subsequent phosphotyrosine immunoblotting (PY20 and PY99) (Fig. 5B-iGo). In unstimulated adipocytes there was no detectable basal or insulin-stimulated tyrosine phosphorylation of FLAG-Y521A-Munc18c mutant. Under these conditions the total cellular FLAG-Y521A-Munc18c and extent of immunoprecipitation remained constant (Fig. 5B-iiGo). We also assessed the potential effect of insulin stimulation on the binding interaction between FLAG-Y521A-Munc18c and Syntaxin 4 (Fig. 5B-iiiGo). In the basal state, immunoprecipitation of FLAG-Y521A-Munc18c resulted in the coimmunoprecipitation of Syntaxin 4. However, after 10-min insulin stimulation, there was no apparent reduction in the amount of Syntaxin 4 coimmunoprecipitation with FLAG-Y521A-Munc18c protein that was remained over the 30-min time course examined (Fig. 5B-iiiGo) (***, P < 0.001 vs. 0 min; **, P < 0.01 vs. 0 min; *, P < 0.05 vs. 0 min).

PDGF but not insulin stimulated Glut4 translocation requires Y521 phosphorylation of Munc18c
Previous studies have demonstrated that overexpression of Munc18c inhibited insulin-stimulated Glut4 translocation by qualitative Glut4 fluorescence (7, 8). We recapitulated these findings using either the Glut4-eGFP single-cell translocation assay by confocal microscope (Fig. 6Go, A and B) or the quantitative Myc-Glut4 colorimetric assay (Fig. 6Go, C and D). Because it is formally possible that PDGF elicited Glut4 translocation predominantly in fibroblasts, whereas insulin functions in adipocytes, we performed a single-cell translocation assay by confocal microscopy (Fig. 6Go, A-i, A-ii, B-i, and B-ii). Under these transfection conditions, insulin stimulation resulted in a 5-fold increase in the plasma membrane translocation of Glut4 in morphologically identified adipocytes. In contrast, overexpression of FLAG-WT-Munc18c resulted in only a 2.6-fold insulin-stimulated Glut4 translocation (Fig. 6Go, B-i and B-ii). Similarly, expression of the FLAG-Y218A-Munc18c and FLAG-Y521A-Munc18c mutants also resulted in a marked inhibition of insulin-stimulated Glut4 translocation, with no significant effect on the basal state level of cell surface Glut4. As previously observed, PDGF stimulation resulted in an approximate 2-fold increase in Glut4 translocation in morphologically identified adipocytes (Fig. 6Go, A-i and A-ii). In contrast to the effect of insulin, overexpression of FLAG-WT-Munc18c and FLAG-Y218A-Munc18c had no inhibitory effect on PDGF-stimulated Glut4 translocation, whereas expression of the FLAG-Y521A-Munc18c mutant significantly inhibited PDGF-stimulated Glut4 translocation. As shown Fig. 6Go, C and D, the quantitative Myc-Glut4 colorimetric assay showed the essentially identical results to that observed in Fig. 6Go, A-ii and B-ii. Together, these data demonstrate that Y521 tyrosine phosphorylation is not involved in the insulin regulation of Glut4 translocation but is required for PDGF stimulation in fully differentiated cultured adipocytes (***, P < 0.001 vs. 0 min; **, P < 0.01 vs. 0 min; *, P < 0.05 vs. 0 min).


Figure 6
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  		FIG. 6. Differential effects of FLAG-WT-Munc18c and FLAG-Y521A-Munc18c on insulin-stimulated and PDGF-stimulated Glut4 translocation. 3T3L1 adipocytes were cotransfected with the Myc-Glut4-eGFP plasmid and either the pcDNA3 empty vector, FLAG-WT-Munc18c or FLAG-Y521A-Munc18c mutant cDNAs. Forty-eight hours later, the cells were either untreated or stimulated with 5 nM PDGF (A and C) or 100 nM insulin (B and D) for 30 min. The cells were then assayed for the plasma membrane translocation of Glut4 by the design of single-cell assay with a confocal microscope (A-i, A-ii, B-i, and B-ii) or by the colorimetric assay using exofacial Myc tag described under Materials and Methods (C and D). Data are means ± SE (n = 5). *, P <0.05 vs. basal (C). C, Endogenous Munc18c level (pcDNA3) and overexpressed Munc18c level (WT-, Y218A-, Y521A) were shown by Munc18c immunoblotting with whole cell lysate samples. AU, Arbitrary unit; WT, wild type.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is well established that insulin stimulation results in a robust increase in glucose uptake and Glut4 translocation in both striated muscle and adipocytes (34, 35). Although primary adipocytes only express the Glut4 isoform, cultured 3T3L1 adipocytes express both the Glut1 as well as the Glut4 isoform (28). Early studies indicated that despite the presence of both Glut4 and Glut1, insulin stimulation primarily resulted in enhanced glucose uptake through Glut4 (36). More recently, it has been suggested that 50% of the insulin-stimulated glucose uptake is contributed by Glut4 and 50% by Glut1 (28). Thus, to avoid any potential contribution of Glut1, we have developed a quantitative spectroscopic method for determining the extent of Glut4 protein extracellular exposure by taking advantage of an exofacial Myc epitope tag (25, 26, 27).

It is well established that glucose uptake and Glut4 translocation are subjected to regulatory control by several other hormones and cellular states. For example, energy depletion and osmotic shock are also potent inducers of Glut4 translocation and glucose uptake (37, 38). In particular, several studies have demonstrated that activation of the PDGF receptor tyrosine kinase also stimulates glucose uptake and Glut4 translocation in adipocytes and myoblasts (29, 30, 31, 32, 33, 39). In this regard, Yuasa et al. (40) overexpressed PDGF receptor specifically in skeletal muscle of mice and observed that PDGF dose not change blood glucose level compared with vehicle-injected mice. However, various mouse knockout models have demonstrated that in vivo maintenance of glucose homeostasis occurs through suppression of hepatic glucose output in addition to peripheral tissue glucose uptake (41, 42, 43). Thus, it remains possible that alternative pathways, including PDGF stimulation, may have some therapeutic advantage with the potential as a substitutive therapy for insulin treatment, particularly in states of insulin resistance.

In any case, to determine the mechanism accounting for the PDGF stimulation, we focused on the Syntaxin 4 inhibitory protein Munc18c because previous studies have suggested a requirement for Munc18c inactivation as a prerequisite for Glut4 translocation (21, 22, 23). In this regard, we have observed that PDGF stimulation resulted in a dissociation of Munc18c from Syntaxin 4, consistent with a relief of Munc18c inhibition on Syntaxin 4 function. Although we have transfected 3T3L1 adipocytes with cDNA levels similar to that used by others (7), it remains formally possible that the data obtained in this study may reflect the degree of overexpression.

Previously, it was observed that Munc18c has a candidate phosphorylation consensus site (T569) for a proline-directed kinase (44). However, T569 phosphorylation had no significant effect on the association state of the Munc18c-Syntaxin 4 complex or on the ability of insulin to stimulate Glut4 translocation. On the other hand, inspection of the Munc18c amino acid sequence using the motif survey program revealed a potential PDGF receptor tyrosine phosphorylation acceptor site at Y218 and Y521. Interestingly, we have been able to coimmunoprecipitate the PDGF receptor with overexpressed Munc18c, suggesting that PDGF receptor phosphorylation may be direct (data not shown). In this regard, similar to the endogenous Munc18c, overexpressed FLAG-WT-Munc18c, and FLAG-Y218A-Munc18c proteins underwent PDGF-stimulated tyrosine phosphorylation. However, expression of the Y521A-Munc18c mutant resulted in a marked attenuation in the extent of PDGF-stimulated tyrosine phosphorylation, suggesting that Y521 is a major PDGF receptor-dependent tyrosine phosphorylation site.

On the other hand, despite that insulin is a more potent stimulator of Glut4 translocation, we were unable to detect the same extent of Munc18c tyrosine phosphorylation. Although we cannot completely eliminate the possibility that PY20 phosphotyrosine antibody failed to detect insulin-induced tyrosine phosphorylation site(s) that may be distinct from PDGF receptor, we obtained identical results using the PY99 phosphotyrosine antibody. These data are more consistent with that a PDGF receptor-mediated tyrosine phosphorylation of Munc18c is responsible for the PDGF-induced dissociation of the Munc18c-Syntaxin 4 complex. Thus, our data are consistent with the model that Munc18c functions as a negative regulator for Syntaxin 4 and that Munc18c dissociation from Syntaxin 4 is a necessary step for Glut4 translocation.

A role of Munc18c tyrosine phosphorylation in the regulation of membrane fusion events has also been suggested for other cell types. In pancreatic β-cells, it was reported that glucose stimulation resulted in the tyrosine phosphorylation of Y219, and dissociation of the Munc18c-Syntaxin 4 complex (45). Another study also reported that insulin stimulated Y521 phosphorylation, but the physiological consequence remained to be identified (46). Although we have not addressed the effect of glucose stimulation, our data demonstrate that extent of tyrosine phosphorylation on Y521 provides an important determinant of Munc18c-Syntaxin 4 interaction, and the relative amount and Munc18c-Syntaxin4 complex amount negatively correlate with Glut4 translocation. Thus, the ability of overexpressed Munc18c to inhibit insulin but not PDGF-stimulated Glut4 translocation is consistent with the model that Munc18c is a negative regulator for Syntaxin 4, and this interaction is inhibited by PDGF, but not insulin, stimulated Y521 phosphorylation.

Based upon these data, we hypothesize that in the basal state, Glut4 vesicles are continually encountering the plasma membrane but are unable to interact with the core SNARE machinery for docking and fusion. This is consistent with recent total internal reflection fluorescence (TIRF) microscopy measurements of Glut4 trafficking, and in vitro reconstitution of Glut4 vesicle fusion with isolated plasma membranes that demonstrated a requirement for intact SNARE proteins, cytosol, and Akt (34, 47). Because Munc18c likely functions in a manner analogous to Munc18a, the association of Munc18c with Syntaxin 4 would maintain Syntaxin 4 in a closed conformation state that is inaccessible to the Glut4 R-SNARE VAMP2. This model would also account for the disparity between the extents of insulin vs. PDGF-induced Glut4 translocation. For example, it is well established that insulin activation of Akt is a necessary event in the translocation of Glut4 (48, 49, 50). Although insulin is a very potent activator of Akt, PDGF stimulation results in a relatively poor and transient activation of Akt (51). Thus, it is likely that PDGF stimulation of Glut4 translocation results from the release of a tonic inhibition, whereas insulin positively activates the translocation machinery. This is also consistent with insulin and PDGF using distinct pathways, and probably pools of Glut4 in a manner analogous to osmotic shock and exercise/contraction in skeletal muscle (38, 52, 53). In summary, our data directly demonstrate that PDGF stimulation induces Munc18c tyrosine phosphorylation at Y521, and this event is necessary for the dissociation of Munc18c from Syntaxin 4 and subsequent Glut4 translocation. Although this pathway might be partly shared by insulin, further studies are now required to determine what are the distinct insulin-regulated mechanisms to release Munc18c inhibitory role on Syntaxin 4 and/or whether insulin uses a different pool of Syntaxin 4 that is regulated by distinct Munc18c function.


   Footnotes
 
Disclosure Statement: The authors have nothing to declare.

First Published Online October 4, 2007

Abbreviations: eGFP, Enhanced green fluorescent protein; Glut4, glucose transporter 4; HRP, horseradish peroxidase; PDGF, platelet-derived growth factor; SNARE, soluble N-ethylmaleimide sensitive factor attachment protein receptor; t-SNARE, target soluble N-ethylmaleimide sensitive factor attachment protein receptor; v-SNARE, vesicle soluble N-ethylmaleimide sensitive factor attachment protein receptor; VAMP, vesicle-associated membrane protein.

Received November 20, 2006.

Accepted for publication September 19, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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