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Endocrinology Vol. 140, No. 8 3826-3834
Copyright © 1999 by The Endocrine Society

ARTICLES

Synergistic Role of the Phosphatidylinositol 3-Kinase and Mitogen-Activated Protein Kinase Cascade in the Regulation of Insulin Receptor Trafficking1

Toshiyasu Sasaoka, Tsutomu Wada, Hajime Ishihara, Yasumitsu Takata, Tetsuro Haruta, Isao Usui, Manabu Ishiki and Masashi Kobayashi

From the First Department of Medicine, Toyama Medical & Pharmaceutical University, Toyama, 930-0194 Japan

Address all correspondence and requests for reprints to: Toshiyasu Sasaoka, M.D., Ph.D., First Department of Medicine, Toyama Medical & Pharmaceutical University, 2630 Sugitani, Toyama, 930-0194, Japan. E-mail: tsasaoka-tym{at}umin.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To examine the molecular mechanism of insulin receptor trafficking, we investigated the intracellular signaling molecules that regulate this process in Rat1 fibroblasts overexpressing insulin receptors. Cellular localization of insulin receptors was assessed by confocal laser microscopy with indirect immunofluorescence staining. Insulin receptors were visualized diffusely in the basal state. Insulin treatment induced the change of insulin receptor localization to perinuclear compartment. This insulin-induced insulin receptor trafficking was not affected by treatment of the cells with PI3-kinase inhibitor (wortmannin), whereas treatment with MEK [mitogen-activated protein (MAP) kinase-Erk kinase] inhibitor (PD98059) partly inhibited the process in a dose-dependent manner. Interestingly, treatment with both wortmannin and PD98059 almost completely inhibited insulin receptor trafficking. The functional importance of PI3-kinase and MAP kinase in the trafficking process was directly assessed by using single cell microinjection analysis. Microinjection of p85-SH2 and/or catalytically inactive MAP kinase ([K71A]Erk1) GST fusion protein gave the same results as treatment with wortmannin and PD98059. Furthermore, to determine the crucial step for the requirement of PI3-kinase and MAP kinase pathways, the effect of wortmannin and PD98059 on insulin receptor endocytosis was studied. Insulin internalization from the plasma membrane and subsequent insulin degradation were not affected by treatment with wortmannin and PD98059. In contrast, insulin receptor down-regulation from the cell surface and insulin receptor degradation, after prolonged incubation with insulin, were markedly impaired by the treatment. These results suggest that PI3-kinase and MAP kinase pathways synergistically regulate insulin receptor trafficking at a step subsequent to the receptor internalization.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE BIOLOGICAL action of insulin is initiated by binding to cell surface insulin receptors, which results in autophosphorylation of the insulin receptor ß-subunit and activation of its intrinsic kinase activity (1, 2). Ligand binding also triggers the recruitment of insulin receptors to clathrin-coated pits, followed by internalization of the insulin-insulin receptor complex and delivery to an acidic compartment where dissociation of the ligand occurs (3, 4, 5). Then, insulin receptors can recycle back to the cell surface, or can be sorted to the lysosome for degradation (4, 5, 6, 7). This latter process results in the down-regulation of cell surface receptors (4, 5, 8). By controlling the number of surface insulin receptors and the degradation of insulin, this process plays a key role in the regulation of insulin signal (4, 5). In contrast, some studies have shown that internalized insulin receptors are phosphorylated and become active to phosphorylate their substrates (9, 10, 11), indicating that internalization of insulin receptors plays an important role in the propagation of the insulin signal. Insulin receptor internalization as an early step of the trafficking process has been shown to require insulin receptor autophosphorylation and kinase activity as well as intact juxtamembrane domain of insulin receptor ß-subunit (12, 13, 14, 15, 16, 17, 18, 19). Thus, previous studies of insulin receptor trafficking have mainly focused on the role of insulin receptor ß-subunit in insulin receptor internalization (12, 13, 14, 15, 16, 17, 18, 19). Activated insulin receptors transmit signal intracellularly (2). One of the earliest intracellular steps of insulin signaling is tyrosine phosphorylation of insulin receptor substrates (IRS) (20). IRS then interacts with various SH2 domain-containing proteins including PI3-kinase (21). Shc is also tyrosine phosphorylated following insulin stimulation (22, 23, 24). Tyrosine phosphorylated Shc binds to the Grb2·Sos complex, which allows for activation of p21ras and leads to the subsequent stimulation of mitogen-activated protein (MAP) kinase cascade (24, 25). While an importance of both PI3-kinase and MAP kinase pathways in insulin’s mitogenesis (26, 27, 28, 29) and a key role of PI3-kinase in insulin’s metabolic action have been reported (30, 31, 32), the exact molecular mechanisms involved in insulin-induced insulin receptor trafficking process are unknown.

In the present study, to examine how signaling may regulate insulin receptor trafficking, we investigated the requirement of PI3-kinase and MAP kinase pathways for the trafficking process employing PI3-kinase inhibitor (wortmannin) and MAP kinase-Erk kinase (MEK) inhibitor (PD98059) in Rat1 cells overexpressing insulin receptors (HIRc). In addition, the functional importance of these molecules was more specifically determined by using single cell microinjection analysis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Porcine insulin was the kind gift of Shimizu Pharmaceutical Co., (Shizuoka, Japan). Molecular cloning and preparation of the GST fusion protein containing the N-terminal SH2 domain of p85 regulatory subunit of PI3-kinase was described previously (26). [125I]insulin (2000 Ci/mmol) and enhanced chemiluminescence reagents were purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). Trans[35S]-label (1022 Ci/mmol) was from ICN Biochemicals, Inc. (Costa Mesa, CA). A catalytically inactive MAP kinase ([K71A]Erk1) GST fusion protein was from Upstate Biotechnology, Inc. (Lake Placid, NY). A monoclonal antiphosphotyrosine antibody (pY20) was from Transduction Laboratories, Inc. (Lexington, KY). A polyclonal rabbit and a mouse monoclonal antiinsulin receptor antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and Cosmo Bio Co. Ltd., (Tokyo, Japan), respectively. Mouse IgG, fluorescein isothiocyanate (FITC)- or rhodamine-conjugated antimouse, and antirabbit IgG antibodies were from Jackson ImmunoResearch Laboratories, Inc., (West Grove, PA). MEK inhibitor (PD98059) was from New England Biolabs, Inc. (Beverly, MA). Wortmannin, LY294002, and all other routine reagents were purchased from Sigma Chemical Co. (St. Louis, MO).

Cell culture
Rat1 fibroblasts expressing 1 x 106 human insulin receptors per cell (HIRc) were kindly supplied from Dr. J. M. Olefsky (University of California San Diego, San Diego, CA), and maintained in DMEM/F12 containing 10% FCS as previously described (13). For cell treatment, serum-starved cells were preincubated with the indicated concentrations of PI3-kinase inhibitor wortmannin and/or MEK inhibitor PD98059 for the indicated times at 37 C.

Microinjection
Cells were grown on glass coverslips and rendered quiescent by starvation for 24 h in serum-free DMEM. GST fusion proteins including 4 mg/ml mouse IgG were solubilized in microinjection buffer consisting of 5 mM NaPO4 and 100 mM KCl, pH 7.4, and then microinjected using glass capillary needles. Approximately 1 x 10-14 l of the buffer was introduced into each cell. The injection included about 1 x 106 molecules of IgG. Three hundred to 500 cells per coverslip were injected. Immunofluorescent staining as described below of the injected cells indicated that about 80% of the cells were successfully microinjected. One hour after microinjection, the cells were used for the following analysis (23, 26).

Immunostaining and confocal laser microscopy
Cells were treated with 17 nM insulin for 20 min at 37 C, and fixed with 3.7% formaldehyde in PBS for 20 min at 22 C. The fixed cells were rinsed twice with PBS, permeabilized with 0.5% NP40 in PBS for 3 min at 22 C, and blocked with a solution containing 2.5% BSA. The cells were incubated with rabbit polyclonal antiinsulin receptor antibody for 90 min at 22 C. The cells were then stained by incubation with rhodamine-labeled donkey antirabbit IgG antibody to detect insulin receptors and FITC-labeled donkey antimouse IgG antibody to detect injected cells for 1 h at 22 C. The samples were visualized with immunofluorescence microscopy and examined with a Carl Zeiss confocal laser fluorescence inverted microscope (LSM 510, Carl Zeiss, Oberkochen, Germany) using simultaneous lasers with excitation wavelength of 543 and 488 nm for red and green, respectively, and detected using red and green narrow band filters. Cells were observed through an oil plan-neofluar x 63/1.3 objective. The optical resolution (0.5 µm in the x-y direction and 0.4 µm in the z direction) allowed the reliable measurement of the fluorescence in the selected area of the cell. Each cell was optically sliced into twenty sections in the z direction through serial optical sectioning, and the central three sections were selected for the image analysis. The three series of individual two-dimensional image (slice) were analyzed by the computer equipped Zeiss LSM software to determine an average of image sequence in the selected areas. The obtained fluorescence intensity throughout the cell was expressed as a function of x-y direction (cell width). To judge the insulin receptor trafficking, cells were scored as positive if the peak height of perinuclear fluorescent intensity was 10 U above the peripheral intensity of the cell. The percentage of cells positive for trafficked insulin receptors was determined in each experiment by analyzing approximately 300 cells.

Immunoprecipitation and Western blotting
Serum-starved cells were treated with wortmannin and/or PD98059 for 30 min and incubated with 17 nM insulin for 5 min. The cells were lysed in a solubilizing buffer containing 30 mM Tris, 150 mM NaCl, 10 mM EDTA, 0.5% sodium deoxycholate, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM Na3VO4, 160 mM NaF, pH 7.4 for 15 min at 4 C. The lysates were separated by 7.5% SDS-PAGE and transferred onto polyvinylidene difluoride membrane by electroblotting. The membranes were blocked with 2.5% BSA and probed with the antiphosphotyrosine antibody (pY20). Enhanced chemiluminescence detection was performed according to the manufacturer’s instructions (Amersham Pharmacia Biotech).

Insulin internalization and degradation
Serum-starved cells were treated without or with 50 nM wortmannin and 50 µM PD98059 for 30 min at 37 C, and incubated with 36 pM [125I]insulin in KRP-HEPES buffer containing 0.5% BSA, pH 7.6, for 2 h at 4 C. The cells were then warmed to 37 C. At the indicated time points, cells were washed three times with ice-cold PBS, then incubated for 6 min at 4 C in KRP-HEPES buffer, pH 3.0. After the buffer was aspirated, the cells were rinsed once again with the acid KRP-HEPES buffer, and the two buffer aspirates were pooled. Cells were solubilized in 1 N NaOH. The aspirated buffer (representing surface bound ligand) and solubilized cells (representing internalized ligand) were counted in a {gamma}-counter (13, 15). Degradation of [125I]insulin in the medium was determined by precipitation with 7.5% trichloroacetic acid (TCA).

Insulin receptor down-regulation
Serum-starved cells were preincubated without or with 50 nM wortmannin and 50 µM PD98059 for 10 min at 37 C, and further incubated with 17 nM insulin at 37 C for the indicated times. Cells were then cooled to 4 C and acid-washed as described above. After the acid wash, cells were washed three times with ice-cold KRP-HEPES buffer, pH 7.6. The cells were incubated with 36 pM [125I]insulin in KRP-HEPES buffer, pH 7.6, for 3 h at 4 C. The cells were washed three times and solubilized in 1 N NaOH, and solubilized cells were counted in a {gamma}-counter (13, 15). In all binding studies, nonspecific binding was less than 12% of the total bound.

Insulin receptor degradation
Cells were incubated in methionine-free DMEM containing 10% FCS and Trans[35S]-label (0.1 mCi/ml) for 18 h and then in DMEM containing 10% FCS and methionine (0.3 mg/ml) for 45 min. Cells were then incubated with 17 nM insulin in the absence or presence of 50 nM wortmannin and 50 µM PD98059 for the indicated times. The cells were washed twice with ice-cold PBS, lysed in the solubilizing buffer, and purified on a WGA column. The eluate was immunoprecipitated with the antiinsulin antibody for 4 h at 4 C and analyzed by 7.5% SDS-PAGE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Visualization and analysis of insulin receptor trafficking
Insulin-induced insulin receptor trafficking from cell surface to cytoplasmic perinuclear portion was visualized by immunofluorescent microscopy using antiinsulin receptor antibody followed by rhodamine-conjugated second antibody. In the basal state, insulin receptors were diffusely distributed throughout the cell (Fig. 1AGo). Following insulin stimulation, the staining pattern markedly changed to prominent intense staining in the perinuclear compartment, accompanied by decreased peripheral insulin receptor staining (Fig. 1BGo), indicating insulin receptor trafficking from the cell surface to cytoplasmic perinuclear region. Thus, immunofluorescence staining clearly distinguished the cell with trafficked insulin receptors from that with cell surface insulin receptors. To more quantitatively assess the immunofluorescence stained cells, the distribution of insulin receptors was further analyzed with confocal laser microscopy. Confocal image analysis of a representative cell demonstrated the diffuse distribution of insulin receptors throughout the cell in the basal state (Fig. 2AGo). After insulin stimulation, the image analysis showed that insulin receptors were distributed in central area with decreased distribution in peripheral area of the cell (Fig. 2BGo). The cells showing that the peak height of perinuclear fluorescent intensity was 10 U above the peripheral intensity were determined as positive for trafficked insulin receptors. Thus, this analysis unambiguously distinguished the cell with trafficked insulin receptors from that with cell surface insulin receptors. Using this analysis, 12.1 ± 3.5% of cells displayed trafficked insulin receptors in the basal state. Following insulin stimulation, 79.0 ± 8.2% of cells were positive for trafficked insulin receptors (Fig. 3Go). This observation was not specifically induced by the antiinsulin receptor antibody used in this study because the same insulin receptor distribution was also observed by using a different antiinsulin receptor antibody raised against different epitope of the receptor. In addition, the results of immunostaining with control IgG showed only a faint background staining, and insulin treatment did not affect the background staining (data not shown).



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  		Figure 1. Visualization of insulin-induced insulin receptor trafficking. Serum-starved cells on glass cover slips were treated with vehicle (A and B), 50 nM wortmannin (C), 100 µM PD98059 (D), or both (E) for 30 min at 37 C. After stimulation without (A) or with 17 nM insulin (B–E) for 20 min at 37 C, the cells were fixed, incubated with a rabbit polyclonal antiinsulin receptor antibody, and stained with rhodamine-conjugated antirabbit IgG. Cells with trafficked insulin receptors are shown by arrows. Representative results visualized with fluorescence microscope are shown.

 


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  		Figure 2. Analysis of insulin receptor localization. The localization of insulin receptors of a representative cell treated without (A) or with (B) insulin was analyzed with confocal laser microscope. The intensity of insulin receptor distribution is shown in the y-axis, and the cell width is shown in the x-axis.

 


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  		Figure 3. Effect of wortmannin and PD98059 on insulin receptor trafficking. Cells grown on glass coverslips were serum-starved for 24 h, and treated with the indicated concentrations of wortmannin (A), PD98059 (B), or both (C) for 30 min at 37 C. After stimulation with 17 nM insulin for 20 min, the cells were fixed, incubated with a polyclonal antiinsulin receptor antibody, and stained with rhodamine-conjugated antirabbit antibody. The cells were scored for trafficked insulin receptors with the fluorescence microscope. Results are expressed as the percent of total cells and are the mean ± SE of four separate experiments.

 
Effect of wortmannin and PD98059 on insulin receptor trafficking
We then used this system to determine whether insulin-induced insulin receptor trafficking requires PI3-kinase and/or MAP kinase pathway. Pretreatment of the cells with wortmannin alone did not significantly affect insulin receptor trafficking at any concentrations (Fig. 1CGo and 3AGo). In contrast, treatment with PD98059 resulted in a partial inhibition of the process in a dose-dependent fashion. Insulin receptor trafficking was inhibited by 40.3 ± 4.5% in 100 µM PD98059 treatment (Fig. 1DGo and 3BGo). Interestingly, pretreatment with both wortmannin and PD98059 enhanced the inhibitory effect. In the presence of 50 nM wortmannin, PD98059 significantly inhibited insulin receptor trafficking in a dose-dependent manner (Fig. 3CGo). Likewise, in the presence of 100 µM PD98059, wortmannin further enhanced the inhibitory effect in a dose-dependent fashion (data not shown). Thus, treatment with 100 µM PD98059 and 50 nM wortmannin resulted in an effective inhibition of insulin receptor trafficking by 96.1 ± 3.4% (Fig. 1EGo and 3CGo). Employment of LY294002 as other available PI3-kinase inhibitor gave the same results as treatment with wortmannin (data not shown).

Microinjection of p85-SH2 and [K71A]Erk1 GST fusion proteins
As with any other study involving the use of inhibitors, the possibility exists that wortmannin and PD98059 may have direct target other than PI3-kinase and MEK, respectively. Therefore, to directly assess the relevance of PI3-kinase and MAP kinase pathways in insulin-induced insulin receptor trafficking, we used single cell microinjection analysis with p85-SH2 GST fusion protein of PI3-kinase and catalytically inactive MAP kinase ([K71A]Erk1) GST fusion proteins. Since previous studies reported that microinjection of the p85-SH2 GST fusion protein inhibited insulin-stimulated both mitogenesis and Glut4 translocation presumably by behaving as an intracellular competitive inhibitor of the binding of endogenous PI3-kinase (26, 30), and since microinjection of the catalytically inactive MAP kinase ([K71A]Erk1) GST fusion protein inhibited insulin-stimulated DNA synthesis in Rat1 fibroblasts (data not shown), microinjection of p85-SH2 and [K71A]Erk1 GST fusion proteins is useful approach to analyze the involvement of PI3-kinase and MAP kinase in insulin receptor trafficking. In accordance with the results using wortmannin and PD98059, microinjection of p85-SH2 GST fusion protein again did not affect insulin receptor trafficking. In addition, microinjection of [K71A]Erk1 GST fusion protein partially (42.1 ± 3.2%) inhibited the process. Importantly, microinjection of both p85-SH2 and [K71A]Erk1 GST fusion proteins inhibited insulin receptor trafficking by 94.0 ± 2.6% (Fig. 4Go).



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  		Figure 4. Effect of microinjection of p85-SH2 and [K71A]Erk1 GST fusion proteins on insulin receptor trafficking. Cells grown on glass coverslips were serum-starved for 24 h, and were microinjected with p85-SH2 GST fusion protein (6 mg/ml) and/or [K71A]Erk1 GST fusion protein (6 mg/ml) including mouse IgG (4 mg/ml). One hour after microinjection, the cells were incubated with 17 nM insulin for 20 min. The cells were fixed and incubated with a polyclonal rabbit antiinsulin receptor antibody. Insulin receptors were visualized by staining with rhodamine-conjugated antirabbit antibody and identification of injected cells was performed by staining with FITC-conjugated antimouse antibody. The cells were scored for trafficked insulin receptors with confocal laser microscope. Results are expressed as the percent of total cells and are the mean ± SE of four separate experiments.

 
Effect of wortmannin and PD98059 on insulin-induced tyrosine phosphorylation of insulin receptor and IRS-1&-2
Insulin binding to cell surface insulin receptors results in autophosphorylation of the receptor ß-subunit and subsequent tyrosine phosphorylation of IRS-1&2. We examined whether wortmannin and/or PD98059 affect these early steps of insulin signaling. Pretreatment with either 50 nM wortmannin or 50 µM PD98059 did not affect insulin-induced tyrosine phosphorylation of insulin receptors and IRS-1&2. Further, the treatment with both wortmannin and PD98059 again did not significantly change insulin stimulation of these tyrosine phosphorylation (Fig. 5Go). Thus, blockade of the PI3-kinase and MAP kinase cascade with wortmannin and PD98059 did not affect the early events of insulin signaling.



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  		Figure 5. Effect of wortmannin and PD98059 on tyrosine phosphorylation of insulin receptor and IRS-1&2. Cells were treated with 50 nM wortmannin and/or 50 µM PD98059 for 30 min at 37 C. The cells were then stimulated with 17 nM insulin for 5 min. Cell lysates were analyzed by immunoblotting with antiphosphotyrosine antibody. Molecular masses of insulin receptor ß-subunit (95 kDa) and IRS-1&2 (180 kDa) are shown by arrows. Results are representative of three separate experiments.

 
Effect of wortmannin and PD98059 on insulin internalization and degradation
After insulin binding of the insulin receptor, several trafficking steps are involved in targeting insulin receptors to perinuclear compartment (4, 5). As an early step of the trafficking pathway, the insulin-insulin receptor complex must clear from the plasma membrane following insulin binding. Therefore, the observed diffuse distribution of insulin receptors by inhibition of PI3-kinase and MAP kinase pathways could result from a defect in the internalization step of insulin receptor trafficking. To examine this issue, we studied the effect of wortmannin and PD98059 treatment on insulin-induced insulin receptor internalization by examing the labeled insulin internalization. After cells had been warmed to 37 C, insulin-insulin receptor complexes were rapidly internalized in a time-dependent manner. By 20 min, the internalized insulin reached a steady state that represented a balance of ongoing internalization, retroendocytosis, and degradation. At 20 min, about 65% of the initially labeled insulin appeared to exist in the intracellular compartment. Pretreatment with both 50 nM wortmannin and 50 µM PD98059 did not affect the step of insulin internalization (Fig. 6Go). Internalized insulin-insulin receptor complexes are transferred to endosomes in which the dissociation of insulin from its receptor occurs. Following this step, a greater part of internalized insulin is degraded. Therefore, the effect of wortmannin and PD98059 on insulin degradation was next analyzed by precipitation with trichloroacetic acid. After insulin-incubated cells were warmed to 37 C, insulin was degraded in a time-dependent manner. The kinetics of insulin degradation closely paralleled the time course of insulin internalization. Pretreatment with both 50 nM wortmannin and 50 µM PD98059 did not affect the step of insulin degradation (Fig. 7Go). Employment of LY294002 gave the same results as treatment with wortmannin, and the treatment of PI3-kinase and MEK inhibitors with either higher concentrations or longer incubation resulted in no significant inhibitory effect on these steps (data not shown).



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  		Figure 6. Effect of wortmannin and PD98059 on insulin internalization. Cells were treated without ({circ}—{circ}) or with (•—•) 50 nM wortmannin and 50 µM PD98059 for 30 min at 37 C, and were incubated with [125I]insulin for 2 h at 4 C. Cells were then warmed to 37 C and were washed to remove unbound insulin at the indicated time points. Cells were then acid washed, and were solubilized after aspiration of the acid buffer. Aspirated buffer (representing surface-bound insulin) and solubilized cells (representing internalized insulin) were then counted in a {gamma}-counter. After correction for nonspecific binding, results are expressed as the percent of cell-associated counts internalized and are the mean ± SE of five separate experiments.

 


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  		Figure 7. Effect of wortmannin and PD98059 on insulin degradation. Cells were treated without ({circ}—{circ}) or with (•—•) 50 nM wortmannin and 50 µM PD98059 for 30 min at 37 C and were incubated with [125I]insulin for 2 h at 4 C. Cells were then warmed to 37 C and further incubated for the indicated times. Degradation of [125I]insulin in the medium was determined by precipitation with 7.5% TCA. Results are expressed as the percent of TCA precipitable (intact) insulin and are the mean ± SE of five separate experiments.

 
Effect of wortmannin and PD98059 on insulin receptor down-regulation and degradation
Previous studies showed that prolonged exposure of cells to insulin led to loss of insulin receptors from the cell surface (4, 5, 8, 9, 10, 12, 13, 15). Time kinetics of insulin-induced down-regulation was measured in HIRc cells. Incubation with insulin resulted in the insulin receptor down-regulation in a time-dependent manner, and cell surface insulin binding was decreased to 28.5 ± 4.6% of control value after exposure of the cells to 17 nM insulin for 5 h as shown in Fig. 8AGo. This insulin-induced down-regulation of insulin receptors indicates that once internalized receptors must move from the early recycling pathway to the lysosomal pathway for degradation. Therefore, the unchanged diffuse distribution of insulin receptors by pretreatment with wortmannin and PD98059 could be due to an impairment in the ability of receptors to proceed beyond the early recycling pathway. To explore this possibility, we next analyzed the effect of wortmannin and PD98059 on insulin receptor down-regulation from the cell surface. Serum-starved cells were treated with wortmannin and/or PD98059 for 10 min and then exposed to insulin for 5 h at 37 C. Pretreatment with wortmannin did not significantly affect the down-regulation. In contrast, PD98059 treatment partly inhibited the decrease of initial binding to 45.2 ± 4.4%. Importantly, the decrease in insulin binding was inhibited to 72.4 ± 6.4% of control value by treatment with both wortmannin and PD98059 (Fig. 8BGo). Furthermore, the effect of wortmannin and PD98059 on insulin receptor degradation was assessed by using independent approach. Treatment of cells with insulin resulted in a time-dependent degradation of labeled insulin receptors as shown in Fig. 9Go. After the cells were incubated with insulin for 6 h, {alpha}- and ß-subunit of insulin receptors were degraded to 62.9 ± 3.2% and 64.1 ± 2.1% of initial values, respectively. In accordance with the results of insulin receptor down-regulation, pretreatment with wortmannin did not apparently affect the degradation of insulin receptors, and employment of LY294002 gave the same results as treatment with wortmannin (data not shown). PD98059 treatment partly inhibited the decrease of {alpha}- and ß-subunit of insulin receptors to 70.2 ± 2.8%, and 69.7 ± 2.4%, respectively. The degradation was effectively inhibited to 83.8 ± 4.1%, and 85.0 ± 3.2% by treatment with both wortmannin and PD98059 (Fig. 9Go). Thus, PI3-kinase and MAP kinase pathways are required for insulin receptors to be degraded and down regulated from the cell surface.



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  		Figure 8. Effect of wortmannin and PD98059 on insulin receptor down-regulation. A, Serum-starved cells were incubated with 17 nM insulin at 37 C for the indicated times. B, Serum-starved cells were treated with 50 nM wortmannin and/or 50 µM PD98059 for 10 min at 37 C, and were further incubated with 17 nM insulin for 5 h at 37 C. After the incubation, cells were cooled, acid washed, and then incubated with 36 pM [125I] insulin for 3 h at 4 C. After washing, cells were solubilized and cell-associated radioactivity was counted in a {gamma}-counter. After correction for nonspecific binding, results are expressed as the percent of cell-associated counts obtained without insulin exposure and are the mean ± SE of four separate experiments.

 


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  		Figure 9. Effect of wortmannin and PD98059 on insulin receptor degradation. HIRc cells were labeled with [35S]methionine and incubated with 17 nM insulin in the presence of 50 nM wortmannin and/or 50 µM PD98059 for the indicated times. Radioactivity incorporated into {alpha}-subunit (A) and ß-subunit (B) of the insulin receptor was analyzed by SDS-PAGE. The signal intensity was quantitated by densitometer. The results are expressed as the percent of initial intensity of insulin receptor subunits and are the mean ± SE of four separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results showed that insulin-induced insulin receptor trafficking was inhibited by blockade of both PI3-kinase and MAP kinase pathways. In yeast, the only known PI3-kinase is Vps34p, which catalyzes the synthesis of PIns (3)P (33). This activity is essential for the sorting of newly synthesized proteins, as well as for endocytosis, into the vacuole (33, 34). Because the catalytic subunit of PI3-kinase has a structural similarity to Vps34p (35, 36), PI3-kinase is assumed to participate in the regulation of vesicular trafficking in mammals. In fact, this enzyme is required for insulin-dependent translocation of the glucose transporter Glut4 to the plasma membrane (30, 31, 32), for PDGF-dependent trafficking of the PDGF receptor to lysosomes (37, 38), and for transferrin receptor recycling (39). In contrast to the previous studies showing that PI3-kinase has a predominant role in PDGF receptor trafficking (37, 38), our results showed that blockade of PI3-kinase alone by either wortmannin treatment or p85-SH2 GST fusion protein microinjection had a minimal effect on insulin receptor trafficking and degradation. Although the reason for the differences between our results in insulin receptors and those in PDGF receptors remains to be elucidated, it may be due to tissue variation in signal transduction. Thus, previous results were obtained from human HepG2 cells (37, 38), whereas our studies were performed using Rat1 fibroblasts. Alternatively, it may arise from the different signal specificity initiated from different membrane receptors. Along this line, PDGF receptors directly bind to PI3-kinase with high affinity (40), whereas insulin and EGF receptors poorly bind to PI3-kinase (41, 42). Instead, insulin and EGF receptors effectively transmit signals to PI3-kinase via IRS and ErbB3, respectively (41, 42). It is of note that blockade of PI3-kinase by wortmannin treatment or p85-SH2 GST fusion protein had no effect on EGF receptor trafficking (43). Thus, the relative importance of PI3-kinase in receptor trafficking may depend on the association specificity of PI3-kinase with receptors.

MAP kinase pathway also appears to be involved in insulin receptor trafficking and down-regulation. MAP kinase cascade has been shown to be important for mitogenic signal transduction initiated by growth factors including insulin (28, 29). Thus, blockade of MAP kinase cascade by either treatment with MEK inhibitor PD98059 or depletion of MAP kinase using antisense strategy inhibited insulin-induced mitogenic signaling (28, 29). Our studies clearly demonstrated that blockade of MAP kinase pathway by either treatment with MEK inhibitor PD98059 or microinjection of catalytically inactive [K71A]Erk1 GST fusion protein partly inhibited insulin-induced insulin receptor trafficking and degradation. These results further strengthen the important role of MAP kinase pathway in insulin signal transduction. The molecular mechanism by which MAP kinase pathway regulates insulin receptor trafficking is unclear. However, 5-hydroxytryptamine (5-HT) has been shown to induce clathrin-coated pit mediated endocytosis of apCAM in sensory neurons (44). Blockade of MAP kinase pathway by either PD98059 or overexpression of dominant negative MAP kinase inhibited 5-HT induced apCAM trafficking and down-regulation (44), indicating the importance of MAP kinase in the process. MAP kinase-induced apCAM interaction with cytoskeletal element has been suggested to be a possible mechanism leading to apCAM trafficking and down-regulation (44). Along this line, MAP kinase has been reported to enhance myosin light chain kinase activity leading to phosphorylation of myosin light chains which is an essential component of cytoskeleton (45). Taken together, it is interesting to speculate that MAP kinase might be involved in insulin receptor trafficking via affecting cytoskeletal architecture.

Interestingly, blockade of both PI3-kinase and MAP kinase pathway by wortmannin and PD98059 treatment effectively inhibited insulin-induced insulin receptor trafficking and degradation. Support for the results come from the employment of microinjection analysis using p85-SH2 and [K71A]Erk1 GST fusion proteins, which gave the same results as treatment with wortmannin and PD98059. Because recent studies have suggested that PI3-kinase and MAP kinase pathways may converge at some points before MEK activation (46, 47), it is possible that wortmannin inhibition of PI3-kinase affects MAP kinase activation. However, wortmannin treatment did not affect insulin-induced MAP kinase activation in Rat1 fibroblasts (data not shown). In addition, the inhibition of MAP kinase pathway alone by either PD98059 treatment or [K71A]Erk1 microinjection only partly inhibited insulin receptor trafficking and degradation, whereas the blockade of both PI3-kinase and MAP kinase pathways effectively inhibited the process. Therefore, to reconcile the above observations, it seems to be likely that redundant pathways exist using PI3-kinase and MAP kinase. Although MAP kinase pathway appears to be dominant, both inputs complement each other to generate the efficient signal leading to insulin receptor trafficking and down-regulation. Furthermore, we cannot rule out the possibility that the observed effects on insulin receptor trafficking might be indirect, resulting from global perturbation of other cellular processes dependent on the two enzyme systems.

It is important to determine the crucial step at which insulin receptor trafficking is regulated by PI3-kinase and MAP kinase pathways. After insulin binding to its receptor on the cell surface, association of insulin-insulin receptor complexes with clathrin-coated pits is followed by the internalization of these complexes through the formation of clathrin-coated vesicle. The complexes are then transferred to endosomes in which acidification of the structure leads to the dissociation of insulin from its receptor. Following this step, the insulin receptor is either recycled back to the cell surface or targeted to lysosomes for the degradation, whereas the greater part of internalized insulin is degraded (5). Our results showed that treatment with both wortmannin and PD98059 impaired insulin-induced insulin receptor trafficking and down-regulation, whereas the early internalization step of insulin receptors from the membrane was not affected by the treatment. In addition, the wortmannin and PD98059 treatment did not affect the time kinetics of 125I-labeled insulin degradation, whereas insulin receptor degradation measured by metabolic labeling was again impaired by the treatment. Thus, the effect of treatment with wortmannin and PD98059 on the intracellular trafficking process appears to be confined to the insulin receptor, and the treatment does not appear to impair intracellular insulin trafficking. Taken together, the observed impairment of efficient insulin receptor trafficking and degradation appears to be due to deficient trafficking of internalized and insulin-dissociated insulin receptors to perinuclear compartment including lysosome. Therefore, PI3-kinase and MAP kinase pathways appear to synergistically regulate insulin receptor trafficking at a relatively late step of the trafficking pathway. The functional step of these kinases in the trafficking pathway assumed from the present study is not specific to the insulin receptor. Previous reports showed that lack of PI3-kinase binding site of PDGF receptor cytoplasmic domain or wortmannin treatment significantly impaired PDGF receptor trafficking without affecting the step of PDGF internalization (37, 38). Likewise, microinjection of p85-SH2 GST fusion protein did not affect, at least, early stage of EGF receptor endocytosis (43). Therefore, although the crucial step of MAP kinase in the trafficking process of other growth factor receptors has not been investigated, PI3-kinase might affect the trafficking step that is common to growth factor receptors.

In summary, MAP kinase cascade and PI3-kinase appear to synergistically regulate insulin-induced insulin receptor trafficking at the step subsequent to the receptor internalization. The elucidation of signaling pathways that regulate insulin receptor trafficking would lead to further insights into the regulatory mechanisms of insulin signal transduction.


   Footnotes
 
1 This work was supported in part by a grant-in-aid for encouragement of young scientists from the Ministry of Education, Science, and Culture (to T.S.). Back

Received October 19, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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