14-3-3 Facilitates Insulin-Stimulated Intracellular Trafficking of Insulin Receptor Substrate 1
Xiaoqin Xiang,
Mingsheng Yuan1,
Ying Song,
Neil Ruderman,
Rong Wen and
Zhijun Luo
Diabetes and Metabolism Research Unit (X.X., M.Y., N.R., Z.L.), Section of Endocrinology, Evans Department of Medicine, and Department of Biochemistry (Z.L.), Boston University School of Medicine, Boston, Massachusetts 02118; and Department of Ophthalmology (Y.S., R.W.), University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
Address all correspondence and requests for reprints to: Dr. Z. Luo, Diabetes and Metabolism Research Unit, Section of Endocrinology, Evans Department of Medicine, Boston University School of Medicine, 650 Albany Street, Room 820, Boston, Massachusetts 02118. E-mail: zluo{at}medicine.bu.edu.
 |
ABSTRACT
|
---|
The appearance of a complex between tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1) and PI3K in a high-speed pellet fraction (HSP) is thought to be a key event in insulin action. Conversely, the disappearance of the IRS-1/PI3K complex from this fraction has been linked to insulin desensitization. The present study examines the role of 14-3-3, a specific phospho-serine binding protein, in mediating the disappearance of IRS-1 from the HSP after insulin treatment. An in vitro pull-down assay using recombinant 14-3-3 revealed that insulin enhances the association of 14-3-3 with IRS-1 in cultured adipocytes and that this is completely inhibited by wortmannin. An association of IRS-1 and 14-3-3 was also observed and was maximal after stimulation by insulin, when endogenous proteins were immunoprecipitated. Epidermal growth factor (EGF), 12-O-tetradecanoylphorbol-13-acetate, and okadaic acid, other agents that cause serine/threonine phosphorylation of IRS-1, also stimulated IRS binding to 14-3-3. The enhancement of IRS-1 binding to 14-3-3 by insulin was accompanied by movement of IRS-1 and the p85 subunit of PI3K from the HSP to the cytosol. In keeping with a key role of 14-3-3 in mediating this redistribution of IRS-1, the complexes of IRS-1 and 14-3-3 were found in the cytosol but not in the HSP of insulin-treated cells. In addition, colocalization of IRS-1 and 14-3-3 was observed in the cytoplasm after insulin treatment by confocal microscopy. Finally, the addition of a phosphorylated 14-3-3 binding peptide to an adipocyte homogenate (to remove 14-3-3 from IRS-1) increased the abundance of IRS-1/PI3K complexes in the HSP and decreased their abundance in the cytosol. These findings strongly suggest that 14-3-3 participates in the intracellular trafficking of IRS-1 by promoting the displacement of serine-phosphorylated IRS-1 from particular structures. They also suggest that 14-3-3 proteins could play an integral role in the process of insulin desensitization.
 |
INTRODUCTION
|
---|
INSULIN STIMULATES A wide array of metabolic events including glucose transport, and glycogen, lipid, and protein synthesis and inhibition of hepatic glucose production (1). Insulin action is largely mediated by activation of PI3K (2). A growing body of evidence has suggested that the subcellular colocalization of PI3K with insulin receptor substrate 1 and 2 (IRS-1 and -2) is a major determinant of the specificity and magnitude of the metabolic action of insulin (3, 4, 5, 6, 7, 8, 9, 10). As first shown in rat adipocytes, a fraction defined ultracentrifugally as low-density intracellular membranes (LDM) (3, 4), but which also contains cytoskeletal components (9, 10), exhibits the greatest increase in PI3K activity in response to insulin stimulation (3, 4). Plasma membranes show a smaller increase in PI3K activity, whereas almost no activation of PI3K occurs in the cytosol, although its total activity is high when assayed in the presence of exogenous substrate because of the abundance of PI3K in this fraction (3, 4). It has also been reported (11, 12) that IRS-1/PI3K complexes translocate to a compartment containing glucose transporter (GLUT-4) vesicles, although these findings are controversial (4, 13). Kinetic studies of the insulin-stimulated localization of the IRS-1/PI3K signaling complexes in 3T3-L1 adipocytes (8) have revealed the following: 1) IRS-1 in the LDM is tyrosine phosphorylated by the insulin receptor tyrosine kinase more rapidly and to a greater extent than it is in the cytosol (3, 4, 8); 2) IRS-1/PI3K complexes decrease in the LDM and increase in the cytosol as IRS-1 is phosphorylated on serine/threonine residues with increasing duration of insulin stimulation; and 3) the relative stoichiometry of tyrosine phosphorylation of IRS-1 in the LDM and cytosol tends to change in opposite directions. IRS-2 shows similar changes in adipocytes, although it differs in that it is predominantly present in the cytosol, and its tyrosine dephosphorylation occurs faster than does that of IRS-1 (8). Overall, these studies suggest that insulin stimulates tyrosine phosphorylation of IRS-1/2 in an ultracentrifugally defined high-speed pellet fraction (HSP) that contains internal membranes (8) and/or cytoskeletal components (9, 10). They also suggest that serine/threonine phosphorylation of IRS-1, leading to its dissociation from the HSP to the cytosol, may be an event in the process by which insulin signaling is down-regulated (8, 10).
The recent demonstration that 14-3-3 can bind to IRS-1 and IRS-2 (14, 15, 16) raises the possibility that it participates in the regulation of insulin signal transduction. 14-3-3 binds to target proteins containing two common phosphorylation motifs, R-[S/Ar]-[+/Ar]-pS-X-P and R-X-[Ar/S]-[+]-pS-X-P (Ar: aromatic amino acids) (17, 18, 19). Sequence alignment indicates that IRS-1 contains at least five putative 14-3-3 binding sites. Three of them are within the amino-terminal portion of the molecule that is important for the interaction of IRS-1 with the insulin receptor and PI3K and might contribute to its subcellular localization. Thus, it is reasonable to hypothesize that binding to 14-3-3 can disrupt the association of IRS-1 and IRS-2 with 1) the insulin receptor; or 2) PI3K; or 3) cytoskeletal or membrane components of the HSP fraction, and that one or more of these events contributes to the down-regulation of insulin signaling. To address these possibilities, in the present study we have characterized the interaction between 14-3-3 and IRS-1 in differentiated adipocytes. The results indicate that insulin stimulates the binding of 14-3-3 to IRS-1, that this occurs in the cytosol, and that it is dependent on the activation of PI3K. They also indicate that removal of 14-3-3 does not affect IRS-1-associated PI3K activity, but that it increases the ability of IRS-1 to remain bound to the HSP. Thus, the data suggest that the binding of 14-3-3 to IRS-1 is a key event in the translocation of IRS-1 from the HSP fraction to the cytosol in cells treated with insulin.
 |
RESULTS
|
---|
Studies Using a Pull-Down Assay
To examine the possibility that an interaction between IRS-1 and 14-3-3 plays a role in the regulation of insulin signaling (14, 15, 16), an in vitro pull-down assay was performed in which cell extracts from adipocytes treated with insulin for various times were incubated with immobilized glutathione-S-transferase (GST)-14-3-3. IRS-1 binding to 14-3-3 and the activities of IRS-1 immunoprecipitated PI3K and its downstream kinase, Akt/protein kinase B (PKB) were assayed. As shown in Fig. 1A
, PI3K activity in 3T3-L1 adipocytes was significantly increased by insulin at 1 min and peaked at 5 min, whereas activation of Akt/PKB was modest at 1 min and peaked at 15 min. Increases in the ability of IRS-1 to bind to 14-3-3 in vitro followed the same time course as the activation of Akt/PKB. In differentiated 3T3-F442a adipocytes incubated with insulin (Fig. 1B
), activation of Akt/PKB occurred earlier (1 min), as did the peak binding of IRS-1 to 14-3-3 (1 min). Wortmannin, a specific inhibitor of PI3K, completely suppressed both insulin-stimulated IRS-1 binding to 14-3-3 and activation of Akt/PKB. Thus, either PI3K or a kinase downstream of PI3K in the insulin signaling cascade such as Akt/PKB appears to be responsible for the phosphorylation of the 14-3-3 binding sites on IRS-1.

View larger version (51K):
[in this window]
[in a new window]
|
Figure 1. In Vitro Pull-Down Assay on IRS-1 Binding to 14-3-3
A, 3T3-L1 adipocytes were stimulated with insulin for 130 min after serum deprivation for 16 h. IRS-1 was immunoprecipitated and PI3K assayed in cell extracts (500 µg protein). To assess Akt/PKB activation and IRS-1 expression, extracts were subjected to SDS-PAGE (7%) and immunoblotted with antibodies to the region containing phosphoserine 473 of Akt/PKB and to IRS-1, respectively. To quantify 14-3-3 binding to IRS-1, cell extracts (500 µg protein) were incubated with GST-14-3-3 , immobilized to GSH affinity beads, and then subjected to Western blotting against IRS-1. B, 14-3-3 binding assay performed with cell extracts of 3T3-F442a adipocytes in the same way as panel A, except that one plate was preincubated with wortmannin (500 nM) before addition of insulin. Results are representative of three independent experiments.
|
|
Effects of Insulin and Other Agents on IRS-1 Binding to 14-3-3 in Vivo
We considered that insulin-stimulated binding of IRS-1 to exogenous 14-3-3 in vitro might not mirror the in vivo association of these molecules for the following reasons: 1) the ability of IRS-1 to bind to exogenous 14-3-3 in vitro could be limited, if it was already complexed with endogenous 14-3-3, and 2) the increased in vitro binding of IRS-1 to 14-3-3 might be a nonspecific consequence of its increased serine phosphorylation. To examine the in vivo binding of IRS-1 to 14-3-3, IRS-1 was immunoprecipitated and quantified by blotting the coprecipitated 14-3-3 in lysates of 3T3-F442a adipocytes that had been incubated with TNF
, EGF, or insulin. As shown in Fig. 2A
, insulin markedly stimulated the association of IRS-1 and 14-3-3, EGF somewhat less so, and TNF
only minimally. Next, we examined whether the binding of 14-3-3 to IRS-1 is affected by other agents that stimulate serine phosphorylation of IRS-1. In this assay, GST-14-3-3
was coexpressed with flag-tagged IRS-1 in COS7 cells, and the association between the two was assessed by immunoblotting IRS-1 after purification of 14-3-3. As shown in Fig. 2B
, IRS-1 bound weakly to 14-3-3 under basal conditions, but binding was significantly enhanced by a short exposure of the cells to okadaic acid, EGF, or 12-O-tetradecanoylphorbol-13-acetate (TPA), agents known to cause serine/threonine phosphorylation of IRS-1 and insulin resistance (20, 21, 22, 23). Thus, both in vitro and in vivo data indicate that IRS-1 binding to 14-3-3 is stimulated by an increase in its serine/threonine phosphorylation.

View larger version (29K):
[in this window]
[in a new window]
|
Figure 2. Regulation of the Interaction Between IRS-1 and 14-3-3
A, 3T3-F442a adipocytes were stimulated with TNF (5 ng/ml), EGF (50 ng/ml), or insulin (100 nM) for 15 min. Cell extracts (4 mg protein) were subjected to immunoprecipitation with IRS-1 antibodies and immunoblotted with 14-3-3ß antibodies. Under the conditions described here, TNF caused the greatest activation of MAPK. This suggests that MAPK does not mediate the effect of these agents on IRS-1/14-3-3 complex formation. This figure represents one of three independent experiments. B, Plasmids encoding flag-IRS-1 and GST-14-3-3 were cotransfected into COS7 cells. Two days after transfection, cells were starved in 0.1% serum for 18 h and treated with okadaic acid (1 µM, 30 min), TPA (1 µM, 15 min), or EGF (50 ng/ml, 15 min). GST-14-3-3 was purified by GSH affinity purification, and the precipitated complex was subjected to SDS-PAGE and immunoblotted with antiflag antibody.
|
|
Time Course of Insulin-Stimulated IRS-1/PI3K Subcellular Distribution
Because serine/threonine-phosphorylated IRS-1 moves from the HSP to the cytosol in cells incubated with insulin (8, 10), we next assessed whether binding to 14-3-3 augments this movement. As a first step, the time course of changes in the subcellular localization of IRS-1 induced by insulin was examined in F442a adipocytes (Fig. 3A
). The abundance of IRS-1 determined by immunoblotting was much greater in the HSP than in either the cytosol or plasma membrane (Figs. 3A
and 6A
). Based on measurements following differential centrifugation, more than 60% of cellular IRS-1 was present in the HSP fraction and about 30% in the cytosol under basal conditions. During a 30-min incubation with insulin, the content of IRS-1 progressively decreased in the HSP and increased in the cytosol. IRS-1 in plasma membranes (
10% of total cellular mass of IRS-1 under basal conditions) increased transiently after 1 min of incubation with insulin and decreased at later time points (Fig. 3A
). Although IRS-1 distribution in the different fractions changed with time of insulin stimulation, the total cellular IRS-1 mass, calculated from the sum of the total densitometric units in each fraction, appeared to remain constant.

View larger version (26K):
[in this window]
[in a new window]
|
Figure 3. Effect of insulin on IRS-1 and PI3K Distribution
Subcellular fractions from 3T3-F442a adipocytes treated with or without insulin for the indicated times were prepared as described in Material and Methods. Twenty-five micrograms of HSP and plasma membrane (PM) and 100 µg of cytosol were subjected to SDS-PAGE and immunoblotted against IRS-1. The immunoblot is one example of three independent experiments (A). The graphs represent averages (±SEM) of IRS-1 distribution (A) and P85 subunit localization in the HSP (B) from three experiments determined by scan densitometry. Values of IRS-1 distribution are expressed as percentage of IRS-1 in the HSP at zero time point (100%). P85 localization in the HSP is represented by arbitrary densitometric unit.
|
|

View larger version (18K):
[in this window]
[in a new window]
|
Figure 6. Incubation with a 14-3-3 Binding Peptide Causes IRS-1 to Relocate from the Cytosol to the HSP
A, HSP, PM, and cytosol from 3T3-F442a adipocytes, treated with insulin, as indicated were prepared in the presence or absence of a 14-3-3 binding peptide (PS) (0.4 mM). Twenty micrograms of each fraction were subjected to SDS-PAGE and then blotted with antibodies to IRS-1 or the p85 subunit of PI3K. Lower panel represents averages (±SEM) of PS-induced IRS-1 mobilization in the HSP (open square) and cytosol (solid square) from three experiments (t test for * and °, P < 0.05). Values are expressed as percentage of the highest (100%) in each fraction. B, HSP was prepared as described in panel A. This fraction and total cell extracts containing 20 µg proteins were immunoblotted for IRS-1 and anti-14-3-3ß.
|
|
The distribution of the p85 subunit of PI3K in the HSP changed as a function of the tyrosine phosphorylation of IRS-1 by the insulin receptor (Fig. 3B
). In contrast to the progressive decrease in concentration of IRS-1 in the HSP during a 30-min incubation with insulin, the abundance of p85 initially increased, reaching a plateau between 1 and 5 min, and then began to decline. At 30 min, it was diminished by 50% from its peak value, but was still higher than 0 min. In contrast, IRS-1 was maximally diminished at this time. These dynamic changes are similar to those described by others (8).
Analysis of Subcellular Localization of IRS-1 and 14-3-3 by Confocal Microscopy
To gain insight into whether IRS-1 interacts with 14-3-3 in intact cells, the colocalization of the two proteins in response to insulin was studied in 3T3 F442a adipocytes. The cells were stained simultaneously with specific antibodies and visualized by immunofluorescence confocal microscopy. As shown in Fig. 4
, in the resting state, IRS-1 was concentrated around the nucleus (stained red by rhodamine). In contrast, 14-3-3 was uniformly distributed in the cytoplasm (green). When the two images for IRS-1 and 14-3-3 were merged with each other, an obvious orange color was not produced, suggesting no detectable colocalization at this stage. After 30 min of treatment of cells with insulin, the perinuclear concentration of IRS-1 had disappeared and it was distributed uniformly in the cytoplasm. When the images of IRS-1 and 14-3-3 were superimposed, scattered orange spots were observed, indicating that a portion of IRS-1 had been colocalized with 14-3-3. Because the majority was still green after merging the two pictures, however, it appeared that substantial amounts of 14-3-3 did not associate with IRS-1.

View larger version (111K):
[in this window]
[in a new window]
|
Figure 4. IRS-1 and 14-3-3 Localization by Immunofluorescence Microscopy
3T3-F442a cells were stained by indirect immunofluorescence and observed by confocal microscopy at 63x, as described in Materials and Methods. Panels ac show 14-3-3 (a, green) and IRS-1 (b, red) localization under basal condition; panels df are images from insulin-stimulated cells stained as ac. Panels c and f represent superimposed images of IRS-1 and 14-3-3. Bar, 10 µm.
|
|
Binding to 14-3-3 Facilitates IRS-1 Translocation from the HSP Fraction to the Cytosol
To examine the ability of IRS-1 to bind to 14-3-3 in the two fractions, the extracts of HSP and cytosol were normalized to contain an equal amount of IRS-1 per unit volume and then incubated with immobilized GST-14-3-3. The association of the two polypeptides was then assessed by Western blotting of the IRS-1 immunoprecipitate. As shown in Fig. 5A
(lane 3), IRS-1 in the cytosol was minimally bound to 14-3-3 under basal conditions; indeed, such binding was even less than it was in the HSP. In contrast, after a 30-min incubation with insulin IRS binding to 14-3-3 was twice as great in the cytosol as in the HSP. Furthermore, this could be an underestimate of the difference in binding between the two fractions because 14-3-3 in the cytosol is much more abundant than in the HSP (Fig. 6B
). Thus, if much of IRS-1 in the cytosol was already complexed with endogenous 14-3-3, this could have diminished its binding to the recombinant GST-14-3-3. To test this possibility, IRS-1 was immunoprecipitated from both the HSP and cytosol, subjected to SDS-PAGE, and then immunoblotted with anti-14-3-3 antibodies. As shown in Fig. 5B
, after insulin stimulation, binding of IRS-1 to endogenous 14-3-3 was observed in the cytosol, but was not detectable in the HSP. No coimmunoprecipitation of 14-3-3 with the HSP IRS-1 could rule against the possibility that the binding of 14-3-3 to the cytosolic IRS-1 occurred during homogenization.

View larger version (40K):
[in this window]
[in a new window]
|
Figure 5. Insulin Stimulates Association of IRS-1 in the Cytosol
A, In vitro binding of IRS-1 to 14-3-3. The HSP and cytosol from 3T3-F442a adipocytes, treated with or without insulin for 30 min, were prepared as in panel A. Extracts of the two fractions containing equal amounts of IRS-1 (12 mg total proteins) were incubated with immobilized GST-14-3-3 , and the IRS-1 content in the precipitated 14-3-3/IRS-1 complex was determined by immunoblotting. B, NP-40 solubilized supernatant from the HSP (1 mg) and cytosol (2 mg) fractions of 3T3-F442a adipocytes were immunoprecipitated with anti-IRS-1 antibodies and subjected to anti-14-3-3ß immunoblotting. Extracts from the HSP (20 µg) and cytosol (50 µg) were used for IRS-1 blot (top panel). Results are representative of three independent experiments.
|
|
The findings presented in Fig. 5
strongly suggest that the binding of serine/threonine-phosphorylated IRS-1 to 14-3-3 facilitates movement of IRS-1 from the HSP to the cytosol. To test this notion further, adipocyte homogenates were incubated with a phosphorylated 14-3-3 binding peptide (derived from the Raf-1 sequence around S621, which effectively removes endogenous 14-3-3) (18), and IRS-1 distribution was reexamined. As shown earlier (Fig. 3A
), after a 30-min incubation with insulin, IRS-1 was diminished in the HSP and increased in the cytosol (Fig. 6A
). When the homogenates were incubated with the phosphorylated 14-3-3 binding peptide, IRS-1 in the HSP was increased to a level comparable to that in the basal condition (lanes 1 and 4), and IRS-1 in the cytosol was correspondingly decreased. Due to the difference in IRS-1 concentration in the two fractions, the intensity of the IRS-1 band in the HSP was much stronger than it was in the cytosol. However, if one takes into account the fact that the cytosol was diluted 10-fold more than the HSP, the increase of IRS-1 in the HSP after addition of the peptide was quantitatively similar to its disappearance from the cytosol.
When the phosphorylated 14-3-3 binding peptide was added to the adipocyte homogenate, p85 abundance in the HSP after 30 min of incubation with insulin was similar to the 1 min value (Fig. 6A
). Likewise, PI3K activity (lipid kinase) in the HSP fraction was substantially increased (data not shown). These results suggest that PI3K stays with IRS-1 as a complex even after movement from the HSP to the cytosol. Addition of phosphorylated 14-3-3 binding peptide did not affect the distribution of IRS-1 in the absence of insulin nor, evidently, the distribution of 14-3-3 itself (Fig. 6B
). Under identical conditions, a peptide containing the same sequence as the binding peptide, but which was not phosphorylated, did not bind to 14-3-3 and failed to affect the distribution of IRS-1 in cells incubated with insulin (data not shown). Collectively, these results strongly suggest that by binding to serine-phosphorylated IRS-1, 14-3-3 causes the IRS-1/PI3K complex to dissociate from the HSP.
IRS-1-Associated PI3K Activity Is Not Inhibited by 14-3-3
We next assessed whether binding of 14-3-3 to IRS-1 inhibits the associated PI3K activity. IRS-1 was purified from 3T3-F442a adipocyte extracts by immunoprecipitation with anti-IRS-1 antibody in the presence of either the phosphorylated 14-3-3 binding peptide or the nonphosphorylated peptide. As shown in Fig. 7A
, IRS-1-associated PI3K activity was the same under the two conditions. Likewise, no difference in PI3K activity was observed when the extracts were immunoprecipitated with anti-p85 antibody in the presence of either peptide (Fig. 7B
). In the latter experiment, no effect of insulin on PI3K activity was evident, possibly reflecting the fact that only a small portion of total cellular p85 associates with IRS-1 in response to insulin stimulation (3). These results strongly suggest that 14-3-3 does not have a direct effect on PI3K activity, although it binds to IRS-1 and possibly to PI3K itself (24).

View larger version (43K):
[in this window]
[in a new window]
|
Figure 7. Removal of 14-3-3 from IRS-1 Does Not Affect Its Associated PI3K Activity
Extracts (500 µg protein) prepared from 3T3-F442a adipocytes, treated with or without insulin for 15 min, were used for immunoprecipitation of IRS-1 (A) or P85 (B) in the presence or absence of phosphorylated (active, PS) or nonphosphorylated (inactive, P) 14-3-3 binding peptides. PI3K activity was assayed as described in Materials and Methods. Results are representative of five independent experiments.
|
|
 |
DISCUSSION
|
---|
It has recently been demonstrated that 14-3-3 can bind to IRS-1 and IRS-2 (14, 15, 16); however, the physiological relevance of such binding for insulin signaling is not clear. The present study indicates that the binding of IRS-1 to 14-3-3 1) is stimulated by insulin and other agents that induce serine/threonine phosphorylation of IRS-1, 2) requires that PI3K or a kinase dependent on PI3K be activated, and 3) is associated with a movement of IRS-1 and PI3K from the HSP to the cytosol that is inhibited when IRS-1 is deprived of 14-3-3. In addition, using confocal microscopy, we have shown that IRS-1 is localized in the perinuclear region and that after insulin stimulation it moves to the cytosol, where it colocalizes with 14-3-3. Overall, these results suggest that 14-3-3 very likely plays a role in the process of insulin desensitization.
Under basal conditions, a substantial amount of cellular IRS-1 is associated with internal membranes or cytoskeleton (3, 4, 5, 6, 7, 8, 9, 10). Ultracentrifugal fractionation suggests that after insulin stimulation IRS-1 moves toward the plasma membrane where it is phosphorylated by the insulin receptor tyrosine kinase and that it then dissociates into the cytosol (3, 4, 5, 6, 7, 8, 9, 10, 27, 28). The results of the present study, in which both biochemical fractionation and immunofluorescence microscopy were used, suggest the movement involves 14-3-3. Thus, it was demonstrated that in the resting state of 3T3-F442a adipocytes, IRS-1 clearly possesses a perinuclear localization and that it does not colocalize with 14-3-3, which is distributed evenly in the cytoplasm (Fig. 4
). The perinuclear localization of IRS-1 is similar to that of the glucose transporter 4 (24), in keeping with the finding that both are present in the high-speed fraction (9). However, their localization is not identical, because further separation by sucrose gradient centrifugation has shown that they are present in different fractions (9). After the insulin stimulation, ultracentrifugal fractionation revealed that IRS-1 moves from the HSP to the cytosol and that this correlates morphologically with redistribution from its perinuclear location to a uniform dispersal throughout the cytoplasm. Interestingly, the latter was associated with the colocalization of IRS-1 and 14-3-3, consistent with biochemical data showing that a 30-min treatment of cells with insulin gives rise to maximal binding of IRS-1 to 14-3-3. Our morphological findings are in agreement with those of the most recent study by Jacobs et al. (26), who have shown that transiently expressed IRS-1 is concentrated in the perinuclear region of COS7 cells and that it moves to the plasma membrane in response to insulin when the cells are cotransfected with insulin receptor. It should be noted that only a portion of 14-3-3 is revealed to be colocalized with IRS-1 after insulin stimulation. This is not surprising because 14-3-3 is an extremely abundant protein, and it associates with great many intracellular signaling molecules. It is suggested that upon insulin stimulation, only a small portion of IRS-1 is phosphorylated on tyrosine residues by the insulin receptor. Thus, it is of interest to investigate whether only this tyrosine-phosphorylated IRS-1 is bound by 14-3-3.
Previous studies of the effects of insulin on IRS binding to 14-3-3 have yielded conflicting results. Thus, Kosaki et al. (14) reported that insulin stimulated the binding of IRS-1 to 14-3-3 in 3T3-L1 adipocytes, whereas Ogihara et al. (16) were unable to demonstrate such an effect in HepG2 hepatoma cells. These discrepant findings could reflect differences in the kinases that phosphorylate 14-3-3 binding sites on IRS-1 in the two cells. Alternatively, the high level of recombinantly expressed IRS-1 in the HepG2 cells could have increased its serine phosphorylation and binding to 14-3-3 by mass action. In this context, we found that insulin-stimulated binding of IRS-1 to 14-3-3 in CHO-T cells overexpressing the insulin receptor occurs only when the expression of recombinant IRS-1 is kept at a very low level (our unpublished data).
The present results suggest that serine/threonine phosphorylation of 14-3-3 binding sites on IRS-1 occurs very rapidly in 3T3-F442a adipocytes, reaching a peak after 1 min of insulin treatment, based on the in vitro pull-down assay (Fig. 1
). In contrast, IRS-1 dissociation from the HSP was limited at this time and only reached its maximum at 10 min. One possible explanation for these findings is that at the later time points after insulin treatment, IRS-1 had already complexed with 14-3-3 in vivo and thus further binding in vitro was limited.
The observation that the abundance of PI3K (p85 subunit), which is complexed with IRS in the HSP, decreases concurrently with IRS in this fraction after insulin treatment suggests that it too is translocated (Figs. 3B
and 6A
). This is in agreement with a previous report by Inoue et al. (8). The novel finding here is that the exit of both molecules from the HSP is reversed by incubation of the cell homogenate with a phosphorylated 14-3-3 binding peptide. This strongly suggests that 14-3-3 is required for the translocation of both molecules. It also suggests that 14-3-3 could play a significant role in the down regulation of insulin signaling, because the translocation of PI3K from the HSP to the cytosol separates it from its phosphoinositide substrates (phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-biphosphate). This would effectively decrease P13K activity in vivo, even when the enzyme retained its assayable activity in a cell lysates. (Note: when assayed in a cell lysate, the ability of PI3K to phosphorylate exogenous phosphoinositides is typically determined.) Alternatively, binding to 14-3-3 could down-regulate insulin signaling by depressing tyrosyl phosphorylation of IRS-1 and/or altering the interaction of IRS-1 with the insulin receptor or a tyrosine phosphatase. The observation that IRS-associated PI3K activity is not decreased in the whole-cell lysate after 30 min of insulin treatment (i.e. after much of the p85 had exited the HSP fraction) (data not shown and Figs. 3B
and 6A
) makes this explanation less likely, however.
Effects of 14-3-3 on the subcellular localization of signaling complexes have been described for molecules other than IRS-1. One of these is Cdc25C, a protein phosphatase that controls the entry of eukaryotic cells into mitosis. When the 14-3-3 binding site on Cdc25C is phosphorylated by the cell cycle checkpoint protein kinase Chk1 (which is activated by DNA damage stress), the resultant binding to 14-3-3 inhibits Cdc25C by causing it to translocate from nucleus to cytosol. This leads to an arrest of the cell cycle in G2 phase (29, 30, 31, 32). Another example is the proapoptotic transcription factor, forkhead protein (DAF16), which is sequestered in the cytosol when it is bound by 14-3-3 (33, 34, 35).
14-3-3 could participate in the process of IRS-1 dissociation from the HSP by at least two mechanisms. First, by directly binding to serine/threonine phosphorylated IRS-1, 14-3-3 could disrupt the association of IRS-1 with a protein in the HSP, thereby allowing the IRS-1/PI3K complex to move into the cytosol and; second, serine-phosphorylated IRS-1 complexed with PI3K could move from the HSP to the cytosol by an as-yet-unknown mechanism, and binding to 14-3-3 would simply act to sequester it in the cytosol. The data of Inoue et al. (8) have led them to propose an alternative theory in which the insulin-stimulated translocation of IRS-1 to the cytosol is caused by serine/threonine phosphorylation of a component of the internal membranes, rather than of IRS-1. Our finding that removal of 14-3-3 allows serine-phosphorylated IRS-1 to rebind to the HSP does not support this theory. It also suggests that serine/threonine phosphorylation of IRS-1 in the absence of 14-3-3 is not sufficient to allow it to remain in the cytosol.
As already noted, by causing the translocation of PI3K from the HSP to the cytosol, 14-3-3 could hypothetically inhibit PI3K activity, either by binding to it (i.e. direct inhibition), or by binding to IRS-1 and causing it to dissociate from PI3K. The latter mechanism was proposed by Kosaki et al. (14), based on their finding that the specific activity of PI3K in a ternary complex of PI3K/IRS-1/14-3-3, purified by anti-14-3-3 immunoprecipitation, was lower than that in an anti-IRS-1-immunoprecipitated IRS-1/PI3K complex, in which they had attempted to immunodeplete 14-3-3. Inhibition of PI3K by 14-3-3 is not supported by the results of the present study, in that we found no increase of PI3K activity when 14-3-3 was removed from the IRS-1-PI3K complex (Fig. 7
).
The results also have implications for the study of insulin resistance. First, in terms of methodology, the in vitro pull-down assay could serve as an efficient and rapid method for assessing the serine/threonine phosphorylation of IRS-1. By using this approach, we have found that the binding of IRS-1 to 14-3-3 is increased in rat skeletal muscle in which IRS-1 serine/threonine phosphorylation and insulin resistance are induced by incubation with phorbol ester (our unpublished data). Second, the following lines of evidence suggest that 14-3-3 could be involved in the pathogenesis of insulin resistance: 1) its binding to IRS-1 is stimulated by EGF, TPA, and okadaic acid, agents that induce insulin resistance in various cell types (20, 21, 22, 23); and 2) chronic treatment of cells with insulin in the presence of glucose, which as shown here increases the association of IRS-1 with 14-3-3, can cause insulin resistance (36, 37, 38).
In summary, the results of the present study strongly suggest that 14-3-3 binding to IRS-1 is set in motion by serine/threonine phosphorylation of IRS-1 in response to the activation of PI3K by insulin. They also suggest that it plays a role in the translocation of the IRS-1/PI3K complex from the HSP to the cytosol and, in this way, could contribute to insulin desensitization. The identity of the 14-3-3 binding sites on IRS-1 remains to be determined. We have mutated five putative 14-3-3 binding sites on IRS-1; however, binding of 14-3-3 to this mutated IRS-1 was unaffected. Because IRS-1 contains more than 30 potential serine/threonine phosphorylation sites, further studies are needed. Also needed will be an effort to identify the kinases that phosphorylate IRS-1 and enhance its binding to 14-3-3. The role of Akt/PKB in this process is unclear. In F442a cells, the apparent maximal binding of IRS-1 to 14-3-3 preceded maximal activation of Akt/PKB. On the other hand, some activation of Akt/PKB had occurred at the time of maximal binding. Thus, at present, PI3K and Akt/PKB both remain candidate kinases.
 |
MATERIALS AND METHODS
|
---|
Materials
Insulin, TPA, wortmannin, and phosphatidylinositol were purchased from Sigma (St. Louis, MO), and tumor necrosis factor
(TNF
), epidermal growth factor (EGF), and okadaic acid were purchased from Calbiochem (San Diego, CA). Polyclonal antibody against IRS-1 was purchased from Upstate Biotechnology, Inc.(Lake Placid, NY) and monoclonal antibody to p85
from Transduction Laboratories, Inc. (Lexington, KY). Polyclonal antibody against 14-3-3ß was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and antibody against phospho-PKB (Akt/PKB) from New England Biolabs, Inc. (Beverly, MA). Rhodamine-conjugated donkey antirabbit IgG and fluorescein isothiocyanate-conjugated donkey antigoat IgG antibodies were from Molecular Probes, Inc. (Eugene, OR). [
-32P]-ATP was purchased from NEN Life Science Products (Boston, MA), and COS7 cells were from ATCC (Manassas, VA); the 3T3-L1 and 3T3-F442a fibroblast cells were generous gifts from Dr. H. Green (Harvard Medical School, Boston, MA).
Transfection
COS7 cells were cultured in DMEM supplemented with 10% FBS. Cells near confluence in a 75-cm2 flask were split into 10-cm plates and allowed to grow overnight to reach 70% confluence before transfection. Plasmids (5 µg) were transfected into the COS7 cells by Lipofectamine according to manufacturers protocol (Life Technologies, Inc., Gaithersburg, MD). Forty-eight hours after transfection, cells were starved in DMEM-0.1% FBS overnight and stimulated by extracellular ligands for a short period of time as indicated in the figure legend.
Adipocyte Differentiation and Subcellular Fractionation
The 3T3-L1 and 3T3-F442a fibroblasts were maintained in DMEM containing 10% calf serum. Two days after confluence, the cells were induced to differentiate into adipocytes (39, 40). Eight to 10 d after the addition of the differentiation reagents, cells were starved in 0.1% FBS for 16 h and incubated with or without insulin as described in the figure legends, and washed with buffer A (250 mM sucrose, 20 mM HEPES, 1 mM EDTA, pH 7.4). Adipocytes from three 10-cm plates were then resuspended in 35 ml buffer A supplemented with 1 mM NaVO3, 50 nM okadaic acid, 2 µg/ml leupeptin, 2 µg/ml pepstatin A, 2 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride, pH 7.4) and homogenized by 10 strokes of a Teflon homogenizer. Cell homogenates were subjected to differential centrifugation for preparation of plasma membranes, an HSP fraction, and cytosol as described by Clancy and Czech (41). The plasma membranes and HSP were resuspended in 200 µl buffer B (same as buffer A except omitting sucrose).
Western Blot and Immunoprecipitation
Protein assays were performed using a Bio-Rad Laboratories, Inc. kit (Hercules, CA). Equal amounts of cell extract were resolved on SDS-PAGE and electrophoretically transferred to Immobilon (Millipore Corp., Bedford, MA). The Immobilon membrane was blocked with Tris-buffered saline-0.1% Tween-20 (TBST) containing 5% nonfat milk and then blotted with specific antibody for 1 h. The membrane was washed three times with TBST, incubated with a horse radish peroxidase-conjugated antibody (Santa Cruz Biotechnology, Inc.) against the first antibody, washed again and developed by the chemiluminescent method. For immunoprecipitation, specific antibody (2 µg) was incubated with cell extracts in a lysis buffer (42) for 216 h and then with 20 µl protein A/G beads for an additional hour. The precipitates were washed several times with or without 0.5 M NaCl according to the purpose of the experiments.
Immunofluorescence Microscopy
3T3 F442a adipocytes were split and grown on a collagen I-coated coverslip overnight. Cells were stimulated with 100 nM insulin after 2 h of serum starvation, washed with Dulbeccos PBS (DPBS), and fixed with 4% paraformaldehyde. The fixed cells were then washed twice with DPBS, permeabilized with 0.1% Triton X-100 in PBS for 3 min, and washed again with DPBS. The slides were blocked with 5% BSA for 1 h, and then simultaneously incubated with rabbit anti-IRS-1 and goat anti-14-3-3ß antibodies (1:50) for 218 h and washed three times with DPBS (5 min/each time). The slides were then stained with rhodamine-conjugated antirabbit IgG and fluorescein isothiocyanate-conjugated antigoat IgG (1:200) for 1 h and washed with PBS three times. Confocal images were obtained with a E600 fluorescence microscope (Nikon, Melville, NY) equipped with a Bio-Rad MRC 1024 confocal laser scanning imaging system from Bio-Rad Laboratories, Inc.
PI3K Assay
Cell extracts prepared in a lysis buffer (3), after treatment of cells with or without insulin, were immunoprecipitated with an antibody against IRS-1, and the immunocomplex was precipitated by adding trisacryl protein A beads (Pierce Chemical Co., Rockford, IL). PI3K was assayed according to Kelly et al. (3).
Assessment of 14-3-3 Binding
To study 14-3-3 binding in vivo, 1) the HSP fraction prepared from 3T3-F442a adipocytes was solubilized in buffer B containing 1% NP-40, 500 mM NaCl and then centrifuged in Eppendorf tubes (Madison, WI) at 13,000 rpm for 15 min. The supernatant from this spin was diluted 3-fold with buffer A, which was used to prepare the cytosol. The extracts of HSP and cytosol were then subjected to immunoprecipitation with anti IRS-1 antibodies and immunoblotted with anti-14-3-3ß antibody; and 2) cell extracts containing transiently expressed recombinant IRS-1 and GST-14-3-3 (fusion protein containing glutathione-S-transferase at the amino terminus and 14-3-3 at the carboxy terminus) were prepared in the lysis buffer (42) and passed through glutathione (GSH) beads. Binding was visualized by immunoblotting with antiflag antibody. To assess 14-3-3 binding in vitro, GST-14-3-3 was immobilized to GSH beads and incubated with adipocyte extracts. The precipitate was subjected to immunoblotting with anti-IRS-1 antibody.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Zheng-Yue Jiang for reagents and Dr. Howard Green for 3T3-L1 and 3T3-F442a preadipocyte cell lines. We also thank Dr. Yasuo Ido and Mr. Jose Cacicedo for technical advice on immunofluorescence and Dr. M.W. Zang for help in preparing figures.
 |
FOOTNOTES
|
---|
This work was supported by research grants from the American Diabetes Association (to Z.J.L.) and Juvenile Diabetes Foundation (to N.R.) and a pilot grant from the Boston Obesity and Nutrition Research Center (to Z.J.L.). X.Q.X. was the recipient of a mentor-based fellowship (to N.R.) from the American Diabetes Association.
1 Present address: Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts 02215. 
Abbreviations: DPBS, Dulbeccos PBS; EGF, epidermal growth factor; GSH, glutathione; GST, glutathione-S-transferase; HSP, high-speed pellet; IRS, insulin receptor substrate; LDM, low-density intracellular membranes; PKB, protein kinase B; TPA, 12-O-tetradecanoylphorbol-13-acetate.
Received for publication March 28, 2001.
Accepted for publication November 21, 2001.
 |
REFERENCES
|
---|
-
Chipkin SR, Kelly KL, Ruderman NB 1994 In: Kahn RC, Weir GC, eds. Joslins diabetes mellitus, ed. 13. Philadelphia: Lea & Febiger; 97115
-
White MF 1998 The IRS-signalling system: a network of docking proteins that mediate insulin action. Mol Cell Biochem 182:311[CrossRef][Medline]
-
Kelly KL, Ruderman NB, Chen KS 1992 Phosphatidylinositol-3-kinase in isolated rat adipocytes. Activation by insulin and subcellular distribution. J Biol Chem 267:34233428[Abstract/Free Full Text]
-
Kelly KL, Ruderman NB 1993 Insulin-stimulated phosphatidylinositol 3-kinase. Association with a 185-kDa tyrosine-phosphorylated protein (IRS-1) and localization in a low density membrane vesicle. J Biol Chem 268:43914398[Abstract/Free Full Text]
-
Heller-Harrison RA, Morin M, Czech MP 1995 Insulin regulation of membrane-associated insulin receptor substrate 1. J Biol Chem 270:2444224450[Abstract/Free Full Text]
-
Ricort JM, Tanti JF, Van Obberghen E, Le Marchand-Brustel Y 1996 Different effects of insulin and platelet-derived growth factor on phosphatidylinositol 3-kinase at the subcellular level in 3T3-L1 adipocytes. A possible explanation for their specific effects on glucose transport. Eur J Biochem 239:1722[Abstract]
-
Kublaoui B, Lee J, Pilch PF 1995 Dynamics of signaling during insulin-stimulated endocytosis of its receptor in adipocytes. J Biol Chem 270:5965[Abstract/Free Full Text]
-
Inoue G, Cheatham B, Emkey R, Kahn CR 1998 Dynamics of insulin signaling in 3T3-L1 adipocytes. Differential compartmentalization and trafficking of insulin receptor substrate (IRS)-1 and IRS-2. J Biol Chem 273:1154811555[Abstract/Free Full Text]
-
Clark SF, Martin S, Carozzi AJ, Hill MM, James DE 1998 Intracellular localization of phosphatidylinositide 3-kinase and insulin receptor substrate-1 in adipocytes: potential involvement of a membrane skeleton. J Cell Biol 140:12111225[Abstract/Free Full Text]
-
Clark SF, Molero, JC, James DE 2000 Release of insulin receptor substrate proteins from an intracellular complex coincides with the development of insulin resistance. J Biol Chem 275:38193826[Abstract/Free Full Text]
-
Heller-Harrison RA, Morin M, Guilherme A, Czech MP 1996 Insulin-mediated targeting of phosphatidylinositol 3-kinase to GLUT4-containing vesicles. J Biol Chem 271:1020010204[Abstract/Free Full Text]
-
Wang QH, Bilan PJ, Tsakiridis T, Henek A, Klip A 1998 Actin filaments participate in the relocalization of phosphatidylinositol3-kinase to glucose transporter-containing compartments and in the stimulation of glucose uptake in 3T3-L1 adipocytes. Biochem J 331:917928[Medline]
-
Del Vecchio RL, Pilch PF 1991 Phosphatidylinositol 4-kinase is a component of glucose transporter (GLUT 4)-containing vesicles. J Biol Chem 266:1327813283[Abstract/Free Full Text]
-
Kosaki A, Yamada K, Suga J, Otaka A, Kuzuya H 1998 14-3-3beta protein associates with insulin receptor substrate 1 and decreases insulin-stimulated phosphatidylinositol 3'-kinase activity in 3T3L1 adipocytes. J Biol Chem 273:940944[Abstract/Free Full Text]
-
Craparo A, Freund R, Gustafson TA 1997 14-3-3 (epsilon) interacts with the insulin-like growth factor I receptor and insulin receptor substrate I in a phosphoserine-dependent manner. J Biol Chem 272:1166311669[Abstract/Free Full Text]
-
Ogihara T, Isobe T, Ichimura T, Taoka M, Funaki M, Sakoda H, Onishi Y, Inukai K, Anai M, Fukushima Y, Kikuchi M, Yazaki Y, Oka Y, Asano T 1997 14-3-3 protein binds to insulin receptor substrate-1, one of the binding sites of which is in the phosphotyrosine binding domain. J Biol Chem 272:2526725274[Abstract/Free Full Text]
-
Aitken A 1996 14-3-3 and its possible role in co-ordinating multiple signaling pathways. Trends Cell Biol 6:341347[CrossRef]
-
Muslin AJ, Tanner JW, Allen PM, Shaw AS 1997 Interaction of 14-3-3 with signaling proteins is mediated by the recognition of phosphoserine. Cell 84:889897
-
Yaffe MB, Rittinger K, Volinia S, Caron PR, Aitken A, Leffers H, Gamblin SJ, Smerdon SJ, Cantley LC 1997 The structural basis for 14-3-3:phosphopeptide binding specificity. Cell 91:961971[Medline]
-
Tanti JF, Gremeaux T, Cormont M, Van Obberghen E, Le Marchand-Brustel Y 1993 Okadaic acid stimulates IGF-II receptor translocation and inhibits insulin action in adipocytes. Am J Physiol 64:E868E873
-
Tanti JF, Gremeaux T, Van Obberghen E, Le Marchand-Brustel Y 1994 Serine/threonine phosphorylation of insulin receptor substrate 1 modulates insulin receptor signaling. J Biol Chem 269:60516057[Abstract/Free Full Text]
-
Chin JE, Liu F, Roth RA 1994 Activation of protein kinase C
inhibits insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1. Mol Endocrinol 8:5158[Abstract]
-
De Fea K, Roth RA 1997 Modulation of insulin receptor substrate-1 tyrosine phosphorylation and function by mitogen-activated protein kinase. J Biol Chem 272:3140031406[Abstract/Free Full Text]
-
Guilherme A, Emoto M, Buxton JM, Bose S, Sabini R, Theurkauf WE, Leszyk J, Czech MP 2000 Perinuclear localization and insulin responsiveness of Glut4 requires cytoskeletal integrity in 3T3L1 adipocytes. J Biol Chem 275:3815138159[Abstract/Free Full Text]
-
Bonnefoy-Berard N, Liu YC, Von Willebrand M, Sung A, Elly C, Mustelin T, Yoshida H, Ishizaka K, Altman A 1995 Inhibition of phosphatidylinositol 3-kinase activity by association with 14-3-3 proteins in T cells. Proc Natl Acad Sci USA 92:1014210146[Abstract]
-
Jacobs AR, LeRoith D, Taylor SI 2001 Insulin receptor substrate-1 PH and PTB domains are both involved in plasma membrane targeting. J Biol Chem 276: 4079540802
-
VanRenterghem B, Morin M, Czech MP, Heller-Harrison RA 1998 Interaction of insulin receptor substrate-1 with the d3A subunit of the adaptor protein complex-3 in cultured adipocytes. J Biol Chem 273:2994229949[Abstract/Free Full Text]
-
Vainshtein I, Kovacina KS, Roth RA 2001 The insulin receptor substrate (IRS)-1 plecktrin homology domain functions in downstream signaling. J Biol Chem 276:80738078[Abstract/Free Full Text]
-
Sanchez Y, Wong C, Thomas RS, Richman R, Wu Z, Piwnica-Qorms H, Elledge SJ 1997 Conservation of the Chk1 checkpoint pathway in mammals: linkage of DNA damage to Cdk regulation through Cdc25. Science 277:14971501[Abstract/Free Full Text]
-
Peng CY, Graves PR, Thomas RS, Wu Z, Shaw AS, Piwnica-Worms H 1997 Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216. Science 277:15011505[Abstract/Free Full Text]
-
Kumagai A, Dunphy WG 1999 Binding of 14-3-3 proteins and nuclear export control the intracellular localization of the mitotic inducer Cdc25. Genes Dev 13:10671072[Abstract/Free Full Text]
-
Lopez-Girona A, Furnari B, Mondersert O, Russell P 1999 Nuclear localization of Cdc25 is regulated by DNA damage and a 14-3-3 protein. Nature 397:172175[CrossRef][Medline]
-
Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, Anderson MJ, Arden KC, Blenis J, Greenberg ME 1999 Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96:857868[Medline]
-
Takaishi H, Konishi H, Matsuzaki H, Ono Y, Shirai Y, Saito N, Kitamura T, Ogawa W, Kasuga M, Kikkawa U, Nishizuka Y 1999 Regulation of nuclear translocation of forkhead transcription factor AFX by protein kinase B. Proc Natl Acad Sci USA 96:1183611841[Abstract/Free Full Text]
-
Biggs III WH, Heisenhelder J, Hunter T, Cavenee WK, Arden KC 1999 Protein kinase B/Akt-mediated phosphorylation promotes nuclear exclusion of the winged helix transcription factor FKHR1. Proc Natl Acad Sci USA 96:74217426[Abstract/Free Full Text]
-
Kurowski TG, Lin Y, Luo ZJ, Tschlis PN, Buse MG, Heydrick SJ, and Ruderman N 1999 Hyperglycemia inhibits insulin activation of AKT/protein kinase B but not phosphatidylinositol 3-kinase in Raf skeletal muscle. Diabetes 48:658663[Abstract]
-
Kozka IJ, Clark AE, Holman GD 1991 Chronic treatment with insulin selectively down-regulates cell-surface GLUT4 glucose transporters in 3T3-L1 adipocytes. J Biol Chem 266:1172611731[Abstract/Free Full Text]
-
Ricort JM, Van Obberghen E, Le Marchand-Brustel Y 1995 Alterations in insulin signalling pathway induced by prolonged insulin treatment of 3T3-L1 adipocytes. Diabetologia 38:11481156[CrossRef][Medline]
-
Frost SC, Lane MD 1985 Evidence for the involvement of vicinal sulfhydryl groups in insulin-activated hexose transport by 3T3-L1 adipocytes. J Biol Chem 260:26462652[Abstract]
-
Djian P, Philips M, Green H 1985 The activation of specific gene transcription in the adipose conversion of 3T3 cells. J Cell Physiol 124:554556[Medline]
-
Clancy BM, Czech MP 1990 Hexose transport stimulation and membrane redistribution of glucose transporter isoforms in response to cholera toxin, dibutyryl cyclic AMP, and insulin in 3T3-L1 adipocytes. J Biol Chem 265:1243412443[Abstract/Free Full Text]
-
Luo ZJ, Zhang XF, Rapp, UR, Avruch J 1995 Identification of the 14.3.3
domains important for self-association and Raf binding. J Biol Chem 270:2368123687[Abstract/Free Full Text]