Phosphorylation of PTP1B at Ser50 by Akt Impairs Its Ability to Dephosphorylate the Insulin Receptor

Lingamanaidu V. Ravichandran1, Hui Chen1, Yunhua Li and Michael J. Quon

Cardiology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Michael J. Quon, M.D., Ph.D., Cardiology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 10, Room 8C-218, 10 Center Drive MSC 1755, Bethesda, Maryland 20892-1755. E-mail: quonm{at}nih.gov


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PTP1B is a protein tyrosine phosphatase that negatively regulates insulin sensitivity by dephosphorylating the insulin receptor. Akt is a ser/thr kinase effector of insulin signaling that phosphorylates substrates at the consensus motif RXRXXS/T. Interestingly, PTP1B contains this motif (RYRDVS50), and wild-type PTP1B (but not mutants with substitutions for Ser50) was significantly phosphorylated by Akt in vitro. To determine whether PTP1B is a substrate for Akt in intact cells, NIH-3T3IR cells transfected with either wild-type PTP1B or PTP1B-S50A were labeled with [32P]-orthophosphate. Insulin stimulation caused a significant increase in phosphorylation of wild-type PTP1B that could be blocked by pretreatment of cells with wortmannin or cotransfection of a dominant inhibitory Akt mutant. Similar results were observed with endogenous PTP1B in untransfected HepG2 cells. Cotransfection of constitutively active Akt caused robust phosphorylation of wild-type PTP1B both in the absence and presence of insulin. By contrast, PTP1B-S50A did not undergo phosphorylation in response to insulin. We tested the functional significance of phosphorylation at Ser50 by evaluating insulin receptor autophosphorylation in transfected Cos-7 cells. Insulin treatment caused robust receptor autophosphorylation that could be substantially reduced by coexpression of wild-type PTP1B. Similar results were obtained with coexpression of PTP1B-S50A. However, under the same conditions, PTP1B-S50D had an impaired ability to dephosphorylate the insulin receptor. Moreover, cotransfection of constitutively active Akt significantly inhibited the ability of wild-type PTP1B, but not PTP1B-S50A, to dephosphorylate the insulin receptor. We conclude that PTP1B is a novel substrate for Akt and that phosphorylation of PTP1B by Akt at Ser50 may negatively modulate its phosphatase activity creating a positive feedback mechanism for insulin signaling.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PTP1B IS A protein tyrosine phosphatase (PTPase) that plays an important physiological role to negatively modulate insulin sensitivity in vivo, at least in part, by directly dephosphorylating both the insulin receptor and insulin receptor substrate 1 (IRS-1) (1, 2, 3, 4). PTP1B knockout mice have increased sensitivity to the metabolic actions of insulin and are resistant to becoming obese (1, 2). In addition, overexpression of PTP1B in rat adipose cells impairs metabolic actions of insulin (5, 6), and alterations in expression of PTP1B in insulin target tissues have been implicated in the pathophysiology of insulin resistance in obesity and diabetes (7, 8). Among the multitude of PTPases identified to date, only a few, including PTP1B, LAR, PTP-{alpha}, and PTP-{epsilon}, are known to dephosphorylate insulin receptors in intact cells (3, 9, 10, 11, 12). This implies a high level of specificity for particular PTPases to dephosphorylate and inactivate the insulin receptor tyrosine kinase (12). Thus, PTP1B is an attractive therapeutic target for the treatment of diabetes and obesity. Since PTP1B is the prototypical PTPase that was the first to be identified (13) and among the first to be cloned (14, 15), it has been the subject of numerous studies (for reviews see Refs. 16 and 17). The crystal structure for PTP1B has been solved (18), and considerable effort has been devoted to designing specific PTP1B inhibitors (6, 19, 20, 21, 22, 23). Nevertheless, mechanisms for regulating PTP1B activity remain poorly understood. The elucidation of potential regulatory mechanisms may lead to novel strategies for manipulating PTP1B function that will be useful for treating diabetes and obesity.

Akt is a ser/thr kinase downstream from PI3K in insulin signaling pathways (24, 25, 26) that has been implicated as an effector of metabolic actions of insulin (27, 28). The discovery of a number of substrates for Akt has been facilitated by the identification of a robust Akt consensus phosphorylation motif RXRXXS/T (for reviews see Refs. 29 and 30). Interestingly, PTP1B contains this motif (RYRDVS50), and amino acids immediately preceding the putative Akt phosphorylation site at Ser50 (Tyr46, Arg47, Asp48 and Val49) play important roles in stabilizing substrates in the catalytic cleft of PTP1B (18, 22, 31, 32, 33, 34). Thus, it is plausible that phosphorylation at Ser50 may alter the ability of PTP1B to engage and dephosphorylate its substrates. In the present study, we evaluated PTP1B as a novel substrate for Akt and investigated the potential role of phosphorylation at Ser50 in regulating PTP1B function related to insulin signaling.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To identify potential regulatory phosphorylation sites on PTP1B, we scanned the PTP1B amino acid sequence for the Akt consensus phosphorylation motif RXRXXS/T and discovered a matching sequence in PTP1B (RYRDVS) with a predicted phosphorylation site at Ser50. Even though this region of PTP1B is highly conserved among PTPases and homologous sequences are contained in many intracellular and receptor-like PTPases, the exact Akt phosphorylation motif is present in this region in only 2 of 30 other PTPases examined (Fig. 1AGo). Moreover, the Akt phosphorylation motif in PTP1B is completely conserved among many different species, raising the possibility that PTP1B may be a bona fide substrate for Akt (Fig. 1BGo). To help evaluate PTP1B as a substrate for Akt, we constructed point mutants with substitutions of Ala or Asp for Ser50, a catalytically inactive substrate trapping mutant with substitution of Ser for Cys215, and constructs that contained mutations at both positions 50 and 215 (Fig. 1CGo).



View larger version (45K):
[in this window]
[in a new window]
 
Figure 1. Akt Consensus Phosphorylation Motif in PTP1B

A, Amino acid sequence alignment of other PTPases with the region of human PTP1B containing the Akt consensus phosphorylation motif (RXRXXS) (adapted from Ref. 32 ). The putative Akt phosphorylation site at Ser50 in PTP1B is present in homologous sequences of only 2 of the other 30 PTPases examined. B, The Akt consensus phosphorylation motif in PTP1B is conserved among different species. C, Illustration of PTP1B point mutants that were constructed to substitute Ala or Asp for Ser50 at the putative Akt phosphorylation site or Ser for Cys215 in the catalytic domain of PTP1B.

 
Akt Phosphorylates PTP1B in Vitro at Ser50
To determine whether PTP1B is capable of functioning as a direct substrate for Akt, we first performed in vitro Akt kinase assays using activated recombinant Akt and purified PTP1B (truncated protein containing amino acids 1–322) in the presence of [32P]-ATP. In these assays, autophosphorylation of Akt was evident in both the presence and absence of PTP1B (Fig. 2AGo, lanes 1 and 3). However, significant phosphorylation of PTP1B was only observed in the presence, but not in the absence, of Akt (Fig. 2AGo, lanes 2–3). We next evaluated Ser50 as a potential Akt phosphorylation site by using wild-type and mutant PTP1B proteins immunoprecipitated from lysates of transfected NIH-3T3IR cells as the substrate in our in vitro kinase assays. Note that little, if any, endogenous PTP1B was detected in PTP1B immunoprecipitates of untransfected cells while comparable amounts of recombinant wild-type and mutant PTP1B were recovered (Fig. 2BGo). Consistent with the previous results, full-length wild-type PTP1B underwent significant phosphorylation in the presence, but not in the absence, of Akt (Fig. 2AGo, lanes 5–6). Similarly, PTP1B-C215S (catalytically inactive mutant with Ser50 intact) was also substantially phosphorylated in the presence of Akt (Fig. 2AGo, lane 9). Importantly, phosphorylation of PTP1B-S50A or PTP1B-S50D (mutants missing the putative Akt phosphorylation site) was negligible when compared with either PTP1B-WT or PTP1B-C215S (Fig. 2AGo, lanes 6–9) and was similar to PTP1B-WT incubated in the absence of Akt (Fig. 2AGo, lane 5). These results suggest that Akt is capable of directly phosphorylating PTP1B at Ser50 and raise the possibility that PTP1B may be a novel substrate for Akt.



View larger version (28K):
[in this window]
[in a new window]
 
Figure 2. Akt Phosphorylates PTP1B in Vitro at Ser50

In vitro kinase assays were carried out in the presence of [{gamma}-32P]-ATP using purified active Akt (lanes 1, 3, 4, and 6–9) and either truncated purified PTP1B (a.a. 1–322) (lanes 2–3) or full-length PTP1B immunoprecipitated from lysates of NIH-3T3IR cells transiently transfected with empty vector, PTP1B-WT, PTP1B-S50A, PTP1B-S50D, or PTP1B-C215S mutants (lanes 4–9). A, Purified PTP1B is phosphorylated only in the presence of Akt (compare lanes 2 and 3). Phosphorylation of immunoprecipitated PTP1B-WT and PTP1B-C215S by Akt (lanes 6 and 9) is significantly greater than that observed for PTP1B-S50A or PTP1B-S50D (lanes 7 and 8). Autophosphorylation of Akt is evident and consistent with the presence of active Akt (lanes 1, 3, 4, and 6–9). B, Anti-PTP1B immunoblot demonstrating comparable recovery of PTP1B in anti-PTP1B immunoprecipitates used for the kinase assays (lanes 5–9). Representative results are shown for experiments that were repeated independently at least four times.

 
Phosphorylation of PTP1B at Ser50 in Intact Cells
To determine whether PTP1B can be phosphorylated at Ser50 in intact cells, we performed in vivo labeling experiments in NIH-3T3IR cells transiently cotransfected with Akt and either PTP1B-WT or PTP1B-S50A. After transfection and labeling with [32P]-orthophosphate, cells were treated without or with insulin, and the amount of label incorporated into PTP1B was assessed in PTP1B immunoprecipitates using a PhosphorImager. Insulin treatment caused a significant 70% increase in phosphorylation of PTP1B-WT in intact cells (Fig. 3Go, lanes 1–2). By contrast, no significant increase in PTP1B phosphorylation was observed with insulin treatment of cells expressing PTP1B-S50A (Fig. 3, lanes 3–4). Note that the PTP1B recovered by immunoprecipitation in these experiments was predominantly recombinant PTP1B since there is very little endogenous PTP1B present in NIH-3T3IR cells (cf. Fig. 2BGo). Since Akt is downstream from PI3K in insulin signaling pathways, we evaluated the effects of pretreatment of cells with the PI3K inhibitor wortmannin. Interestingly, we found that the insulin-stimulated increase in phosphorylation of PTP1B-WT was completely inhibited by wortmannin (data not shown). To examine the insulin-stimulated phosphorylation of PTP1B in a more physiological context, we repeated the in vivo labeling studies in untransfected HepG2 cells. Importantly, insulin also stimulated a significant increase in phosphorylation of endogenous PTP1B in this liver cell line (Fig. 4Go, lanes 1–2) that was inhibited by wortmannin pretreatment (Fig. 4Go, lanes 3–4). Although a previous report suggested that PTP1B undergoes tyrosine phosphorylation in response to insulin stimulation in transfected rat 1 fibroblasts (35), we were unable to detect any increased tyrosine phosphorylation of PTP1B under our experimental conditions by immunoblotting with an antiphosphotyrosine antibody (data not shown). We next evaluated the effects of constitutively active and dominant inhibitory mutants of Akt on insulin-stimulated phosphorylation of PTP1B. In control cells transfected with only PTP1B-WT (no Akt cotransfection), insulin stimulation resulted in an increase in phosphorylated PTP1B (Fig. 5, lanes 1–2). This phosphorylation was inhibited by coexpressing the dominant inhibitory mutant Akt-AAA (Fig. 5Go, lanes 3–4). Coexpression of the constitutively active Akt-myr resulted in robust phosphorylation of PTP1B in both the absence and presence of insulin (Fig. 5Go, lanes 5–6). Taken together with our in vitro data, these results in intact cells suggest that phosphorylation of PTP1B at Ser50 by Akt can occur in vivo and that this phosphorylation may be regulated by insulin.



View larger version (31K):
[in this window]
[in a new window]
 
Figure 3. Insulin Stimulates Phosphorylation of PTP1B at Ser50 in Intact Cells

NIH-3T3IR cells transiently cotransfected with Akt and either PTP1B-WT or PTP1B-S50A were labeled with [32P]-orthophosphate and then stimulated without or with insulin (100 nM, 10 min). PTP1B immunoprecipitated from cell lysates was subjected to 10% SDS-PAGE and Phosphor-Imager analysis. A, PhosphorImager scan of a representative in vivo labeling experiment showing that insulin stimulates phosphorylation of PTP1B-WT (lanes 1 and 2) but not PTP1B-S50A (lanes 3 and 4). B, Anti-PTP1B immunoblot demonstrating comparable recovery of PTP1B in all anti-PTP1B immunoprecipitates. C, Quantification of results by PhosphorImager were normalized for recovery of PTP1B in the immunoprecipitates. Results shown are the mean ± SEM of three independent experiments.

 


View larger version (37K):
[in this window]
[in a new window]
 
Figure 4. Insulin-Stimulated Phosphorylation of Endogenous PTP1B in Untransfected HepG2 Cells Is Blocked by Wortmannin Pretreatment

HepG2 cells were labeled with [32P]-orthophosphate and pretreated without or with wortmannin. Cells were then stimulated without or with insulin (100 nM, 10 min) and PTP1B immunoprecipitates of cell lysates were subjected to 10% SDS-PAGE and PhosphorImager analysis. A, Phosphor-Imager scan of representative in vivo labeling experiment showing that insulin stimulates phosphorylation of PTP1B only in the absence of wortmannin pretreatment (lanes 1 and 2) but not after wortmannin pretreatment (lanes 3 and 4). B, Anti-PTP1B immunoblot demonstrating comparable recovery of PTP1B in all anti-PTP1B immunoprecipitates. C, Quantification of results by PhosphorImager was normalized for recovery of PTP1B in the immunoprecipitates. Results shown are the mean ± SEM of three independent experiments.

 


View larger version (29K):
[in this window]
[in a new window]
 
Figure 5. Effect of Akt Mutants on Phosphorylation of PTP1B

NIH-3T3IR cells transiently transfected with PTP1B-WT and an empty vector (control), Akt-AAA, or Akt-myr were labeled with [32P]-orthophosphate and stimulated without or with insulin (100 nM, 10 min). PTP1B immunoprecipitated from cell lysates was subjected to 10% SDS-PAGE and PhosphorImager analysis. A, Image from PhosphorImager analysis of a representative in vivo labeling experiment that was repeated independently five times. B, Anti-PTP1B immunoblot demonstrating comparable recovery of PTP1B in the immunoprecipitates for each group.

 
Role of Ser50 in Regulating Function of PTP1B
To examine functional consequences of phosphorylation of PTP1B by Akt in response to insulin stimulation, we used an in vitro phosphatase assay to directly assess PTP1B activity. NIH-3T3IR cells cotransfected with PTP1B-WT and either a control vector or Akt-myr were stimulated without or with insulin, and anti-PTP1B immunoprecipitates were tested for their ability to dephosphorylate a tyrosine-phosphorylated peptide substrate derived from the insulin receptor. Interestingly, the activity of PTP1B to dephosphorylate the peptide substrate was significantly decreased by approximately 25% after either insulin stimulation or cotransfection of Akt-myr (Fig. 6Go). Thus, phosphorylation of PTP1B by Akt in response to insulin stimulation may impair the catalytic activity of PTP1B.



View larger version (32K):
[in this window]
[in a new window]
 
Figure 6. Catalytic Activity of PTP1B Is Impaired After Insulin Stimulation or Cotransfection with Constitutively Active Akt

NIH-3T3IR cells transiently cotransfected with PTP1B-WT and either a control vector or constitutively active Akt (Akt-myr) were serum starved overnight and then treated without or with insulin (100 nM, 5 min). Phosphatase activity in PTP1B immunoprecipitates was assessed using a tyrosine-phosphorylated insulin receptor peptide as described in Materials and Methods. A, Phosphatase activity was normalized for PTP1B recovery and plotted as a percent of the sample from untreated control cells (mean ± SEM of three independent experiments). Both insulin treatment and coexpression of Akt-myr resulted in a significant decrease in PTP1B catalytic activity (P < 0.03). B, Anti-PTP1B immunoblot demonstrating comparable recovery of PTP1B in the immunoprecipitates for each group.

 
We next investigated the ability of wild-type and mutant PTP1B to dephosphorylate the insulin receptor in intact cells. Cos-7 cells have low levels of endogenous insulin receptor and PTP1B. By transiently cotransfecting these cells with human insulin receptor and various PTP1B constructs, we were able to evaluate effects of recombinant PTP1B on receptor phosphorylation in the transfected cells without interference from untransfected cells. Cell lysates of cotransfected cells were immunoblotted with antiphosphotyrosine antibody to evaluate autophosphorylation of the insulin receptor (Fig. 7Go). In control cells overexpressing only human insulin receptor, insulin treatment caused robust receptor autophosphorylation (Fig. 7Go, lanes 1–2). As expected, coexpression of wild-type PTP1B significantly decreased insulin-stimulated receptor phosphorylation while overexpression of the catalytically inactive PTP1B-C215S mutant had no detectable effect (Fig. 7Go, lanes 3–4 and 9–10). Overexpression of PTP1B-S50A, a mutant with substitution of Ala in the putative Akt phosphorylation site at Ser50, resulted in decreased insulin-stimulated receptor phosphorylation similar to that observed with overexpression of PTP1B-WT (Fig. 7Go, lanes 7–8). By contrast, overexpression of PTP1B-S50D, a mutant designed to mimic phosphorylation at Ser50, was associated with a level of receptor phosphorylation intermediate between that observed in control cells and cells expressing PTP1B-WT or PTP1B-S50A (Fig. 7Go, lanes 5–6).



View larger version (27K):
[in this window]
[in a new window]
 
Figure 7. Mutations at Ser50 Affect the Ability of PTP1B to Dephosphorylate the Insulin Receptor

Cos-7 cells transiently cotransfected with human insulin receptor (hIR) and either empty vector (control) or various PTP1B constructs were treated without (-) or with (+) insulin (100 nM, 3 min). Whole-cell lysates (30 µg total protein) were subjected to 10% SDS-PAGE and immunoblotted with antibodies against phosphotyrosine, insulin receptor, or PTP1B. Representative blots are shown for experiments that were repeated independently four times.

 
In addition to the insulin receptor, IRS-1 is also a substrate for PTP1B (4). Therefore, we evaluated the ability of our PTP1B mutants to affect insulin-stimulated tyrosine phosphorylation of IRS-1. In experiments similar to those shown in Fig. 7Go, hemagglutinin (HA)-tagged IRS-1 was immunoprecipitated from lysates of Cos-7 cells transiently cotransfected with PTP1B constructs, insulin receptor, and IRS1-HA; these samples were then immunoblotted with antiphosphotyrosine antibody (Fig. 8Go). As with the insulin receptor, insulin treatment of control cells resulted in significant tyrosine phosphorylation of IRS1-HA (Fig. 8, lanes 1–2). This phosphorylation was significantly decreased in the presence of PTP1B-WT or PTP1B-S50A (Fig. 8Go, lanes 3–4 and 7–8). IRS1-HA in insulin-stimulated cells overexpressing PTP1B-S50D was largely, but not completely, dephosphorylated (~15% of the level observed in the insulin-stimulated control) (Fig. 8Go, lanes 5–6). Thus, PTP1B-S50D may have an impaired ability to dephosphorylate both the insulin receptor and IRS-1.



View larger version (24K):
[in this window]
[in a new window]
 
Figure 8. Mutations at Ser50 Affect the Ability of PTP1B to Dephosphorylate IRS-1

Cos-7 cells transiently cotransfected with human insulin receptor (hIR), HA-tagged IRS-1 (IRS1-HA), and either empty vector (control) or various PTP1B constructs were treated without (-) or with (+) insulin (100 nM, 3 min). Anti-HA immunoprecipitates of cell lysates (400 µg total protein) were subjected to 8% SDS-PAGE and immunoblotted with antibodies against phosphotyrosine or HA. Representative blots are shown for experiments that were repeated independently three times.

 
We, and others, have previously shown that the catalytically inactive substrate trapping mutant PTP1B-C215S can coimmunoprecipitate with the phosphorylated insulin receptor (6, 35). To determine whether interactions between PTP1B and the insulin receptor can be altered by manipulations at Ser50, we examined the ability of S50A/C215S and S50D/C215S mutants to coimmunoprecipitate with the phosphorylated insulin receptor in NIH-3T3IR cells. As expected, significant coimmunoprecipitation of the insulin receptor and PTP1B-C215S was observed in samples derived from insulin-stimulated cells (Fig. 9Go, lanes 1–2). Similar results were obtained with the S50A/C215S mutant. By contrast, the insulin receptor did not coimmunoprecipitate to the same extent with the S50D/C215S mutant (Fig. 9Go, lanes 5–6). These results suggest that phosphorylation at Ser50 may interfere with the ability of PTP1B to interact with its substrates. Moreover, these results are also consistent with the impaired ability of PTP1B-S50D to dephosphorylate the insulin receptor and IRS-1.



View larger version (21K):
[in this window]
[in a new window]
 
Figure 9. Mutations at Ser50 Affect the Ability of PTP1B to Bind Phosphorylated Insulin Receptor

NIH-3T3IR cells transiently transfected with PTP1B-C215S, S50A/C215S, S50D/C215S, or empty vector (control) were treated without (-) or with (+) insulin (100 nM, 3 min). Anti-PTP1B immunoprecipitates of cell lysates (500 µg total protein) were subjected to 10% SDS-PAGE and immunoblotted with antibodies against the insulin receptor or PTP1B (lanes 1–7). Immunoblots of cell lysates are shown in lanes 8–14. Representative blots are shown for experiments that were repeated independently four times.

 
To provide further evidence that phosphorylation of PTP1B at Ser50 by Akt is important for regulating PTP1B activity, we coexpressed various Akt constructs along with both the insulin receptor and either PTP1B-WT or PTP1B-S50A in Cos-7 cells. As previously demonstrated in Fig. 7Go, insulin-stimulated phosphorylation of the insulin receptor was significantly decreased by overexpression of either PTP1B-WT or PTP1B-S50A alone (Fig. 10Go, lanes 1–4 and 9–12). Coexpression of a dominant inhibitory Akt mutant (Akt-AAA) had no detectable effect on the ability of either PTP1B-WT or PTP1B-S50A to dephosphorylate the insulin receptor (Fig. 10Go, lanes 7–8 and 15–16). Importantly, coexpression of the constitutively active Akt mutant (Akt-myr) significantly impaired the ability of PTP1B-WT, but not PTP1B-S50A, to dephosphorylate the insulin receptor (compare Fig. 10Go. lanes 5–6 with lanes 13–14). Taken together with data from experiments using PTP1B-S50D and S50D/C215S shown in Figs. 7–9GoGoGo, these results are consistent with the possibility that phosphorylation of PTP1B at Ser50 by Akt may negatively regulate catalytic activity of PTP1B and impair its ability to dephosphorylate the insulin receptor. To confirm the functional significance of these results in a more physiological context, we examined insulin-stimulated tyrosine phosphorylation of the insulin receptor in the absence and presence of wortmannin in untransfected HepG2 cells. Consistent with an Akt-dependent positive feedback mechanism involving regulation of PTP1B activity, wortmannin pretreatment completely inhibited insulin-stimulated phosphorylation of Akt (Fig. 11CGo) and resulted in a small, but statistically significant, 25% decrease in insulin receptor autophosphorylation (Fig. 11Go, A and B).



View larger version (25K):
[in this window]
[in a new window]
 
Figure 10. Action of Activated Akt to Inhibit Dephosphorylation of the Insulin Receptor by PTP1B Requires Ser50

Cos-7 cells transiently cotransfected with human insulin receptor, a PTP1B construct, and either empty vector (control) or the indicated Akt constructs were treated without (-) or with (+) insulin (100 nM, 3 min). Whole-cell lysates (30 µg total protein) were subjected to 10% SDS-PAGE and immunoblotted with antibodies against phosphotyrosine, insulin receptor, PTP1B, or Akt. Note that coexpression of the constitutively active Akt-myr significantly inhibits the ability of PTP1B-WT to dephosphorylate the insulin receptor (lanes 5 and 6) but has no effect on the ability of PTP1B-S50A to dephosphorylate the insulin receptor (lanes 13 and 14). Representative blots are shown from experiments that were repeated independently four times.

 


View larger version (28K):
[in this window]
[in a new window]
 
Figure 11. Insulin-Stimulated Autophosphorylation of the Insulin Receptor Is Impaired by Wortmannin Pretreatment in Untransfected HepG2 Cells

Cells were pretreated without or with wortmannin and then stimulated without or with insulin (100 nM, 10 min). A, Insulin receptor immunoprecipitates of cell lysates were subjected to 10% SDS-PAGE followed by immunoblotting with antiphosphotyrosine or antiinsulin receptor antibodies. A representative immunoblot is shown from experiments that were repeated independently four times. B, Quantification of antiphosphotyrosine immunoblots (mean ± SEM of four independent experiments normalized for insulin receptor recovery). C, Akt and phospho-Akt immunoblot of cell lysates demonstrating inhibition of Akt phosphorylation by wortmannin pretreatment.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The importance of PTP1B in negatively regulating metabolic actions of insulin has been unequivocally demonstrated by the presence of increased insulin sensitivity, enhanced insulin receptor phosphorylation, and resistance to obesity in PTP1B knockout mice (1, 2). Thus, it is of interest to identify novel mechanisms for regulating catalytic activity and function of PTP1B, such as phosphorylation of critical residues by known kinases. Akt is a ser/thr kinase downstream from PI3K that plays essential roles in cell growth, differentiation, proliferation, and survival (30). With respect to insulin signaling, Akt has been implicated in both metabolic actions of insulin to promote glucose transport (27, 28, 36) as well as vasodilator actions of insulin mediated by nitric oxide (37). Known substrates of Akt that participate in insulin signaling pathways and whose functions are regulated by Akt phosphorylation include glycogen synthase kinase-3 (GSK-3) (25), phosphofructokinase-2 (38), mammalian target of rapamycin (39, 40), forkhead transcription factor FHKR (41, 42), insulin receptor substrate-1 (IRS-1) (43, 44), cyclic nucleotide phosphodiesterase 3B (45, 46), and endothelial nitric oxide synthase (47, 48). The identification of many of these Akt substrates has been facilitated by the existence of a robust consensus phosphorylation motif (30). We found that PTP1B contains this motif [amino acids (a.a.) 45–50] in a region that forms important stabilizing contacts with amino acids immediately upstream from the phosphotyrosine residue of PTP1B substrates. Thus, phosphorylation of Ser50 in this region might be predicted to have functionally important consequences. Interestingly, only two other PTPases (TC-PTP and PTP-MEG1) contain this exact Akt phosphorylation motif despite high homology of this region in general among many PTPases. The absolute conservation of this region in PTP1B among different species is also consistent with its potential role as an Akt phosphorylation site.

PTP1B Is a Novel Substrate for Akt Both in Vitro and in Intact Cells
We demonstrated that PTP1B can function as a direct substrate for Akt under in vitro conditions using both a commercially available purified truncated PTP1B (a.a. 1–322) as well as full-length PTP1B immunoprecipitated from transfected cells as a substrate. The phosphorylation of PTP1B by Akt when Ser50 was present (PTP1B-WT or PTP1B-C215S) was significantly greater than when Ser50 was replaced by either Ala or Asp. The small amount of phosphorylation observed with PTP1B-S50A and PTP1B-S50D may be due to nonspecific incorporation of [{gamma}-32P]-ATP or the presence of other cryptic Akt phosphorylation sites. Alternatively, it is possible that known sites of phosphorylation on PTP1B such as Tyr66, Tyr152, Tyr153 (35), Ser352, Ser378, Ser386 (49), or other unknown sites may undergo weak phosphorylation by kinases that coimmunoprecipitate with PTP1B. Nevertheless, substantial phosphorylation of PTP1B by Akt occurs only when Ser50 is intact. This strongly suggests that Ser50 is a bona fide target for direct phosphorylation by Akt in vitro.

Results from our in vivo labeling experiments were also consistent with the presence of an Akt phosphorylation site in PTP1B at Ser50 and suggest that PTP1B can function as a substrate for Akt in intact cells. Insulin treatment of NIH-3T3IR cells cotransfected with PTP1B and Akt caused a significant increase in phosphorylation of PTP1B-WT, but not PTP1B-S50A. Furthermore, this in vivo phosphorylation of PTP1B-WT was blocked by pretreatment of cells with the PI3K inhibitor wortmannin and did not require coexpression of Akt. Importantly, we also observed similar results in untransfected HepG2 cells, suggesting that endogenous PTP1B can be phosphorylated by Akt in response to insulin stimulation in a more physiologically relevant cell type. Moreover, a dominant inhibitory Akt mutant blocked the phosphorylation of PTP1B in response to insulin. We, and others, have previously documented that the mutant Akt used in this assay inhibits wild-type Akt activity (46, 50). Finally, coexpression of a constitutively active Akt mutant resulted in robust phosphorylation of PTP1B. Interestingly, the phosphorylation of PTP1B appeared to be much greater in response to cotransfection of Akt-myr than in response to insulin stimulation. This suggests that not all PTP1B molecules are phosphorylated in response to insulin stimulation. One possible explanation for these observations is that the amount of label incorporated into PTP1B during the 10-min insulin stimulation is likely to be much less than during the lengthy period of time transfected cells are exposed to elevated levels of Akt-myr. It is also possible that Akt-myr is a potent kinase that can phosphorylate PTP1B at additional sites. When we directly assessed PTP1B activity using an in vitro phosphatase assay, we observed a significant inhibition of PTP1B activity in both PTP1B immunoprecipitates derived from insulin-treated cells or cells cotransfected with Akt-myr. Thus, it is plausible that insulin stimulation may result in impaired PTP1B activity under physiological conditions. It seems unlikely that the phosphorylation of PTP1B we observed in response to insulin stimulation represents tyrosine phosphorylation because we did not observe any significant increase in tyrosine-phosphorylated PTP1B by immunoblotting under our experimental conditions. Taken together, our results suggest that Ser50 is an insulin- and PI3K-dependent Akt phosphorylation site on PTP1B in vivo, and it is possible that phosphorylation of PTP1B on Ser50 by Akt may contribute to feedback regulation of insulin signaling.

Role of Phosphorylation at Ser50 in Regulating Function of PTP1B
We hypothesized that phosphorylation at Ser50 by Akt impairs the ability of PTP1B to dephosphorylate its substrates. We evaluated this possibility by comparing the ability of wild-type and mutant forms of PTP1B to dephosphorylate physiological substrates such as the insulin receptor and IRS-1 in intact cells. Insulin-stimulated receptor autophosphorylation was greatly decreased (compared with results from control cells) in cells overexpressing wild-type PTP1B. Since this was not observed with the catalytically inactive PTP1B-C215S, we conclude that wild-type PTP1B is effective at dephosphorylating the insulin receptor as previously described (3). Overexpression of PTP1B-S50A (mutant unable to undergo phosphorylation at Ser50 resulted in dephosphorylation of the insulin receptor to a similar extent as wild-type PTP1B. By contrast, PTP1B-S50D (mutant designed to mimic phosphorylation at Ser50 had an impaired ability to dephosphorylate the insulin receptor. We observed similar results with respect to the effects of our PTP1B constructs on insulin-stimulated phosphorylation of IRS-1. Since IRS-1 is a substrate for PTP1B (4), decreased levels of insulin-stimulated IRS-1 phosphorylation in the presence of PTP1B-WT or PTP1B-S50A may represent both direct effects of PTP1B on IRS-1 as well as secondary effects of PTP1B mediated by dephosphorylation of the insulin receptor. Although we cannot entirely rule out the possibility that our results with IRS-1 are completely secondary to dephosphorylation of the insulin receptor, this seems less likely because the relative impairment of PTP1B-S50D with respect to IRS-1 dephosphorylation is less than that observed for insulin receptor dephosphorylation (compare Fig. 7Go, lanes 2 and 6 with Fig. 8Go, lanes 2 and 6). Thus, data regarding the ability of our mutant PTP1B constructs to dephosphorylate the insulin receptor and IRS-1 in intact cells are consistent with the idea that phosphorylation of PTP1B at Ser50 negatively regulates its function. In addition, data from coimmunoprecipitation experiments suggest that this impairment in PTP1B function may be the result of decreased binding of PTP1B to its substrates when Ser50 is phosphorylated (mimicked by the S50D mutant).

Our experiments examining insulin receptor autophosphorylation in cells coexpressing constitutively active Akt (Akt-myr) with PTP1B further support the idea that phosphorylation of Ser50 in PTP1B by Akt is functionally relevant. Results from cells coexpressing Akt-myr and PTP1B-WT were similar to results from cells overexpressing PTP1B-S50D, suggesting that phosphorylation of PTP1B by Akt impairs PTP1B function. In addition, these results argue against the possibility that the impaired function of PTP1B-S50D is due to misfolding of the mutant protein. More importantly, the impairment of PTP1B function caused by coexpression of Akt-myr was not observed with PTP1B-S50A, strongly suggesting that it is the ability of Akt to phosphorylate Ser50 that results in impaired PTP1B function. Finally, the impairment of insulin receptor autophosphorylation caused by wortmannin pretreatment of untransfected HepG2 cells is consistent with the idea that endogenous Akt may be phosphorylating endogenous PTP1B and impairing its ability to dephosphorylate the insulin receptor under more physiological conditions. This may represent a novel mechanism for negatively regulating PTP1B activity.

Implications for Insulin Signaling
Previously identified sites on PTP1B in the noncatalytic region at Ser352, Ser378, and Ser386 undergo phosphorylation in response to 12-O-tetradecanoyl phorbol-13-acetate stimulation or changes in cell cycle (49). Phosphorylation at these sites has been suggested to alter subcellular targeting of PTP1B and is associated with modest decreases in PTPase activity. Bandyopadhyay et al. have previously identified Tyr66, Tyr152, and Tyr153 as sites on PTP1B that can be phosphorylated by the insulin receptor and enhance interactions between PTP1B and the insulin receptor (35). It is possible that differences in cell type may account for our inability to detect tyrosine phosphorylation of PTP1B in our experiments. We have now identified a novel Akt phosphorylation site on PTP1B at Ser50. Our data suggest that phosphorylation of PTP1B at Ser50 by Akt negatively modulates the ability of PTP1B to dephosphorylate important physiological targets of PTP1B. There are multiple phosphorylated tyrosine residues on both the insulin receptor and IRS proteins. The exact sites on the insulin receptor that are targets for PTP1B are not known, but it seems likely that a majority of the sites are dephosphorylated by PTP1B since significant effects can be detected by antiphosphotyrosine immunoblotting. The observed decreases in IRS-1 tyrosine phosphorylation may represent both direct effects of PTP1B and secondary effects of reduced insulin receptor activity.

Since Akt is a downstream effector of insulin action and PTP1B acts at upstream sites to inhibit insulin action, the ability of Akt to impair PTP1B function may represent a positive feedback mechanism. This is not unprecedented since Akt participates in positive feedback mechanisms for insulin signaling at the level of IRS-1 (43). In addition to the putative pathophysiological role of PTP1B in insulin-resistant states such as diabetes and obesity, the recent identification of PTP1B as the major PTPase that dephosphorylates and activates c-src in human breast cancer cell lines suggests that excessive PTP1B activity may also play a role in breast cancer and other malignancies (51). Identification of novel mechanisms for negatively regulating PTP1B activity and function, such as phosphorylation by Akt, may lead to critical insights for the development of therapies for a variety of important public health problems.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Reagents
Reagents were obtained from the following sources: monoclonal anti-PTP1B antibody from Oncogene Science, Inc. (Boston, MA); recombinant PTP1B protein, phosphorylated insulin receptor peptide substrate (IR 5, 9, 10), and Biomol Green reagent from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA); rabbit polyclonal anti-PTP1B antibody, purified recombinant activated Akt, and GSK peptide from Upstate Biotechnology, Inc. (Lake Placid, NY); antiinsulin receptor and antiphosphotyrosine antibodies (PY20) from Transduction Laboratories, Inc. (Lexington, KY); anti-HA antibody (HA-11) from BabCO (Richmond, CA); wortmannin from Sigma (St. Louis, MO); protein G agarose and LipofectAMINE PLUS from Life Technologies, Inc. (Gaithersburg, MD); rabbit polyclonal anti-phospho-Akt (Ser473) from New England Biolabs, Inc. (Beverly, MA); and [{gamma}-32P]-ATP and [32P]-orthophosphate from ICN Biomedicals, Inc. (Irvine, CA).

Expression Plasmids
pCIS2.
pC152 is a parental expression vector with a CMV promoter/enhancer (52, 53).

PTP1B-WT.
cDNA for human PTP1B was ligated into the multiple cloning region of pCIS2 as described (5).

PTP1B-C215S.
cDNA for a catalytically inactive mutant PTP1B with substitution of Ser for Cys215 was ligated into the multiple cloning region of pCIS2 as described (5).

PTP1B-S50D.
A point mutant derived from PTP1B-WT with Asp substituted for Ser50 was constructed using the mutagenic oligonucleotide 5'-GG TAC AGA GAC GTG GAT CCC TTT GAC CAT AG-3' and the Morph Mutagenesis kit (5 Prime to 3 Prime, Boulder, CO). This mutagenesis also introduced a silent mutation at the position encoding Val49 creating a new BamHI site.

PTP1B-S50A.
A point mutant derived from PTP1B-S50D with Ala substituted at position 50 was constructed using the mutagenic oligonucleotide 5'-GG TAC AGA GAC GTG GCT CCC TTT GAC CAT AG-3' and the Morph kit.

S50D/C215S.
A mutant PTP1B containing both substitutions of Asp for Ser50 and Ser for Cys215 was derived from PTP1B-C215S using the mutagenic oligonucleotide pairs 5'-CGA AAT AGG TAC AGA GAC GTG GAT CCC TTT GAC CAT AGT CGG-3' and 5'-CCG ACT ATG GTC AAA GGG ATC CAC GTC TCT GTA CCT ATT TCG-3' with the QuikChange mutagenesis kit (Stratagene, La Jolla, CA). This also introduced a silent mutation at the position encoding Val49, creating a new BamHI site.

S50A/C215S.
A mutant PTP1B containing both substitutions of Ala for Ser50 and Ser for Cys215 was derived from PTP1B-C215S using the mutagenic oligonucleotide pairs 5'-CGA AAT AGG TAC AGA GAC GTG GCT CCC TTT GAC CAT AGT CGG-3' and 5'-CCG ACT ATG GTC AAA GGG AGC CAC GTC TCT GTA CCT ATT TCG-3' with the QuikChange kit. The presence of the desired mutations in all PTP1B constructs was verified by direct sequencing.

Akt-WT.
cDNA for mouse Akt-1 was ligated into multiple cloning region of pCIS2 as described previously (28).

Akt-AAA.
A dominant inhibitory mutant of Akt with substitutions of Ala for Lys179 in the ATP binding domain, as well as for the regulatory phosphorylation sites, Thr308 and Ser473, was created and subcloned into pCIS2 as described (46).

Akt-myr.
cDNA for mouse Akt-1 with a myristoylation sequence from pp60 c-src (54) fused in-frame with the N terminus of Akt (generous gift from Drs. P. N. Tsichlis and K. Datta) was ligated into the multiple cloning region of pCIS2 as described (28).

pCIS-hIR.
cDNA for the human insulin receptor was ligated into the multiple cloning site of pCIS2 as described (55).

IRS1-HA.
cDNA for human IRS-1 was subcloned into pCIS2 with a sequence coding for an HA-epitope tag fused to the C terminus as described (56).

Cell Culture and Transfection
NIH-3T3 fibroblasts overexpressing human insulin receptors (NIH-3T3IR) or Cos-7 cells were maintained in DMEM containing 10% FBS, L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 µg/ml), in a humidified atmosphere with 5% CO2 at 37 C. Cells were transiently transfected with various constructs using LipofectAMINE PLUS according to the manufacturer’s instructions. HepG2 cells were maintained in DMEM containing 100 mM glucose, 10% FBS, L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 µg/ml).

In Vitro Akt Kinase Assays
In vitro assays using purified activated Akt as the kinase and PTP1B as substrate were carried out at 30 C for 30 min in kinase assay buffer containing 50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM dithiothreitol, 50 µM ATP, and 2.5 µCi [{gamma}-32P]-ATP/assay. Reactions were stopped by adding Laemmli sample buffer and boiling for 10 min. Samples were subjected to 10% SDS-PAGE and a PhosphorImager (Molecular Dyamics, Inc., Sunnyvale, CA) was used to detect phosphorylated PTP1B and autophosphorylated Akt. In addition, gel contents were transferred to nitrocellulose and immunoblotted with anti-PTP1B antibody. Finally, activity of Akt in each assay was independently verified using a peptide substrate derived from GSK-3 (RPRAATF) as described (29, 57). For assays using purified PTP1B (truncated protein containing amino acids 1–322), 2.5 µg of PTP1B, and approximately 0.3 µg of Akt (specific activity,154 nmol phosphate transferred to GSK-3 peptide/min/mg protein) were used. In some experiments, full-length recombinant wild-type and mutant PTP1B proteins immunoprecipitated from lysates of transfected NIH-3T3IR cells were used as substrate. One day after transfection, cell lysates were prepared using lysis buffer A (50 mM Tris-HCl, pH 7.4, 125 mM NaCl, 1% Triton X-100, 0.5% NP-40, 1 mM Na3VO4, 50 mM NaF, 0.1 mM okadaic acid, and complete protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN). Lysates (500 µg total protein) were precleared with protein G agarose beads for 1 h at 4 C and then incubated with anti-PTP1B antibody (2.5 µg) and protein G agarose beads for 2 h at 4 C. The immunocomplexes were washed four times with kinase assay buffer and used in the in vitro kinase assays as described above.

In Vivo Phosphorylation Experiments
Untransfected HepG2 cells or NIH-3T3IR cells transiently transfected with various PTP1B and Akt constructs were serum starved overnight and then labeled for 4 h with [32P]-orthophosphate (75 µCi/ml in KRB buffer, pH 7.4, containing 1% BSA) as described previously (56). After labeling, cells were treated without or with insulin (100 nM, 10 min) and washed four times with PBS, after which whole-cell lysates were prepared as described above. Lysates were immunoprecipitated with anti-PTP1B antibody as described above, and samples were separated by 10% SDS-PAGE. Phosphorylated PTP1B was detected and quantified using a PhosphorImager. In addition, gel contents were transferred to nitrocellulose for immunoblotting with anti-PTP1B antibody. In some experiments, transfected cells were pretreated with wortmannin (100 nM) 2.5 h after initiation of labeling with [32P]-orthophosphate (90 min before insulin treatment).

In Vitro PTP1B Phosphatase Assay
NIH-3T3IR cells transiently cotransfected with PTP1B-WT and either a control vector or Akt-myr were serum starved overnight and then treated without or with insulin (100 nM, 5 min) and washed with PBS, and whole-cell lysates were prepared as described above using Buffer A without Na3VO4 and NaF. Lysates were immunoprecipitated with anti-PTP1B antibody and Protein G as described above, and the immunoprecipitates were washed four times with phosphatase assay buffer (50 mM HEPES, pH 7.2, 1 mM EDTA, 1 mM dithiothreitol, 0.05% NP-40). The Protein G beads were then suspended in 55 µl of assay buffer and 5 µl of 1.5 mM phosphorylated insulin receptor peptide (residues 1142–1153 of the insulin receptor phosphorylated at Tyr 1146, 1150, and 1151) were added as the substrate. After incubation at 30 C for 30 min, 25 µl of the clear supernatant were transferred to half-size 96-well plates, and 100 µl of Biomol Green reagent were added to each well. Samples were gently agitated for 20–30 min, and the free phosphate liberated was determined by absorbance at 620 nM using a microtiter-plate spectrophotometer. A standard curve was generated for each experiment to quantify results.

Dephosphorylation of Insulin Receptor and IRS-1 by PTP1B
The ability of wild-type and mutant PTP1B to dephosphorylate the insulin receptor was evaluated by immunoblotting whole-cell lysates derived from Cos-7 cells transiently cotransfected with human insulin receptor and either pCIS2 (empty vector) or PTP1B constructs. The day after transfection, cells were serum-starved overnight and then treated without or with insulin (100 nM) for 3 min. Whole-cell lysates were made as previously described (58) using lysis buffer B (50 mM Tris, pH 7.4, 300 mM NaCl, 1% Triton X-100, 1 mM Na3VO4, and complete protease inhibitor cocktail) and centrifuged at 6,000 x g for 3 min at 4 C to pellet the remaining cellular debris. An aliquot from each group was resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies against phosphotyrosine, insulin receptor, or PTP1B. In some experiments cells were also cotransfected with various Akt constructs. In addition, similar experiments were performed on anti-HA immunoprecipitates of cell lysates derived from cells also cotransfected with IRS1-HA. For immunoprecipitation, an aliquot of each lysate was incubated with 2 µg of HA-11 antibody overnight at 4 C followed by incubation with washed protein G-conjugated agarose beads for 2 h at 4 C on a rotating wheel. Immunocomplexes were washed twice with lysis buffer B (without protease inhibitors), once with buffer C (20 mM Tris, pH 7.4, 150 mM NaCl), and then subjected to SDS-PAGE and immunoblotting. Coimmunoprecipitation of insulin receptor with PTP1B was assessed in cell lysates derived from NIH-3T3IR cells transiently transfected with PTP1B mutants C215S, S50A/C215S, or S50D/C215S that were treated without or with insulin (100 nM) for 3 min. Samples were immunoprecipitated with anti-PTP1B antibody followed by immunoblotting with antiinsulin receptor antibody.

Assessment of Insulin Receptor Autophosphorylation and Akt Phosphorylation in HepG2 Cells
Confluent HepG2 cells were serum starved overnight, pretreated without or with wortmannin (100 nM) for 90 min, and then stimulated without or with insulin (100 nM, 10 min). Insulin receptor and PTP1B were immunoprecipitated from whole-cell lysates (500 µg total protein) using antiinsulin receptor and anti-PTP1B antibodies, respectively, and samples were immunoblotted with antiphosphotyrosine antibody (PY20), antiinsulin receptor antibody, or anti-PTP1B antibodies. Akt and phospho-Akt immunoblots were also performed using whole-cell lysates (100 µg total protein).


    ACKNOWLEDGMENTS
 
We thank Dr. Monica Montagnani and Fredly Bataille for technical assistance with some experiments.


    FOOTNOTES
 
1 These authors made equal contributions to this work. Back

Abbreviations: a.a., Amino acids; GSK-3, glycogen synthase kinase-3, HA, hemagglutinin; IRS-1, insulin receptor substrate 1; PTPase, protein tyrosine phosphatase; PTP1B-WT, wild-type PTP1B.

Received for publication March 13, 2001. Accepted for publication June 19, 2001.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Elchebly M, Payette P, Michaliszyn E, et al. 1999 Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene. Science 283:1544–1548[Abstract/Free Full Text]
  2. Klaman LD, Boss O, Peroni OD, et al. 2000 Increased energy expenditure, decreased adiposity, and tissue-specific insulin sensitivity in protein-tyrosine phosphatase 1B-deficient mice. Mol Cell Biol 20:5479–5489[Abstract/Free Full Text]
  3. Kenner KA, Anyanwu E, Olefsky JM, Kusari J 1996 Protein-tyrosine phosphatase 1B is a negative regulator of insulin- and insulin-like growth factor-I-stimulated signaling. J Biol Chem 271:19810–19816[Abstract/Free Full Text]
  4. Goldstein BJ, Bittner-Kowalczyk A, White MF, Harbeck M 2000 Tyrosine dephosphorylation and deactivation of insulin receptor substrate-1 by protein-tyrosine phosphatase 1B. Possible facilitation by the formation of a ternary complex with the Grb2 adaptor protein. J Biol Chem 275:4283–4289[Abstract/Free Full Text]
  5. Chen H, Wertheimer SJ, Lin CH, et al. 1997 Protein-tyrosine phosphatases PTP1B and syp are modulators of insulin-stimulated translocation of GLUT4 in transfected rat adipose cells. J Biol Chem 272:8026–8031[Abstract/Free Full Text]
  6. Chen H, Cong LN, Li Y, et al. 1999 A phosphotyrosyl mimetic peptide reverses impairment of insulin-stimulated translocation of GLUT4 caused by overexpression of PTP1B in rat adipose cells. Biochemistry 38:384–389[CrossRef][Medline]
  7. Byon JC, Kusari AB, Kusari J 1998 Protein-tyrosine phosphatase-1B acts as a negative regulator of insulin signal transduction. Mol Cell Biochem 182:101–108[CrossRef][Medline]
  8. Goldstein BJ, Ahmad F, Ding W, Li PM, Zhang WR 1998 Regulation of the insulin signalling pathway by cellular protein-tyrosine phosphatases. Mol Cell Biochem 182:91–99[CrossRef][Medline]
  9. Ahmad F, Goldstein BJ 1997 Functional association between the insulin receptor and the transmembrane protein-tyrosine phosphatase LAR in intact cells. J Biol Chem 272:448–457[Abstract/Free Full Text]
  10. Moller NP, Moller KB, Lammers R, et al. 1995 Selective down-regulation of the insulin receptor signal by protein-tyrosine phosphatases {alpha} and {epsilon}. J Biol Chem 270:23126–23131[Abstract/Free Full Text]
  11. Lammers R, Moller NP, Ullrich A 1997 The transmembrane protein tyrosine phosphatase {alpha} dephosphorylates the insulin receptor in intact cells. FEBS Lett 404:37–40[CrossRef][Medline]
  12. Lammers R, Bossenmaier B, Cool DE, et al. 1993 Differential activities of protein tyrosine phosphatases in intact cells. J Biol Chem 268:22456–22462[Abstract/Free Full Text]
  13. Tonks NK, Diltz CD, Fischer EH 1988 Purification of the major protein-tyrosine-phosphatases of human placenta. J Biol Chem 263:6722–6730[Abstract/Free Full Text]
  14. Charbonneau H, Tonks NK, Kumar S, et al. 1989 Human placenta protein-tyrosine-phosphatase: amino acid sequence and relationship to a family of receptor-like proteins. Proc Natl Acad Sci USA 86:5252–5256[Abstract]
  15. Chernoff J, Schievella AR, Jost CA, Erikson RL, Neel BG 1990 Cloning of a cDNA for a major human protein-tyrosine-phosphatase. Proc Natl Acad Sci USA 87:2735–2739[Abstract]
  16. Li L, Dixon JE 2000 Form, function, and regulation of protein tyrosine phosphatases and their involvement in human diseases. Semin Immunol 12:75–84[CrossRef][Medline]
  17. Elchebly M, Cheng A, Tremblay ML 2000 Modulation of insulin signaling by protein tyrosine phosphatases. J Mol Med 78:473–482[CrossRef][Medline]
  18. Barford D, Flint AJ, Tonks NK 1994 Crystal structure of human protein tyrosine phosphatase 1B. Science 263:1397–1404[Medline]
  19. Burke Jr TR, Ye B, Yan X, et al. 1996 Small molecule interactions with protein-tyrosine phosphatase PTP1B and their use in inhibitor design. Biochemistry 35:15989–15996[CrossRef][Medline]
  20. Groves MR, Yao ZJ, Roller PP, Burke Jr TR, Barford D 1998 Structural basis for inhibition of the protein tyrosine phosphatase 1B by phosphotyrosine peptide mimetics. Biochemistry 37:17773–17783[CrossRef][Medline]
  21. Zhang YL, Yao ZJ, Sarmiento M, Wu L, Burke Jr TR, Zhang ZY 2000 Thermodynamic study of ligand binding to protein-tyrosine phosphatase 1B and its substrate-trapping mutants. J Biol Chem 275:34205–34212[Abstract/Free Full Text]
  22. Sarmiento M, Wu L, Keng YF, et al. 2000 Structure-based discovery of small molecule inhibitors targeted to protein tyrosine phosphatase 1B. J Med Chem 43:146–155[CrossRef][Medline]
  23. Iversen LF, Andersen HS, Branner S, et al. 2000 Structure-based design of a low molecular weight, nonphosphorus, nonpeptide, and highly selective inhibitor of protein-tyrosine phosphatase 1B. J Biol Chem 275:10300–10307[Abstract/Free Full Text]
  24. Kohn AD, Kovacina KS, Roth RA 1995 Insulin stimulates the kinase activity of RAC-PK, a pleckstrin homology domain containing ser/thr kinase. EMBO J 14:4288–4295[Abstract]
  25. Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA 1995 Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378:785–789[CrossRef][Medline]
  26. Alessi DR, Andjelkovic M, Caudwell B, et al. 1996 Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J 15:6541–6551[Abstract]
  27. Kohn AD, Summers SA, Birnbaum MJ, Roth RA 1996 Expression of a constitutively active Akt Ser/Thr kinase in 3T3–L1 adipocytes stimulates glucose uptake and glucose transporter 4 translocation. J Biol Chem 271:31372–31378[Abstract/Free Full Text]
  28. Cong LN, Chen H, Li Y, et al. 1997 Physiological role of Akt in insulin-stimulated translocation of GLUT4 in transfected rat adipose cells. Mol Endocrinol 11:1881–1890[Abstract/Free Full Text]
  29. Alessi DR, Caudwell FB, Andjelkovic M, Hemmings BA, Cohen P 1996 Molecular basis for the substrate specificity of protein kinase B; comparison with MAPKAP kinase-1 and p70 S6 kinase. FEBS Lett 399:333–338[CrossRef][Medline]
  30. Vanhaesebroeck B, Alessi DR 2000 The PI3K-PDK1 connection: more than just a road to PKB. Biochem J 346:561–576[CrossRef][Medline]
  31. Zhang ZY, Walsh AB, Wu L, McNamara DJ, Dobrusin EM, Miller WT 1996 Determinants of substrate recognition in the protein-tyrosine phosphatase, PTP1. J Biol Chem 271:5386–5392[Abstract/Free Full Text]
  32. Sarmiento M, Zhao Y, Gordon SJ, Zhang ZY 1998 Molecular basis for substrate specificity of protein-tyrosine phosphatase 1B. J Biol Chem 273:26368–26374[Abstract/Free Full Text]
  33. Andersen HS, Iversen LF, Jeppesen CB, et al. 2000 2-(Oxalylamino)-benzoic acid is a general, competitive inhibitor of protein-tyrosine phosphatases. J Biol Chem 275:7101–7108[Abstract/Free Full Text]
  34. Sarmiento M, Puius YA, Vetter SW, et al. 2000 Structural basis of plasticity in protein tyrosine phosphatase 1B substrate recognition. Biochemistry 39:8171–8179[CrossRef][Medline]
  35. Bandyopadhyay D, Kusari A, Kenner KA, et al. 1997 Protein-tyrosine phosphatase 1B complexes with the insulin receptor in vivo and is tyrosine-phosphorylated in the presence of insulin. J Biol Chem 272:1639–1645[Abstract/Free Full Text]
  36. Kohn AD, Barthel A, Kovacina KS, et al. 1998 Construction and characterization of a conditionally active version of the serine/threonine kinase Akt. J Biol Chem 273:11937–11943[Abstract/Free Full Text]
  37. Zeng G, Nystrom FH, Ravichandran LV, et al. 2000 Roles for insulin receptor, PI3-kinase, and Akt in insulin-signaling pathways related to production of nitric oxide in human vascular endothelial cells. Circulation 101:1539–1545[Abstract/Free Full Text]
  38. Deprez J, Vertommen D, Alessi DR, Hue L, Rider MH 1997 Phosphorylation and activation of heart 6-phosphofructo-2-kinase by protein kinase B and other protein kinases of the insulin signaling cascades. J Biol Chem 272:17269–17275[Abstract/Free Full Text]
  39. Scott PH, Brunn GJ, Kohn AD, Roth RA, Lawrence Jr JC 1998 Evidence of insulin-stimulated phosphorylation and activation of the mammalian target of rapamycin mediated by a protein kinase B signaling pathway. Proc Natl Acad Sci USA 95:7772–7777[Abstract/Free Full Text]
  40. Nave BT, Ouwens M, Withers DJ, Alessi DR, Shepherd PR 1999 Mammalian target of rapamycin is a direct target for protein kinase B: identification of a convergence point for opposing effects of insulin and amino-acid deficiency on protein translation. Biochem J 344:427–431[CrossRef][Medline]
  41. Nakae J, Park BC, Accili D 1999 Insulin stimulates phosphorylation of the forkhead transcription factor FKHR on serine 253 through a wortmannin-sensitive pathway. J Biol Chem 274:15982–15985[Abstract/Free Full Text]
  42. Guo S, Rena G, Cichy S, He X, Cohen P, Unterman T 1999 Phosphorylation of serine 256 by protein kinase B disrupts transactivation by FKHR and mediates effects of insulin on insulin-like growth factor-binding protein-1 promoter activity through a conserved insulin response sequence. J Biol Chem 274:17184–17192[Abstract/Free Full Text]
  43. Paz K, Liu YF, Shorer H, et al. 1999 Phosphorylation of insulin receptor substrate-1 (IRS-1) by protein kinase B positively regulates IRS-1 function. J Biol Chem 274:28816–28822[Abstract/Free Full Text]
  44. Li J, DeFea K, Roth RA 1999 Modulation of insulin receptor substrate-1 tyrosine phosphorylation by an Akt/phosphatidylinositol 3-kinase pathway. J Biol Chem 274:9351–9356[Abstract/Free Full Text]
  45. Kitamura T, Kitamura Y, Kuroda S, et al. 1999 Insulin-induced phosphorylation and activation of cyclic nucleotide phosphodiesterase 3B by the serine-threonine kinase Akt. Mol Cell Biol 19:6286–6296[Abstract/Free Full Text]
  46. Ahmad F, Cong LN, Stenson Holst L, et al. 2000 Cyclic nucleotide phosphodiesterase 3B is a downstream target of protein kinase B and may be involved in regulation of effects of protein kinase B on thymidine incorporation in FDCP2 cells. J Immunol 164:4678–4688[Abstract/Free Full Text]
  47. Fulton D, Gratton JP, McCabe TJ, et al. 1999 Regulation of endothelium-derived nitric oxide production by the protein kinase Akt [published erratum appears in Nature 1999 Aug 19;400(6746):792]. Nature 399:597–601[CrossRef][Medline]
  48. Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM 1999 Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 399:601–605[CrossRef][Medline]
  49. Flint AJ, Gebbink MF, Franza Jr BR, Hill DE, Tonks NK 1993 Multi-site phosphorylation of the protein tyrosine phosphatase, PTP1B: identification of cell cycle regulated and phorbol ester stimulated sites of phosphorylation. EMBO J 12:1937–1946[Abstract]
  50. Wang Q, Somwar R, Bilan PJ, et al. 1999 Protein kinase B/Akt participates in GLUT4 translocation by insulin in L6 myoblasts. Mol Cell Biol 19:4008–4018[Abstract/Free Full Text]
  51. Bjorge JD, Pang A, Fujita DJ 2000 Identification of PTP1B as the major tyrosine phosphatase activity capable of dephosphorylating and activating c-Src in several human breast cancer cell lines. J Biol Chem 275:41439–41446[Abstract/Free Full Text]
  52. Choi T, Huang M, Gorman C, Jaenisch R 1991 A generic intron increases gene expression in transgenic mice. Mol Cell Biol 11:3070–3074[Medline]
  53. Quon MJ, Zarnowski MJ, Guerre-Millo M, de la Luz Sierra M, Taylor SI, Cushman SW 1993 Transfection of DNA into isolated rat adipose cells by electroporation: evaluation of promoter activity in transfected adipose cells which are highly responsive to insulin after one day in culture. Biochem Biophys Res Commun 194:338–346[CrossRef][Medline]
  54. Klippel A, Reinhard C, Kavanaugh WM, Apell G, Escobedo MA, Williams LT 1996 Membrane localization of phosphatidylinositol 3-kinase is sufficient to activate multiple signal-transducing kinase pathways. Mol Cell Biol 16:4117–4127[Abstract]
  55. Quon MJ, Guerre-Millo M, Zarnowski MJ, et al. 1994 Tyrosine kinase-deficient mutant human insulin receptors (Met1153– >Ile) overexpressed in transfected rat adipose cells fail to mediate translocation of epitope-tagged GLUT4. Proc Natl Acad Sci USA 91:5587–5591[Abstract]
  56. Ravichandran LV, Esposito DL, Chen J, Quon MJ 2001 PKC-{zeta} phosphorylates IRS-1 and impairs its ability to activate PI 3-kinase in response to insulin. J Biol Chem 276:3543–3549[Abstract/Free Full Text]
  57. Cohen P, Alessi DR, Cross DA 1997 PDK1, one of the missing links in insulin signal transduction? FEBS Lett 410:3–10[CrossRef][Medline]
  58. Nystrom FH, Chen H, Cong LN, Li Y, Quon MJ 1999 Caveolin-1 interacts with the insulin receptor and can differentially modulate insulin signaling in transfected Cos-7 cells and rat adipose cells. Mol Endocrinol 13:2013–2024[Abstract/Free Full Text]