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PTEN Does Not Modulate GLUT4 Translocation in Rat Adipose Cells under Physiological Conditions
Biochemical and Biophysical Research Communications 288, 1011–1017 (2001) doi:10.1006/bbrc.2001.5876, available online at http://www.idealibrary.com on
PTEN Does Not Modulate GLUT4 Translocation in Rat Adipose Cells under Physiological Conditions Valerie A. Mosser, Yunhua Li, and Michael J. Quon 1 Cardiology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892
Received September 26, 2001
PTEN is a 3ⴕ-inositol lipid phosphatase that dephosphorylates products of PI 3-kinase. Since PI 3-kinase is required for many metabolic actions of insulin, we investigated the role of PTEN in insulin-stimulated translocation of GLUT4. In control rat adipose cells, we observed a ⬃2-fold increase in cell surface GLUT4 upon maximal insulin stimulation. Overexpression of wild-type PTEN abolished this response to insulin. Translocation of GLUT4 in cells overexpressing PTEN mutants without lipid phosphatase activity was similar to that observed in control cells. Overexpression of PTEN-CBR3 (mutant with disrupted membrane association domain) partially impaired translocation of GLUT4. In Cos-7 cells, overexpression of wild-type PTEN had no effect on ERK2 phosphorylation in response to acute insulin stimulation. However, Elk-1 phosphorylation in response to chronic insulin treatment was significantly decreased. Thus, when PTEN is overexpressed, both its lipid phosphatase activity and subcellular localization play a role in antagonizing metabolic actions of insulin that are dependent on PI 3-kinase but independent of MAP kinase. However, because translocation of GLUT4 in cells overexpressing a dominant inhibitory PTEN mutant (C124S) was similar to that of control cells, we conclude that endogenous PTEN may not modulate metabolic functions of insulin under normal physiological conditions. © 2001 Academic Press Key Words: metabolism; signal transduction; insulin resistance; phosphatase; glucose.
Insulin-stimulated glucose transport requires activation of phosphatidylinositol 3-kinase (PI3K) and its downstream ser/thr kinases effectors including PDK-1, PKC-, and Akt (1–5). One mechanism to control PI3Kdependent actions involves modulation of upstream 1
To whom correspondence should be addressed at Cardiology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 10, Room 8C-218, 10 Center Drive MSC 1755, Bethesda, MD 20892-1755. Fax: (301) 402-1679. E-mail: [email protected].
inputs regulating generation of lipid products of PI3K. Downstream control by 3⬘- and 5⬘-inositol lipid phosphatases that dephosphorylate lipid products of PI3K (e.g., PTEN and SHIP-1 and -2) may also regulate PI3K-dependent functions (6 –11). Since SHIP-1, -2, and PTEN can all dephosphorylate the PI3K product PI(3–5)P 3, these lipid phosphatases might be predicted to have similar actions. However, differential effects of PTEN and SHIP-2 may be determined by distinct lipid products of PI3K that are substrates for only 3⬘- or only 5⬘-phosphatases. The potential for inositol lipid phosphatases to modulate metabolic insulin signaling pathways was first shown by Vollenweider et al. who demonstrated that overexpression of SHIP-1 in 3T3-L1 adipocytes inhibits insulin-stimulated translocation of the insulin responsive glucose transporter GLUT4 (12). However, overexpression of a catalytically inactive SHIP-1 mutant did not affect GLUT4 translocation (12). The related SHIP-2 has a wider tissue distribution and SHIP-2 “knockout” mice have increased insulin sensitivity with respect to GLUT4 translocation and glucose transport. Thus, it is likely that SHIP-2 plays an important role in regulating metabolic functions of insulin (13). PTEN is a tumor suppresser that is among the most commonly mutated genes in human cancer (14). The 3⬘-inositol lipid phosphatase activity of PTEN is critical to its role as a tumor suppressor (15). In addition to its lipid phosphatase activity, PTEN also has protein tyrosine phosphatase activity that has been implicated in regulation of growth factor and integrin mediated signaling through MAP kinase, focal adhesion kinase (FAK), and Shc (16 –20). Homozygous deletion of the PTEN gene in mice is embryonic lethal (21, 22) while PTEN heterozygotes have phenotypes related to neoplasia in multiple tissues (23) and autoimmune disorders (24). A recent study has concluded that PTEN plays a role to negatively modulate metabolic actions of insulin (25). In that report, overexpression of PTEN in 3T3-L1 adipocytes inhibited insulinstimulated GLUT4 translocation and glucose uptake while microinjection of anti-PTEN antibody enhanced
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basal and insulin-stimulated translocation of GLUT4. Nevertheless, abnormal metabolic phenotypes in either patients with naturally occurring mutations in PTEN or in heterozygous PTEN knockout mice are not generally observed (22–24, 26, 27). Furthermore, polymorphisms and mutations at the PTEN locus did not correlate with type 2 diabetes or other metabolic phenotypes in a Danish cohort (28). Thus, the contribution of PTEN to metabolic actions of insulin under physiological conditions has not been firmly established and appears somewhat controversial. To address this issue in the present study, we overexpressed both wild-type and mutant forms of PTEN in rat adipose cells in primary culture. METHODS Expression Plasmids pCIS2. (29, 30).
Parental expression vector with CMV promoter/enhancer
PTEN-WT. The full-length cDNA for murine PTEN was cloned into a TOPO TA cloning vector (Invitrogen, Carlsbad, CA) by PCR from a mouse testis Marathon-Ready cDNA library (Clontech, Palo Alto, CA) (generous gift from Dr. Feng Liu). The forward and reverse primers used were: 5⬘-GCC ACA GGC TCC CAG ACA TGA CAG-3⬘ and 5⬘-CAG ACT TTT GTA ATT TGT GAA TGC-3⬘, respectively. After verification by direct DNA sequencing, an EcoR1 fragment containing the cDNA for PTEN was blunt-ended and ligated into the Hpa1 site of pCIS2 in the sense orientation. PTEN-C124S. Catalytically inactive point mutant (Ser substituted for Cys 124) derived from PTEN-WT using mutagenic oligonucleotide 5⬘-GCA GCA ATT CAC AGT AAA GCT GG-3⬘. All mutants were derived from PTEN-WT using the Morph mutagenesis kit (3prime–5-prime, Inc.; Boulder, CO). PTEN-G129E. A PTEN mutant (Glu substituted for Gly 129) lacking lipid phosphatase activity (protein phosphatase activity intact) was derived from PTEN-WT using mutagenic oligonucleotide 5⬘GCA ATT CAC TGT AAG GCT GGA AAG GAA CGG ACT GG-3⬘. This mutagenesis also disrupted an upstream Alu1 site. 129
PTEN-G129R. A PTEN mutant (Arg substituted for Gly ) that lacks lipid and protein phosphatase activity was derived from PTEN-WT using the mutagenic oligonucleotide 5⬘-GCA ATT CAC TGT AAA GCT GGA AAG CGG CGG ACT GG-3⬘. This mutagenesis also created an additional EclX1 site. PTEN-CBR3. A PTEN mutant with multiple substitutions replacing basic/hydrophobic residues in the CBR3 domain (putative membrane association domain) was derived from PTEN-WT by substituting Ala for Lys at positions 263, 266, 267, and 269, Ala for Met 264, and Gly for Leu 265 ( 263KMLKKDK 269 to AAGAADA) using mutagenic oligonucleotide 5⬘-C TTC CAC AAA CAG AAC GCG GCG GGC GCA GCG GAC GCA ATG TTT CAC TTT TGG G-3⬘. This mutagenesis also created an additional MspA1I site. PTEN-D92N. A PTEN mutant (Asn substituted for Asp 92) was derived from PTEN-WT using mutagenic oligonucleotide 5⬘-GCA CAG TAT CCT TTT GAA AAC CAT AAC C-3⬘. This mutagenesis also disrupted a BpuA1 site. This is a potential substrate trapping mutant similar to the D92A mutant (16, 31) hIR. cDNA for the human insulin receptor was subcloned into pCIS2 as described (32). ERK2-HA. expression vector for HA-tagged ERK2 (gift from M. Cobb).
Akt-HA. cDNA for HA-tagged Akt (gift from P.N. Tsichlis and K. Datta) was subcloned into pCIS2. GLUT4-HA. cDNA for HA-tagged GLUT4 was subcloned into pCIS2 as described (32).
Antibodies Murine monoclonal antibodies against PTEN were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). MAPK and phosphoMAPK antibodies were from New England Biolabs Inc. (Beverly, MA). Murine monoclonal antibodies against the HA epitope (HA-11) were from BabCO (Berkeley, CA). Akt antibody was from Upstate Biotechnology (Lake Placid, NY) and phospho-Akt antibody (against Ser 473) was from Cell Signaling (Beverly, MA).
Transfection of Rat Adipose Cells Rat adipose cells in primary culture were prepared from epididymal fat pads and transfected by electroporation as described (30). Each experimental group was transfected with a total of 5 g DNA/ cuvette (1 g GLUT4-HA plus 4 g of either the various PTEN constructs or pCIS2). After transfected cells were processed and cultured (33), cells were stimulated with insulin (0 – 60 nM) for 25 min and cell surface epitope-tagged GLUT4 was measured using a double antibody binding assay (33). In addition, whole cell lysates and membrane fractions were prepared for immunoblotting with anti-HA or anti-PTEN antibodies (33).
ERK2 Phosphorylation Assay Cos-7 cells were grown as described (34). LipofectAMINE Plus (Life Technologies Inc., Gaithersburg, MD) was used to co-transfect cells with ERK2-HA, hIR, and either pCIS2 or PTEN-WT. After transfection and serum starvation overnight the cells were treated without or with insulin (100 nM, 3 min). Anti-HA immunoprecipitates of cell lysates were separated by SDS–PAGE and immunoblotted with anti-ERK and anti-phospho-ERK antibodies (34). In addition, cell lysates were immunoblotted with anti-PTEN antibodies. Quantification of phospho-ERK2 blots was performed using a laser scanning densitometer (Molecular Dynamics, Inc.; Sunnyvale, CA) and results were normalized for ERK2 expression.
Akt Phosphorylation Assay NIH-3T3 IR cells were maintained as described for Cos-7 cells (35). Cells were transiently co-transfected with Akt-HA and pCIS2, PTEN-WT, or PTEN-C124S using lipofectAMINE. One day after transfection, cells were serum starved overnight and then treated without or with insulin (100 nM, 3 min). Anti-HA immunoprecipitates of cell lysates were separated by SDS–PAGE and immunoblotted with anti-phospho-Akt antibodies, and anti-Akt antibodies. Cell lysates were also immunoblotted with anti-PTEN antibodies. Phospho-Akt blots were quantified and normalized for recovery of Akt.
Elk-1 Phosphorylation Assay The Path-Detect system (Stratagene, La Jolla, CA) was used to assess effects of PTEN constructs on the phosphorylation of an Elk-1 reporter in Cos-7 cells as described (34). After transfection and serum starvation overnight (as described in the legend to Fig. 4) cells were treated without or with insulin (100 nM, 7 h) and cell lysates were assayed for luciferase activity.
Statistical Analysis Dose–response curves for GLUT4 translocation were compared using MANOVA. Paired t tests were used to compare results from ERK2, Elk-1 and Akt phosphorylation experiments. P values less than 0.05 were considered to represent statistical significance.
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FIG. 1. Overexpression of recombinant PTEN and GLUT4-HA in rat adipose cells. Cells were co-transfected with GLUT4-HA (1 g/cuvette) and pCIS2, PTEN-WT, PTEN-D92N, PTEN-CBR3, PTEN-G129E or PTEN-G129R (4 g/cuvette). Whole cell lysates (upper panel) or membrane fractions (lower panel) were subjected to SDS–PAGE and immunoblotted with anti-PTEN or anti-HA antibody.
RESULTS Role of PTEN in Insulin-Stimulated Translocation of GLUT4 To evaluate the role of PTEN in insulin-stimulated translocation of GLUT4 in a bona fide insulin target cell we co-transfected wild-type or mutant forms of PTEN along with HA-tagged GLUT4 in rat adipose cells. Comparable overexpression of the various PTEN constructs was confirmed by immunoblotting cell lysates (Fig. 1, upper panel). Since the transfection efficiency of adipose cells by electroporation is ⬃5% (32), we estimate that recombinant PTEN constructs were overexpressed approximately 60-fold in the transfected cells relative to endogenous PTEN levels (Fig. 1, compare lanes 1–2 with lanes 3–7). As expected, comparable expression of GLUT4-HA was also observed in the co-transfected cells (Fig. 1, lower panel). In control cells co-transfected with the empty expression vector pCIS2 and GLUT4-HA, we observed an ⬃2-fold increase in cell surface GLUT4 upon maximal insulin stimulation (Fig. 2). Overexpression of wildtype PTEN abolished the ability of insulin to stimulate translocation of GLUT4 without significantly affecting basal levels of cell surface GLUT4 in the absence of insulin (Fig. 2A). Interestingly, overexpression of PTEN-CBR3 (a mutant with a disrupted putative membrane association domain) only partially impaired translocation of GLUT4 to the cell surface in both the absence and the presence of insulin (Fig. 2B). These results suggest that subcellular localization of PTEN may be critical for its ability to inhibit metabolic actions of insulin. By contrast, overexpression of PTENG129R (a lipid and protein phosphatase inactive mutant) did not significantly alter insulin-stimulated translocation of GLUT4 when compared with results from paired control cells (Fig. 2C). We obtained similar results with overexpression of either PTEN-G129E (a lipid phosphatase inactive mutant that retains protein phosphatase activity) or PTEN-C124S (an enzymatically inactive molecule resulting from a point mutation
of the catalytic cysteine residue) (data not shown). To confirm that the PTEN-C124S mutant acts in a dominant inhibitory fashion as previously described (36), we examined effects of overexpression of PTEN-C124S on activation of Akt by co-transfecting HA-tagged Akt and either PTEN-WT or PTEN-C124S in NIH-3T3 IR cells and assessing the levels of phosphorylated Akt-HA in response to insulin stimulation (Fig. 3). As expected, insulin stimulation caused a significant increase in phospho-Akt levels in control cells transfected with the empty expression vector pCIS2 (Fig. 3, lanes 1 and 2) that was substantially inhibited by overexpression of wild-type PTEN (Fig. 3, lanes 3 and 4). By contrast, overexpression of PTEN-C124S significantly increased both basal and insulin-stimulated phospho-Akt levels above those observed in control cells (Fig. 3, lanes 5 and 6). These results are consistent with a dominant inhibitory effect of PTEN-C124S on endogenous PTEN activity. Taken together, results from Figs. 2 and 3 suggest that the lipid phosphatase activity of PTEN is responsible for inhibiting insulin-stimulated translocation of GLUT4 when PTEN is overexpressed but that endogenous PTEN may not contribute to regulation of translocation of GLUT4 in rat adipose cells in primary culture. Effects of Overexpression of PTEN on ERK2 and Elk-1 Phosphorylation Since wild-type PTEN has protein phosphatase activity in addition to its 3⬘-inositol lipid phosphatase activity, we also evaluated effects of overexpression of PTEN on acute and chronic insulin signaling related to MAP kinase pathways. Cos-7 cells transiently cotransfected with human insulin receptor, HA-tagged ERK2 and either pCIS2 (empty vector control) or PTEN-WT were treated without or with insulin for 3 min. PTEN overexpression was confirmed by immunoblotting (Fig. 4A). The effect of overexpressed PTEN on phosphorylated ERK2 levels was evaluated by immunoblotting anti-HA immunoprecipitates with a phos-
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overexpressed PTEN to inhibit metabolic actions of insulin appears to be independent of insulin’s effects on ERK2 phosphorylation. We evaluated effects of overexpression of PTEN on activation of MAP kinase pathways over longer time periods by assessing Elk-1 phosphorylation in response to chronic insulin treatment (Fig. 5). Stimulation of control cells with insulin for 7 h resulted in a 3-fold increase in phosphorylation of the Elk-1 reporter. Overexpression of wild-type PTEN significantly reduced both basal and insulin-stimulated Elk-1 phosphorylation. By contrast, cells overexpressing the D92N, CBR3, or G129R PTEN mutants had responses similar to the control cells. Interestingly, overexpression of G129E (lipid phosphatase inactive but protein phosphatase active) caused a small, but statistically significant, increase in both basal and insulinstimulated Elk-1 phosphorylation. Thus, both the lipid and protein phosphatase activity of PTEN may contribute to modulation of MAP kinase pathways in response to chronic insulin stimulation. DISCUSSION The importance of PTEN as a tumor suppressor that antagonizes PI3K-dependent functions (15, 37) is well
FIG. 2. Effect of overexpression of PTEN on insulin-stimulated translocation of GLUT4. Rat adipose cells were co-transfected with GLUT4-HA (1 g/cuvette) and pCIS2 (E), PTEN-WT (F, A), PTENCBR3 (■, B), or PTEN-G129R (Œ, C) constructs (4 g/cuvette) and treated with insulin for 25 min (0 – 60 nM). Data are expressed as the percentage of cell surface GLUT4-HA in the control group treated with a maximally effective insulin concentration. Overexpression of PTEN-WT completely inhibited insulin-stimulated translocation of GLUT4 (P ⬍ 0.0001) while PTEN-G129R was without effect (P ⬎ 0.84) and PTEN-CBR3 had a partial inhibitory effect (P ⬍ 0.0008). Results are the mean ⫾ SEM of at least five independent experiments.
pho-specific ERK antibody and normalizing these results for total ERK2-HA expression (Fig. 4). In control cells, acute insulin stimulation resulted in a ⬃10-fold increase in phospho-ERK levels. Overexpression of PTEN did not significantly alter either basal or insulin-stimulated phospho-ERK levels (Fig. 4). These results suggest that overexpression of PTEN does not modulate activation of MAP kinase pathways in response to acute insulin stimulation. Thus, the ability of
FIG. 3. PTEN-C124S is a dominant inhibitory mutant. NIH3T3 IR cells were transiently co-transfected with Akt-HA (2 g/plate), and either pCIS2 (control), PTEN-WT or PTEN-C124S (2 g/plate). After overnight serum starvation, cells were treated without or with insulin (100 nM, 3 min). Cell lysates were immunoprecipitated with an anti-HA antibody followed by immunoblotting with antibodies against either phospho-Akt or Akt. Lysates were also immunoblotted with anti-PTEN antibody. (A) Representative immunoblot from an experiment that was repeated independently six times. (B) Mean ⫾ SEM of six independent experiments quantified by scanning densitometry and normalized for Akt expression. Insulin stimulated a significant increase in phospho-Akt levels in control cells (P ⬍ 0.001). Overexpression of PTEN-WT inhibited the insulin response (P ⬍ 0.003). Overexpression of PTEN-C124S increased both basal and insulin-stimulated phospho-Akt above the levels observed in control cells (P ⬍ 0.05).
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in a single patient with a functionally significant PTEN mutation (43) and PTEN knockout mice do not appear to have an abnormal metabolic phenotype. This raises the possibility that PTEN may not have a physiological role to regulate PI3K-dependent metabolic actions of insulin under normal conditions. Role of PTEN in GLUT4 Translocation
FIG. 4. Effect of overexpression of PTEN on insulin-stimulated ERK-2 phosphorylation in Cos-7 cells. Cells were transiently cotransfected with ERK2-HA (1 g/plate), hIR (1 g/plate), and either pCIS2 (control) or PTEN-WT (3 g/plate). After overnight serum starvation, cells were treated without or with insulin (100 nM, 3 min). (A) ERK2-HA immunoprecipitated from cell lysates with an anti-HA antibody was immunoblotted with antibodies against either ERK-2 or phospho-ERK-2. Cell lysates were immunoblotted for PTEN. (B) Insulin-stimulated increases in phospho-ERK-2 were quantified by scanning densitometry and normalized for ERK2-HA expression (mean ⫾ SEM of three independent experiments). Insulin stimulated a significant increase in phospho-ERK2 levels in cells transfected with either pCIS2 or PTEN-WT (P ⬍ 0.02). There was no statistically significant difference in phospho-ERK2 levels between the pCIS2 and PTEN-WT groups in either the basal or insulinstimulated state (P ⬎ 0.13).
established in patients with naturally occurring mutations in PTEN (15, 26, 27) as well as in heterozygous PTEN knockout mice (22, 23). Since PI3K is essential for insulin-stimulated translocation of GLUT4 in adipose cells (1) it is possible that PTEN may also have a physiological role to negatively regulate metabolic actions of insulin. Several studies suggest that PTEN may enhance metabolic insulin signaling pathways by opposing inhibitory actions of TNF-␣ on IRS-1 and NF-B (38, 39). In Caenorhabditis elegans, genetic evidence supports a role for the PTEN homolog DAF-18 in regulation of metabolism (40, 41). In addition, previous studies in mammalian cells have demonstrated that overexpression of PTEN, SHIP-1, and SHIP-2 can inhibit metabolic functions of insulin (12, 25, 42). However, results from experiments where a signaling protein is overexpressed may not necessarily be relevant to understanding the function of the protein under normal physiological conditions. Since SHIP-2 knockout mice have increased insulin sensitivity and glucose tolerance, SHIP-2 appears to play an important role in regulating basal metabolic actions of insulin (13). By contrast, a metabolic phenotype has only been reported
Overexpression of wild-type PTEN in rat adipose cells completely inhibited insulin-stimulated translocation of GLUT4 suggesting that high level expression of PTEN can effectively block this metabolic action of insulin. Previous studies in 3T3-L1 adipocytes demonstrated only partial inhibition of GLUT4 translocation and glucose uptake with overexpression of PTEN (25). The quantitative differences between our results and previously published studies may reflect the unique properties of the different cell types or differences in the levels of overexpression for PTEN. A C2 domain exists in PTEN homologous to domains in PLC␦1, PKC, and phospholipase A2 that are involved in Ca 2⫹dependent membrane association (44). The CBR3 loop in the C2 domain of PTEN has been implicated in localization of PTEN to membranes (44) and this may be important for its function (45– 47). When compared with wild-type PTEN, overexpression of a PTEN mutant containing a disrupted putative membrane target-
FIG. 5. Effect of overexpression of PTEN on phosphorylation of Elk-1 in Cos-7 cells. Elk-1 phosphorylation was assessed using the Path-Detect luciferase assay. Cells were transiently co-transfected in 6-well dishes with pFA-Elk (0.025 g/well), pFR-luc (0.5 g/well), hIR (0.5 g/well) and either pCIS2 (control) or PTEN construct (0.5 g/well). After overnight serum starvation, cells were treated without or with insulin (100 nM, 7 hr) and luciferase activity was determined in cell lysates. (A) Results (mean ⫾ SEM of n independent experiments performed in quintuplicate) were normalized to a paired insulin-stimulated control group (pCIS2). Overexpression of PTEN-WT significantly inhibited both basal and insulin-stimulated Elk-1 phosphorylation (P ⬍ 0.006). Overexpression of PTEN-G129E slightly enhanced both basal and insulin-stimulated Elk-1 phosphorylation (P ⬍ 0.03) while overexpression of the other PTEN mutants had no significant effect on Elk-1 phosphorylation. (B) Cell lysates derived from experiments in A were immunoblotted with anti-PTEN antibody.
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ing domain (PTEN-CBR3) had a partially impaired ability to inhibit insulin-stimulated translocation of GLUT4. Thus, membrane localization of PTEN may be important for its ability to inhibit translocation of GLUT4. By contrast with wild-type PTEN, overexpression of PTEN mutants lacking lipid phosphatase activity (G129R, G129E, or C124S) had no effect on either basal or insulin-stimulated translocation of GLUT4. G129R is a naturally occurring PTEN mutant present in glioblastomas (48) that abolishes both the protein and lipid phosphatase activity of PTEN (15). G129E is a naturally occurring PTEN mutant associated with Cowden’s syndrome that lacks lipid phosphatase activity but is still able to dephosphorylate protein substrates (15, 37, 49). C124S is a dominant inhibitory PTEN mutant (36). Since the G129E mutant has no lipid phosphatase activity but retains protein phosphatase activity, we conclude that it is the lipid phosphatase activity that accounts for the inhibitory effects of overexpressed wild-type PTEN protein. Moreover, the protein phosphatase activity of PTEN implicated in integrin mediated signaling via dephosphorylation of FAK and Shc (16, 20) does not appear to have a role in antagonizing insulin-mediated translocation of GLUT4. Using an Akt phosphorylation assay to assess effects of PTEN to modulate PI3K-dependent insulin signaling, we confirmed that wild-type PTEN inhibited insulin-stimulated phosphorylation of Akt while the C124S mutant opposed endogenous PTEN activity and enhanced both basal and insulin-stimulated phosphorylation of Akt. Since overexpression of the C124S dominant inhibitory mutant was without effect on either basal or insulin-stimulated levels of GLUT4 at the cell surface, we conclude that endogenous PTEN probably does not play a significant role in regulating translocation of GLUT4 in rat adipose cells. Our results are in keeping with the absence of a metabolic phenotype in the overwhelming majority of patients with naturally occurring PTEN mutations (15, 26, 27) and PTEN knockout mice (22, 23). However, our results do not agree with a previous study in 3T3-L1 adipocytes that showed microinjection of antibodies against PTEN enhanced GLUT4 translocation (25). It is possible that microinjection of PTEN antibodies may be having other non-specific effects in addition to inhibiting PTEN activity.
PTEN in glioblastoma cells expressing endogenous mutated PTEN resulted in decreased MAP kinase activation in response to integrins, EGF, or PDGF stimulation (19). We examined effects of PTEN to modulate both acute and chronic effects of insulin on MAPK pathways. By contrast with the inhibitory effects of PTEN on PI3K-dependent metabolic actions of insulin, overexpression of wild-type PTEN had no effect on the acute action of insulin to stimulate phosphorylation of ERK1/2. Our results do not agree with a previously published study showing that overexpression of PTEN did inhibit insulin-stimulated phosphorylation of MAPK in MCF-7 breast cancer cells (50). It is possible that differences in cell type as well as in the duration of insulin treatment (3 min vs 30 min) and other experimental conditions may account for the discordance between our results. It is also possible that the results from transfected HA-tagged ERK may not reflect what happens to endogenous ERK. Despite the absence of an effect of PTEN on acute effects of insulin to stimulate MAPK pathways, we observed that chronic effects of insulin on phosphorylation of Elk-1 were inhibited by overexpression of PTEN. These inhibitory effects of PTEN were absent when the G129E, G129R, CBR3, and D92N mutants were expressed. Interestingly, the G129E mutant that has no lipid phosphatase activity but retains protein phosphatase activity also caused a slight increase in both basal and insulin-stimulated phosphorylation of Elk-1. Thus, both the lipid and protein phosphatase activity of PTEN may contribute to its ability to modulate Elk-1 phosphorylation. In summary, effects of overexpressed PTEN to inhibit insulin-stimulated translocation of GLUT4 in rat adipose cells depends upon its lipid phosphatase activity. PTEN does not appear to influence the acute effects of insulin on MAPK-dependent pathways although both the lipid and protein phosphatase activity of PTEN may modulate chronic effects of insulin of these pathways. Finally, it is unlikely that endogenous PTEN plays a necessary role to regulate important metabolic functions of insulin such as increased glucose transport in adipose cells.
Role of PTEN in MAPK-Dependent Insulin Signaling Pathways
REFERENCES
ACKNOWLEDGMENT We thank Dr. Feng Liu for providing the PTEN cDNA and for helpful discussions.
The role of PTEN in MAPK signaling is controversial. Sun et al. compared embryonic stem cells from homozygous PTEN knockout mice with cells from normal mice and found no difference in basal levels of MAP kinase or FAK phosphorylation (7). By contrast, Gu et al. demonstrated that expression of wild-type 1016
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PTEN Does Not Modulate GLUT4 Translocation in Rat Adipose Cells under Physiological Conditions Valerie A. Mosser, Yunhua Li, and Michael J. Quon 1 Cardiology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892
Received September 26, 2001
PTEN is a 3ⴕ-inositol lipid phosphatase that dephosphorylates products of PI 3-kinase. Since PI 3-kinase is required for many metabolic actions of insulin, we investigated the role of PTEN in insulin-stimulated translocation of GLUT4. In control rat adipose cells, we observed a ⬃2-fold increase in cell surface GLUT4 upon maximal insulin stimulation. Overexpression of wild-type PTEN abolished this response to insulin. Translocation of GLUT4 in cells overexpressing PTEN mutants without lipid phosphatase activity was similar to that observed in control cells. Overexpression of PTEN-CBR3 (mutant with disrupted membrane association domain) partially impaired translocation of GLUT4. In Cos-7 cells, overexpression of wild-type PTEN had no effect on ERK2 phosphorylation in response to acute insulin stimulation. However, Elk-1 phosphorylation in response to chronic insulin treatment was significantly decreased. Thus, when PTEN is overexpressed, both its lipid phosphatase activity and subcellular localization play a role in antagonizing metabolic actions of insulin that are dependent on PI 3-kinase but independent of MAP kinase. However, because translocation of GLUT4 in cells overexpressing a dominant inhibitory PTEN mutant (C124S) was similar to that of control cells, we conclude that endogenous PTEN may not modulate metabolic functions of insulin under normal physiological conditions. © 2001 Academic Press Key Words: metabolism; signal transduction; insulin resistance; phosphatase; glucose.
Insulin-stimulated glucose transport requires activation of phosphatidylinositol 3-kinase (PI3K) and its downstream ser/thr kinases effectors including PDK-1, PKC-, and Akt (1–5). One mechanism to control PI3Kdependent actions involves modulation of upstream 1
To whom correspondence should be addressed at Cardiology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 10, Room 8C-218, 10 Center Drive MSC 1755, Bethesda, MD 20892-1755. Fax: (301) 402-1679. E-mail: [email protected].
inputs regulating generation of lipid products of PI3K. Downstream control by 3⬘- and 5⬘-inositol lipid phosphatases that dephosphorylate lipid products of PI3K (e.g., PTEN and SHIP-1 and -2) may also regulate PI3K-dependent functions (6 –11). Since SHIP-1, -2, and PTEN can all dephosphorylate the PI3K product PI(3–5)P 3, these lipid phosphatases might be predicted to have similar actions. However, differential effects of PTEN and SHIP-2 may be determined by distinct lipid products of PI3K that are substrates for only 3⬘- or only 5⬘-phosphatases. The potential for inositol lipid phosphatases to modulate metabolic insulin signaling pathways was first shown by Vollenweider et al. who demonstrated that overexpression of SHIP-1 in 3T3-L1 adipocytes inhibits insulin-stimulated translocation of the insulin responsive glucose transporter GLUT4 (12). However, overexpression of a catalytically inactive SHIP-1 mutant did not affect GLUT4 translocation (12). The related SHIP-2 has a wider tissue distribution and SHIP-2 “knockout” mice have increased insulin sensitivity with respect to GLUT4 translocation and glucose transport. Thus, it is likely that SHIP-2 plays an important role in regulating metabolic functions of insulin (13). PTEN is a tumor suppresser that is among the most commonly mutated genes in human cancer (14). The 3⬘-inositol lipid phosphatase activity of PTEN is critical to its role as a tumor suppressor (15). In addition to its lipid phosphatase activity, PTEN also has protein tyrosine phosphatase activity that has been implicated in regulation of growth factor and integrin mediated signaling through MAP kinase, focal adhesion kinase (FAK), and Shc (16 –20). Homozygous deletion of the PTEN gene in mice is embryonic lethal (21, 22) while PTEN heterozygotes have phenotypes related to neoplasia in multiple tissues (23) and autoimmune disorders (24). A recent study has concluded that PTEN plays a role to negatively modulate metabolic actions of insulin (25). In that report, overexpression of PTEN in 3T3-L1 adipocytes inhibited insulinstimulated GLUT4 translocation and glucose uptake while microinjection of anti-PTEN antibody enhanced
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basal and insulin-stimulated translocation of GLUT4. Nevertheless, abnormal metabolic phenotypes in either patients with naturally occurring mutations in PTEN or in heterozygous PTEN knockout mice are not generally observed (22–24, 26, 27). Furthermore, polymorphisms and mutations at the PTEN locus did not correlate with type 2 diabetes or other metabolic phenotypes in a Danish cohort (28). Thus, the contribution of PTEN to metabolic actions of insulin under physiological conditions has not been firmly established and appears somewhat controversial. To address this issue in the present study, we overexpressed both wild-type and mutant forms of PTEN in rat adipose cells in primary culture. METHODS Expression Plasmids pCIS2. (29, 30).
Parental expression vector with CMV promoter/enhancer
PTEN-WT. The full-length cDNA for murine PTEN was cloned into a TOPO TA cloning vector (Invitrogen, Carlsbad, CA) by PCR from a mouse testis Marathon-Ready cDNA library (Clontech, Palo Alto, CA) (generous gift from Dr. Feng Liu). The forward and reverse primers used were: 5⬘-GCC ACA GGC TCC CAG ACA TGA CAG-3⬘ and 5⬘-CAG ACT TTT GTA ATT TGT GAA TGC-3⬘, respectively. After verification by direct DNA sequencing, an EcoR1 fragment containing the cDNA for PTEN was blunt-ended and ligated into the Hpa1 site of pCIS2 in the sense orientation. PTEN-C124S. Catalytically inactive point mutant (Ser substituted for Cys 124) derived from PTEN-WT using mutagenic oligonucleotide 5⬘-GCA GCA ATT CAC AGT AAA GCT GG-3⬘. All mutants were derived from PTEN-WT using the Morph mutagenesis kit (3prime–5-prime, Inc.; Boulder, CO). PTEN-G129E. A PTEN mutant (Glu substituted for Gly 129) lacking lipid phosphatase activity (protein phosphatase activity intact) was derived from PTEN-WT using mutagenic oligonucleotide 5⬘GCA ATT CAC TGT AAG GCT GGA AAG GAA CGG ACT GG-3⬘. This mutagenesis also disrupted an upstream Alu1 site. 129
PTEN-G129R. A PTEN mutant (Arg substituted for Gly ) that lacks lipid and protein phosphatase activity was derived from PTEN-WT using the mutagenic oligonucleotide 5⬘-GCA ATT CAC TGT AAA GCT GGA AAG CGG CGG ACT GG-3⬘. This mutagenesis also created an additional EclX1 site. PTEN-CBR3. A PTEN mutant with multiple substitutions replacing basic/hydrophobic residues in the CBR3 domain (putative membrane association domain) was derived from PTEN-WT by substituting Ala for Lys at positions 263, 266, 267, and 269, Ala for Met 264, and Gly for Leu 265 ( 263KMLKKDK 269 to AAGAADA) using mutagenic oligonucleotide 5⬘-C TTC CAC AAA CAG AAC GCG GCG GGC GCA GCG GAC GCA ATG TTT CAC TTT TGG G-3⬘. This mutagenesis also created an additional MspA1I site. PTEN-D92N. A PTEN mutant (Asn substituted for Asp 92) was derived from PTEN-WT using mutagenic oligonucleotide 5⬘-GCA CAG TAT CCT TTT GAA AAC CAT AAC C-3⬘. This mutagenesis also disrupted a BpuA1 site. This is a potential substrate trapping mutant similar to the D92A mutant (16, 31) hIR. cDNA for the human insulin receptor was subcloned into pCIS2 as described (32). ERK2-HA. expression vector for HA-tagged ERK2 (gift from M. Cobb).
Akt-HA. cDNA for HA-tagged Akt (gift from P.N. Tsichlis and K. Datta) was subcloned into pCIS2. GLUT4-HA. cDNA for HA-tagged GLUT4 was subcloned into pCIS2 as described (32).
Antibodies Murine monoclonal antibodies against PTEN were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). MAPK and phosphoMAPK antibodies were from New England Biolabs Inc. (Beverly, MA). Murine monoclonal antibodies against the HA epitope (HA-11) were from BabCO (Berkeley, CA). Akt antibody was from Upstate Biotechnology (Lake Placid, NY) and phospho-Akt antibody (against Ser 473) was from Cell Signaling (Beverly, MA).
Transfection of Rat Adipose Cells Rat adipose cells in primary culture were prepared from epididymal fat pads and transfected by electroporation as described (30). Each experimental group was transfected with a total of 5 g DNA/ cuvette (1 g GLUT4-HA plus 4 g of either the various PTEN constructs or pCIS2). After transfected cells were processed and cultured (33), cells were stimulated with insulin (0 – 60 nM) for 25 min and cell surface epitope-tagged GLUT4 was measured using a double antibody binding assay (33). In addition, whole cell lysates and membrane fractions were prepared for immunoblotting with anti-HA or anti-PTEN antibodies (33).
ERK2 Phosphorylation Assay Cos-7 cells were grown as described (34). LipofectAMINE Plus (Life Technologies Inc., Gaithersburg, MD) was used to co-transfect cells with ERK2-HA, hIR, and either pCIS2 or PTEN-WT. After transfection and serum starvation overnight the cells were treated without or with insulin (100 nM, 3 min). Anti-HA immunoprecipitates of cell lysates were separated by SDS–PAGE and immunoblotted with anti-ERK and anti-phospho-ERK antibodies (34). In addition, cell lysates were immunoblotted with anti-PTEN antibodies. Quantification of phospho-ERK2 blots was performed using a laser scanning densitometer (Molecular Dynamics, Inc.; Sunnyvale, CA) and results were normalized for ERK2 expression.
Akt Phosphorylation Assay NIH-3T3 IR cells were maintained as described for Cos-7 cells (35). Cells were transiently co-transfected with Akt-HA and pCIS2, PTEN-WT, or PTEN-C124S using lipofectAMINE. One day after transfection, cells were serum starved overnight and then treated without or with insulin (100 nM, 3 min). Anti-HA immunoprecipitates of cell lysates were separated by SDS–PAGE and immunoblotted with anti-phospho-Akt antibodies, and anti-Akt antibodies. Cell lysates were also immunoblotted with anti-PTEN antibodies. Phospho-Akt blots were quantified and normalized for recovery of Akt.
Elk-1 Phosphorylation Assay The Path-Detect system (Stratagene, La Jolla, CA) was used to assess effects of PTEN constructs on the phosphorylation of an Elk-1 reporter in Cos-7 cells as described (34). After transfection and serum starvation overnight (as described in the legend to Fig. 4) cells were treated without or with insulin (100 nM, 7 h) and cell lysates were assayed for luciferase activity.
Statistical Analysis Dose–response curves for GLUT4 translocation were compared using MANOVA. Paired t tests were used to compare results from ERK2, Elk-1 and Akt phosphorylation experiments. P values less than 0.05 were considered to represent statistical significance.
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FIG. 1. Overexpression of recombinant PTEN and GLUT4-HA in rat adipose cells. Cells were co-transfected with GLUT4-HA (1 g/cuvette) and pCIS2, PTEN-WT, PTEN-D92N, PTEN-CBR3, PTEN-G129E or PTEN-G129R (4 g/cuvette). Whole cell lysates (upper panel) or membrane fractions (lower panel) were subjected to SDS–PAGE and immunoblotted with anti-PTEN or anti-HA antibody.
RESULTS Role of PTEN in Insulin-Stimulated Translocation of GLUT4 To evaluate the role of PTEN in insulin-stimulated translocation of GLUT4 in a bona fide insulin target cell we co-transfected wild-type or mutant forms of PTEN along with HA-tagged GLUT4 in rat adipose cells. Comparable overexpression of the various PTEN constructs was confirmed by immunoblotting cell lysates (Fig. 1, upper panel). Since the transfection efficiency of adipose cells by electroporation is ⬃5% (32), we estimate that recombinant PTEN constructs were overexpressed approximately 60-fold in the transfected cells relative to endogenous PTEN levels (Fig. 1, compare lanes 1–2 with lanes 3–7). As expected, comparable expression of GLUT4-HA was also observed in the co-transfected cells (Fig. 1, lower panel). In control cells co-transfected with the empty expression vector pCIS2 and GLUT4-HA, we observed an ⬃2-fold increase in cell surface GLUT4 upon maximal insulin stimulation (Fig. 2). Overexpression of wildtype PTEN abolished the ability of insulin to stimulate translocation of GLUT4 without significantly affecting basal levels of cell surface GLUT4 in the absence of insulin (Fig. 2A). Interestingly, overexpression of PTEN-CBR3 (a mutant with a disrupted putative membrane association domain) only partially impaired translocation of GLUT4 to the cell surface in both the absence and the presence of insulin (Fig. 2B). These results suggest that subcellular localization of PTEN may be critical for its ability to inhibit metabolic actions of insulin. By contrast, overexpression of PTENG129R (a lipid and protein phosphatase inactive mutant) did not significantly alter insulin-stimulated translocation of GLUT4 when compared with results from paired control cells (Fig. 2C). We obtained similar results with overexpression of either PTEN-G129E (a lipid phosphatase inactive mutant that retains protein phosphatase activity) or PTEN-C124S (an enzymatically inactive molecule resulting from a point mutation
of the catalytic cysteine residue) (data not shown). To confirm that the PTEN-C124S mutant acts in a dominant inhibitory fashion as previously described (36), we examined effects of overexpression of PTEN-C124S on activation of Akt by co-transfecting HA-tagged Akt and either PTEN-WT or PTEN-C124S in NIH-3T3 IR cells and assessing the levels of phosphorylated Akt-HA in response to insulin stimulation (Fig. 3). As expected, insulin stimulation caused a significant increase in phospho-Akt levels in control cells transfected with the empty expression vector pCIS2 (Fig. 3, lanes 1 and 2) that was substantially inhibited by overexpression of wild-type PTEN (Fig. 3, lanes 3 and 4). By contrast, overexpression of PTEN-C124S significantly increased both basal and insulin-stimulated phospho-Akt levels above those observed in control cells (Fig. 3, lanes 5 and 6). These results are consistent with a dominant inhibitory effect of PTEN-C124S on endogenous PTEN activity. Taken together, results from Figs. 2 and 3 suggest that the lipid phosphatase activity of PTEN is responsible for inhibiting insulin-stimulated translocation of GLUT4 when PTEN is overexpressed but that endogenous PTEN may not contribute to regulation of translocation of GLUT4 in rat adipose cells in primary culture. Effects of Overexpression of PTEN on ERK2 and Elk-1 Phosphorylation Since wild-type PTEN has protein phosphatase activity in addition to its 3⬘-inositol lipid phosphatase activity, we also evaluated effects of overexpression of PTEN on acute and chronic insulin signaling related to MAP kinase pathways. Cos-7 cells transiently cotransfected with human insulin receptor, HA-tagged ERK2 and either pCIS2 (empty vector control) or PTEN-WT were treated without or with insulin for 3 min. PTEN overexpression was confirmed by immunoblotting (Fig. 4A). The effect of overexpressed PTEN on phosphorylated ERK2 levels was evaluated by immunoblotting anti-HA immunoprecipitates with a phos-
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overexpressed PTEN to inhibit metabolic actions of insulin appears to be independent of insulin’s effects on ERK2 phosphorylation. We evaluated effects of overexpression of PTEN on activation of MAP kinase pathways over longer time periods by assessing Elk-1 phosphorylation in response to chronic insulin treatment (Fig. 5). Stimulation of control cells with insulin for 7 h resulted in a 3-fold increase in phosphorylation of the Elk-1 reporter. Overexpression of wild-type PTEN significantly reduced both basal and insulin-stimulated Elk-1 phosphorylation. By contrast, cells overexpressing the D92N, CBR3, or G129R PTEN mutants had responses similar to the control cells. Interestingly, overexpression of G129E (lipid phosphatase inactive but protein phosphatase active) caused a small, but statistically significant, increase in both basal and insulinstimulated Elk-1 phosphorylation. Thus, both the lipid and protein phosphatase activity of PTEN may contribute to modulation of MAP kinase pathways in response to chronic insulin stimulation. DISCUSSION The importance of PTEN as a tumor suppressor that antagonizes PI3K-dependent functions (15, 37) is well
FIG. 2. Effect of overexpression of PTEN on insulin-stimulated translocation of GLUT4. Rat adipose cells were co-transfected with GLUT4-HA (1 g/cuvette) and pCIS2 (E), PTEN-WT (F, A), PTENCBR3 (■, B), or PTEN-G129R (Œ, C) constructs (4 g/cuvette) and treated with insulin for 25 min (0 – 60 nM). Data are expressed as the percentage of cell surface GLUT4-HA in the control group treated with a maximally effective insulin concentration. Overexpression of PTEN-WT completely inhibited insulin-stimulated translocation of GLUT4 (P ⬍ 0.0001) while PTEN-G129R was without effect (P ⬎ 0.84) and PTEN-CBR3 had a partial inhibitory effect (P ⬍ 0.0008). Results are the mean ⫾ SEM of at least five independent experiments.
pho-specific ERK antibody and normalizing these results for total ERK2-HA expression (Fig. 4). In control cells, acute insulin stimulation resulted in a ⬃10-fold increase in phospho-ERK levels. Overexpression of PTEN did not significantly alter either basal or insulin-stimulated phospho-ERK levels (Fig. 4). These results suggest that overexpression of PTEN does not modulate activation of MAP kinase pathways in response to acute insulin stimulation. Thus, the ability of
FIG. 3. PTEN-C124S is a dominant inhibitory mutant. NIH3T3 IR cells were transiently co-transfected with Akt-HA (2 g/plate), and either pCIS2 (control), PTEN-WT or PTEN-C124S (2 g/plate). After overnight serum starvation, cells were treated without or with insulin (100 nM, 3 min). Cell lysates were immunoprecipitated with an anti-HA antibody followed by immunoblotting with antibodies against either phospho-Akt or Akt. Lysates were also immunoblotted with anti-PTEN antibody. (A) Representative immunoblot from an experiment that was repeated independently six times. (B) Mean ⫾ SEM of six independent experiments quantified by scanning densitometry and normalized for Akt expression. Insulin stimulated a significant increase in phospho-Akt levels in control cells (P ⬍ 0.001). Overexpression of PTEN-WT inhibited the insulin response (P ⬍ 0.003). Overexpression of PTEN-C124S increased both basal and insulin-stimulated phospho-Akt above the levels observed in control cells (P ⬍ 0.05).
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in a single patient with a functionally significant PTEN mutation (43) and PTEN knockout mice do not appear to have an abnormal metabolic phenotype. This raises the possibility that PTEN may not have a physiological role to regulate PI3K-dependent metabolic actions of insulin under normal conditions. Role of PTEN in GLUT4 Translocation
FIG. 4. Effect of overexpression of PTEN on insulin-stimulated ERK-2 phosphorylation in Cos-7 cells. Cells were transiently cotransfected with ERK2-HA (1 g/plate), hIR (1 g/plate), and either pCIS2 (control) or PTEN-WT (3 g/plate). After overnight serum starvation, cells were treated without or with insulin (100 nM, 3 min). (A) ERK2-HA immunoprecipitated from cell lysates with an anti-HA antibody was immunoblotted with antibodies against either ERK-2 or phospho-ERK-2. Cell lysates were immunoblotted for PTEN. (B) Insulin-stimulated increases in phospho-ERK-2 were quantified by scanning densitometry and normalized for ERK2-HA expression (mean ⫾ SEM of three independent experiments). Insulin stimulated a significant increase in phospho-ERK2 levels in cells transfected with either pCIS2 or PTEN-WT (P ⬍ 0.02). There was no statistically significant difference in phospho-ERK2 levels between the pCIS2 and PTEN-WT groups in either the basal or insulinstimulated state (P ⬎ 0.13).
established in patients with naturally occurring mutations in PTEN (15, 26, 27) as well as in heterozygous PTEN knockout mice (22, 23). Since PI3K is essential for insulin-stimulated translocation of GLUT4 in adipose cells (1) it is possible that PTEN may also have a physiological role to negatively regulate metabolic actions of insulin. Several studies suggest that PTEN may enhance metabolic insulin signaling pathways by opposing inhibitory actions of TNF-␣ on IRS-1 and NF-B (38, 39). In Caenorhabditis elegans, genetic evidence supports a role for the PTEN homolog DAF-18 in regulation of metabolism (40, 41). In addition, previous studies in mammalian cells have demonstrated that overexpression of PTEN, SHIP-1, and SHIP-2 can inhibit metabolic functions of insulin (12, 25, 42). However, results from experiments where a signaling protein is overexpressed may not necessarily be relevant to understanding the function of the protein under normal physiological conditions. Since SHIP-2 knockout mice have increased insulin sensitivity and glucose tolerance, SHIP-2 appears to play an important role in regulating basal metabolic actions of insulin (13). By contrast, a metabolic phenotype has only been reported
Overexpression of wild-type PTEN in rat adipose cells completely inhibited insulin-stimulated translocation of GLUT4 suggesting that high level expression of PTEN can effectively block this metabolic action of insulin. Previous studies in 3T3-L1 adipocytes demonstrated only partial inhibition of GLUT4 translocation and glucose uptake with overexpression of PTEN (25). The quantitative differences between our results and previously published studies may reflect the unique properties of the different cell types or differences in the levels of overexpression for PTEN. A C2 domain exists in PTEN homologous to domains in PLC␦1, PKC, and phospholipase A2 that are involved in Ca 2⫹dependent membrane association (44). The CBR3 loop in the C2 domain of PTEN has been implicated in localization of PTEN to membranes (44) and this may be important for its function (45– 47). When compared with wild-type PTEN, overexpression of a PTEN mutant containing a disrupted putative membrane target-
FIG. 5. Effect of overexpression of PTEN on phosphorylation of Elk-1 in Cos-7 cells. Elk-1 phosphorylation was assessed using the Path-Detect luciferase assay. Cells were transiently co-transfected in 6-well dishes with pFA-Elk (0.025 g/well), pFR-luc (0.5 g/well), hIR (0.5 g/well) and either pCIS2 (control) or PTEN construct (0.5 g/well). After overnight serum starvation, cells were treated without or with insulin (100 nM, 7 hr) and luciferase activity was determined in cell lysates. (A) Results (mean ⫾ SEM of n independent experiments performed in quintuplicate) were normalized to a paired insulin-stimulated control group (pCIS2). Overexpression of PTEN-WT significantly inhibited both basal and insulin-stimulated Elk-1 phosphorylation (P ⬍ 0.006). Overexpression of PTEN-G129E slightly enhanced both basal and insulin-stimulated Elk-1 phosphorylation (P ⬍ 0.03) while overexpression of the other PTEN mutants had no significant effect on Elk-1 phosphorylation. (B) Cell lysates derived from experiments in A were immunoblotted with anti-PTEN antibody.
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ing domain (PTEN-CBR3) had a partially impaired ability to inhibit insulin-stimulated translocation of GLUT4. Thus, membrane localization of PTEN may be important for its ability to inhibit translocation of GLUT4. By contrast with wild-type PTEN, overexpression of PTEN mutants lacking lipid phosphatase activity (G129R, G129E, or C124S) had no effect on either basal or insulin-stimulated translocation of GLUT4. G129R is a naturally occurring PTEN mutant present in glioblastomas (48) that abolishes both the protein and lipid phosphatase activity of PTEN (15). G129E is a naturally occurring PTEN mutant associated with Cowden’s syndrome that lacks lipid phosphatase activity but is still able to dephosphorylate protein substrates (15, 37, 49). C124S is a dominant inhibitory PTEN mutant (36). Since the G129E mutant has no lipid phosphatase activity but retains protein phosphatase activity, we conclude that it is the lipid phosphatase activity that accounts for the inhibitory effects of overexpressed wild-type PTEN protein. Moreover, the protein phosphatase activity of PTEN implicated in integrin mediated signaling via dephosphorylation of FAK and Shc (16, 20) does not appear to have a role in antagonizing insulin-mediated translocation of GLUT4. Using an Akt phosphorylation assay to assess effects of PTEN to modulate PI3K-dependent insulin signaling, we confirmed that wild-type PTEN inhibited insulin-stimulated phosphorylation of Akt while the C124S mutant opposed endogenous PTEN activity and enhanced both basal and insulin-stimulated phosphorylation of Akt. Since overexpression of the C124S dominant inhibitory mutant was without effect on either basal or insulin-stimulated levels of GLUT4 at the cell surface, we conclude that endogenous PTEN probably does not play a significant role in regulating translocation of GLUT4 in rat adipose cells. Our results are in keeping with the absence of a metabolic phenotype in the overwhelming majority of patients with naturally occurring PTEN mutations (15, 26, 27) and PTEN knockout mice (22, 23). However, our results do not agree with a previous study in 3T3-L1 adipocytes that showed microinjection of antibodies against PTEN enhanced GLUT4 translocation (25). It is possible that microinjection of PTEN antibodies may be having other non-specific effects in addition to inhibiting PTEN activity.
PTEN in glioblastoma cells expressing endogenous mutated PTEN resulted in decreased MAP kinase activation in response to integrins, EGF, or PDGF stimulation (19). We examined effects of PTEN to modulate both acute and chronic effects of insulin on MAPK pathways. By contrast with the inhibitory effects of PTEN on PI3K-dependent metabolic actions of insulin, overexpression of wild-type PTEN had no effect on the acute action of insulin to stimulate phosphorylation of ERK1/2. Our results do not agree with a previously published study showing that overexpression of PTEN did inhibit insulin-stimulated phosphorylation of MAPK in MCF-7 breast cancer cells (50). It is possible that differences in cell type as well as in the duration of insulin treatment (3 min vs 30 min) and other experimental conditions may account for the discordance between our results. It is also possible that the results from transfected HA-tagged ERK may not reflect what happens to endogenous ERK. Despite the absence of an effect of PTEN on acute effects of insulin to stimulate MAPK pathways, we observed that chronic effects of insulin on phosphorylation of Elk-1 were inhibited by overexpression of PTEN. These inhibitory effects of PTEN were absent when the G129E, G129R, CBR3, and D92N mutants were expressed. Interestingly, the G129E mutant that has no lipid phosphatase activity but retains protein phosphatase activity also caused a slight increase in both basal and insulin-stimulated phosphorylation of Elk-1. Thus, both the lipid and protein phosphatase activity of PTEN may contribute to its ability to modulate Elk-1 phosphorylation. In summary, effects of overexpressed PTEN to inhibit insulin-stimulated translocation of GLUT4 in rat adipose cells depends upon its lipid phosphatase activity. PTEN does not appear to influence the acute effects of insulin on MAPK-dependent pathways although both the lipid and protein phosphatase activity of PTEN may modulate chronic effects of insulin of these pathways. Finally, it is unlikely that endogenous PTEN plays a necessary role to regulate important metabolic functions of insulin such as increased glucose transport in adipose cells.
Role of PTEN in MAPK-Dependent Insulin Signaling Pathways
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ACKNOWLEDGMENT We thank Dr. Feng Liu for providing the PTEN cDNA and for helpful discussions.
The role of PTEN in MAPK signaling is controversial. Sun et al. compared embryonic stem cells from homozygous PTEN knockout mice with cells from normal mice and found no difference in basal levels of MAP kinase or FAK phosphorylation (7). By contrast, Gu et al. demonstrated that expression of wild-type 1016
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