Regulation of Phosphoinositide Metabolism, Akt Phosphorylation, and Glucose Transport by PTEN (Phosphatase and Tensin Homolog Deleted on Chromosome 10) in 3T3-L1 Adipocytes

Hiraku Ono, Hideki Katagiri, Makoto Funaki, Motonobu Anai, Kouichi Inukai, Yasushi Fukushima, Hideyuki Sakoda, Takehide Ogihara, Yukiko Onishi, Midori Fujishiro, Masatoshi Kikuchi, Yoshitomo Oka and Tomoichiro Asano

Third Department of Internal Medicine (H.K., M.F., K.I., Y.F., T.O., M.F., T.A.) Faculty of Medicine, University of Tokyo, Tokyo 113, Japan; The Institute for Adult Disease (H.O., M.A., H.S., Y.O., M.K.), Asahi Life Foundation, Tokyo 160, Japan; and The Third Department of Internal Medicine (Y.O.), Yamaguchi University School of Medicine, Yamaguchi 755, Japan

Address all correspondence and requests for reprints to: Dr. Tomoichiro Asano, Third Department of Internal medicine, University of Tokyo 7–3-1, Hongo, Bunkyo-ku, Tokyo, Japan 113-8655. E-mail: asano-tky{at}umin.ac.jp


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To investigate the roles of PTEN (phosphatase and tensin homolog deleted on chromosome 10) in the regulation of 3-position phosphorylated phosphoinositide metabolism as well as insulin-induced Akt phosphorylation and glucose metabolism, wild-type PTEN and its phosphatase-dead mutant (C124S) with or without an N-terminal myristoylation tag were overexpressed in Sf-9 cells and 3T3-L1 adipocytes using baculovirus and adenovirus systems, respectively. When expressed in Sf-9 cells together with the p110{alpha} catalytic subunit of phosphoinositide 3-kinase, myristoylated PTEN markedly reduced the accumulations of both phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate induced by p110{alpha}. In contrast, overexpression of the C124S mutants apparently increased these accumulations.

In 3T3-L1 adipocytes, insulin-induced accumulations of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate were markedly suppressed by overexpression of wild-type PTEN with the N-terminal myristoylation tag, but not by that without the tag. On the contrary, the C124S mutants of PTEN enhanced insulin-induced accumulations of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate. Interestingly, the phosphorylation level of Akt at Thr308 (Akt2 at Thr309), but not at Ser473 (Akt2 at Ser474), was revealed to correlate well with the accumulation of phosphatidylinositol 3,4,5-trisphosphate modified by overexpression of these PTEN proteins. Finally, insulin-induced increases in glucose transport activity were significantly inhibited by the overexpression of myristoylated wild-type PTEN, but were not enhanced by expression of the C124S mutant of PTEN. Therefore, in conclusion, 1) PTEN dephosphorylates both phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate in vivo, and the C124S mutants interrupt endogenous PTEN activity in a dominant-negative manner. 2) The membrane targeting process of PTEN may be important for exerting its function. 3) Phosphorylations of Thr309 and Ser474 of Akt2 are regulated differently, and the former is regulated very sensitively by the function of PTEN. 4) The phosphorylation level of Ser474, but not that of Thr309, in Akt2 correlates well with insulin-stimulated glucose transport activity in 3T3-L1 adipocytes. 5) The activity of endogenous PTEN may not play a major role in the regulation of glucose transport activity in 3T3-L1 adipocytes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
INOSITOL PHOSPHOLIPIDS PHOSPHORYLATED at the D3-position of their inositol rings (3-phosphoinositides) are known to play important roles in various cellular events. Phosphatidylinositol 3-phosphate [PI(3)P], recently shown to interact with FYVE domain-containing proteins (1, 2, 3), has been implicated in vesicular trafficking functions (4). Although the function of phosphatidylinositol 3,5-bisphosphate [PI(3, 5)P2] remains unclear, phosphatidylinositol 3,4-bisphosphate [PI(3, 4)P2] and phosphatidylinositol 3,4,5-trisphosphate [PI(3, 4, 5)P3], the cellular contents of which rapidly increase in response to stimulation with various hormones and growth factors (5, 6, 7, 8, 9), reportedly act as second messengers by associating with PH domain-containing proteins (10, 11) for a variety of downstream effects including cell survival (12), cell proliferation (13), chemotaxis (14), and membrane ruffling and glucose metabolism (15, 16). Thus, the regulation mechanism of 3-phosphoinositides has been intensively investigated, as have their downstream targets. Phosphoinositide 3-kinases (PI 3-kinases) are the enzymes that synthesize these 3-phosphoinositides. They are categorized into three classes on the basis of primary structure, regulation, and in vitro lipid substrate specificity (17, 18). Many inhibition experiments using gene mutation, dominant-negative enzymes, and pharmacological inhibitors have shown these enzymes to have critical functions involving downstream targets. Mutation of class III PI 3-kinase, which produces only PI(3)P, impairs vesicular morphology and the endocytic pathway (1, 19). Studies using a dominant-negative form of class I PI 3-kinase have implicated PI(3, 4)P2 and/or PI(3, 4, 5)P3 in DNA synthesis (20), membrane ruffling (21), and glucose metabolism (22, 23, 24, 25).

Recently, not only the producers of 3-phosphoinositides, but also proteins that destroy 3-phosphoinositides, have received attention, as they are also important for regulating the cellular content of 3-position phosphorylated phosphoinositides. SH2 domain-containing inositol 5'-phosphatase is known to have a specific catalytic activity, dephosphorylating 5-phosphate from PI(3, 4, 5)P3 (26). Another protein that has recently received considerable attention is phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Having strong and specific enzyme activity, PTEN dephosphorylates 3-phosphate from 3-phosphoinositides (27). The evidence that cells which have a mutated PTEN gene have increased cellular 3-phosphoinositide content (28, 29) and that they show tumorigenicity (30, 31, 32) indicates that the degradation of 3-phosphoinositides is necessary for maintaining normal cellular functions.

The critical roles of PI 3-kinase in insulin signal transduction are well established. In blocking PI 3-kinase activation with specific inhibitors or overexpressing the dominant-negative form of p85, various insulin effects, such as GLUT4 translocation and DNA synthesis, were shown to be inhibited (15, 24, 33). However, whether a 3-phosphoinositide degradation mechanism is involved in the regulation of insulin actions remains unclear. Herein, we demonstrate the roles of PTEN in the regulation of 3-position phosphorylated phosphoinositide metabolism in experiments using Sf-9 insect cells as well as 3T3-L1 adipocytes. In addition, the effects of PTEN on insulin-induced Akt phosphorylation and insulin-stimulated glucose uptake were investigated in 3T3-L1 adipocytes.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Quantification of 3-Phosphoinositides from Sf-9 Cells
To investigate the in vivo effect of PTEN, baculoviruses for the four constructs of PTEN, as illustrated in Fig. 1Go, were prepared. Virus that overexpressed LacZ from Escherichia coli was used as a control. Sf-9 cells were infected with one of the five baculoviruses with or without baculovirus overexpressing the p110{alpha} subunit of PI 3-kinase. Successful expressions of wild-type and mutated PTEN proteins are shown in Fig. 2AGo and that of p110{alpha} in Fig. 2BGo. These Sf-9 cells were labeled with [32P]-orthophosphate for 4 h, and [32P]-labeled phosphoinositides from the cells were deacylated and quantified using HPLC. The radioactivities of quantified glycerophosphoinositol 3,4-bisphosphate from PI(3, 4)P2 and glycerophosphoinositol 3,4,5-trisphosphate from PI(3, 4, 5)P3 are shown in Fig. 2Go, C and D, respectively. As previously reported (34), overexpressing the p110{alpha} subunit of PI 3-kinase increased PI(3, 4)P2 and PI(3, 4, 5)P3 by 7.4-fold and 2.4-fold, respectively, as compared with Sf-9 cells overexpressing LacZ.



View larger version (17K):
[in this window]
[in a new window]
 
Figure 1. DNA Constructs of Viruses

We made the four illustrated DNA constructs for baculovirus and adenovirus. WT, Wild-type PTEN with N-terminal FLAG tag; C124S, construct with cysteine 124 mutated to serine, which is known as a catalytic-dead mutant; Myr-WT, wild-type PTEN with myristoylation signal sequence added to the N-terminal; Myr-C124S, cysteine 124 was mutated to serine in the myr-WT construct.

 


View larger version (34K):
[in this window]
[in a new window]
 
Figure 2. Quantification of 3-Phosphoinositides from Sf-9 Cells

PTEN and its mutants or LacZ were expressed with or without the p110{alpha} subunit of PI 3-kinase in Sf-9 cells by baculoviral infection. Cell lysates were electrophoresed and immunoblotted with anti-PTEN antibody (A) or anti-T56 (C-terminal GLUT2 tag, added to p110{alpha}) antibody (B). Phospholipids in the cells were labeled with [32P]-orthophosphate, extracted, and deacylated, and the radioactivities from glycerophosphoinositol 3,4-bisphosphate (C) and glycerophosphoinositol 3,4,5-trisphosphate (D), which were created from PI(3 4 )P2 and PI(3 4 5 )P3, respectively, were analyzed by HPLC and quantified with an on-line solid scintillation counter. Data are expressed as the fold increase over the basal level (LacZ, without p110{alpha} coexpression). Panels A and B are representative immunoblots. Values in panels C and D are means ± SE of three separate experiments, each of which was performed in triplicate.

 
Co-overexpression of wild-type PTEN decreased the PI(3, 4)P2 content slightly to 80% of that from LacZ+p110{alpha}, but no significant difference was observed in the amount of PI(3, 4, 5)P3. On the other hand, overexpression of the N-terminal myristoylation signal tagged PTEN decreased the cellular contents of PI(3, 4)P2 and PI(3, 4, 5)P3 markedly to 21% and 40%, respectively, of those from LacZ+p110{alpha}. The overexpression of either C124S phosphatase-dead mutant, with or without the N-terminal myristoylation signal, increased the cellular contents of both PI(3, 4)P2 and PI(3, 4, 5)P3 markedly in the presence of coexpressed p110{alpha}, but without affecting the quantities of 3-phosphoinositides in the absence of coexpressed p110{alpha}. This result suggests that these catalytic-dead PTEN mutants exert a dominant-negative effect against endogenous PTEN in Sf-9 cells, in terms of the catalytic activity on 3-phosphoinositides. No significant effects were observed in the cellular contents of PI(3)P, phosphatidylinositol 4-phosphate [PI (4)P] and phosphatidylinositol 4,5-bisphosphate [PI (4, 5)P2] by overexpressing either wild-type or mutant PTEN (data not shown).

Quantification of 3-Phosphoinositides from 3T3-L1 Adipocytes
Subsequently, wild-type PTEN and its mutants were overexpressed in 3T3-L1 adipocytes by adenoviral infection. LacZ from E. coli and {Delta}p85{alpha} of PI 3-kinase were used as controls. 3T3-L1 adipocytes were infected with these adenoviruses, and expressions of wild-type PTEN and its mutants were observed by immunoblotting (Fig. 3AGo). These cells were prelabeled with [32P]-orthophosphate for 2 h and incubated with or without insulin. Then, [32P]-labeled phosphoinositides from the cells were deacylated and quantified using HPLC. The radioactivities of the quantified glycerophosphoinositol 3,4-bisphosphate from PI(3, 4)P2 and glycerophosphoinositol 3,4,5-trisphosphate from PI(3, 4, 5)P3 extracted from cells stimulated for 1 min with insulin are shown in Fig. 3Go, B and C, respectively. Without insulin stimulation, the levels of PI(3, 4)P2 and PI(3, 4, 5)P3 were very low even when catalytic-dead PTEN mutants were overexpressed. Insulin stimulation markedly increased the synthesis of PI(3, 4)P2 and PI(3, 4, 5)P3, and the overexpression of myristoylated PTEN markedly attenuated insulin-induced accumulations of both PI(3, 4)P2 and PI(3, 4, 5)P3. On the other hand, the effect of wild-type PTEN was revealed to be far smaller than that of myristoylated PTEN, and only a partial decrease in insulin-induced synthesis of PI(3, 4)P2 was observed in cells overexpressing wild-type PTEN. In addition, the effect of myristoylated PTEN was very similar to that of overexpressed {Delta}p85{alpha} in terms of the suppression of insulin-induced accumulations of PI(3, 4)P2 and PI(3, 4, 5)P3.



View larger version (46K):
[in this window]
[in a new window]
 
Figure 3. Quantification of 3-Phosphoinositides from 3T3-L1 Adipocytes

PTEN and its mutants, LacZ or {Delta}p85{alpha}, were expressed in 3T3-L1 adipocytes by adenoviral infection. Cell lysates were electrophoresed and immunoblotted with anti-PTEN antibody (A). Cells were labeled with [32P]-orthophosphate for 2 h, and after 100 nM insulin stimulation for 1 min (B and C) or 20 min (D and E), phospholipids extracted from the cells were deacylated and the radioactivities from glycerophosphoinositol 3,4-bisphosphate and glycerophosphoinositol 3,4,5-trisphosphate, which were created from PI(3 4 )P2 and PI(3 4 5 )P3, respectively, were analyzed by HPLC and quantified with a liquid scintillation counter using the fraction-collection method as described in Materials and Methods. Data are expressed as the fold increase over the basal level (LacZ without insulin). Panel A is a representative immunoblot. Values in panels B, C, D, and E are means ± SE of three separate experiments, each of which was performed in triplicate.

 
On the other hand, overexpression of the catalytic-dead mutants significantly enhanced insulin-induced accumulations of both PI(3, 4)P2 and PI(3, 4, 5)P3, irrespective of addition of the myristoylation tag. This effect was more marked at 20 min after insulin stimulation. As shown in Fig. 3Go, D and E, accumulations of PI(3, 4)P2 and PI(3, 4, 5)P3 recovered to basal levels in the control LacZ expressing 3T3-L1 adipocytes after 20 min of insulin stimulation. However, in the cells overexpressing myristoylated catalytic-dead PTEN, insulin-induced increases in PI(3, 4)P2 and PI(3, 4, 5)P3 were shown to persist beyond 20 min after insulin stimulation. Based on these data obtained at 20 min, it appears that the myristoylated catalytic-dead PTEN was more potent than its nonmyristoylated counterpart, in terms of the inhibitory effect on endogenous PTEN. Overexpression of either PTEN or its mutants had no significant effects on the level of PI(3)P (data not shown).

Overexpression of PTEN Did Not Affect Insulin-Induced PI 3-Kinase Activation
As shown in Fig. 3Go, overexpression of PTEN and its mutants affects insulin-stimulated accumulations of PI(3, 4)P2 and PI(3, 4, 5)P3. To investigate whether overexpression of PTEN and its mutants affects insulin-induced PI 3-kinase activation, we examined the in vitro PI 3-kinase activities of antiphosphotyrosine antibody immunoprecipitants from 3T3-L1 adipocytes overexpressing control LacZ and PTEN proteins. As shown in Fig. 4Go, neither PTEN nor its mutants altered PI 3-kinase activities in the antiphosphotyrosine antibody immunoprecipitates, regardless of insulin stimulation. In contrast, the overexpression of {Delta}p85{alpha} markedly inhibited insulin-induced PI 3-kinase activation, as we reported previously (33).



View larger version (50K):
[in this window]
[in a new window]
 
Figure 4. PI 3-Kinase Activities from Antiphosphotyrosine Antibody Immunoprecipitants

3T3-L1 adipocytes infected with adenoviruses were lysed and immunoprecipitated with antiphosphotyrosine antibody. The PI 3-kinase activities from the immunoprecipitants were quantified as described in Materials and Methods. A, Representative PI(3 )P spots on TLC plates. B, The intensities of the spots on the TLC plates were measured. Data are expressed as the fold increase over the basal level (LacZ without insulin). Values are means ± SE of three separate experiments, each of which were performed in triplicate. Statistical analysis showed no significant differences between insulin-stimulated control cells and any of the insulin-stimulated PTEN construct-expressing cells.

 
Subcellular Localization of PTEN and Its Mutants with/without Insulin Stimulation
As shown above, myristoylated PTEN and myristoylated catalytic-dead PTEN exhibited much stronger effects on the contents of inositol phospholipids than their nonmyristoylated counterparts. Although several groups have reported that PTEN has domains associating with membranes (35, 36), PTEN was shown to be present mainly in the cytoplasm (37, 38). Thus, it has been unclear whether PTEN actually associates with membranes to exert its dephosphorylation activity. Herein, we investigated what percentages of PTEN and its mutants were present in the membrane fraction in the presence or absence of insulin stimulation (Fig. 5Go). The amount of endogenous PTEN in membrane fractions from LacZ-expressing control cells was very small and was almost undetectable by immunoblotting, irrespective of insulin stimulation (data not shown). With overexpression of PTEN proteins, their existence in the membrane fraction was clearly detected by immunoblotting; approximately 0.4–0.5% of overexpressed nonmyristoylated PTEN and its catalytic-dead mutant was collected in the membrane fraction in the basal state. On the other hand, approximately 5–6% of the myristoylated types was collected in the membrane fraction.



View larger version (54K):
[in this window]
[in a new window]
 
Figure 5. Membrane-Fractionated PTEN and Its Mutants with/without Insulin Stimulation

3T3-L1 adipocytes infected with adenoviruses were stimulated with/without 100 nM insulin for 1, 5, or 15 min. After stimulation, one-tenth of the cells was prepared for SDS-PAGE as the total cellular lysate. The remaining nine-tenths was treated as described in Materials and Methods to obtain the total membrane fraction, which was also prepared for SDS-PAGE. These samples were immunoblotted with anti-PTEN antibody. A, Representative bands of wild-type and myristoylated PTEN in the membrane fraction and total cell lysate in the absence of insulin. B, Upper bands show membrane-fractionated PTEN. For these bands, the film exposure time of nonmyristoylated PTEN was longer than that of myristoylated PTEN. The PTEN level in the total cell lysate was not significantly altered in response to insulin stimulation (not shown). The lower graphs show the ratios of membrane-fractionated PTEN to total cellular PTEN. Intensities of the membrane-fractionated and total cellular PTEN bands were quantified using NIH image, and the ratios were calculated. The upper part of each panel shows representative immunoblots. Values shown in the lower part of each panel are means ± SE of three separate experiments, each of which was performed in triplicate. Values without symbols are not significantly different from values obtained without insulin.

 
The percent ratios of wild-type PTEN and catalytic-dead PTEN in the membrane fraction were observed to be significantly increased, by 1.8-fold and 1.7-fold, respectively, 1–5 min after insulin stimulation. On the other hand, the membrane-fractionated protein ratio of the myristoylated PTEN was not significantly affected by insulin stimulation.

Effects of Wild-Type and Mutant PTEN on Akt Phosphorylation
The phosphorylation and activation of Akt are reportedly regulated by the lipid products of PI 3-kinase (reviewed in Refs. 39, 40, 41). Thus, we investigated the effects of overexpressing wild-type and mutant PTEN proteins on Akt phosphorylation in the presence or absence of insulin stimulation. Using anti-Akt antibody, we confirmed that the endogenous Akt protein level did not significantly change with the adenoviral expression of LacZ, PTEN or its mutants, or {Delta}p85{alpha} (Fig. 6AGo). Figure 6Go (panels B and C) shows the phosphorylation levels of Akt, which were demonstrated with antiphospho-Thr308-Akt antibody and antiphospho-Ser473-Akt antibody (New England Biolabs, Beverly, MA), which recognize both phospho-Thr308 of Akt1 and phospho-Thr309 of Akt2, and both phospho-Ser473 of Akt1 and phospho-Ser474 of Akt2, respectively (42). Akt2 expression in 3T3-L1 cells reportedly increases during differentiation from fibroblasts into adipocytes, and in 3T3-L1 adipocytes, Akt2 is dominantly expressed as compared with Akt1 (42, 43). Thus, in 3T3-L1 adipocytes, antiphospho-Thr308 Akt antibody and antiphospho-Ser473 Akt antibody are considered to detect mainly phospho-Thr309 and phospho-Ser474 of Akt2, respectively.



View larger version (49K):
[in this window]
[in a new window]
 
Figure 6. Akt Phosphorylation in 3T3-L1 Adipocytes

3T3-L1 adipocytes infected with adenoviruses were stimulated without/with insulin for 5 or 15 min. The cells were lysed, electrophoresed, and immunoblotted with anti-Akt (A, without insulin stimulation), antiphospho-Thr308 (B), or antiphospho-Ser473 (C) Akt antibody. The intensities of the bands were quantified with NIH image. The graphs in panels B and C show the intensities of bands without/with 15 min of insulin stimulation. Upper parts of panels B and C are representative immunoblots. Data shown in the graphs in panels B and C are expressed as the fold increase over the intensities from LacZ, insulin (+). Values are means ± SE of three separate experiments, each of which was performed in triplicate.

 
First, overexpression of the wild-type and mutated PTEN proteins did not significantly affect basal phosphorylation levels of Thr309 or Ser474 of Akt2 in 3T3-L1 adipocytes. The phosphorylation level of Thr309 of Akt2 in the insulin-stimulated state was only partially (25%) decreased in wild-type PTEN overexpressing 3T3-L1 adipocytes. The overexpression of myristoylated PTEN more markedly inhibited insulin-induced Akt phosphorylation than did that of nonmyristoylated PTEN, and the degree of inhibition was similar to that observed with the overexpression of {Delta}p85{alpha}. In contrast, overexpressions of nonmyristoylated and myristoylated C124S-PTEN proteins enhanced insulin-induced Thr309 phosphorylation by 2.6-fold and 5.3-fold, respectively.

Interestingly, the effects of these overexpressed PTEN proteins on insulin-induced phosphorylation were revealed to be much smaller for Ser474 than for Thr309 of Akt2. While overexpression of myristoylated PTEN significantly, although only partially, reduced Ser474 phosphorylation, overexpression of phosphatase-dead types did not increase the Ser474 phosphorylation level. Overexpression of {Delta}p85{alpha} strongly inhibited insulin-induced phosphorylation of both Thr309 and Ser474.

Effects of Overexpressing PTEN and Its Mutants on Insulin-Stimulated Glucose Uptake by 3T3-L1 Adipocytes
Finally, we investigated how PTEN and its mutants affect insulin-induced glucose transport activation of 3T3-L1 adipocytes, which is thought to be an action occurring downstream from PI 3-kinase and presumably downstream from Akt activations (reviewed in Refs. 44 and 45). Overexpression of myristoylated PTEN decreased 2-deoxyglucose uptake by insulin-stimulated 3T3-L1 adipocytes by 47%, while nonmyristoylated PTEN had no effect (Fig. 7Go). In addition, phosphatase-dead PTEN mutants did not affect insulin-induced glucose transport activation, despite enhancing insulin-induced accumulations of PI(3, 4)P2 and PI(3, 4, 5)P3 and insulin-induced phosphorylation of the Thr309 of Akt2. In contrast to the weak effect of PTEN, {Delta}p85{alpha} strongly inhibited the insulin-induced increase in glucose transport activity, i.e. by approximately 90%.



View larger version (33K):
[in this window]
[in a new window]
 
Figure 7. 2-Deoxyglucose Uptake of 3T3-L1 Adipocytes

Glucose uptake was assayed in adenoviral infected 3T3-L1 adipocytes as described in Materials and Methods. The radioactivities from 2-deoxy-D-[3H]-glucose taken into the cells were quantified using a liquid scintillation counter. Data are expressed as the fold increase over the basal level (LacZ without insulin). Values are means ± SE of three separate experiments, each of which was performed in triplicate. Statistical analysis showed no significant difference between the value from insulin-stimulated control cells and the values from WT, C124S, or myr-C124S insulin (+).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Over the past decade, numerous oncogenic genes have been identified, and most of their protein products were shown to be tyrosine kinases. Such tyrosine kinase proteins reportedly induce cellular proliferation via the activations of PI 3-kinase, ras, and other signal transduction molecules. Recently, PTEN was demonstrated to be a tumor suppressor gene involved in oncogenesis of a wide range of cancers (reviewed in Refs. 46, 47, 48). Interestingly, PTEN contains a conserved catalytic motif found in multiple tyrosine phosphatases (30) and has been shown to dephosphorylate the D-3 position of phosphoinositide (27, 28, 29, 49). Thus, PTEN has been regarded as a negative regulator of PI 3-kinase.

In the field of insulin action, activation of PI 3-kinase has been demonstrated to be essential for various insulin-induced glucose metabolic functions including glucose uptake and glycogen synthesis as well as insulin-induced mitogenesis. For example, it was shown that overexpression of a catalytic subunit of PI 3-kinase, p110{alpha}, induced GLUT4 translocation to the cell surface resulting in increased glucose uptake, irrespective of insulin stimulation (50), while overexpression of the dominant negative form of the regulatory subunit, p85{alpha}, inhibited this action (24, 33). Thus, taking the above-mentioned facts and several reports concerning the oncogenic role of PI 3-kinase and the tumor-suppressing property of PTEN into consideration, it is reasonable to speculate that PTEN plays a role in insulin-induced glucose metabolism as important as that of PI 3-kinase. Indeed, one previous report (51) described overexpression of PTEN as significantly attenuating insulin-induced GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes. However, it remains unclear whether or not endogenous PTEN is indeed involved in the regulation of glucose metabolism. To answer this question, we prepared wild-type as well as the dominant negative forms of PTEN constructs with or without an N-terminal myristoylation tag.

In this study, first of all, we investigated whether overexpressing PTEN and its mutants affects cellular quantities of 3-phosphoinositides in Sf-9 cells as well as 3T3-L1 adipocytes. Several reports have shown that PTEN has strong and specific phosphatase activity for 3-phosphoinositides in vitro (27, 28, 36, 49), but few reports have focused on PTEN activity toward each type of 3-phosphoinositide in the physiological in vivo environment of cells. Our results confirmed that not only PI(3, 4, 5)P3, but also PI(3, 4)P2, is regulated by PTEN, while the cellular PI(3)P content was unaffected by PTEN and its mutants expressed in Sf-9 insect cells as well as 3T3-L1 adipocytes.

Contrary to our expectations, wild-type PTEN without the myristoylation tag did not markedly decrease 3-position phosphorylated phosphoinositides in Sf-9 cells, despite the very high level of expression induced by the baculovirus system. Similarly, the effect of overexpressing wild-type PTEN without the myristoylation tag in 3T3-L1 adipocytes was revealed to be minimal also in terms of the reductions in PI(3, 4)P2 and PI(3, 4, 5)P3, despite its overexpression level exceeding 50-fold as compared with that of endogenous PTEN.

Several studies have shown that, in PTEN-deficient cells, levels of 3-phosphoinositides are increased and that reintroduction of wild-type PTEN normalizes these levels (29). Thus, our results suggest that when some amount of PTEN protein comparable to that of endogenous PTEN exists, the effects on 3-phosphoinositides are already adequate and saturated at least in Sf-9 cells and 3T3-L1 adipocytes. On the other hand, PTEN with the myristoylation tag did significantly and strongly reduce the accumulations of PI(3, 4)P2 and PI(3, 4, 5)P3 in both Sf-9 cells and 3T3-L1 adipocytes. Several reports have suggested that PTEN exists mainly in the cytoplasm (37, 38). Although it has been speculated that PTEN moves to the membrane when activated (35, 36), this hypothesis has not yet been demonstrated. In this study, it was shown that the membrane-associated portions of wild-type and catalytic dead PTEN temporarily increased in response to insulin stimulation. On the other hand, in the case of myristoylated PTEN, a much higher level of overexpressed protein existed in the membrane fraction, even in the basal state, than in the case of nonmyristoylated PTEN. Thus, the myristoylated-PTEN used in this study is regarded as a constitutively active form of the PTEN mutant. Although the functional regulatory mechanism of endogenous PTEN is unclear at present, taking into consideration that the myristoylated PTEN functions as a constitutively active form, the membrane targeting process of PTEN is likely to be important. Further study is needed to elucidate the mechanism of PTEN translocation to the membranes.

The C124S point mutation of PTEN, which corresponds to the catalytic pocket that grasps the inositol ring of 3-phosphoinositides (36), has been identified in various tumor cells. Overexpression of the C124S mutant of PTEN markedly enhanced the increases in 3-phosphoinositides induced by p110{alpha} overexpression in Sf-9 cells and by insulin stimulation in 3T3-L1 adipocytes. These results indicate that this mutant protein is not only unable to catalyze 3-phosphoinositides but also has an apparent dominant negative effect against the activity of endogenous PTEN. A functional difference between C124S mutants of PTEN with and without the N-terminal myristoylation tag was observed for the phosphoinositide content 20 min after insulin stimulation. Overexpression of the C124S mutant of PTEN with, but not that without, the myristoylation tag maintained elevated cellular PI(3, 4)P2 and PI(3, 4, 5)P3 contents induced by insulin stimulation for at least 20 min. As shown in Fig. 5Go, C124S PTEN without the myristoylation tag temporarily and rapidly moves to membranes after insulin stimulation, similar to wild-type PTEN, but subsequently returns to the cytoplasm. This is the most likely reason for the C124S mutant of PTEN without the myristoylation tag not being able to continue to exert a dominant-negative effect against endogenous PTEN, unlike that with the N-terminal myristoylation tag. These results, obtained with C124S mutants of PTEN, indicate that endogenous PTEN does play a role in the dephosphorylation of PI(3, 4)P2 and PI(3, 4, 5)P3 in Sf-9 cells as well as 3T3-L1 adipocytes.

Much remains unknown regarding the pathway between PI 3-kinase activation and GLUT4 translocation. At present, Akt is regarded as a possible mediator of insulin-induced GLUT4 translocation (52), although this speculation has not been substantiated (44, 45). Akt is a serine/threonine kinase located downstream from PI 3-kinase, and its activation is reportedly mediated by phosphorylation at Thr308 (corresponding to Thr309 of Akt2) and Ser473 (corresponding to Ser474 of Akt2) (41). Phosphorylation of Thr308 is mediated by 3-phosphoinositide-dependent kinase 1 (PDK1), the activity of which is dependent upon PI(3, 4, 5)P3 or both PI(3, 4)P2 and (2) PI(3, 4, 5)P3. On the other hand, the kinase responsible for the phosphorylation of Ser473 has been suggested to be 3-phosphoinositide-dependent kinase 2 (PDK2), or possibly also PDK1 (53), but remains to be determined.

Comparing Fig. 3Go with Fig. 6Go, our data revealed a close relationship between the cellular PI(3, 4, 5)P3 content and the level of phosphorylation at Thr309 of Akt2. This correlation between PI(3, 4, 5)P3 and Thr309 phosphorylation of Akt agrees with the hypothesis that both Akt and PDK1 are recruited onto membranes by PI(3, 4, 5)P3, where Akt is phosphorylated by PDK1. However, interestingly, the phosphorylation level of Ser474 of Akt2 did not show a good correlation with the cellular content of either PI(3, 4)P2 or (2) PI(3, 4, 5)P3. This discrepancy appears to strongly support the hypothesis that phosphorylation of Ser474 is mediated by mechanisms other than phosphorylation by PDK1, although phosphorylation of Ser474 is likely to be mediated by the activation of PI 3-kinase since overexpression of the dominant negative form of the p85{alpha} regulatory subunit of PI 3-kinase inhibited phosphorylation at both Thr309 and Ser474. Further study is necessary to clarify the regulation of the phosphorylation level of Ser474 of Akt.

In addition, a very important finding of the current study is that the phosphorylation of Ser474, but not that of Thr309, which is highly dependent on the synthesis of PI(3, 4, 5)P3, in Akt2 correlates well with an increase in glucose transport activity. Overexpression of PTEN with the N-terminal myristoylation tag suppresses insulin-induced phosphorylation of Thr309 and Ser474 in Akt2 to 21% and 40%, respectively, and decreases insulin-induced glucose uptake to 53%. Overexpression of the C124S mutant of PTEN enhanced neither the phosphorylation of Ser474 of Akt2 nor insulin-induced glucose uptake by 3T3-L1 adipocytes, despite a 1.7- to 4.1-fold increase in the synthesis of PI(3, 4, 5)P3 and also a 2.6- to 5.3-fold increase in phosphorylation of Thr309 of Akt2. Such phenomena were also observed in experiments employing stimulation with insulin concentrations lower than 100 nM (data not shown). Therefore, it may be important to determine which kinase phosphorylates Ser474 in Akt2 and which protein binds to this C-terminal portion of Akt, and we are currently performing experiments with these aims. However, since the dominant negative mutants of PTEN did not affect (enhance) glucose uptake in either the presence or the absence of insulin, endogenous PTEN may not play a major role in the regulation of glucose transport activity in 3T3-L1 adipocytes, despite endogenous PTEN clearly affecting the accumulations of PI(3, 4)P2 and PI(3, 4, 5)P3.

In conclusion, 1) PTEN dephosphorylates both PI(3, 4)P2 and PI(3, 4, 5)P3 in vivo, and C124S mutants interrupted endogenous PTEN activity in a dominant-negative manner. 2) Membrane targeting processes of PTEN may be important for this function. 3) Phosphorylations of Thr309 and Ser474 of Akt2 were regulated differently, and the former regulation was very sensitive to the function of PTEN. 4) The phosphorylation level of Ser474, but not that of Thr309, in Akt2 correlates well with insulin-stimulated glucose transport activity in 3T3-L1 adipocytes. 5) The activity of endogenous PTEN may not play a major role on the regulation of glucose transport activity in 3T3-L1 adipocytes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Reagents
Phosphatidylinositol, PI (4)P, PI (4, 5)P2, dexamethasone, and phloretin were purchased from Sigma (St. Louis, MO). 3-Isobutyl-1-methylxanthine and 2-deoxy-D-glucose were from Wako Pure Chemical Industries Ltd. (Osaka, Japan). The enhanced chemiluminescence (ECL) detection system and Protein G Sepharose 6MB were from Amersham Pharmacia Biotech (Arlington Heights, IL). [32P]-orthophosphate, [{gamma}-32P]-ATP, and 2-deoxy-D-[3H]-glucose were from ICN Biochemicals, Inc./MORAVEK (Costa Mesa/Brea, CA). All other reagents from commercial sources were of analytical grade.

DNA Constructs
PTEN cDNA of Rattus norvegicus was amplified by PCR from its brain cDNA library with primers corresponding to sequences already reported and cloned into pCR2.1 plasmid vector (Invitrogen, San Diego, CA). The Cys124 to Ser mutant form (C124S) of PTEN was constructed using PCR with mutagenic oligonucleotides 5'-CCA AGG CTG GGA AAG GAC GGA CTG-3' and 5'-CCT TGG AGT GAA TTG CTG CAA CAT G-3' as PCR primers and ligated with the newly integrated StyI restriction enzyme site. Nucleotides for the N-terminal FLAG tag and myristoylation signal peptide were added using oligonucleotides 5'-ATG GAC TAC AAG GAC GAC GAT GAC AAG ACA GCC ATC ATC AAA GAG-3' and 5'-ATG GGA TGT TGG TGT AGC AGC AAT CCC GAA GAC GAC GAC GAC TAC AAG GAC GAC-3', respectively. All sequences were confirmed using a 370A DNA sequencer (PE Applied Biosystems, Norwalk, CT).

Cell Culture
Sf-9 insect cells were maintained in TC-100 medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% FBS (Life Technologies, Inc.) at 27 C. Sf-9 cells were harvested 48 h after baculoviral infection for subsequent experiments. 3T3-L1 fibroblasts were maintained in DMEM containing 10% donor calf serum (Life Technologies, Inc.) under an atmosphere of 10% CO2/90% air at 37 C. Two days after the fibroblasts reached confluence, differentiation was induced by incubating these cells for 48 h in DMEM containing 0.5 mM 3-isobutyl-1-methylxanthine, 4 mg/ml dexamethasone, and 10% FBS. Thereafter, the cells were maintained in DMEM supplemented with 10% FBS, which was renewed every other day. The cells were infected with the indicated adenoviruses on day 3 after inducing differentiation, and the experiments were conducted on day 6 when >90% of cells expressed the adipocyte phenotype and GLUT4 was almost fully expressed.

Antibodies
Antiphosphotyrosine antibody 4G10 was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). The anti-PTEN antibody was raised in rabbits against synthetic peptides corresponding to 18 C-terminal amino acids (DPENEPFDEDQHSQITKV) of the PTEN protein. Antibody was affinity purified on Affi-Gel-10 (Bio-Rad Laboratories, Inc., Hercules, CA) columns to which the corresponding peptide had been coupled, and extensively dialyzed against PBS (PBS) and concentrated with Centricon 30 (Amicon Inc., Beverly, MA). Antibodies against Akt, the phospho-Thr308 and phospho-Ser473 of Akt, were purchased from New England Biolabs, Inc. (Beverly, MA). Antibody against the C-terminal GLUT2 tag of p110{alpha} was prepared as described previously (50, 54).

Gene Transduction
To obtain recombinant baculoviruses, all DNA constructs (Fig. 1Go) were subcloned into pBacPAK8 and transfected with the parental viral genome into Sf-9 insect cells as instructed by the manufacturer (CLONTECH Laboratories, Inc., Palo, Alto, CA). Baculoviruses overexpressing LacZ and the GLUT2-tagged p110{alpha} subunit of PI 3-kinase were made as previously described (34). Viral titers for protein overexpression were adjusted so that the expression levels of the wild-type and mutant PTEN proteins were similar, irrespective of co-overexpression of p110{alpha}. Similarly, the expression levels of p110{alpha} were adjusted so as to be similar, irrespective of co-overexpression of PTEN or other co-overexpressed proteins. The experiments described below were executed 48 h after baculoviral infection.

To obtain recombinant adenoviruses, each DNA construct (Fig. 1Go) was subcloned into a pAdexCAwt cosmid cassette and transfected with the parental viral genome into HEK293 cells as described previously (50). Adenoviruses overexpressing LacZ and {Delta}p85{alpha} were prepared as described previously (33, 34). In the case of adenoviruses, maximal overexpression of myristoylation signal-tagged mutants was lower than that of non-myristoylation-tagged mutants. To maximize the effects of overexpressed PTEN mutants, the levels of overexpressed proteins were adjusted so as to be similar in wild-type PTEN and the C124S mutant, and in myr-wild-type PTEN and the myr-C124S mutant. The expression level of nonmyristoylation mutants was approximately 3 times that of myristoylation signal-added mutants in the experiments described below. The subsequent experiments were executed 72 h after adenoviral infection of 3T3-L1 adipocytes.

Western Blotting
Cells were washed with ice-cold PBS twice, and collected with Laemmli sample buffer containing 100 mM dithiothreitol, and then subjected to SDS-PAGE. Immunoblotting was performed with the ECL-plus (for anti-phosphoThr308-Akt antibody immunoblotting) or the ECL (for other antibodies) system, according to the manufacturer’s instructions. The images of bands on films were scanned with a film-scanner (EPSON, Nagano, Japan) and the intensities were quantified by NIH Image.

In Vivo 32P-Labeling of Phosphoinositides
Sf-9 cells infected with baculovirus were phosphate-starved with phosphorus-free DMEM (Sigma) with 5 mM HEPES adjusted to pH 6.2 for 18 h and then labeled with [32P]-orthophosphate (0.5 mCi/ml) for 4 h. Thereafter, the cells were washed with ice-cold PBS, collected with 750 µl of methanol-1 N HCl (1:1) solution, and mixed well and the lipids were extracted by adding 380 µl of chloroform.

Similarly, 3T3-L1 adipocytes infected with adenovirus were phosphate starved for 14 h with phosphorus-free DMEM, followed by serum starvation for 1 h. [32P]-orthophosphate (0.5 mCi/ml) was added, and the cells were cultured for an additional 2 h. After the labeling period, the cells were incubated with or without 100 nM insulin for the indicated times. The reaction was then terminated with an ice-cold PBS wash, followed by the addition of methanol-1 N HCl (1:1) and finally lipid extraction with chloroform.

HPLC Analysis
The extracted lipid was deacylated and subjected to anion-exchange HPLC using a Partisphere strong anion-exchange column (Whatman, Clifton, NJ) as described previously (55). The radioactivity was detected with an on-line radiochemical detector in the case of experiments using Sf-9 cells. The samples from labeled 3T3-L1 adipocytes were subjected to HPLC analysis, after which fractionation was carried out for every 20-sec elution from the HPLC column and the radioactivity of each fraction was counted using a liquid scintillation counter. [32P]-labeled PI(3)P, PI(3, 4)P2, and PI(3, 4, 5)P3 generated in vitro as described previously (34) were deacylated and used as internal standards. Each sample and standard was coinjected with the nonradioactive nucleotides ADP and ATP for each HPLC analysis to confirm the peak of each phosphorylated lipid.

PI 3-Kinase Activity Assay
3T3-L1 adipocytes infected with adenovirus were serum starved for 3 h in DMEM containing 0.2% BSA. Then, the cells were stimulated with 100 nM insulin for 15 min, washed once with ice-cold PBS, and lysed with PBS containing 1% Nonidet P-40, 0.35 mg/ml phenylmethylsulfonyl fluoride, and 100 mM sodium vanadate. Insoluble materials were eliminated by centrifugation, and the supernatants were immunoprecipitated with antiphosphotyrosine antibodies and protein G-Sepharose. The PI 3-kinase activity in the immunoprecipitates was measured as described previously (50).

Subcellular Fractionation of 3T3-L1 Adipocytes
The method of obtaining the total membrane fraction from 3T3-L1 adipocytes was as described previously (56) with some modifications. 3T3-L1 adipocytes were serum-starved for 3 h in DMEM containing 0.2% BSA and incubated with or without 100 nM insulin for the indicated times. Cells were washed twice with ice-cold HES buffer (20 mM HEPES, pH 7.4, 1 mM EDTA, 22.5 mM sucrose), and then detached from the dish using a rubber policemen and collected in 0.6 ml of homogenizing buffer (HES buffer containing 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 40 mM ß-glycerophosphate, 50 mM sodium fluoride). The cells were homogenized by passage through a 29-gauge needle 16 times. One tenth of the suspension was set aside, sonicated, and prepared for SDS-PAGE as the cell lysate. The remaining nine tenths of the suspension was centrifuged at 600 x g for 10 min to remove mainly nuclei and mitochondria as the sediment. The supernatant was centrifuged at 250,000 x g for 90 min at 4 C to precipitate the total membrane particulate fraction. The supernatant was removed, and the precipitate was resuspended with homogenizing buffer and again centrifuged at 250,000 x g for 90 min. After the supernatant had been removed, the precipitant was lysed with 50 µl of lysis buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 10 mM EDTA, 10% glycerol, 1% Nonidet P-40, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 40 mM ß-glycerophosphate, 50 mM sodium fluoride). The lysate was centrifuged at 15,000 x g for 10 min, and the supernatant was prepared for SDS-PAGE as the membrane fraction.

2-Deoxyglucose Uptake Assay
3T3-L1 adipocytes in a 12-well culture dish were serum starved for 3 h in DMEM containing 0.2% BSA, and glucose-free incubation was performed for 45 min in Krebs-Ringer phosphate buffer (15). Cells were then incubated with or without 100 NM insulin for 15 min, and 2-deoxy-D-[3H]-glucose uptake was measured as described previously (57).

Statistical Analysis
Data are expressed as means ± SE. Comparisons were made using the unpaired Student’s t test; P < 0.05 was considered a statistically significant difference. The representative immunoblots from one experiment were shown in some figures.


    FOOTNOTES
 
Abbreviations: ECL, Enhanced chemiluminescence; HES, 20 mM HEPES, pH 7.4, 1 mM EDTA, 22.5 mM sucrose; PI(3 )P, phosphatidylinositol 3-phosphate; PI(4 )P, phosphatidylinositol 4-phosphate; PI(3 4 )P2 , phosphatidylinositol 3,4-bisphosphate; PI(3 5 )P2, phosphatidylinositol 3,5-bisphosphate; PI(4 5 )P2, phosphatidylinositol 4,5-bisphosphate; PI(3 4 5 )P3, phosphatidylinositol 3,4,5-trisphosphate; PI 3-kinase, phosphoinositide 3-kinase; PDK1, 3-phosphoinositide-dependent kinase 1; PDK2, 3-phosphoinositide-dependent kinase 2; PTEN, phosphatase and tensin homolog deleted on chromosome 10.

Received for publication September 27, 2000. Accepted for publication May 15, 2001.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Burd CG, Emr SD 1998 Phosphatidylinositol(3)-phosphate signaling mediated by specific binding to RING FYVE domains. Mol Cell 2:157–162[Medline]
  2. Gaullier JM, Simonsen A, D’Arrigo A, Bremnes B, Stenmark H, Aasland R 1998 FYVE fingers bind PtdIns(3)P. Nature 394:432–433[CrossRef][Medline]
  3. Patki V, Lawe DC, Corvera S, Virbasius JV, Chawla A 1998 A functional PtdIns(3)P-binding motif. Nature 394:433–434[CrossRef][Medline]
  4. Schu PV, Takegawa K, Fry MJ, Stack JH, Waterfield MD, Emr SD 1993 Phosphatidylinositol 3-kinase encoded by yeast VPS34 gene essential for protein sorting. Science 260:88–91[Medline]
  5. Auger KR, Serunian LA, Soltoff SP, Libby P, Cantley LC 1989 PDGF-dependent tyrosine phosphorylation stimulates production of novel polyphosphoinositides in intact cells. Cell 57:167–175[Medline]
  6. Traynor-Kaplan AE, Thompson BL, Harris AL, Taylor P, Omann GM, Sklar LA 1989 Transient increase in phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol trisphosphate during activation of human neutrophils. J Biol Chem 264:15668–15673[Abstract/Free Full Text]
  7. Kucera GL, Rittenhouse SE 1990 Human platelets form 3-phosphorylated phosphoinositides in response to {alpha}-thrombin, U46619, or GTP {gamma} S. J Biol Chem 265:5345–5348[Abstract/Free Full Text]
  8. Pignataro OP, Ascoli M 1990 Epidermal growth factor increases the labeling of phosphatidylinositol 3,4-bisphosphate in MA-10 Leydig tumor cells. J Biol Chem 265:1718–1723[Abstract/Free Full Text]
  9. Kelly KL, Ruderman NB 1993 Insulin-stimulated phosphatidylinositol 3-kinase. Association with a 185-kDa tyrosine-phosphorylated protein (IRS-1) and localization in a low density membrane vesicle. J Biol Chem 268:4391–4398[Abstract/Free Full Text]
  10. Salim K, Bottomley MJ, Querfurth E, et al. 1996 Distinct specificity in the recognition of phosphoinositides by the pleckstrin homology domains of dynamin and Bruton’s tyrosine kinase. EMBO J 15:6241–6250[Abstract]
  11. Alessi DR, James SR, Downes CP, et al. 1997 Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase B{alpha}. Curr Biol 7:261–269[Medline]
  12. Yao R, Cooper GM 1995 Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor. Science 267:2003–2006[Medline]
  13. Leevers SJ, Weinkove D, MacDougall LK, Hafen E, Waterfield MD 1996 The Drosophila phosphoinositide 3-kinase Dp110 promotes cell growth. EMBO J 15:6584–6594[Abstract]
  14. Okada T, Sakuma L, Fukui Y, Hazeki O, Ui M 1994 Blockage of chemotactic peptide-induced stimulation of neutrophils by wortmannin as a result of selective inhibition of phosphatidylinositol 3-kinase. J Biol Chem 269:3563–3567[Abstract/Free Full Text]
  15. Cheatham B, Vlahos CJ, Cheatham L, Wang L, Blenis J, Kahn CR 1994 Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation. Mol Cell Biol 14:4902–4911[Abstract]
  16. Evans JL, Honer CM, Womelsdorf BE, Kaplan EL, Bell PA 1995 The effects of wortmannin, a potent inhibitor of phosphatidylinositol 3-kinase, on insulin-stimulated glucose transport, GLUT4 translocation, antilipolysis, and DNA synthesis. Cell Signal 7:365–376[CrossRef][Medline]
  17. Vanhaesebroeck B, Leevers SJ, Panayotou G, Waterfield MD 1997 Phosphoinositide 3-kinases: a conserved family of signal transducers. Trends Biochem Sci 22:267–272[CrossRef][Medline]
  18. Anderson RA, Boronenkov IV, Doughman SD, Kunz J, Loijens JC 1999 Phosphatidylinositol phosphate kinases, a multifaceted family of signaling enzymes. J Biol Chem 274:9907–9910[Free Full Text]
  19. Stack JH, DeWald DB, Takegawa K, Emr SD 1995 Vesicle-mediated protein transport: regulatory interac- tions between the Vps15 protein kinase and the Vps34 PtdIns 3-kinase essential for protein sorting to the vacuole in yeast. J Cell Biol 129:321–334[Abstract]
  20. Jhun BH, Rose DW, Seely BL, et al. 1994 Microinjection of the SH2 domain of the 85-kilodalton subunit of phosphatidylinositol 3-kinase inhibits insulin-induced DNA synthesis and c-fos expression. Mol Cell Biol 14:7466–7475[Abstract]
  21. Wennstrom S, Hawkins P, Cooke F, et al. 1994 Activation of phosphoinositide 3-kinase is required for PDGF-stimulated membrane ruffling. Curr Biol 4:385–393[Medline]
  22. Hara K, Yonezawa K, Sakaue H, et al. 1994 1-Phosphatidylinositol 3-kinase activity is required for insulin-stimulated glucose transport but not for RAS activation in CHO cells. Proc Natl Acad Sci USA 91:7415–7419[Abstract]
  23. Kotani K, Carozzi AJ, Sakaue H, et al. 1995 Requirement for phosphoinositide 3-kinase in insulin-stimulated GLUT4 translocation in 3T3–L1 adipocytes. Biochem Biophys Res Commun 209:343–348[CrossRef][Medline]
  24. Sakaue H, Ogawa W, Takata M, et al. 1997 Phosphoinositide 3-kinase is required for insulin-induced but not for growth hormone- or hyperosmolarity-induced glucose uptake in 3T3–L1 adipocytes. Mol Endocrinol 11:1552–1562[Abstract/Free Full Text]
  25. Mothe I, Delahaye L, Filloux C, Pons S, White MF, Van Obberghen E 1997 Interaction of wild type and dominant-negative p55PIK regulatory subunit of phosphatidylinositol 3-kinase with insulin-like growth factor-1 signaling proteins. Mol Endocrinol 11:1911–1923[Abstract/Free Full Text]
  26. Damen JE, Liu L, Rosten P, et al. 1996 The 145-kDa protein induced to associate with Shc by multiple cytokines is an inositol tetraphosphate and phosphatidylinositol 3,4,5-triphosphate 5-phosphatase. Proc Natl Acad Sci USA 93:1689–1693[Abstract/Free Full Text]
  27. Maehama T, Dixon JE 1998 The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem 273:13375–13378[Abstract/Free Full Text]
  28. Stambolic V, Suzuki A, de la Pompa JL, et al. 1998 Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN. Cell 95:29–39[Medline]
  29. Haas-Kogan D, Shalev N, Wong M, Mills G, Yount G, Stokoe D 1998 Protein kinase B (PKB/Akt) activity is elevated in glioblastoma cells due to mutation of the tumor suppressor PTEN/MMAC. Curr Biol 8:1195–1198[Medline]
  30. Li J, Yen C, Liaw D, et al. 1997 PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 275:1943–1947[Abstract/Free Full Text]
  31. Steck PA, Pershouse MA, Jasser SA, et al. 1997 Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers. Nat Genet 15:356–362[Medline]
  32. Whang YE, Wu X, Suzuki H, et al. 1998 Inactivation of the tumor suppressor PTEN/MMAC1 in advanced human prostate cancer through loss of expression. Proc Natl Acad Sci USA 95:5246–5250[Abstract/Free Full Text]
  33. Katagiri H, Asano T, Inukai K, et al. 1997 Roles of PI 3-kinase and Ras on insulin-stimulated glucose transport in 3T3–L1 adipocytes. Am J Physiol 272:E326–331
  34. Funaki M, Katagiri H, Kanda A, et al. 1999 p85/p110-type phosphatidylinositol kinase phosphorylates not only the D-3, but also the D-4 position of the inositol ring. J Biol Chem 274:22019–22022[Abstract/Free Full Text]
  35. Wu X, Hepner K, Castelino-Prabhu S, et al. 2000 Evidence for regulation of the PTEN tumor suppressor by a membrane-localized multi-PDZ domain containing scaffold protein MAGI-2. Proc Natl Acad Sci USA 97:4233–4238[Abstract/Free Full Text]
  36. Lee JO, Yang H, Georgescu MM, et al. 1999 Crystal structure of the PTEN tumor suppressor: implications for its phosphoinositide phosphatase activity and membrane association. Cell 99:323–334[Medline]
  37. Li DM, Sun H 1997 TEP1, encoded by a candidate tumor suppressor locus, is a novel protein tyrosine phosphatase regulated by transforming growth factor ß. Cancer Res 57:2124–2129[Abstract]
  38. Furnari FB, Lin H, Huang HS, Cavenee WK 1997 Growth suppression of glioma cells by PTEN requires a functional phosphatase catalytic domain. Proc Natl Acad Sci USA 94:12479–12484[Abstract/Free Full Text]
  39. Toker A, Cantley LC 1997 Signalling through the lipid products of phosphoinositide-3-OH kinase. Nature 387:673–676[CrossRef][Medline]
  40. Shepherd PR, Withers DJ, Siddle K 1998 Phosphoinositide 3-kinase: the key switch mechanism in insulin signalling. Biochem J 333:471–490[Medline]
  41. Coffer PJ, Jin J, Woodgett JR 1998 Protein kinase B (c-Akt): a multifunctional mediator of phosphatidylinositol 3-kinase activation. Biochem J 335:1–13[Medline]
  42. Hill MM, Clark SF, Tucker DF, Birnbaum MJ, James DE, Macaulay SL 1999 A role for protein kinase Bß/Akt2 in insulin-stimulated GLUT4 translocation in adipocytes. Mol Cell Biol 19:7771–7781[Abstract/Free Full Text]
  43. Summers SA, Whiteman EL, Cho H, Lipfert L, Birnbaum MJ 1999 Differentiation-dependent suppression of platelet-derived growth factor signaling in cultured adipocytes. J Biol Chem 274:23858–23867[Abstract/Free Full Text]
  44. Alessi DR, Downes CP 1998 The role of PI 3-kinase in insulin action. Biochim Biophys Acta 1436:151–164[Medline]
  45. Summers SA, Yin VP, Whiteman EL, Garza LA, Cho H, Tuttle RL, Birnbaum MJ 1999 Signaling pathways mediating insulin-stimulated glucose transport. Ann NY Acad Sci 892:169–186[Abstract/Free Full Text]
  46. Myers MP, Tonks NK 1997 PTEN: sometimes taking it off can be better than putting it on. Am J Hum Genet 61:1234–1238[CrossRef][Medline]
  47. Cantley LC, Neel BG 1999 New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway. Proc Natl Acad Sci USA 96:4240–4245[Abstract/Free Full Text]
  48. Dahia PL 2000 PTEN, a unique tumor suppressor gene. Endocr Relat Cancer 7:115–129[Abstract/Free Full Text]
  49. Myers MP, Pass I, Batty IH, et al. 1998 The lipid phosphatase activity of PTEN is critical for its tumor suppressor function. Proc Natl Acad Sci USA 95:13513–13518[Abstract/Free Full Text]
  50. Katagiri H, Asano T, Ishihara H, et al. 1996 Overexpression of catalytic subunit p110{alpha} of phosphatidylinositol 3-kinase increases glucose transport activity with translocation of glucose transporters in 3T3–L1 adipocytes. J Biol Chem 271:16987–16990[Abstract/Free Full Text]
  51. Nakashima N, Sharma PM, Imamura T, Bookstein R, Olefsky JM 2000 The tumor suppressor PTEN negatively regulates insulin signaling in 3T3–L1 adipocytes. J Biol Chem 275:12889–12895[Abstract/Free Full Text]
  52. 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]
  53. Balendran A, Casamayor A, Deak M, et al. 1999 PDK1 acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2. Curr Biol 9:393–404[CrossRef][Medline]
  54. Katagiri H, Asano T, Ishihara H, et al. 1992 Replacement of intracellular C-terminal domain of GLUT1 glucose transporter with that of GLUT2 increases Vmax and Km of transport activity. J Biol Chem 267:22550–22555[Abstract/Free Full Text]
  55. Serunian LA, Auger KR, Cantley LC 1991 Identification and quantification of polyphosphoinositides produced in response to platelet-derived growth factor stimulation. Methods Enzymol 198:78–87[Medline]
  56. Asano T, Shibasaki Y, Ohno S, et al. 1989 Rabbit brain glucose transporter responds to insulin when expressed in insulin-sensitive Chinese hamster ovary cells. J Biol Chem 264:3416–3420[Abstract/Free Full Text]
  57. Asano T, Takata K, Katagiri H, et al. 1992 Domains responsible for the differential targeting of glucose transporter isoforms. J Biol Chem 267:19636–19641[Abstract/Free Full Text]