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Originally published In Press as doi:10.1074/jbc.M600393200 on June 2, 2006

J. Biol. Chem., Vol. 281, Issue 31, 21588-21593, August 4, 2006
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Fine Tuning PDK1 Activity by Phosphorylation at Ser163*

Ramon A. Riojas{ddagger}, Chintan K. Kikani{ddagger}, Changhua Wang§, Xuming Mao§, Lijun Zhou{ddagger}, Paul R. Langlais{ddagger}, Derong Hu§, James L. Roberts§, Lily Q. Dong§||, and Feng Liu{ddagger}§1

From the Departments of {ddagger}Biochemistry, §Pharmacology, and ||Cellular and Structural Biology and the Barshop Center for Longevity and Aging Studies, University of Texas Health Science Center, San Antonio, Texas 78229

Received for publication, January 17, 2006 , and in revised form, May 22, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
3-Phosphoinositide-dependent protein kinase-1 (PDK1) mediates phosphorylation and activation of members of the AGC protein kinase family and plays an essential role in insulin signaling and action. However, whether and how PDK1 activity is regulated in cells remains largely uncharacterized. In the present study, we show that PDK1 undergoes insulin-stimulated and phosphatidylinositol 3-kinase-dependent phosphorylation at Ser244 in the activation loop and at a novel site: Ser163 in the hinge region between the two lobes of the kinase domain. Sequence alignment studies revealed that the residue corresponding to Ser163 of PDK1 in all other AGC kinases is glutamate, suggesting that a negative charge at this site may be important for PDK1 function. Replacing Ser163 with a negatively charged residue, glutamate, led to a 2-fold increase in PDK1 activity. Molecular modeling studies suggested that phosphorylated Ser163 may form additional hydrogen bonds with Tyr149 and Gln223. In support of this, mutation of Tyr149 to Ala is sufficient to reduce PDK1 activity. Taken together, our results suggest that PDK1 phosphorylation of Ser163 may provide a mechanism to fine-tune PDK1 activity and function in cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
3-Phosphoinositide-dependent kinase-1 (PDK1)2 is a member of the AGC kinase family (named for the similar Ser/Thr kinases, cAMP-dependent protein kinase, cGMP-dependent protein kinase and protein kinase C) and functions downstream of PI 3-kinase in the insulin signaling pathway. PDK1 phosphorylates many important kinases such as Akt/PKB, p70 S6 kinase, and protein kinase C isoforms in their respective activation loops and plays an important role in insulin-mediated biological events in cells (1, 2).

It remains to be determined whether and how PDK1 activity is regulated in cells. Unlike its substrates such as Akt and p70 S6 kinase, the in vitro kinase activity of PDK1 immunoprecipitated from cells is not significantly affected by prior treatment of the cells with either growth factors or pharmacological inhibitors of PI 3-kinase (3, 4). These results lead to the current view that PDK1 is a constitutively active kinase. However, PDK1 activity has been found to be stimulated by insulin (5, 6) or sphingosine (7), suggesting that the activity of the kinase may be regulated in certain cell types.

The molecular mechanisms that regulate PDK1 activity in cells remain unknown. Multiple phosphorylation sites have been identified in human PDK1, including Ser241 in the activation loop (Ser244 for mouse PDK1) (8, 9). While phosphorylation in the activation loop has been shown to be essential for PDK1 activity, it remains to be determined whether phosphorylation at this site is regulated in cells. In addition to Ser244, PDK1 undergoes growth factor-stimulated phosphorylation at additional site(s) (10), suggesting a potential mechanism for regulating PDK1 activity.

In the present study, we show that phosphorylation of endogenous mouse PDK1 at Ser244 is stimulated by insulin. In addition, we identified Ser163 in the catalytic domain of mouse PDK1 as a novel insulin-stimulated phosphorylation site. Together these data suggest that PDK1 activity can be stimulated in cells and phosphorylation plays an important role in the regulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—cDNAs encoding hemagglutinin- or Myc-tagged PDK1 were described previously (11). PDK1 mutants were generated by single-stranded site-directed mutagenesis using the Kunkel method (12).

Cell Lines—Chinese hamster ovary cells stably expressing the human insulin receptor (CHO-IR) were grown in Ham's F-12 medium (Invitrogen) supplemented with 10% newborn calf serum and 1% penicillin/streptomycin. The murine hepatocyte cell line, a gift of Dr. D. Accili (13), was maintained in {alpha}-minimal essential medium supplemented with 1 mM L-glutamine, 200 nM dexamethasone, and 4% fetal bovine serum at 33 °C with 5% CO2. GT1–7 cells are immortalized mouse hypothalamic neuronal cells and have a gonadotropin-releasing hormone-secreting phenotype (14). These cells were maintained in 1:1 Dulbecco's modified Eagle's medium/F-12 medium with 1% penicillin/streptomycin, and 10% fetal bovine serum. All cell culture experiments were conducted at 37 °C with 5% CO2.

Transfection, Immunoprecipitation, and Western Blot—Transfections were normally performed in 100- or 60-mm plates with 10 or 5 µg of recombinant plasmid per plate, respectively, using Lipofectamine or LipofectAmine2000 reagent according to the manufacturer's protocol (Invitrogen). Twenty-four hours after transfection, cells were serum-starved and treated with or without insulin as indicated in the figure legends. Cells were then lysed in buffer containing 50 mM Hepes, pH 7.6, 150 mM NaCl, 1% Triton X-100, 1 mM NaF, 20 mM sodium pyrophosphate, 20 mM beta-glycerol phosphate, 1 mM sodium orthovanadate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 µM microcystin-LR, and 1 mM phenylmethylsulfonyl fluoride. Homogenates were centrifuged (10,000 x g, 4 °C, 10 min), and supernatants were used for immunoprecipitation or Western analysis, using similar procedures as described previously (11). Antibodies to phospho-PDK1S241, phospho-AktT308, phospho-ERK1/2, and ERK1/2 were from Cell Signaling Technologies (Danvers, MA). Antibody to Akt was from BIOSOURCE (Camarillo, CA). Anti-PDK1 antibody was described previously (4).


Figure 1
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  		FIGURE 1.
GT1–7 cells are highly insulin (Ins) responsive. GT1–7 cells were serum-starved and treated with the indicated concentrations of insulin for 15 min. Whole cell lysates were analyzed by Western blotting with anti-phospho-AktS473 (upper panel) and anti-Akt (lower panel) (A) and anti-phospho-MAPK (upper panel) and anti-p44/42 MAPK (lower panel)(B).

 
In Vitro Kinase Assay—PDK1 in vitro activity assay was performed as described previously, using a synthetic peptide derived from the activation loop of Akt (KTFCGTPEYLAPEVRR) as a substrate (4). PDK1 kinase activity was normalized to PDK1 protein levels determined by Western blotting using the LI-COR Odyssey system (LI-COR Biosciences, Lincoln, NE).

In Vivo Labeling and Phosphoamino Acid Analysis—Radiolabeling of cells with [32P]orthophosphate and phosphoamino acid analysis were performed as reported previously (10).

Molecular Modeling—Various structures described in the text were obtained using their Protein Data Bank codes from the Protein Data Bank. Structural modulations were performed using Swiss-PDB viewer program (15) and were refined using Pymol (DeLano Scientific, San Carlos, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Insulin Stimulates Autophosphorylation of PDK1 at Ser244— We previously found that insulin had no significant effect on PDK1 phosphorylation at Ser244 when the kinase is overexpressed in cells (10). To exclude the possibility that the increased basal Ser244 phosphorylation was caused by overexpression, we studied endogenous PDK1 phosphorylation in mouse hypothalamic GT1–7 cells. As shown in Fig. 1, treating GT1–7 cells with 10 nM insulin is sufficient to induce significant phosphorylation of endogenous Akt (Fig. 1A) and MAPK (Fig. 1B). These results suggest that GT1–7 cells are insulin responsive and are an excellent cell system to study the effect of insulin on endogenous PDK1.


Figure 2
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  		FIGURE 2.
Endogenous PDK1 undergoes insulin-stimulated phosphorylation at Ser244. A, GT1–7 cells were serum-starved for 6 h and treated with 10 nM insulin for the time indicated. Endogenous proteins were analyzed by Western analyses with the anti-phospho-hPDK1S241 (homologous to mPDK1S244) antibody as described under "Materials and Methods" (upper panel). The membrane was stripped and reprobed with the anti-PDK1 antibody (lower panel). B, serum-starved GT1–7 cells were treated with 50 µM LY294002 or 50 nM wortmannin for 1 h and then with 10 nM insulin for 10 min. Proteins from cell lysates were analyzed by Western blot analyses with the anti-phospho-hPDK1S241 antibody (upper panel) or the anti-PDK1 antibody (lower panel). C, densitometry calculations were performed using the Scion Image software, and data are presented as the mean fold ± S.E. (n = 5, **, p < 0.01; *, p < 0.05, Student's t test).

 
We examined insulin-stimulated PDK1 phosphorylation in GT1–7 cells by Western blot using a phospho-specific antibody to Ser241 of human PDK1 (Ser244 of mouse PDK1). We found that insulin greatly stimulated mouse PDK1 phosphorylation at Ser244 in a time-dependent manner (Fig. 2A). The insulin-stimulated PDK1S244 phosphorylation was inhibited by two PI 3-kinase-selective inhibitors, LY294002 and wortmannin (Fig. 2B and C). Insulin-stimulated PDK1S244 phosphorylation was also observed in other cell lines such as 3T3-L1 and C2C12 cells, albeit at higher doses of insulin treatment (data not shown). Since phosphorylation of PDK1 at Ser244 is essential for its activity, these data provide evidence that the activity of endogenous PDK1 in intact cells can be stimulated. However, we found that in certain cell lines such as HEK293 cells, insulin has no significant effect on endogenous PDK1 phosphorylation at Ser244 (data not shown), suggesting that the stimulatory effect of insulin on PDK1 phosphorylation may be cell type-specific.

Insulin-stimulated Phosphorylation of PDK1 at a Site Other than Ser244—We attempted to determine whether insulin stimulates PDK1 phosphorylation at a site(s) in addition to Ser244. To this end, we generated a double mutant of PDK1 in which Lys114 in the ATP-binding site is replaced with Gly and Ser244 in the activation loop is replaced with Ala (PDK1K114G/S244A). In vivo labeling of mouse hepatocytes showed that insulin-stimulated PDK1K114G/S244A phosphorylation in a time-dependent manner (Fig. 3A). Unlike phosphorylation of wild-type PDK1 at Ser244 in GT1–7 cells, the insulin-stimulated phosphorylation of the PDK1K114G/S244A double mutant was not evident until 10 min. However, the insulin-stimulated PDK1K114G/S244A phosphorylation was also inhibited by pretreatment of cells with the PI 3-kinase inhibitor LY294002 (Fig. 3, B and C). Phosphoamino acid analyses revealed that the insulin-simulated PDK1K114G/S244A phosphorylation occurred exclusively on serine residues (Fig. 3D). Since PDK1K114G/S244A is kinase-inactive, these results suggest that insulin-stimulated phosphorylation of PDK1 is mediated by a heterologous kinase.


Figure 3
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  		FIGURE 3.
Insulin stimulates phosphorylation of PDK1 at a site other than Ser244 in a PI 3-kinase-dependent manner. A, mouse hepatocytes were transfected with Myc-tagged PDK1K114G/S244A, radiolabeled with [32P]orthophosphate, and treated with 100 nM insulin for the times indicated. PDK1K114G/S244A samples were immunoprecipitated and analyzed by autoradiography (upper panel) and Western blotting (WB) with anti-Myc antibodies (lower panel). Data are representative of three independent experiments. B, CHO-IR cells transiently expressing PDK1 were radiolabeled with [32P]orthophosphate and pretreated with or without 50 µM LY294002 and then with or without 10 nM insulin for 10 min. Myc-tagged PDK1 proteins were immunoprecipitated and analyzed by autoradiography (upper panel) and Western blotting with an anti-Myc antibody (lower panel). Data are representative of four independent experiments. C, densitometry analyses were performed using the Scion Image software (n = 4, *, p < 0.05, Student's t test). D, in vivo labeled PDK1K114G/S244A proteins from previous experiments were analyzed by phosphoamino acid analysis as described previously (10).

 
Identification of Ser163 as the Novel Insulin-stimulated Phosphorylation Site—Based on the finding that insulin-stimulated phosphorylation of PDK1K114G/S244A is inhibited by LY294002 (Fig. 3B), we hypothesized that a kinase downstream of PI 3-kinase, such as an AGC kinase, may play a role in phosphorylating PDK1. AGC kinases prefer substrates with a consensus phosphorylation sequence RXRXXS/T, where R is either Arg or Lys, X is any amino acid, and S/T is the potential phosphorylation site (16, 17). A sequence analysis suggested that there are several potential AGC kinase phosphorylation sites in mouse PDK1, including Ser50, Ser95, and Ser163. To test whether any of these sites is phosphorylated in cells in response to insulin stimulation, triple mutants of PDK1, including PDK1K114G/S244A/S50A, PDK1K114G/S244A/S95A, PDK1K114G/S244A/S163D, were generated. In vivo labeling of hepatocytes revealed that mutations at Ser50 or Ser95 had no effect on insulin-stimulated PDK1 phosphorylation. On the other hand, replacing Ser163 with Asp greatly reduced insulin-stimulated phosphorylation of PDK1 (Fig. 4A).

To provide further evidence that Ser163 of PDK1 is phosphorylated in cells, we replaced Ser163 with Thr (PDK1S163T). In vivo labeling and phosphoamino acid analyses revealed a small but notable threonine phosphorylation of the PDK1S163T mutant but not the wild-type PDK1 (Fig. 4B). These findings suggest that Ser163, which is conserved between mouse, human, and Drosophila PDK1 (Fig. 4C), is a potential insulin-stimulated phosphorylation site. Interestingly, all other AGC kinases have a glutamic acid residue at the site corresponding to Ser163 of mouse PDK1 (Fig. 4C), suggesting that a negative charge at this site may be important for PDK1 function.

Replacing Ser163 with a Negatively Charged Amino Acid Residue Increases PDK1 Activity—To investigate the potential role of PDK1 phosphorylation at Ser163, mutants of PDK1 were generated in which the residue was changed to either glutamic acid (S163E) to mimic phosphorylation or to alanine (S163A) to disrupt phosphorylation. Wild-type (PDK1WT), PDK1S163E, or PDK1S163A were expressed in CHO-IR cells, purified by immunoprecipitation, and analyzed by in vitro kinase assays using an Akt peptide as a substrate. PDK1S163E, but not PDK1S163A, displayed a 2-fold increase in kinase activity compared with PDK1WT (Fig. 5A). However, replacing Ser163 with alanine had no significant effect on PDK1 kinase activity, suggesting that phosphorylation at this site may play a role in fine tuning but not indispensable for PDK1 activity. We also examined whether mutation at Ser163 affects PDK1 activity in intact cells. Significantly more insulin-stimulated Akt phosphorylation was observed in cells overexpressing PDK1S163E compared with cells expressing PDK1WT or the PDK1S163A mutant (Fig. 5, B and C). Consistent with the finding that replacing Ser163 with alanine had no significant effect on PDK1 activity in vitro (Fig. 5A), overexpression of the PDK1S163A mutant did not suppress insulin-stimulated Akt phosphorylation in intact cells (Fig. 5, B and C), suggesting that this mutant does not function as a dominant negative in cells.


Figure 4
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  		FIGURE 4.
Insulin stimulates PDK1 phosphorylation at Ser163 in the kinase domain. A, mouse hepatocytes transiently expressing Myc-tagged double mutant (PDK1K114G/S244A)(lanes 1 and 2) or triple mutants of PDK1 (lanes 3–5) were radiolabeled with [32P]orthophosphate and then treated with or without 100 nM insulin for 10 min. PDK1 proteins were immunoprecipitated with an anti-Myc antibody and analyzed by autoradiography (upper panel) or Western blotting (WB) with an anti-Myc antibody (lower panel). B, in vivo labeled PDK1WT or PDK1S163T were detected by phosphoamino acid analysis and analyzed by using a PhosphorImager system (GE Healthcare). C, sequence alignment of mouse and human PDK1 with other AGC kinases.

 
Structural Modeling of PDK1S163E—To gain insight into how mutation of PDK1S163E may function to increase PDK1 activity, we employed molecular modeling using the crystal structure of PDK1WT (Protein Data Bank ID: 1H1W (18)). In the PDK1WT structure, Ser163 (human PDK1 Ser160) is located on the beta5-strand in the hinge region of the kinase domain. The backbone oxygen of Ser163 forms a hydrogen bond with the ATP molecule. Interestingly, we noticed a sulfate ion occupying electron density in proximity to Ser163, and the sulfate ion appears to form hydrogen bonds with the adjacent residues Tyr149 and Gln223 (hPDK1 Tyr146 and Gln220) (Fig. 6A). Following Ser163 to Glu mutation and energy minimization, the Glu side chain occupied the position equivalent to the sulfate ion and formed hydrogen bond contacts with Tyr149 and Gln223 (Fig. 6B). This model suggests that phosphorylation of Ser163 may stabilize the active conformation by contacting Tyr149 and Gln223. To test this, we mutated either Tyr149 or Gln223 to Ala and analyzed PDK1 kinase activity in vitro. Replacing either Tyr149 or Gln223 to Ala led to reduced PDK1 autophosphorylation in vitro (data not shown). In addition, a double mutation of Tyr149 to Ala and Ser163 to Glu (PDK1Y149A/S163E) was tested for its ability to phosphorylate Akt in hepatocytes. Compared with PDK1S163E, PDK1Y149A/S163E reduced the activity of PDK1 on Akt phosphorylation (Fig. 6, C and D), suggesting that the hydrogen bond contact between Glu163 and Tyr149 may be required for the increase in PDK1 activity.


Figure 5
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  		FIGURE 5.
Replacing Ser163 with glutamate increases PDK1 activity. A, PDK1 wild type (WT) or mutant proteins (S163E and S163A) were transiently expressed in CHO-IR cells and purified by immunoprecipitation. Purified PDK1 proteins were used for an in vitro kinase activity assay as described under "Materials and Methods." Data are shown as mean fold ± S.E. (n = 3, *, p < 0.05, Student's t test). B, mouse hepatocytes transiently expressing wild-type or mutants of PDK1 were serum-starved, treated with 10 nM insulin for 15 min, and lysed. Cell lysates were analyzed by Western blot using the indicated antibodies. C, Akt phosphorylation as shown in B was normalized to protein levels and quantified using the LI-COR Odyssey. Data are shown as mean fold ± S.E. (n = 4, *, p < 0.05, Student's t test).

 


Figure 6
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  		FIGURE 6.
Glu163 substitution may form additional hydrogen bonds with Tyr149. A, PDK1WT structure (Protein Data Bank ID: 1H1W) is displayed showing hydrogen bonds (dotted lines) with a sulfate ion and adjacent residues. B, structural modeling of Ser163 substituted with Glu suggests that hydrogen bonds (dotted lines) are formed with Tyr149 and Gln223. C, mouse hepatocytes transiently expressing the indicated mutants of PDK1 were serum-starved, treated with 10 nM insulin for 15 min, and lysed. Cell lysates were analyzed by Western blot using the indicated antibodies. D, Akt phosphorylation as shown in C was normalized to protein levels and quantified using the LI-COR Odyssey. Data are shown as mean fold ± S.E. (n = 3, *, p < 0.05, Student's t test).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
PDK1 is well characterized as a master kinase that activates multiple AGC kinases, yet how the kinase itself is regulated remains largely unknown. In the present study, we show that insulin stimulated autophosphorylation of endogenous PDK1 at Ser244 in GT1–7 cells (Fig. 2) as well as other cell lines such as 3T3-L1 and C2C12 cells, although at higher doses (data not show). While these results suggest that PDK1 activity may be stimulated in cells, it has previously been reported that in vitro kinase activity of PDK1 is not sensitive to insulin treatment (4, 9). One possible explanation for this discrepancy may be that PDK1 undergoes dimerization and autophosphorylation in vitro, which leads to auto-activation of the kinase during the in vitro kinase assay processes. Consistent with this idea, it has been shown that insulin stimulates endogenous PDK1 activity in mouse retina (19) and human skeletal muscle (6).

In addition to Ser244, we found that insulin stimulated PDK1 phosphorylation at Ser163. Our results suggest that a heterologous kinase is involved in insulin-stimulated phosphorylation of PDK1 at Ser163. However, the identity of this kinase is currently unknown. Phosphoamino acid analysis studies revealed that insulin-stimulated phosphorylation of PDK1K114G/S244A occurred primarily on serine residues (Fig. 3D), suggesting that a Ser/Thr kinase is involved in the phosphorylation. The sequence surrounding Ser163 is similar to the hydrophobic motifs of p70 S6 kinase and Akt (20), which are phosphorylated by mTOR (21, 22). Thus, one potential PDK1 Ser163 kinase is mTOR, which is activated by insulin and inhibited by PI 3-kinase inhibitors (23). However, we found that overexpression of mTOR did not significantly stimulate PDK1 phosphorylation (data not shown). Further studies are needed to characterize the serine kinase(s) that mediates insulin-stimulated PDK1 phosphorylation at Ser163.

To determine whether Ser163 is phosphorylated in cells, we replaced this residue with threonine and carried out in vivo labeling and phosphoamino acid analysis. Using a similar approach, we previously identified Ser244 as a trans-autophosphorylation site (10). We detected a low but notable insulin-stimulated threonine phosphorylation of the PDK1S163T mutant but not wild-type PDK1 (Fig. 4B). The low stoichiometry of PDK1S163T threonine phosphorylation is consistent with our previous findings that Ser244 and Ser399 are major phosphorylation sites (8, 10). However, we cannot exclude the possibilities that Ser163 is a better target than Thr163 for the heterologous kinase or that only a fraction of PDK1 is phosphorylated at this site in cells.

Structurally, PDK1S163 lies near the ATP-binding pocket and the equivalent residue in other AGC protein kinases is a negatively charged amino acid residue, glutamate (Fig. 4C). Computer modeling studies of a mutated PDK1 Ser163 to Glu suggest that additional hydrogen bonds are formed with Tyr149 on the beta4 strand and Gln223. Gln223 is located in the Mg2+-binding loop and is structurally conserved between most AGC kinases. According to our model, phosphorylation of Ser163 may stabilize the active conformation by forming hydrogen bonds with Tyr149 and Gln223. We found that replacing Ser163 with alanine had no significant effect on PDK1 activity, suggesting that phosphorylation at this site may act to fine-tune PDK1 activity and/or specificity.

In addition to insulin-stimulated phosphorylation of PDK1, other mechanisms of regulating PDK1 activity have been proposed. Insulin/IGF-1 treatments stimulate plasma membrane (24, 25) and nuclear (26, 27) localization of PDK1. Tyrosine phosphorylation has also been shown to activate PDK1 (28, 29). In vitro autophosphorylation of PDK1 is increased by deletion of the pleckstrin homology domain (8) or by interacting with the hydrophobic motifs of RSK2 or PKC{zeta} (30, 31), suggesting that substrate binding may induce allosteric activation of PDK1. More recently, it was found that STRAP (serine/threonine kinase receptor-associated protein) binds the kinase domain of PDK1 and stimulates its activity in vitro (32). Taken together, these findings suggest that PDK1 activity can be regulated by multiple mechanisms in vivo, which is not surprising since PDK1 plays a key role in the activation of numerous downstream kinases (2, 33).

In conclusion, our studies suggest that PDK1 activity is regulated at multiple levels and phosphorylation plays an important role in the regulation. In addition to autophosphorylation in the activation loop, PDK1 activity is also regulated by phosphorylation at Ser163 via a heterologous kinase. Phosphorylation of these sites may provide a novel mechanism to fine-tune PDK1 activity.


   FOOTNOTES
 
* This work was supported by a Research Award from the American Diabetes Association (to F. L.) and an National Institutes of Health Predoctoral Fellowship F31DK068874 (to R. A. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Pharmacology, UTHSCSA, 7703 Floyd Curl Dr., San Antonio, TX 78229. E-mail: liuf{at}uthscsa.edu.

2 The abbreviations used are: PDK1, 3-phosphoinositide-dependent protein kinase-1; PI, phosphatidylinositol; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; CHO, Chinese hamster ovary. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Domenico Accili for the murine hepatocyte cell line and Dr. P. John Hart and Dr. Alex Taylor for helpful discussions regarding the structural models.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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