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J. Biol. Chem., Vol. 277, Issue 19, 16632-16638, May 10, 2002

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Substitution of the Autophosphorylation Site Thr⁵¹⁶ with a Negatively Charged Residue Confers Constitutive Activity to Mouse 3-Phosphoinositide-dependent Protein Kinase-1 in Cells^*

Michael J. Wick, KeriLyn R. Wick^§, Hui Chen^¶, Huili He, Lily Q. Dong, Michael J. Quon^¶, and Feng Liu^**

From the Departments of  Pharmacology and  Biochemistry, The University of Texas Health Science Center, San Antonio, Texas 78229 and the ^¶ Cardiology Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, December 27, 2001, and in revised form, February 6, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

3-Phosphoinositide-dependent protein kinase-1 (PDK-1)is a serine/threonine kinase that has been found to phosphorylate and activate several members of the AGC protein kinase family including protein kinase B (Akt), p70 S6 kinase, and protein kinase C. However, the mechanism(s) by which PDK-1 is regulated remains unclear. Here we show that mouse PDK-1 (mPDK-1) undergoes autophosphorylation in vitro on both serine and threonine residues. In addition, we have identified Ser³⁹⁹ and Thr⁵¹⁶ as the major mPDK-1 autophosphorylation sites in vitro. Furthermore, we have found that these two residues, as well as Ser²⁴⁴ in the activation loop, are phosphorylated in cells and demonstrated that Ser²⁴⁴ is a major in vivo phosphorylation site. Abolishment of phosphorylation at Ser²⁴⁴, but not at Ser³⁹⁹ or Thr⁵¹⁶, led to a significant decrease of mPDK-1 autophosphorylation and kinase activity in vitro, indicating that autophosphorylation at Ser³⁹⁹ or Thr⁵¹⁶ is not essential for mPDK-1 autokinase activity. However, overexpression of mPDK-1^T516E, but not of mPDK-1^S244E or mPDK-1^S399D, in Chinese hamster ovary and HEK293 cells was sufficient to induce Akt phosphorylation at Thr³⁰⁸ to a level similar to that of insulin stimulation. Furthermore, this increase in phosphorylation was independent of the Pleckstrin homology domain of Akt. Taken together, our results suggest that mPDK-1 undergoes autophosphorylation at multiple sites and that this phosphorylation may be essential for PDK-1 to interact with and phosphorylate its downstream substrates in vivo.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

3-Phosphoinositide-dependent protein kinase-1 (PDK-1)¹ is a recently identified protein kinase that functions downstream from PI 3-kinase and upstream of protein kinase B (Akt) in receptor tyrosine kinase signal transduction pathways (1). PDK-1 phosphorylates Thr³⁰⁸ in the activation loop of Akt and contributes to the activation of the enzyme (2, 3). In addition to Akt, PDK-1 also phosphorylates and activates several other downstream effectors of PI 3-kinase including p70 S6 kinase (4, 5), protein kinase C isoforms (6-9), serum and glucocorticoid-inducible kinase (10-12), and protein kinase C-related kinases 1 and 2 (13, 14). These kinases play versatile roles in numerous cellular events; thus, the activation state of PDK-1 toward these substrates may play an important physiological role in the regulation of a variety of biological activities including cell proliferation, differentiation, and apoptosis.

Although the activation of several PDK-1 substrates has been characterized, the mechanism by which PDK-1 activity is regulated in cells is largely unknown. Phosphorylation of protein kinase C and protein kinase C by PDK-1 in vivo has been shown to be inhibited by the PI 3-kinase inhibitor LY294002 (6), suggesting that PDK-1 activity is mediated by a PI 3-kinase-dependent mechanism. Consistent with this finding, it has recently been found that insulin stimulates the activity of PDK-1 toward Akt and that this activation is blocked by wortmannin, another inhibitor of PI 3-kinase (15). However, others have found PDK-1 to be constitutively activated (4, 8), and PDK-1-mediated phosphorylation of p70 S6 kinase in cells has been shown to be unaffected by wortmannin treatment (5).

Although activation of PI 3-kinase may be important for the activity of PDK-1 toward some of its downstream substrates, differential phosphorylation of PDK-1 itself may also play an important role in regulating the kinase activity of the enzyme. A recent study identified Ser²⁵, Ser²⁴¹, Ser³⁹³, Ser³⁹⁶, and Ser⁴¹⁰ on human PDK-1 (hPDK-1) as phosphorylated sites in cells (17). Of these sites, phosphorylation at Ser²⁴¹ was found to be essential for hPDK-1 activity in vitro (17). However, the functional role(s) of PDK-1 phosphorylation at the other sites is unknown.

Recently, we found that purified mPDK-1 underwent significant autophosphorylation in vitro (8, 18). However, the identity of these sites and the extent to which phosphorylation of these residues played a role in PDK-1 function remained unclear. In the present study, we report the identification of two major in vitro autophosphorylation sites on mPDK-1: Thr⁵¹⁶ and Ser³⁹⁹. Interestingly, we found that Ser²⁴⁴ in the activation loop of mPDK-1 is only marginally phosphorylated in vitro. On the other hand, we demonstrate that Ser²⁴⁴, Ser³⁹⁹, and Thr⁵¹⁶ of mPDK-1 are phosphorylated in cells and that Ser²⁴⁴ is a major in vivo phosphorylation site. Overexpression of mPDK-1^T516E, but not mPDK-1^S244E, mPDK-1^S399D, or mPDK-1^T516A, resulted in Akt Thr³⁰⁸ phosphorylation to a level similar to that of insulin stimulation. Furthermore, this increase in phosphorylation is independent of the PH domain of Akt. Taken together, our results suggest that phosphorylation of PDK-1 at multiple sites plays distinct and important roles in the regulation of PDK-1 function in vivo under various cellular conditions.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Buffers-- Buffer A consisted of 50 mM Hepes, pH 7.6, 150 mM NaCl, and 0.1% Triton X-100. Buffer B consisted of 50 mM Hepes, pH 7.6, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 1 mM NaF, 1 mM sodium pyrophosphate, 1 mM Na₃VO₄, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride. Buffer C consisted of 50 mM Tris-HCl, pH 7.5, 5 mM MgCl₂, 1 mM Na₃VO₄, 1 mM sodium pyrophosphate, 1 mM NaF, and 1 mM phenylmethylsulfonyl fluoride.

Reagents-- cDNAs encoding Myc-tagged full-length mPDK-1, mPDK-1^PH, mPDK-1^A280V, Myc-tagged Akt-1, and FLAG-tagged full-length and Akt-1^PH were described previously (18). Polyclonal anti-Akt and anti-phospho-Akt (Thr³⁰⁸) antibodies were obtained from New England Biolabs. Monoclonal anti-Myc was obtained from Santa Cruz Biotechnology, Inc. The polyclonal antibody to PDK-1 was described previously (8).

Site-directed Mutagenesis-- All mPDK-1 mutants were generated using single-stranded site-directed mutagenesis according to the protocol described by Kunkel et al. (19) using customized primers. All constructs were verified by restriction mapping and DNA sequencing.

Cell Culture, Transfection, Immunoprecipitation, and Western Blot-- Chinese hamster ovary cells overexpressing the insulin receptor (CHO/IR), and human embryonic kidney (HEK293) cells were used in all experiments. Transfections were performed using LipofectAMINE (Invitrogen) according to the manufacturer's protocol. The cells were lysed in Buffer B. The cell lysates were centrifuged at 12,000 × g for 10 min at 4 °C, and the clarified supernatant was used for either immunoprecipitation or Western blot analysis. The proteins in cell lysates were immunoprecipitated by incubation with the primary antibody conjugated to protein G-Sepharose beads for 6-18 h at 4 °C. The immunoprecipitates were washed three times with ice-cold Buffer A. For immunoblot analysis, the cell lysates or immunoprecipitates were separated by SDS-PAGE using 10 or 15% polyacrylamide gels. Following electrophoresis, the proteins were transferred onto nitrocellulose membranes, and the bound proteins were detected by blotting with primary antibody, followed by horseradish peroxidase- or alkaline phosphatase-conjugated secondary antibodies (Promega, Madison, WI).

In Vitro Phosphorylation Assays-- CHO/IR cells transiently expressing Myc-tagged wild type or mutant PDK-1 were washed once with phosphate-buffered saline followed by serum starvation for 1 h at 37 °C. The cells were lysed in Buffer B, and the proteins were immunoprecipitated using antibody to the tag as described above. The bound protein was washed twice with ice-cold Buffer A and once with Buffer C. Phosphorylation was initiated with the addition of 30 µl of Buffer C plus 2 µCi of [-³²P]ATP (PerkinElmer Life Sciences) and incubated for 20 min at 30 °C. The bound protein was eluted by heating at 95 °C for 3 min in SDS-PAGE sample buffer. The proteins were separated by SDS-PAGE and blotted to nitrocellulose or polyvinylidene difluoride membrane. Phosphorylation of PDK-1 was visualized by autoradiography, and the bands corresponding to PDK-1 were excised and used in various assays.

mPDK-1 in Vitro Kinase Activity Assay-- CHO/IR cells overexpressing wild type or mutant mPDK-1 were washed with phosphate-buffered saline and serum starved for 1 h. The cells were lysed in Buffer B, and Myc-tagged PDK-1 was immunoprecipitated using antibody to the tag. Immunoprecipitates were washed twice with ice-cold Buffer A and once with Buffer C. The activity of mPDK-1 was determined by incubation of the immunoprecipitates with 40 µl of Buffer C containing 2 µCi of [-³²P]ATP and PDK1 substrate peptide corresponding to the sequence surrounding Thr³⁰⁸ of Akt-1 (KTFCGTPEYLAPEVRR) for 30 min at 30 °C. Phosphorylated peptide substrate was precipitated with trichloroacetic acid, spotted on p81 Whatman paper, and washed three times in 1% H₃PO₄. Peptide phosphorylation by PDK-1 was determined by scintillation counting.

In Vivo Metabolic ³²P Labeling of mPDK-1-- CHO/IR cells overexpressing Myc-tagged wild type or mutant PDK-1 were washed once and incubated with warmed Krebs-Ringer bicarbonate buffer for 30 min at 37 °C. The cells were labeled with 400-800 µCi of [³²P]orthophosphate (PerkinElmer Life Sciences) for 4 h and then left untreated or treated with 10 nM insulin for 5 min at 37 °C. The cells were lysed in Buffer B and immunoprecipitated as described above. The bound protein was washed two times with high and low salt wash buffers (20 mM Na₂HPO₄, pH 8.6, 0.5% Triton X-100, 0.1% SDS, 0.02% NaN₃, and either 1 or 0.15 M NaCl) and eluted by heating at 95 °C for 3 min in SDS-PAGE sample buffer. The proteins were separated by SDS-PAGE, blotted to nitrocellulose or polyvinylidene difluoride membrane, and utilized as described above.

Phosphopeptide and Phosphoamino Acid Analysis-- In vitro or metabolically ³²P-labeled proteins were separated by SDS-PAGE, transferred to nitrocellulose (phosphopeptide mapping) or polyvinylidene difluoride (phosphoamino acid analysis) membranes, and excised as described above. The bound protein was blocked with 0.5% polyvinylpyrrolidone-360 (Sigma) in 100 mM acetic acid for 30 min at 37 °C. The protein was washed twice with double distilled H₂O, washed once with 50 mM ammonium bicarbonate, and subjected to trypsin digest for 18 h at 37 °C. The digests were collected and lyophilized, and the samples were desalted using C₁₈ ZipTips (Millipore, Bedford, MA) according to a modified protocol by the manufacturer. The samples were loaded on TLC cellulose plates and subjected to electrophoresis and liquid chromatography as described previously (20). Phosphopeptides were visualized by autoradiography using x-ray film. Phosphoamino acid analysis was carried out using a protocol described previously (21).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

mPDK-1 Undergoes Autophosphorylation on Both Serine and Threonine Residues in Vitro-- We (8, 18) and others (22) have previously demonstrated that PDK-1 undergoes significant autophosphorylation in vitro. In an attempt to identify PDK-1 autophosphorylation sites, we carried out phosphoamino acid analysis and two-dimensional phosphopeptide mapping studies on in vitro phosphorylated wild type mPDK-1. Our results showed that mPDK-1 autophosphorylated in vitro on both serine and threonine residues at approximately a 1:1 ratio (Fig. 1A, left panel). No in vitro phosphorylation was observed for a kinase-inactive mutant of mPDK-1 (mPDK-1^K114G) (data not shown). Phosphopeptide analysis of wild type mPDK-1 revealed three major (Fig. 1A, right panel, 1-3) and nine minor (Fig. 1A, right panel, a-i) phosphopeptides, suggesting that PDK-1 autophosphorylates multiple sites in vitro. Phosphoamino acid analysis of phosphopeptides 1-3 revealed that phosphopeptides 1 and 2 contained exclusively phosphothreonine, whereas phosphopeptide 3 contained only phosphoserine (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Phosphoamino acid analysis and two-dimensional phosphopeptide mapping of in vitro autophosphorylated mPDK-1. A, mPDK-1 autophosphorylation occurs on both serine and threonine residues. Myc-tagged wild type mPDK-1 overexpressed in CHO/IR cells was immunoprecipitated and autophosphorylated in vitro in the presence of [-32P]ATP. In vitro autophosphorylated mPDK-1 was analyzed by phosphoamino acid analysis (PAA, left panel) or by two-dimensional mapping studies (2D, right panel). Phosphoamino acids and phosphopeptides were visualized by autoradiography. B and C, effect of Ser244 phosphorylation on mPDK-1 autophosphorylation (B) and kinase activity (C) in vitro. Myc-tagged wild type (WT) or Ser244 mutants of mPDK-1 were immunoprecipitated from serum-starved CHO/IR cells transiently expressing these proteins. Bound mPDK-1 proteins were autophosphorylated in vitro in the presence of [-32P]ATP (B) or assayed for kinase activity (C) as described under "Materials and Methods." mPDK-1 autophosphorylation was examined by autoradiography (Autorad, B, upper panel), and relative protein amounts were determined by Western blot (WB) analysis using antibody to the Myc tag (B, lower panel). D and E, phosphoamino acid analysis and two-dimensional mapping of autophosphorylated mPDK-1S244A (D) or mPDK-1S244T (E). Experiments were carried out as described in A. Phosphorylation of mPDK-1 Ser244 mutants was visualized by autoradiography. All of the results are representative of at least four experiments with similar results.

We have recently shown that Ser²⁴⁴ of mPDK-1 is important for PDK-1 function in cells (15). To determine whether Ser²⁴⁴ is an in vitro autophosphorylation site, we examined the phosphorylation of wild type and Ser²⁴⁴ mutants of mPDK-1 (Fig. 1B). We found that wild type mPDK-1 underwent significant autophosphorylation in vitro. Replacing Ser²⁴⁴ of mPDK-1 with alanine resulted in a dramatic decrease in the in vitro phosphorylation of the protein. This decrease in phosphorylation was partially recovered by replacing Ser²⁴⁴ with a negatively charged glutamate. Interestingly, substitution of Ser²⁴⁴ with a phosphorylatable threonine residue resulted in an overall increase in autophosphorylation compared with wild type. Replacing Ser²⁴⁴ of mPDK-1 with alanine also led to a significant decrease of the in vitro kinase activity of the enzyme, which was partially recovered by substitution of this residue with either glutamate or threonine (Fig. 1C). These findings further support Ser²⁴⁴ as an autophosphorylation site and demonstrate that phosphorylation at this site is important for mPDK-1 autokinase activity.

To further characterize the role of Ser²⁴⁴ in mPDK-1 in vitro autophosphorylation, we performed phosphoamino acid analysis and two-dimensional phosphopeptide mapping studies on in vitro autophosphorylated mPDK-1^S244A. Our results showed that although overall autophosphorylation of the mutant PDK-1 was significantly decreased compared with wild type (Fig. 1B), there was no significant change in the overall serine:threonine ratio (Fig. 1, D, left panel versus A, left panel), suggesting that Ser²⁴⁴ is not a major autophosphorylation site in vitro. Consistent with this, two-dimensional phosphopeptide mapping studies revealed that despite the significant decrease in overall phosphorylation of this mutant (Fig. 1B), mutating Ser²⁴⁴ to alanine did not abolish the in vitro autophosphorylation of the three major peptides (peptides 1-3) (Fig. 1, D, right panel versus A, right panel). On the other hand, replacing Ser²⁴⁴ of mPDK-1 with threonine led to a notable increase in threonine:serine autophosphorylation compared with the wild type enzyme (Fig. 1, E, left panel versus A, left panel). Two-dimensional phosphopeptide mapping studies of the mPDK-1^S244T mutant showed a significant increase in autophosphorylation of phosphopeptide e compared with wild type protein (Fig. 1, E, right panel versus A, right panel). Phosphoamino acid analysis of phosphopeptide e revealed that this peptide contained only phosphoserine for the wild type and exclusively phosphothreonine for the S244T mutant of mPDK-1 (data not shown). Taken together, these results identified phosphopeptide e of wild type mPDK-1 as a Ser²⁴⁴-containing peptide.

Identification of Ser³⁹⁹ as a Major Site of mPDK-1 Autophosphorylation in Vitro-- Because Ser²⁴⁴ of mPDK-1 was found to be only marginally phosphorylated in vitro, we attempted to identify the major mPDK-1 in vitro autophosphorylation sites. At least five serine residues in hPDK-1, including Ser²⁵, Ser²⁴¹, Ser³⁹³, Ser³⁹⁶, and Ser⁴¹⁰ were found phosphorylated in cells (17). To test whether the corresponding sites in mPDK-1 are phosphorylated in vitro, we generated mutants of mPDK-1 in which the corresponding sites, Ser²⁵, Ser²⁴⁴, Ser³⁹⁶, Ser ³⁹⁹, and Ser⁴¹³, were replaced with either alanine or glycine. Mutant mPDK-1 proteins were expressed in CHO/IR cells, purified by immunoprecipitation, and autophosphorylated in vitro in the presence of [-³²P]ATP. Phosphoamino acid analysis of the in vitro phosphorylated mPDK-1 mutants revealed that mutations at Ser²⁵, Ser²⁴⁴, Ser³⁹⁶, and Ser⁴¹³ had no significant effect on mPDK-1 in vitro autophosphorylation (data not shown). On the other hand, replacing Ser³⁹⁹ with glycine (mPDK-1^S399G) significantly decreased phosphoserine content compared with that of wild type (Fig. 2, left panel versus Fig. 1A, left panel). Phosphopeptide mapping studies revealed that mutation at this site resulted in the loss of phosphopeptide 3 (Fig. 2, right panel), implicating Ser³⁹⁹ of mPDK-1 as a major in vitro autophosphorylation site.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Identification of Ser399 as a major mPDK-1 serine autophosphorylation site in vitro. Myc-tagged mPDK-1S399G was immunoprecipitated from serum-starved CHO/IR cells transiently expressing the protein and autophosphorylated in vitro in the presence of [-32P]ATP. Phosphoamino acid analysis (PAA, left panel) and two-dimensional mapping studies (2D, right panel) were carried out as described the legend to in Fig. 1A and visualized by autoradiography. The results are representative of three experiments with similar results.

Identification of Thr⁵¹⁶ as a Major mPDK-1 in Vitro Autophosphorylation Site-- In an attempt to localize potential threonine autophosphorylation site(s), we examined autophosphorylation of a mPDK-1 mutant lacking the carboxyl-terminal PH domain (mPDK-1^PH, residues 1-459). Phosphoamino acid analysis revealed that deletion of the PH domain resulted in a complete abolishment of threonine autophosphorylation (Fig. 3A, left panel). Phosphopeptide mapping studies of mPDK-1^PH revealed a loss of both phosphopeptides 1 and 2 (Fig. 3A, right panel), suggesting that the major mPDK-1 threonine autophosphorylation site(s) might be localized in the PH domain.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Identification of Thr516 as a major mPDK-1 threonine autophosphorylation site in vitro. A, deletion of the PH domain abolished mPDK-1 threonine autophosphorylation. The PH domain-deleted mutant of mPDK-1 (mPDK-1PH) was immunoprecipitated from serum-starved CHO/IR cells transiently expressing the protein, autophosphorylated in vitro in the presence of [-32P]ATP and subjected to phosphoamino acid analysis (PAA, left panel) or two-dimensional phosphopeptide mapping studies (2D, right panel). B, diagram of the domain structure of mPDK-1 and potential threonine autophosphorylation sites in the PH domain. C, mutation of Thr516 to alanine-abolished mPDK-1 threonine autophosphorylation in vitro. Myc-tagged mPDK-1T516A was purified from serum-starved CHO/IR cells transiently expressing the protein, autophosphorylated in vitro in the presence of [-32P]ATP, and analyzed by phosphoamino acid analysis (left panel) or two-dimensional phosphopeptide mapping (right panel).

Sequence analysis of mPDK-1 revealed the presence of four threonine residues in the PH domain: Thr⁴⁸², Thr⁵¹⁶, Thr⁵²¹, and Thr⁵²⁵ (Fig. 3B). To identify potential mPDK-1 threonine autophosphorylation sites, we mutated these threonine residues individually to alanine. The mPDK-1 threonine mutants were then expressed in CHO/IR cells, purified by immunoprecipitation, and autophosphorylated in vitro in the presence of [-³²P]ATP. We found that mutations at Thr⁴⁸², Thr⁵²¹, and Thr⁵²⁵ had no significant effect on mPDK-1 autophosphorylation in vitro (data not shown). On the other hand, replacing Thr⁵¹⁶ with alanine significantly decreased phosphothreonine content compared with that of the wild type enzyme (Fig. 3C, left panel versus Fig. 1A, left panel). Interestingly, mutating Thr⁵¹⁶ to alanine resulted in the loss of both phosphothreonine-containing peptides 1 and 2 in the two-dimensional phosphopeptide map of this mutant (Fig. 3C, right panel). One possible explanation for this observation may be that two phosphopeptides were generated because of incomplete digestion of the Thr⁵¹⁶-containing tryptic peptide. Consistent with this idea, Thr⁵¹⁶ is immediately flanked on the amino terminus by a lysine residue (Lys⁵¹⁵). Phosphorylation at Thr⁵¹⁶ could potentially impair the ability of trypsin to cleave the peptide bond between Lys⁵¹⁵ and Thr⁵¹⁶, resulting in two Thr⁵¹⁶-containing phosphopeptides. To confirm this, we mutated Thr⁵¹⁶ of mPDK-1 to serine. mPDK-1^T516S was expressed in CHO/IR cells, immunoprecipitated, autophosphorylated in vitro, and subjected to two-dimensional phosphopeptide mapping analysis. Phosphopeptide mapping showed a significant decrease in phosphorylation at phosphopeptides 1 and 2. Subsequent phosphoamino acid analysis revealed that both phosphopeptides 1 and 2 contained exclusively phosphoserine (data not shown). These results provide further evidence that phosphopeptides 1 and 2 are due to incomplete tryptic digestion of the Thr⁵¹⁶-containing peptide.

Ser²⁴⁴, Ser³⁹⁹, and Thr⁵¹⁶ of mPDK-1 Are Phosphorylated in Cells-- To determine whether Ser²⁴⁴, Ser³⁹⁹, and Thr⁵¹⁶ are phosphorylated in cells, phosphopeptide mapping studies were performed on wild type and point-mutated mPDK-1 overexpressed and metabolically labeled in CHO/IR cells. Two-dimensional phosphopeptide mapping of the metabolically labeled wild type mPDK-1 revealed the presence of phosphopeptides 1-3 (Fig. 4, panel a), indicating that both Ser³⁹⁹ and Thr⁵¹⁶ are phosphorylated in intact cells. Consistent with the results from in vitro autophosphorylation and two-dimensional phosphopeptide mapping studies, mutation of Thr⁵¹⁶ to alanine resulted in a loss of both phosphopeptides 1 and 2 for the metabolically labeled mPDK-1 (Fig. 4, panel b). Similarly, mutating Ser³⁹⁹ to glycine resulted in the loss of phosphopeptide 3 (Fig. 4, panel c). Our results also showed phosphopeptide e to be heavily phosphorylated in vivo, indicating that Ser²⁴⁴ is one of the major phosphorylation sites in cells (Fig. 4). Phosphoamino acid analysis of phosphopeptide e recovered from the maps of metabolically labeled wild type or the S244T mutant of mPDK-1 indicated that this peptide contained solely phosphoserine or phosphothreonine, respectively (data not shown). Interestingly, phosphoamino acid analysis of phosphopeptides f and i recovered from the maps of metabolically labeled wild type or mPDK-1^S244T indicated that these two peptides also contained only phosphoserine or phosphothreonine, respectively (data not shown). These findings implicate Ser²⁴⁴ as a major phosphorylation site of mPDK-1 in cells and suggest that phosphopeptides e, f, and i are most likely due to incomplete digestion of Ser²⁴⁴-containing peptide. Interestingly, we found that the in vivo phosphorylation pattern of kinase-inactive PDK-1^K114G is similar to that of the wild type enzyme, suggesting that PDK-1 may undergo transphosphorylation in vivo.²

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Ser244, Ser399, and Thr516 of mPDK-1 are phosphorylated in cells. Myc-tagged, wild type (a) and T516A (b) and S399G (c) mutants of mPDK-1 were expressed in CHO/IR cells and metabolically labeled with [32P]orthophosphate, immunoprecipitated, and analyzed by two-dimensional phosphopeptide mapping as described under "Materials and Methods." The results are representative of four independent experiments with similar results.

Mutation at Ser²⁴⁴ and Thr⁵¹⁶ of mPDK-1 Affects Phosphorylation of mPDK-1 in CHO/IR Cells-- We have recently found that insulin stimulates mPDK-1 phosphorylation in NIH3T3 cells and rat adipocytes (15). To test whether a mutation at Ser³⁹⁹ or Thr⁵¹⁶ affects the overall phosphorylation of mPDK-1 in vivo, we examined basal and insulin-stimulated phosphorylation of wild type and mPDK-1 phosphorylation site mutants in CHO/IR cells. Consistent with our recent findings (15), insulin treatment resulted in a significant increase in mPDK-1 phosphorylation (Fig. 5A, lane 2 versus lane 1). Interestingly, we found that despite an overall decrease in phosphorylation, the mPDK-1^S244A mutant was still phosphorylated in cells, and the phosphorylation was enhanced in response to insulin stimulation (Fig. 5A, lanes 3 and 4 versus lanes 1 and 2). These findings suggest that insulin stimulates the phosphorylation at a site(s) in addition to or other than Ser²⁴⁴ in CHO/IR cells. Although mutating Ser³⁹⁹ to glycine or aspartate had no significant effect on mPDK-1 phosphorylation in vivo (data not shown), replacing Thr⁵¹⁶ of mPDK-1 with glutamate (Fig. 5A, lane 7 versus lane 2), but not alanine (Fig. 5A, lanes 5 versus lane 2), resulted in an increase in the basal phosphorylation of mPDK-1 to the same level as that of the insulin-stimulated wild type protein. Furthermore, this phosphorylation was not further increased following insulin treatment (Fig. 5A, lane 8 versus lane 7). Interestingly, the recently identified constitutively active mutant of mPDK-1, mPDK-1^A280V (18), also showed an enhanced and insulin-independent increase in basal phosphorylation (Fig. 5A, lanes 9 and 10 versus lanes 1 and 2). These findings suggest that phosphorylation of Thr⁵¹⁶ may result in a similar conformational change induced by the alanine to valine mutation at position 280, which subsequently leads to enhanced phosphorylation of mPDK-1.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Phosphorylation at Thr affects mPDK-1 phosphorylation and activity in cells. A, replacing Thr516 with glutamate leads to an increase in mPDK-1 phosphorylation in vivo. CHO/IR cells transiently expressing Myc-tagged wild type (WT) or various mutants of mPDK-1 were metabolically labeled with [32P]orthophosphate and then treated with (+) or without () insulin. 32P-Labeled mPDK-1 proteins were immunoprecipitated using antibody to the tag, separated by SDS-PAGE, and detected by autoradiography (upper panel). Expression of mPDK-1 proteins was determined by Western blot (WB) analysis using antibody to the tag (lower panel). B, overexpression of PDK-1T516E stimulates Akt phosphorylation at Thr308. CHO/IR co-expressing Akt and wild type (lanes 1 and 7) or mutant (lanes 3-6 and 8-12) mPDK-1 were serum-starved and then left untreated (lanes 1-6) or treated with insulin (lanes 7-12). The cell lysates were resolved by SDS-PAGE and transferred to nitrocellulose, and Akt Thr308 phosphorylation was determined by Western blot using a phospho-specific antibody to Thr308 (top panel). The expression of Akt (middle panel) and PDK-1 (bottom panel) was determined by Western blot analysis using antibodies specific to these proteins. C, mPDK-1T516E-mediated phosphorylation of Thr308 does not require the PH domain of Akt. FLAG-tagged full-length (lanes 1-4) or PH domain-deleted mutant (lanes 5-8) of Akt were transiently expressed in CHO/IR cells together with either wild type or mutants of mPDK-1. The cells were serum-starved and treated with (+) or without () insulin. Akt phosphorylation at Thr308 was determined by Western blot using a phospho-specific antibody. The expression of FLAG-tagged Akt, PH-Akt, and Myc-tagged PDK-1 was determined by Western blot using antibodies to FLAG (middle panel) or PDK-1 (bottom panel), respectively.

Mutating Thr⁵¹⁶ of mPDK-1 to Glutamate Constitutively Activates mPDK-1 in Cells-- To determine whether autophosphorylation plays a role in the function of mPDK-1, we examined the kinase activity of mPDK-1, both in vitro and in cells. In vitro, we found that mutation of Ser³⁹⁹ of mPDK-1 to glycine or aspartate, or Thr⁵¹⁶ to alanine or glutamate, had no significant effect of mPDK-1 kinase activity (data not shown). To examine the effect of PDK-1 autophosphorylation on its kinase activity in cells, phosphorylation of the PDK-1 substrate Akt by wild type and autophosphorylation mutants was examined. As expected, insulin treatment resulted in a significant increase in Akt phosphorylation at Thr³⁰⁸ (Fig. 5B, lane 7 versus lane 1). Although co-expression of mPDK-1^S244E or mPDK-1^S399D mutants had no effect on Akt phosphorylation at Thr³⁰⁸ under basal conditions (Fig. 5B, lanes 2 and 3 versus lane 1), co-expression of mPDK-1^T516E resulted in a significant increase in Akt phosphorylation to a level similar to that induced by insulin stimulation (Fig. 5B, lane 5 versus lane 7). Under this condition, only a small increase in Akt phosphorylation at Thr³⁰⁸ was observed in cells co-expressing mPDK-1^T516A (Fig. 5B, lane 4 versus lane 1), suggesting that a negatively charged residue at this site plays a critical role in promoting PDK-1 to phosphorylate Akt in cells. Furthermore, insulin stimulation did not increase the elevated Akt Thr³⁰⁸ phosphorylation in cells co-expressing mPDK-1^T516E (Fig. 5B, lane 11 versus lane 5). Similar results were obtained for mPDK-1^T516E co-expressed with Akt in CHO and HEK-293 cells (data not shown). In agreement with our recent study (18), co-expression of mPDK-1^A280V with Akt also resulted in a similar increase in basal Akt phosphorylation at Thr³⁰⁸ (Fig. 5B, lane 6).

Previous studies have suggested that translocation of Akt to the plasma membrane is required for full phosphorylation and activation of the enzyme (23, 24). Studies have also shown that this translocation requires an intact PH domain (25). To better understand the mechanism by which autophosphorylation of PDK-1 regulates its activation of downstream substrates, we co-expressed FLAG-tagged wild type Akt-1 or the PH domain deletion mutant (Akt-1^PH) with either wild type or mutant mPDK-1 in CHO/IR cells. Insulin stimulation (Fig. 5C, lane 2 versus lane 1) or co-expression of either mPDK-1^T516E (Fig. 5C, lane 3 versus lane 1) or mPDK-1^A280V (Fig. 5C, lane 4 versus lane 1) resulted in significant Akt phosphorylation at Thr³⁰⁸. The PH domain-deleted mutant of Akt was not phosphorylated on Thr³⁰⁸ in response to insulin stimulation (Fig. 5C, lane 6). However, co-expression of PDK-1^T516E was sufficient to restore Akt-1^PH phosphorylation at Thr³⁰⁸ to the same level as that of insulin-stimulated, full-length Akt (Fig. 5C, lane 7 versus lane 2). It is of interest to note that the insulin-independent and Akt PH domain-independent phosphorylation of Akt at Thr³⁰⁸ can be mediated by both PDK-1^T516E and PDK-1^A280V mutants (Fig. 5, B, lanes 5 and 6 and C, lanes 7 and 8). These data suggest that substituting Val²⁸⁰ with alanine or Thr⁵¹⁶ with glutamate may lead to a similar conformational change of mPDK-1, which is essential for interaction and phosphorylation of downstream substrates such as Akt in cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The mechanism(s) by which PDK-1 activity is regulated has remained, for a large part, unclear. Subcellular localization and substrate conformation have been hypothesized as the primary means for regulating PDK-1 activity enzyme (22, 26, 27); however, some studies have indicated that phosphorylation of PDK-1 may also play a role (8, 15). PDK-1 is phosphorylated both in vitro and in cells (2, 17), and treatment of PDK-1-expressing cells with insulin or hydrogen peroxide has been shown to increase phosphorylation of PDK-1 on specific residues (15, 28). Several studies have focused on Ser²⁴⁴ (mPDK-1) or Ser²⁴¹ (hPDK-1) in the activation loop as a critical phosphorylation site, because phosphorylation upon this residue is essential for PDK-1 kinase activity (15, 17). Some studies have indicated that phosphorylation at Ser²⁴¹ is not regulated by growth factor stimulation (17). On the other hand, we have found that in certain cell types, mPDK-1 phosphorylation and activity increase following insulin stimulation in a Ser²⁴⁴-dependent manner (15). However, in CHO/IR cells, we found that the mPDK-1^S244A mutant still underwent insulin-stimulated phosphorylation (Fig. 5A). These findings suggest that insulin-stimulated phosphorylation of mPDK-1 occurs at sites other than or in addition to Ser²⁴⁴, and this phosphorylation may function in conjunction with Ser²⁴⁴ to regulate mPDK-1 activity in cells.

In the present study, we identify Ser³⁹⁹ and Thr⁵¹⁶ on mPDK-1 as two major in vitro autophosphorylation sites, which can also be phosphorylated in cells. This finding is in agreement with a recent study by Park et al. (28), who showed the Thr⁵¹⁶-corresponding site in hPDK-1 to be phosphorylated in vitro. However, our results also show that phosphorylation at these sites may play different roles in PDK-1 function in cells. Indeed, substitution of Thr⁵¹⁶ with glutamate increases basal PDK-1 phosphorylation in cells to that seen with insulin stimulation, whereas replacing Ser³⁹⁹ with aspartate does not significantly affect either basal or insulin-stimulated phosphorylation of the enzyme. In addition, overexpression of mPDK-1^T516E, but not mPDK-1^S399D, led to Akt phosphorylation at Thr³⁰⁸ to the same level as that induced by insulin treatment. Taken together, these findings suggest that autophosphorylation at Thr⁵¹⁶, but not Ser³⁹⁹, regulates PDK-1 phosphorylation upon additional residues in cells and subsequently functions to regulate the activity of PDK-1 toward its substrates.

Previous studies have shown that hPDK-1 isolated from cells is constitutively active in vitro, presumably as a result of autophosphorylation upon Ser²⁴¹ (4, 8, 22). In agreement with this, we have found that a mutation of the corresponding Ser²⁴⁴ on mPDK-1 renders the kinase inactive in vitro (Fig. 1C) and in cells (Ref. 15 and data not shown). Interestingly, although we found the kinase activity of mPDK-1 to be significantly reduced by a mutation at Ser²⁴⁴, we found the ratio of serine:threonine autophosphorylation unchanged in its absence. This suggests that Ser²⁴⁴ is not a major site for in vitro autophosphorylation. Low autophosphorylation at Ser²⁴⁴ may result from phosphorylation at this site in cells that remains stable in vitro. In support of this, phosphorylation at Ser²⁴⁴ is less stable if the residue is replaced with threonine, resulting in an increase in autophosphorylation at this site in vitro (Fig. 1B). Although Ser²⁴⁴ is a minor phosphorylation site, phosphopeptide maps of metabolically labeled wild type and S244T mutant of mPDK-1 indicated that Ser²⁴⁴ is highly phosphorylated in CHO/IR cells, which is in agreement with our recent studies showing that Ser²⁴⁴ is a major phosphorylation site in NIH-3T3 cells (15). In contrast, autophosphorylation upon the major in vitro sites, Ser³⁹⁹ and Thr⁵¹⁶, is less stable in cells, which indicates that phosphorylation upon these residues is reversible and may be tightly regulated in cells.

Insulin stimulation has been shown to increase PDK-1 phosphorylation in both NIH-3T3 and rat adipose cells (15). Consistent with this, we observed insulin-stimulated phosphorylation of mPDK-1 when overexpressed in CHO/IR cells (Fig. 5A); however, we found that this insulin-stimulated increase could occur in the absence of Ser²⁴⁴ phosphorylation. Mutations at Ser³⁹⁹ and Thr⁵¹⁶ did not significantly affect insulin-stimulated mPDK-1 phosphorylation (Fig. 5A and data not shown), suggesting the presence of other insulin-stimulated phosphorylation site(s). On the other hand, replacement of Thr⁵¹⁶ with a negatively charged glutamate residue resulted in a significant increase in basal phosphorylation. The increase in basal mPDK-1^T516E phosphorylation also correlates with an increase in mPDK-1^T516E-mediated phosphorylation of Akt at Thr³⁰⁸ (Fig. 5B), which is in agreement with our recent finding that insulin-stimulated phosphorylation of mPDK-1 correlates with an increase in activity (15).

It has been previously shown that growth factor-mediated phosphorylation and activation of Akt requires a translocation event via an intact PH domain (23-25, 29). Recently, we (18) and others (22, 30) have shown that phosphorylation at Thr³⁰⁸ of Akt by PDK-1 is facilitated by an intact PDK-1-PH domain, whereas others have suggested that translocation of PDK-1 to the membrane may play a role in the phosphorylation and activation of the enzyme (31). In the present study, we have found that mutating Thr⁵¹⁶ to glutamate allows PDK-1 to phosphorylate Thr³⁰⁸ not only in a growth factor-independent fashion but also without the requirement for the PH domain of Akt (Fig. 5C). These data suggest that the T516E mutation of mPDK-1 bypasses the normal requirement for translocation and allows it to interact with and subsequently phosphorylate Akt at Thr³⁰⁸. One possible explanation for this observation may be that the introduction of a negatively charged residue at Thr⁵¹⁶ results in a conformational change in mPDK-1, which normally occurs after growth factor-mediated translocation of the enzyme to the plasma membrane. This resulting conformation may result in an increase in autophosphorylation at Ser²⁴⁴ and a subsequent increase in mPDK-1 activity. Consistent with this, a mPDK-1^S244A/T516E mutant was unable to increase phosphorylation of mPDK-1 or Thr³⁰⁸ of Akt in cells (data not shown), suggesting that phosphorylation at Thr⁵¹⁶ modifies activity of mPDK-1 through a Ser²⁴⁴ phosphorylation-dependent mechanism.

In Caenorhabditis elegans, a natural point mutation (A277V) allows the mammalian PDK-1 homolog to bypass the need for PI 3-kinase for its activation (16). We recently reported that a corresponding mutation in mPDK1 (A280V) resulted in an increase in the in vitro autophosphorylation of mPDK-1 and constitutive activation of the kinase toward Akt in cells (18). Whereas this mutation has not been found to occur naturally in higher species, it is interesting to note the similarity between mPDK-1^A280V and mPDK-1^T516E. We found that although neither had any significant effect on mPDK-1 activity in vitro (data not shown), both elevated mPDK-1 basal phosphorylation in cells (Fig. 5A). Furthermore, overexpression of either mPDK-1^A280V or mPDK-1^T516E was sufficient to elevate Akt phosphorylation at Thr³⁰⁸ in a growth factor- and Akt PH domain-independent manner (Fig. 5, B and Fig. C). These findings suggest that each mutation may result in a conformational change and/or cellular relocalization of mPDK1, which allows the enzyme to interact with and phosphorylate its downstream substrates such as Akt.

In conclusion, our results suggest that phosphorylation at Thr⁵¹⁶ plays an important role in the regulation of mPDK-1 in vivo activity toward its substrates. Although it remains to be established whether phosphorylation upon Thr⁵¹⁶ is directly regulated by insulin stimulation, autophosphorylation at this site may specifically direct the extent of phosphorylation at other site(s) and subsequently the activity of mPDK-1 in response to insulin or other growth factors. Autophosphorylation at Thr⁵¹⁶ may be specifically regulated in cells by various cellular events, such as conformational changes of the kinase following growth factor stimulation. Alternatively, Thr⁵¹⁶ may be constitutively autophosphorylated in cells, and this phosphorylation may be tightly controlled by a serine/threonine phosphatase, allowing dynamic regulation of this pivotal kinase in response to growth factor stimulation. Further studies will be needed to test these possibilities.

	FOOTNOTES

^* This study is supported by National Institutes of Health Grants DK56166 (to F. L. and L. Q. D.) and a Research Award from the American Diabetes Association (to F. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Supported by a National Institutes of Health Postdoctoral Training Grant 2T32AG00205-11.

^** To whom correspondence should be addressed. E-mail: liuf@uthscsa.edu.

Published, JBC Papers in Press, February 27, 2002, DOI 10.1074/jbc.M112402200

² Wick, M. J., and Liu, F., manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: PDK-1, 3-phosphoinositide-dependent protein kinase-1; mPDK-1, mouse PDK-1; hPDK-1, human PDK-1; CHO, Chinese hamster ovary; IR, insulin receptor; PH, pleckstrin homology, PI, phosphatidylinositol.

	REFERENCES

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