PDK1 Acquires PDK2 Activity in the Presence of a Synthetic Peptide

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Research Paper 393

PDK1 acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2 Anudharan Balendran*†, Antonio Casamayor*†, Maria Deak†, Andrew Paterson†, Piers Gaffney‡, Richard Currie§, C. Peter Downes§ and Dario R. Alessi†

Background: Protein kinase B (PKB) is activated by phosphorylation of Thr308 Addresses: *MRC Protein Phosphorylation Unit, and of Ser473. Thr308 is phosphorylated by the 3-phosphoinositide-dependent Department of Biochemistry, University of Dundee, ‡ protein kinase-1 (PDK1) but the identity of the kinase that phosphorylates Dundee DD1 5EH, UK. Ludwig Institute of Cancer Research, London W1P 8BY, UK. §Department of Ser473 (provisionally termed PDK2) is unknown. Biochemistry, University of Dundee, Dundee DD1 5EH, UK. Results: The kinase domain of PDK1 interacts with a region of protein kinase C-related kinase-2 (PRK2), termed the PDK1-interacting fragment (PIF). PIF is Correspondence: Dario R. Alessi E-mail: [email protected] situated carboxy-terminal to the kinase domain of PRK2, and contains a consensus motif for phosphorylation by PDK2 similar to that found in PKBα, †A.B. and A.C. contributed equally to this work. except that the residue equivalent to Ser473 is aspartic acid. Mutation of any of the conserved residues in the PDK2 motif of PIF prevented interaction of PIF Received: 12 January 1999 Revised: 18 February 1999 with PDK1. Remarkably, interaction of PDK1 with PIF, or with a synthetic Accepted: 3 March 1999 peptide encompassing the PDK2 consensus sequence of PIF, converted PDK1 from an enzyme that could phosphorylate only Thr308 of PKBα to one that Published: 8 April 1999 phosphorylates both Thr308 and Ser473 of PKBα in a manner dependent on Current Biology 1999, 9:393–404 phosphatidylinositol (3,4,5) trisphosphate (PtdIns(3,4,5)P3). Furthermore, the http://biomednet.com/elecref/0960982200900393 interaction of PIF with PDK1 converted the PDK1 from a form that is not directly © Elsevier Science Ltd ISSN 0960-9822 activated by PtdIns(3,4,5)P3 to a form that is activated threefold by PtdIns(3,4,5)P3. We have partially purified a kinase from brain extract that α phosphorylates Ser473 of PKB in a PtdIns(3,4,5)P3-dependent manner and that is immunoprecipitated with PDK1 antibodies.

Conclusions: PDK1 and PDK2 might be the same enzyme, the substrate specificity and activity of PDK1 being regulated through its interaction with another protein(s). PRK2 is a probable substrate for PDK1.

Background carboxy-terminal tail. The catalytic domain of PKB belongs The first steps in insulin signal transduction lead to the to the AGC subfamily of protein kinase domains (where activation of phosphoinositide (PI) 3-kinase and the for- AGC is short for ‘the cyclic AMP-dependent protein kinase, mation of phosphatidylinositol (3,4,5) trisphosphate cyclic GMP-dependent protein kinase and protein kinase C

(PtdIns(3,4,5)P3) at the plasma membrane [1–3], which family) and was originally identified because of its similarity can then be converted to PtdIns(3,4)P2 [4] or to to that of protein kinase C (PKC) isoforms and protein PtdIns(4,5)P2 [5,6] by specific phosphatases. It has been kinase A (PKA) [13]. Recent work has led to a greater established that PtdIns(3,4,5)P3 (and/or PtdIns(3,4)P2) is a understanding of the mechanism by which PKB is acti- key second messenger for insulin signalling (reviewed in vated. PKB interacts (through its PH domain) with

[1–3]). Protein kinase B (PKB, also known as Akt) is PtdIns(3,4,5)P3, the product of PI 3-kinase activation. This rapidly activated in response to insulin and its activation is results in the translocation of PKB to the plasma membrane prevented by inhibitors of PI 3-kinase [7]. Further studies but does not directly stimulate PKB activity [2,3,13]. have revealed that PKB is likely to mediate many of the Instead, PKB is activated at the membrane by the phospho- effects of insulin in cells, including the stimulation of rylation of two residues, Thr308 and Ser473. Both of these glycogen synthesis in skeletal muscle [8], the stimulation residues need to be phosphorylated for maximal activation of glycolysis in cardiac muscle [9] and certain effects that of PKB, and their phosphorylation in vivo is prevented by insulin has on protein synthesis [10–12]. inhibitors of PI 3-kinase [14]. Thr308 is located between subdomains VII and VIII of the kinase domain, a region in PKB contains an amino-terminal pleckstrin-homology (PH) which many kinases that are activated by phosphorylation domain followed by a kinase catalytic domain and then a are phosphorylated. Ser473 is located carboxy-terminal to 394 Current Biology, Vol 9 No 8

the catalytic domain, in a region that nevertheless displays and Ser473 being regulated through its interaction with high homology between different AGC family members. other cellular components. Interestingly, other members of the AGC subfamily of protein kinases, including p70 S6 kinase [15], PKC isoforms Results [16] and the serum-and glucocorticoid-inducible kinase The carboxy-terminal region of PRK2 interacts specifically (SGK) [17,18] also possess residues lying in equivalent with PDK1 sequences to Thr308 and Ser473 of PKB, and phosphoryla- A yeast two-hybrid screen was carried out to identify pro- tion of these residues is necessary for activation of these teins expressed in human skeletal muscle that interact kinases in vivo. The residues equivalent to Thr308 lie in a with PDK1. We identified a clone corresponding to the Thr–Phe–Cys–Gly–Thr–Xaa–Glu–Tyr consensus motif carboxy-terminal 77 amino acids of PRK2 that yielded a (where the italicised Thr corresponds to Thr308 and Xaa is positive interaction with full-length PDK1 as well as with a variable residue). The residues surrounding Ser473 lie in a the isolated kinase domain (residues 51–404), but not with Phe–Xaa–Xaa–Phe–Ser/Thr–Phe/Tyr consensus motif. We the PH domain of PDK1 (Figure 1a–d). We have termed [19,20] and others [21,22] have identified a protein kinase this region of PRK2 the ‘PDK1-interacting fragment’ termed 3-phosphoinositide-dependent protein kinase-1 (PIF). PRK2 is a Rho-dependent and lipid-dependent

(PDK1) which, in the presence of PtdIns(3,4,5)P3 or protein kinase [29,30] that, like PDK1 and PKB, belongs PtdIns(3,4)P2, phosphorylates PKB at Thr308. Subsequent to the AGC subfamily of protein kinases. The PIF region work has also shown that PDK1 phosphorylates the equiva- in PRK2 lies immediately carboxy-terminal to the kinase lent residues on PKC isoforms [23–25], p70 S6 kinase catalytic domain, in a region where there is high sequence [26,27], SGK [17] and PKA [28]. The finding that PDK1 homology between members of the AGC subfamily does not phosphorylate PKB significantly at Ser473, in vitro (Figure 1e). Most notably, the carboxyl terminus of PIF or in cotransfection experiments, suggested that a distinct contains the consensus sequence for PDK2 phosphoryla- protein kinase, which has provisionally been termed PDK2, tion (Phe–Xaa–Xaa–Phe–Ser/Thr–Phe/Tyr), except that might catalyse this reaction [19]. Here, we present findings in PRK2 the Ser/Thr residue equivalent to Ser473 of PKB that indicate this might not be the case. In particular, we is replaced with a negatively charged aspartic acid residue demonstrate that PDK1 can be converted to a form that (Asp978), perhaps mimicking a phosphorylated state. phosphorylates both Ser473 and Thr308 via interaction with a small peptide corresponding to the carboxy-terminal In order to confirm that PDK1 interacted with PIF, we region of protein kinase C-related protein kinase-2 (PRK2). expressed PIF as a glutathione-S-transferase (GST) fusion These observations suggest that PDK1 and PDK2 may be protein in 293 cells (human embryonic kidney cells) the same enzyme, the specificity of PDK1 towards Thr308 together with Myc-epitope-tagged PDK1. The cells were

Figure 1

Two-hybrid interaction of PIF with the kinase domain of PDK1. (a) Yeast were (a) PH domain PH domain (b) transformed with a pGAD10 plasmid (LEU2) + PIF – ∆ ∆ harbouring the carboxy-terminal 77 amino – PH-PDK1 PH-PDK1 Growth on acids of PRK2 (termed PIF) in the presence PIF + PIF – SD –Leu –Trp of either a pAS2-1 plasmid (TRP1) coding PDK1 PDK1 for the expression of the wild-type PDK1, the + PIF – kinase domain of PDK1 (∆PH-PDK1; residues 1–404) or the PH domain of PDK1 (c) (d) (residues 428–556) or the empty pAS2-1 Growth on β-galactosidase vector as a control (denoted by –). The SD –Leu –Trp activation position on the plate of each yeast strain is –Ura –His +3-AT indicated. In (b) the yeast are grown on SD Leu- and Trp-deficient media in which all the strains of yeast are capable of growing, (e) Region A Region B whether or not PIF is interacting with PDK1. PRK2 908 EDVKKHPFFRLIDWSALMDKKVKPPFIPTIRGREDVSNFDDEFTSEAPILTPP--REPRILSEEEQEM---FRDFDYIADWC 984 An interaction between PIF and PDK1 leads PRK1 403 EDVKKQPFFRTLGWEALLARRLPPPFVPTLSGRTDVSNFDEEFTGEAPTLSPP--RDARPLTAAEQAA---FLDFDFVAGGC 479 PKBα 400 KEIMQHRFFAGIVWQHVYEKKLSPPFKPQVTSETDTRYFDEEFTAQMITITPPDQDDSMECVDSERRP—-HFPQFSYSASTA 480 to the expression of reporter genes coding p70 S6K 321 GEVQAHPFFRHINWEELLARKVEPPFKPLLQSEEDVSQFDSKFTRQTPVDSP----DDSTLSESANQV---FLGFTYVAPSV 395 SGK 347 MEIKSHVFFSLINWDDLINKKITPPFNPNVSGPNELRHFDPEFTEEPVPNSIGKSPDSVLVTASVKEAAEAFLGFSYAPPTD 428 for URA3 and HIS1 that will enable growth PKCζ 510 SDIKSHAFFRSIDWDLLEKKQALPPFQPQITDDYGLDNFDTQFTSEPVQLTPD---DEDAIKRIDQSE---FEGFEYINPLL 585 on (c) SD –Leu –Trp –Ura –His +3-AT PKCα 589 RDVREHAFFRRIDWEKLENREIQPPFKPKVCG-KGAENFDKFFTRGQPVLTPP---DQLVIANIDQSD---FEGFSYVNPQF 663 PKAβ 290 SDIKTHKWFATTDWIAIYQRKVEAPFIPKFGRSGDTSNFDDYEEEDIRV------SITECAKEK---FGEF 351 medium and (d) induction of β-galactosidase gene expression. (e) Alignment of the amino- Current Biology PDK2 Motif acid sequence of the carboxy-terminal 77 amino acids of PRK2 with the equivalent Identical residues are denoted by white residues by grey background. region of AGC subfamily kinases indicated. letters on black background, and similar Research Paper Acquisition of PDK2 activity by PDK1 Balendran et al. 395

Figure 2 (a) Specific interaction of GST–PIF with PDK1 in 293 cells. (a,b) Cells were kDa 97 transiently transfected with DNA constructs expressing GST or GST–PIF 67 Myc–PDK1 (1–556) Myc–PDK1 (51–404) together with either wild-type Myc–PDK1, catalytically inactive (kinase- 43 dead) Myc–PDK1 (K111A, D223A) or the kinase domain of Myc–PDK1 GST–PIF GST (residues 51–404). The cells were lysed (36 h post transfection) and the GST or GST–PIF purified from 350 µg of lysate by (a) affinity – – + + – – + + – – + + GST–PIF chromatography of the lysate on glutathione–Sepharose beads or (b) the + + – – + + – – + + – – GST Myc–PDK1 proteins immunoprecipitated using anti-Myc antibodies Wild-type Kinase-dead Wild-type covalently attached to protein-G–Sepharose. The total protein from each Myc–PDK1 Myc–PDK1 Myc–PDK1 purification was electrophoresed on a 10% SDS–polyacrylamide gel and (1–556) (1–556) (51–404) stained with Coomassie blue. The position of the molecular mass (b) markers, glycogen phosphorylase (97 kDa), bovine serum albumin kDa (67 kDa) and ovalbumin (43 kDa) are indicated. (c) Cells were transiently 97 transfected with DNA constructs expressing either GST–PIF, GST alone, 67 Myc–PDK1 (1–556) GST–p90RSK1, GST–MSK1, GST–p70 S6 kinase (p70 S6K) lacking 43 Myc–PDK1 (51–404) α GST–PIF the carboxy-terminal 104 residues, or GST–PKB . The cells were lysed 30 36 h post transfection and the GST fusion proteins purified by affinity chromatography on glutathione–Sepharose beads. Each GST fusion – – + + – – + + – – + + GST–PIF protein (2 µg) was incubated for 30 min at 30°C with + + – – + + – – + + – – GST GST–S473D PKBα and Mg–ATP in the presence or absence of Wild-type Kinase-dead Wild-type µ phospholipid vesicles containing 10 M 1,2-SAD-PtdIns(3,4,5)P3, and Myc–PDK1 Myc–PDK1 Myc–PDK1 the increase in specific activity of GST–S473D PKBα was determined (1–556) (1–556) (51–404) relative to a control incubation in which the GST–S473D PKBα fusion (c)

protein was omitted (average for six determinations, three independent α experiments). The basal activity of GST–S473D PKBα was 3.5 U/mg. 100 PKB Each protein (2 µg) was electrophoresed on a 10% SDS–polyacrylamide 80 gel and immunoblotted with an antibody raised against the PDK1 protein 60 to detect any endogenous PDK1 associated with the 40 Increase in glutathione–Sepharose pull downs. activity (U/mg) 20 GST–S473D –+ –+ –+ –+ –+ –+ PtdIns(3,4,5)P3 GST–PIF GST RSK1 MSK1 p70 S6K PKBα lysed and the GST–PIF fusion protein purified by affinity 67 kDa chromatography on glutathione–Sepharose (Figure 2a), or the Myc–PDK1 isolated by immunoprecipitation using an Current Biology anti-Myc antibody (Figure 2b). The samples were electrophoresed on an SDS–polyacrylamide gel and the pro- blotting. In 293 cells, two PDK1 immunoreactive bands teins visualised by staining with Coomassie blue (Figure 2). were observed, at 63 kDa and 66 kDa, and both were A near-stoichiometric complex was observed between the observed to copurify with GST–PIF (Figure 2c).The GST–PIF fusion protein and wild-type PDK1, with the PDK1 associated with GST–PIF (0.5 µg) was active isolated kinase domain of PDK1 or with a catalytically inac- because in the presence of Mg–ATP and PtdIns(3,4,5)P3 it tive mutant. The complex formed between PDK1 and greatly activated a GST–S473D PKBα mutant (Figure 2c). GST–PIF could not be dissociated by lithium bromide (a Thus, PDK1 bound to PIF can phosphorylate PKB at strong chaotrophic agent) at concentrations up to 2 M or by Thr308, and the interaction between PIF and the kinase incubation with 1% (by volume) Triton X-100 (data not domain of PDK1 does not interfere with the activation of shown), indicating a very strong interaction. No interaction PKB. In contrast, PKB, p70 S6 kinase, p90 ribosomal S6 was observed either between GST and PDK1 (Figure 2) or kinase 1 (RSK1) or mitogen- and stress-activated protein between GST–PIF and epitope-tagged PKB, p70 S6 kinase 1 (MSK1), when expressed in 293 cells as GST kinase or 6-phosphofructo-2-kinase when each of these was fusion proteins, were not associated with any endogenous coexpressed with PIF in 293 cells (data not shown). Analy- PDK1 after their precipitation on glutathione–Sepharose, sis of the interaction using surface plasmon resonance con- as judged by immunoblotting and failure to activate firmed that the GST–PIF fusion protein was capable of GST–S473D PKBα (Figure 2c). associating directly with purified His-tagged PDK1 with an estimated equilibrium dissociation constant of 600 nM (see The ‘PDK2 substrate motif’ mediates the interaction Supplementary material published with this paper on the between PIF and PDK1 internet). No association between GST–PIF and We have termed the amino-terminal 53 residues of PIF His–PKBα was detected using this method. ‘region A’ and the carboxy-terminal 24 residues ‘region B’ (Figure 1e). In order to identify the amino-acid residues in PIF binds specifically to the endogenous PDK1 in 293 cells PIF that were required for the interaction of PIF with GST–PIF purified from 293 cells was found to be associ- PDK1, we mutated many of the residues in PIF that are ated with the endogenous PDK1 as judged by western conserved between the AGC subfamily of protein kinases 396 Current Biology, Vol 9 No 8

to alanine. The mutant GST–PIF proteins were expressed Figure 3 in 293 cells at similar levels, and the amount of endogenous PDK1 activity and PDK1 protein associated with (a) 80 each of the purified constructs was determined (Figure 3a). α 70 PKB Mutation of the conserved residues in region A did not 60 prevent the association of PDK1 with GST–PIF. Remark- 50 ably, however, mutation to alanine of any of the conserved aromatic residues in the PDK2 phosphorylation site motif 40 in region B (Phe974, Phe977 and Tyr979) completely abol- 30 ished the association of endogenous PDK1 with these 20 catalytic activity (U/mg) GST–PIF mutants (Figure 3a). Furthermore, mutation of 10 Increase in GST–S473D Asp978 (the residue equivalent to Ser473 in PKB) to either 0 alanine or serine also abolished the association of PDK1 PtdIns(3,4,5)P3 –+ –+ –+ –+ –+ –+ –+ –+ –+ –+ –+ –+ with PIF. In contrast, mutation of the residues located two PDK1 and three amino acids amino-terminal to Asp978 (Asp976 (Western) or Arg975), which are not conserved between the AGC GST–PIF subfamily members, did not affect the association of PDK1 (Coomassie)

with PIF (Figure 3a). A yeast two-hybrid screen was also PIF constructs

F974A

F977A

Y979A

T951A

F946A

R975A D978A

P932A

D978S H913A used to verify these findings (Figure 3b). D976G

Wild type Wild

Region A Region B A strong interaction (Kd 800 nM) between His–PDK1 and an immobilised and biotinylated 24 residue synthetic (b) peptide corresponding to region B was detected using

PIF constructs Wild type Wild

F974A Y979A surface plasmon resonance measurements. This interac- F977A D978A tion was competed by the addition of free peptide, and µ Vector analysis of this competition revealed a Kd of 1.5 M. In contrast, the interaction between PDK1 and a mutant peptide in which the residue corresponding to Asp978 was PDK1 vector changed to alanine (termed D978A region B peptide) interacted with PDK1 more than tenfold less strongly (see Current Biology Supplementary material). Incubation of purified GST–PIF expressed in 293 cells with the wild-type Mapping the residues on PIF that are required for interaction with µ PDK1. (a) Cells were transiently transfected with DNA constructs region B peptide (100 M) resulted in dissociation of the expressing either wild-type GST–PIF or the indicated point mutants of endogenous PDK1 from GST–PIF, whereas a D978A this protein. The 293 cells were lysed and the GST fusion proteins region B peptide was incapable of inducing the dissocia- purified by affinity chromatography on glutathione–Sepharose beads. tion of PDK1 from PIF (data not shown). Each GST-fusion protein was incubated for 30 min at 30°C with GST–S473D PKBα and Mg–ATP in the presence or absence of µ phospholipid vesicles containing 10 M 1,2-SAD-PtdIns(3,4,5)P3, and GST–PIF converts PDK1 to a form that is able to the increase in specific activity of GST–S473D PKBα was determined phosphorylate Ser473 as well as Thr308 in PKBα relative to a control incubation in which the GST fusion protein was α When purified GST–PIF associated with the endogenous omitted. The basal activity of GST–S473D PKB was 3.5 U/mg. Each protein (2 µg) was electrophoresed on a 10% SDS–polyacrylamide gel PDK1 of 293 cells (Figure 2c) was incubated with and either immunoblotted using an anti-PDK1 antibody raised against GST–PKBα in the presence of Mg–ATP and whole protein to detect any endogenous PDK1, or stained with α PtdIns(3,4,5)P3, we noticed that GST–PKB could be Coomassie blue to establish the level of expression of the GST–PIF activated to a threefold higher maximum specific activity protein. Similar results were obtained in five separate experiments using two or more preparations of the wild-type and mutant GST–PIF (about 90 U/mg) than is achievable by incubation of proteins. The single letter amino-acid code is used in construct names. GST–PKBα with far higher concentrations of His–PDK1 (b) Yeast harbouring the pAS2-1 wild-type PDK1 vector (PDK1 vector) alone (about 30 U/mg; Figure 4a). In contrast, the mutant or the empty pAS2-1 vector (vector) were transformed with pGAD10 form of PKBα in which the Ser473 phosphorylation site is plasmid coding for the expression of the wild-type PIF or the indicated mutated to aspartic acid (GST–S473D PKBα) was acti- mutants of PIF. The resulting yeast strain was grown on SD –Leu –Trp –Ura –His +3-AT medium, which permits growth of only yeast vated to the same maximum specific activity by either constructs in which GST–PIF and PDK1 are capable of interacting. PDK1 or PIF plus PDK1 (Figure 4b). These findings raised the possibility that GST–PIF plus PDK1 had induced phosphorylation of PKBα at Ser473, as well as Ser473. These antibodies did not recognise GST–PKBα Thr308. In order to investigate this possibility, we pre- that had been incubated with GST–PIF and Mg–ATP in pared phospho-specific antibodies (termed P-473) that the absence of PtdIns(3,4,5)P3 or PtdIns(3,4)P2, nor did recognise only PKBα that has been phosphorylated at PKBα become phosphorylated under these conditions Research Paper Acquisition of PDK2 activity by PDK1 Balendran et al. 397

α (Figure 4c). Following the addition of PtdIns(3,4,5)P3 or The PtdIns(3,4,5)P3-dependent phosphorylation of PKB at α PtdIns(3,4)P2, however, GST–PKB became strongly Ser473 is mediated by PDK1 phosphorylated and recognised by the P-473 antibody. The results presented in Figure 4 could be explained in Incubation of the P-473 antibody with the phosphory- two different ways. Firstly, the interaction of PIF with lated Ser473 peptide immunogen used to raise the anti- PDK1 might have allowed the latter to phosphorylate body (but not with the dephosphorylated peptide, or a Ser473 as well as Thr308. Secondly, purified GST–PIF phosphopeptide corresponding to the sequence sur- might be complexed to two different protein kinases, rounding Thr308) abolished its recognition of namely PDK1 and PDK2. In order to distinguish between GST–PKBα (Figure 4c). In contrast, a much higher con- these possibilities we immunodepleted the PDK1 from centration of His–PDK1 that was not bound to PIF GST–PIF using two different antibodies. We then tested induced no phosphorylation of Ser473 in the presence or the ability of the supernatant to phosphorylate PKBα at absence of PtdIns(3,4,5)P3. Ser473. Strikingly, both PDK1 antibodies completely removed all the Ser473 kinase activity associated with To identify the residue(s) in PKBα that are phosphory- GST–PIF (Figure 5a) as well as removing the Thr308 lated by the GST–PIF plus PDK1 complex, the 32P- kinase activity (data not shown). A peptide immunogen to labelled GST–PKBα was digested with trypsin and which one of the antibodies was raised prevented immun- 32 chromatographed on a C18 column [14]. Two major P- odepletion of the PDK2 activity associated with PIF labelled peptides were observed that eluted at 24% and (Figure 5a). 26% acetonitrile (Figure 4d). These peptides co-eluted with the 32P-labelled tryptic peptides containing the residues Ser473 and Thr308, respectively [14,18], and the (a) (b) identity of these peptides and the location of the phospho-

α α α rylated residues (Thr308 and Ser473) was established by GST–wild-type PKB 100 GST–S473D PKB PKB matrix associated laser desorption ionization and time of 90 flight mass spectrometry (MALDI-TOF MS) and solid phase amino-acid sequencing (data not shown). 60 50 Figure 4 30 α Increase in GST–PKB specific activity (U/mg) Incubation of PIF with PKBα induces phosphorylation of PKBα at both specific activity (U/mg) α α Thr308 and Ser473. (a) GST–PKB or (b) GST–S473D PKB was Increase in GST–S473D incubated at 30°C for 1 h with Mg–ATP in the presence of either PtdIns(3,4,5)P3 –+–+ –+–+ His–PDK1 (0.1 µg) or GST–PIF purified from 293 cells, which is His–PDK1 PIF + PDK1 His–PDK1 PIF + PDK1 associated with endogenous PDK1 (1 µg; termed PIF + PDK1), in the (c) GST–PIF ++++–+ presence or absence of phospholipid vesicles containing 10 µM GST–PKBα +++++– 1,2-SAD-PtdIns(3,4,5)P . The increase in specific activity of 3 1,2-SAD-PtdIns(3,4,5)P –+––++ GST–PKBα was determined relative to a control incubation in which 3 C -PtdIns(3,4,5)P ––+––– His–PDK1 or GST–PIF was omitted (average for six determinations, 16 3 C -PtdIns(3,4)P –––+–– two independent experiments). The basal activity of GST–PKBα was 16 2 1.0 U/mg, and of GST–S473D PKBα was 3.5 U/mg. Under the No peptide conditions used, GST–PKBα (a) and GST–S473D PKBα (b) were + De-phospho-473 peptide activated maximally because no further increase occurred if the GST–PKBα incubation period was prolonged or by increasing the concentration of phospho-473 blot + Phospho-473 peptide His–PDK1 or GST–PIF. (c) GST–PKBα was incubated with GST–PIF µ plus PDK1 (1.0 g) as in (a), in the presence or absence of + Phospho-308 peptide µ phospholipid vesicles containing 10 M 1,2-SAD-PtdIns(3,4,5)P3, 32 C16-PtdIns(3,4,5)P3 or C16-PtdIns(3,4)P2 (where C16 stands for P autoradiograph DiC16:0 dipalmitoyl). The reactions were terminated with 1% SDS, the samples subjected to SDS–PAGE and immunoblotted using the α P-473 antibody incubated in the presence or absence of the synthetic (d) GST–PIF + GST–PKB + 1,2-SAD PtdIns(3,4,5)P peptides (all at 10 µg/ml) corresponding to residues 467–477 of 3 30 PKBα phosphorylated at Ser473 (phospho-473 peptide), the same Ser473 Thr308 peptide but not phosphorylated (de-phospho-473 peptide), and a Acetonitrile (%) α 10

peptide corresponding to residues 301–313 of PKB phosphorylated –3 7. 5 at Thr308 (phospho-308 peptide). (d) As in (a), except that radioactive 25 Mg–[γ-32P]ATP (106 cpm per nmol) was employed, the samples x 10 5 alkylated with 4-vinylpyridine, electrophoresed on a 10% Cpm polyacrylamide gel, and the phosphorylated GST–PKBα was digested 2.5 with trypsin and applied to a C18 column as described previously [19]. The 32P-labelled peptides were identified by mass spectrometry and 0 20 120 140 160 180 Edman sequencing as the PKBα tryptic peptides phosphorylated at Fraction number Ser473 and Thr308, respectively. Current Biology 398 Current Biology, Vol 9 No 8

Figure 5

The ability of GST–PIF to phosphorylate PKBα at Ser473 is dependent upon the (a) PDK1 peptide Non-specific PDK1 protein PDK1 peptide antibody presence of PDK1. GST–PIF purified from Control antibody antibody antibody + peptide 293 cells, which is associated with 1,2-SAD-PtdIns(3,4,5)P – + – – – + – – – + – – – + – – – + – – µ µ 3 endogenous PDK1 (20 g in 50 l), was C -PtdIns(3,4,5)P – – + – – – + – – – + – – – + – – – + – µ 16 3 incubated with protein-G–Sepharose (10 l) C16-PtdIns(3,4)P2 – – – + – – – + – – – + – – – + – – – + conjugated to an anti-MSK1 antibody A [42] (5 µg, non-specific antibody), an anti-PDK1 antibody raised against the whole protein (5 µg), an anti-PDK1 peptide antibody raised (b) Identity of mutants H913A P932A F946A T951A Q971A F974A R975A D976G F977A D978A D978S Y979A against the 16 carboxy-terminal amino acid 1,2-SAD-PtdIns(3,4,5)P3 –+–+–+–+–+–+–+–+–+–+–+–+ residues of PDK1 (5 µg, peptide antibody) or no antibody (control) in the presence or PKBα phospho-473 blot absence of the PDK1 carboxy-terminal peptide immunogen (0.2 mM). After incubation on a shaking platform for 60 min at 4°C, the Region A Region B 974-FRDFDY-979 suspension was centrifuged and GST–PIF in PDK2 motif in PRK2 the supernatant (2.0 µg) was incubated with Current Biology GST–PKBα and Mg–ATP for 60 min at 30°C in the presence or absence of phospholipid µ vesicles containing 10 M 1,2-SAD- Identical results were obtained in two separate 1,2-SAD-PtdIns(3,4,5)P3, and the degree of α PtdIns(3,4,5)P3, C16-PtdIns(3,4,5)P3 or C16- experiments. (b) The wild-type and mutant phosphorylation of PKB at Ser473 was µ PtdIns(3,4)P2. The reactions were terminated GST–PIF proteins (2.0 g) used in Figure 3 established by immunoblotting. Similar results by making the solutions 1% in SDS, and the were incubated with GST–PKBα and were obtained in four separate experiments samples were subjected to SDS–PAGE and Mg–ATP for 60 min at 30°C in the presence using two different preparations of the wild- immunoblotted using the P-473 antibody. or absence of lipid vesicles containing 10 µM type and mutant GST–PIF proteins.

These results indicated that the PtdIns(3,4,5)P3-depen- that it phosphorylates Ser473 (data not shown). Moreover, dent Ser473 kinase activity associated with GST–PIF was the ability of GST–PIF* to convert PDK1 into a Ser473 catalysed by PDK1 rather than by a separate enzyme. kinase was dependent upon PDK1’s catalytic activity, Consistent with this notion, all the mutant GST–PIF pro- since a catalytically inactive GST–PDK1 mutant was not teins purified from 293 cells that were able to interact with capable of phosphorylating PKBα at Ser473 in the pres- the endogenous PDK1 (Figure 3) were able to induce the ence of GST–PIF* (see Supplementary material). phosphorylation of PKBα at Ser473 (Figure 5b). In contrast, the point mutations in GST–PIF that abolish its The above experiments do not exclude the possibility that association with PDK1 also prevented the phosphorylation another protein associated with PIF (rather than PIF itself) of PKBα at Ser473 (Figure 5b). might be capable of interacting with PDK1 and altering its substrate specificity. This possibility was eliminated by the Conversion of PDK1 into a kinase that phosphorylates demonstration that a 24 residue synthetic peptide corre- Thr308 and Ser473 of PKBα sponding to region B of PIF was capable of inducing It could still be argued that the phosphorylation of Ser473 His–PDK1 to phosphorylate PKBα at Ser473 in a 3-phos- was catalysed by a protein kinase that is distinct from phoinositide-dependent manner (Figure 6). In contrast, a PDK1, but that this kinase was tightly complexed with region B peptide in which Asp978 was mutated to alanine PDK1 and not dissociated by chaotrophic reagents or was far less effective at inducing this alteration in the speci- detergents, and that this kinase was therefore also removed ficity of PDK1 than the nonmutated peptide. The region B from GST–PIF by immunodepletion of PDK1. In order to peptide converts the wild-type GST–PDK1, but not the address this point, we removed PDK1 from purified kinase-dead GST–PDK1, into a kinase that can phosphory- GST–PIF by immunodepletion and added the PDK1- late Ser473 (data not shown). Also, GST–PDK1 expressed depleted PIF (termed GST–PIF*) to purified wild-type in bacteria was capable of phosphorylating GST–PKBα at GST–PDK1 (see Supplementary material) or His–PDK1 Ser473 in the presence of the region B peptide and

(data not shown). This converted PDK1 from a kinase that PtdIns(3,4,5)P3 (data not shown), indicating that no other phosphorylates PKBα at Thr308 only, to a kinase that protein kinase expressed in eukaryotic cells which might phosphorylates Ser473 as well as Thr308 in a be associated with PDK1 is required for this reaction.

PtdIns(3,4,5)P3- or PtdIns(3,4)P2-dependent manner (see Supplementary material). Initial-rate studies demonstrated Lipid specificity of Ser473 phosphorylation that, under the conditions used, the GST–PIF*–PDK1 We compared the ability of a panel of PtdIns derivatives complex phosphorylates Thr308 of PKBα at twice the rate to stimulate the phosphorylation of Thr308 and Ser473 by Research Paper Acquisition of PDK2 activity by PDK1 Balendran et al. 399

Figure 6

Conversion of PDK1 into an enzyme that can (a) phosphorylate both Thr308 and Ser473 of D978A Region B Region B Region A PKB by addition of a synthetic peptide. peptide peptide peptide No peptide (a) His–PDK1 (20 ng) was incubated in the 1,2-SAD-PtdIns(3,4,5)P3 – + – + – + – + presence or absence of the following C16-PtdIns(3,4,5)P3 – + – + – + – + peptides (all at 20 µM); region B peptide, C16-PtdIns(3,4)P2 – + – + – + – + residues 969–984 of PIF; D978A region B peptide, in which Asp978 is mutated to Phospho-Ser473 blot alanine; or region A peptide, residues 927–951 of PIF. After allowing to stand for (b) (c) (d) 1 h at 4°C, GST–PKBα, Mg–ATP and the PDK1 + 1,2-SAD-PtdIns(3,4,5)P PDK1 + 1,2-SAD-PtdIns(3,4,5)P3 PDK1 + 1,2-SAD-PtdIns(3,4,5)P 3 indicated phospholipid vesicles containing + region B peptide + D978A region B peptide + no peptide µ 15 30 20 30 12 30 either 10 M 1,2-SAD-PtdIns(3,4,5)P3, C16- Thr308 Acetonitrile (%) Acetonitrile (%) Thr308 Acetonitrile (%) PtdIns(3,4,5)P or C -PtdIns(3,4)P were Thr308 3 16 2 15 added. After 1 h at 30°C, the reactions were -3 10 Ser473 -3 -3 8 10 10 10 terminated by making the solutions 1% in x 25 x 25 x 25 10 SDS, following which the samples were 5 Ser473 4 Ser473 subjected to SDS–PAGE and immunoblotted Cpm Cpm 5 Cpm using the P-473 antibody. Identical results 20 20 were obtained in three separate experiments. 0 20 120 140 160 180 120 140 160 180 120 140 160 180 (b–d) His–PDK1 was incubated for 1 h on ice Fraction number as above in the presence of (b) region B Fraction number Fraction number peptide, (c) D978A region B peptide or (d) no Current Biology peptide and then incubated with Mg–[γ-32P]ATP and phospholipid vesicles µ α containing 10 M 1,2-SAD-PtdIns(3,4,5)P3. terminated and the residues on PKB that conditions were established as described in After 1 h at 30°C, the reactions were became phosphorylated under these the legend to Figure 4.

the GST–PIF plus PDK1 complex. Synthetic sn-1- its ability to activate a PKB mutant lacking the PH domain stearoyl, 2-arachidonoyl D-PtdIns(3,4,5,)P3 (1,2-SAD- [20,31]. This is confirmed in Figure 8, which shows that PtdIns(3,4,5)P3), the predominant form of PtdIns(3,4,5)P3 the rate at which His–PDK1 phosphorylates and activates a that occurs naturally (lipid 8, Figure 7), and synthetic 2,3- mutant form of PKBα lacking the PH domain (GST–∆PH α SAD-PtdIns(3,4,5,)P3 (lipid 10, Figure 7) were highly PKB ) is the same in the presence or absence of effective in inducing both the phosphorylation of PtdIns(3,4,5)P3 (Figure 8a). Intriguingly, the addition of GST–PKBα at Ser473 (Figure 7b) and the phosphoryla- GST–PIF* to His–PDK1 halves the rate at which PDK1 tion of Thr308 (measured by the activation of activates GST–∆PH PKBα in the absence of α) GST–S473D PKB , indicating that the enantiomeric PtdIns(3,4,5)P3. The addition of PtdIns(3,4,5)P3 results in configuration of the glycerol moiety is not an important a 3–4-fold enhancement of the rate at which His–PDK1 determinant of specificity. The L enantiomers of these plus GST–PIF* can activate GST–∆PH PKBα (Figure 8a). lipids (lipids 9 and 11, Figure 7) failed to induce a signifi- Furthermore, the GST–PIF plus PDK1 complex purified cant phosphorylation of PKBα at Ser473 or activation of from 293 cells also activates GST-∆PH PKBα at an approx- GST–S473D PKBα. Rac-1,2-dilinoleoyl phosphatidyl D/L- imately fourfold higher rate in the presence of either myo-inositol 3,4,5-trisphosphate (linoleic acid is C18:2; PtdIns(3,4,5)P3 or PtdIns(3,4)P2 than in their absence. lipid 7, Figure 7), sn-1,2-dipalmitoyl D-PtdIns(3,4,5)P3 PtdIns(4,5)P2 or PtdIns(3)P do not increase the rate at (lipid 6, Figure 7) or sn-1,2-dipalmitoyl PtdIns(3,4)P2 which the GST–PIF plus PDK1 complex phosphorylates (lipid 3, Figure 7) were also effective at inducing the PIF GST–∆PH PKBα (Figure 8b). plus PDK1 complex to phosphorylate PKBα at Ser473 and . Thr308 PtdIns-3P (lipid 2, Figure 7), PtdIns(3,5)P2 (lipid Identification of a PtdIns(3,4,5)P3-dependent Ser473 kinase 4, Figure 7) and PtdIns(4,5)P2 (lipid 5, Figure 7), however, activity as PDK1 did not induce the PIF plus PDK1 complex to phosphory- We fractionated rat brain extracts by batchwise chro- late PKBα at Ser473 or Thr308. In the absence of the PIF matography on Q-Sepharose, followed by gradient plus PDK1 complex, none of the PtdIns derivatives tested elution from heparin–Sepharose (see Materials and induced any phosphorylation of PKBα (data not shown). methods). As reported previously [19], PDK1 (assayed as

a PtdIns(3,4,5)P3-dependent enzyme that activates Maximal activation of PKBα by PDK1 plus PIF requires GST–S473D PKBα) was eluted from heparin–Sepharose

PtdIns(3,4,5)P3 to interact with PDK1 as a broad peak at 0.75M NaCl (Figure 9a). A Although PDK1 binds to PtdIns(3,4,5)P3 with high affinity PtdIns(3,4,5)P3-dependent PDK2 activity that phospho- through its PH domain, this interaction does not influence rylated PKBα at Ser473 was also identified in the 400 Current Biology, Vol 9 No 8

Figure 7 Figure 8

(a) (a) 150 α 20 PKB

100 15

activity (U/mg) 50

specific activity (U/mg) 5 Increase in GST–S473D PKB α Increase in GST–S473D ∆ PH Increase in GST– Lipid: 123456 7891011 (b) PtdIns(3,4,5)P3 –+–+–+–+ Experiment 1 Buffer His–PDK1 PIF*+PIF* His–PDK1 Experiment 2 (b) 30

Current Biology α 25 PKB Lipid specificity of phosphoinositide-dependent PIF plus PDK1-induced phosphorylation of Thr308 and Ser473 of PKBα. GST–PKBα was 20 incubated for 60 min at 30°C with GST–PIF (1.0 µg) purified from 293 cells (which is associated with endogenous PDK1, see Figure 3), 15 Mg–ATP and phospholipid vesicles containing various PtdIns lipids (numbered 1–11, see below) all at a final concentration of 10 µM in the 10 assay. (a) The reactions were terminated by the addition of Triton X-100 [19] and the increase in specific activity of GST–S473D PKBα specific activity (U/mg) 5 (in U/mg) determined, relative to a control incubation in which the ∆ PH Increase in GST– GST–PIF plus PDK1 complex was omitted (average for six determinations, two independent experiments). The basal activity of PIF–PDK1++++++ GST–S473D PKBα was 3.5 U/mg. (b) The reactions were terminated No lipid + by making the solutions 1% in SDS, the samples were subjected to PtdIns 3P + SDS–PAGE and the extent of PKBα phosphorylation was established 1,2-SAD-PtdIns(4,5)P2 + using the phospho-specific antibody that recognises PKBα 1,2-SAD-PtdIns(3,4,5)P3 + phosphorylated at Ser473. In (b) the results of two separate C16-PtdIns(3,4,5)P3 + experiments with different preparations of lipid are shown. Lipid 1, C16-PtdIns(3,4)P2 + buffer control; lipid 2, PtdIns 3P; lipid 3, sn-1,2-dipalmitoyl Current Biology PtdIns(3,4)P2; lipid 4, PtdIns(3,5)P2; lipid 5, PtdIns(4,5)P2; lipid 6, sn-1,2-dipalmitoyl D-PtdIns(3,4,5)P3; lipid 7, rac-1,2-dilinoleoyl phosphatidyl D/L-myo-inositol 3,4,5-trisphosphate; lipid 8, 1,2-SAD- PDK1 complexed to PIF is directly activated by PtdIns(3,4,5)P3. ∆ α PtdIns(3,4,5)P3; lipid 9, sn-1-stearoyl, 2-arachidonoyl L-PtdIns(3,4,5)P3; (a) GST– PH PKB was incubated at 30°C for 1 h with Mg–ATP in µ lipid 10, sn-2-stearoyl, 3-arachidonoyl D-PtdIns(3,4,5)P3; lipid 11, the presence of either buffer (control), His–PDK1 (0.1 g), or µ µ sn-2-stearoyl, 3-arachidonoyl L-PtdIns(3,4,5)P3. His–PDK1 (0.1 g) plus GST–PIF* (1.0 g, from which all the PDK1 had been removed by immunodepletion), or GST–PIF* alone (1.0 µg) in the presence or absence of phospholipid vesicles containing 10 µM

1,2-SAD-PtdIns(3,4,5)P3. (b) GST–PIF purified from 293 cells, which heparin–Sepharose eluate, and this was present in the is associated with endogenous PDK1 (2 µg, termed PIF–PDK1), was tailing half of the PDK1 activity (Figure 9a). The peak incubated with GST–∆PH PKB as in (a), with the indicated phosphoinositides (10 µM). The increase in specific activity of containing the PDK2 activity (fractions 7 and 8) was GST–∆PH PKBα was determined, relative to a control incubation in pooled, PDK1 removed by immunoprecipitation and the which His–PDK1 or GST–PIF was omitted. The basal activity of supernatant re-assayed for PDK1 and PDK2 activity. GST–∆PH PKBα was 3.0 U/mg. The average results for a single Strikingly, the antibody completely removed not only the experiment performed in triplicate are shown; similar results were PDK1 (Thr308 kinase) activity but also the PDK2 obtained in three other separate experiments. (Ser473 kinase) activity (Figure 9b). This result indicates that a fraction of the PDK1 in the heparin–Sepharose eluate possesses PDK2 activity. carboxy-terminal to the PDK1 phosphorylation site. Phos- phorylation of both the PDK1 and PDK2 sites is required Discussion for maximal activation of PKB and p70 S6 kinase and/or for Most of the PDK1 substrates identified to date, apart from stability of PKC isoforms [32]. In this paper, we demon- PKA, possess a PDK2 consensus motif about 160 residues strate that the kinase domain of PDK1 interacts specifically Research Paper PDK1 acquires PDK2 activity in the presence of a synthetic peptide Balendran et al. 401

with a modified PDK2 phosphorylation consensus motif Figure 9 present at the carboxyl terminus of PRK2, in which the phosphorylated serine or threonine residue is replaced by (a) 1.5 an aspartic acid (Asp978). Mutation of any of the conserved aromatic residues in the PDK2 consensus sequence to 100 alanine, or mutation of Asp978 to alanine or serine, com- 1 pletely abolished the interaction of PDK1 with PIF 75 (Figure 3). 50 NaCl (M) 0.5 PRK2 itself is likely to be a substrate for PDK1 as it pos- 25 sesses a perfect PDK1 consensus sequence between sub- Thr308 kinase activity (U/mg) domains VII and VIII of the kinase domain 0 (TFCGTPEFL, in single letter amino acid code where the Fraction number 123456 7891011 italicised threonine residue corresponds to Thr816, the putative site of PDK1 phosphorylation). In the case of No PtdIns(3,4,5)P PRK2, it is possible that the role of the PIF domain is to 3 interact directly with PDK1, ensuring that Thr816 is phos- + PtdIns(3,4,5)P3 phorylated efficiently by PDK1. As PRK2 [30] (and its close relative PRK1 [33,34]) might be dependent on the Ser473 kinase activity GTPase Rho for activity, it is possible that the interaction between PRK2 and Rho complexed to GTP is also (b) required for the phosphorylation of PRK2 by PDK1 to take 75 place. One scenario is that the carboxyl terminus of PRK2 50 is exposed upon the formation of a complex between 25 Thr308 kinase PRK2 and Rho–GTP. This may enable interaction with, activity (U/mg) and phosphorylation by, PDK1, leading to the activation of PtdIns(3,4,5)P PRK2. PRK2 is, however, known to become proteolysed 3 – +–+–+ Non- PDK1 during apoptosis as a result of cleavage by caspase-3 imme- Control specific antibody diately carboxy terminal to Asp117 and Asp700 [35]. PRK2 antibody is highly susceptible to proteolysis when purified or over- PtdIns(3,4,5)P3 –+– –+– –+– expressed in cells [36]. One possibility therefore is that a PtdIns(3,4)P2 ––+ ––+––+ carboxy-terminal fragment of PRK2, that is devoid of any catalytic activity, is present in cells, and that this interacts Ser473 kinase activity with PDK1. It is also possible that a fragment of PRK2 may Current Biology be generated by an alternative splicing mechanism or by the use of alternative promoters. Future work will need to (a) Brain extract was fractionated by batchwise chromatography on Q-Sepharose followed by gradient elution from heparin–Sepharose (see address whether PDK1 is complexed with PRK2 or frag- Materials and methods). The column fractions were assayed for their ments of this enzyme in intact cells. ability to phosphorylate PKBα at Thr308 (measured by the increase in GST–S473D PKBα activity) and Ser473 (using the P-473 antibody), in µ SGK and the p70 S6 kinase are far better substrates for the presence (open squares) or absence (closed squares) of 10 M 1,2-SAD-PtdIns(3,4,5)P3. The broken line indicates the salt gradient. PDK1 if the PDK2 phosphorylation site is mutated to an (b) Fractions 7 and 8 of the heparin–Sepharose column were pooled acidic residue [17,26,27]. Thus, PDK1 might be able to and aliquots (0.1 ml) incubated with protein-G–Sepharose (10 µl) interact directly with SGK or the p70 S6 kinase when the conjugated to an MSK1 antibody A [42] (5 µg; non-specific antibody), PDK2 site is phosphorylated, enabling PDK1 to phospho- an anti-PDK1 antibody raised against the whole protein (5 µg), or no antibody (control). After incubation on a shaking platform for 60 min at rylate these enzymes at the PDK1 site in the activation 4°C, the suspension was centrifuged, and an aliquot of the supernatant loop. Future work will need to address whether or not (5 µl) incubated for 60 min at 30°C with either GST–S473D PKBα PDK1 complexed with another protein is capable of induc- (PDK1 assay) or GST–PKBα (PDK2 assay) and Mg–ATP in the µ ing phosphorylation of the PDK2 site of these enzymes. presence or absence of 10 M 1,2-SAD-PtdIns(3,4,5)P3 or C16- α PtdIns(3,4)P2. The phosphorylation of PKB at Ser473 and Thr308 was α then measured as in (a). Similar results were obtained in experiments A catalytically inactive mutant of PKB becomes phos- from two separate purifications. The PDK2 activity was also phorylated at Ser473 in response to insulin, whereas immunoprecipitated using an anti-PDK1 peptide antibody raised against PKBα phosphorylated at Thr308 alone does not become the 16 carboxy-terminal residues of PDK1 (data not shown). phosphorylated at Ser473 in vitro upon incubation with

Mg–ATP in the presence of PtdIns(3,4,5)P3 [14,19]. These observations indicate that phosphorylation of event catalysed by PKBα itself. We have reported previ- PKBα at Ser473 is unlikely to be an autophosphorylation ously that Ser473 is phosphorylated in vitro by MAP 402 Current Biology, Vol 9 No 8

kinase-activated protein kinase-2 (MAPKAP-K2) [14], but Stokoe et al. [21] observed that a PDK1-like activity this enzyme does not mediate the phosphorylation of obtained after chromatography of a brain extract on a Ser473 in vivo in response to insulin, insulin-like growth MonoQ column activated ∆PH PKBα at a threefold higher factor 1 (IGF1), heat shock or hydrogen peroxide [37]. rate in the presence of PtdIns(3,4,5)P3 than in its absence. Furthermore, the phosphorylation of PKBα at Ser473 by It is possible that the partially purified PDK1 used in MAPKAP-K2 in vitro is not dependent upon the presence these experiments was complexed with a PIF-like protein. of PtdIns(3,4,5)P3 [14]. It has also been claimed that the This subunit might have been lost during subsequent integrin-linked kinase (ILK) is capable of phosphorylating purification of the protein. Ser473 of PKBα in vitro [38]. We have thus far not been able to demonstrate that ILK phosphorylates Ser473 of The results presented in this paper raise the strong possi- PKBα (S. Dowler and D.R.A., unpublished observations). bility that PDK1 itself might be the enzyme responsible Indeed, it is uncertain as to whether ILK is actually an for mediating the phosphorylation of PKBα at both active protein kinase, as it lacks certain motifs found in Thr308 and Ser473. It is possible that PRK2, a fragment of every other protein kinase. It has also been claimed that PRK2, or a related peptide/protein(s), might interact with mTOR (mammalian target of rapamycin) can phosphory- the PDK1, converting it into an enzyme that can phospho- late the PDK2 site of p70 S6 kinase [39]. We have been rylate both Ser473 and Thr308 of PKBα, as well as permit- unable to reproduce this finding and furthermore were ting the direct activation of PDK1 by PtdIns(3,4,5)P3. unable to phosphorylate PKBα at Ser473 with mTOR Consistent with this hypothesis, we have identified a

(D.R.A. and P. Shepherd, unpublished observations). PtdIns(3,4,5)P3-dependent Ser473 kinase activity in brain that is immunoprecipitated with a PDK1-specific antibody Given that the insulin/IGF1-induced phosphorylation of (Figure 9). This form of PDK1 might be complexed to Ser473, like that of Thr308, is prevented by inhibitors of PI PRK2 or its carboxy-terminal fragment or to another 3-kinase, it is likely that phosphorylation of Ser473 is also peptide or protein with similar properties. catalysed by a PtdIns(3,4,5)P3-dependent protein kinase. In this study we have made the surprising observation that Conclusions the interaction of PIF with the kinase domain of PDK1 This study provides the first evidence that PDK1 and converts PDK1 from an enzyme that only phosphorylates PDK2 might be the same enzyme and that the specificity PKBα at Thr308 to a form that phosphorylates both of this enzyme for two different phosphorylation sites on α Thr308 and Ser473 in a PtdIns(3,4,5)P3- and PKB might be modulated by the interaction with one or PtdIns(3,4,)P2-dependent manner. The requirement for more accessory proteins. Our results also suggest that PtdIns(3,4,5)P3 or PtdIns(3,4,)P2 is extremely specific, when PDK1 interacts with molecules such as PIF its because only the D enantiomers of PtdIns(3,4,5)P3 are activity may become directly stimulatable by the inter- effective, and many other PtdIns phospholipids, including action of the PH domain of PDK1 with PtdIns(3,4,5)P3 PtdIns(4,5)P2, are ineffective. The residues that surround or PtdIns(3,4)P2. Thr308 and Ser473 are distinct, the only common feature being an aromatic residue immediately carboxy terminal to Materials and methods the phosphorylation site. This suggests that PDK1 pos- Materials The yeast two-hybrid human skeletal muscle library (in pGAD10 vector) sesses two distinct specificities when complexed with PIF. and the pAS2-1 vector were purchased from Clontech. Expression constructs for GST–PKBα [19], GST–PDK1, Myc–PDK1 and other If a mutant of PKBα lacking the PH domain (which cannot mutants of PDK1 [20], GST–p90 RSK1 [42], GST–MSK1 [42] and therefore interact with PtdIns(3,4,5)P ) is expressed in cells, GST–p70 S6 kinase lacking the carboxy terminal 104 residues [26] 3 were generated as described previously. Synthesis of 1,2-SAD- it nevertheless undergoes a wortmannin-sensitive activation PtdIns(3,4,5)P3 and its stereoisomers is described [43]; all lipids were and phosphorylation at Thr308 and Ser473 [40]. This obtained from sources described previously [19]. All the experiments implies that PDK1 is activated by PtdIns(3,4,5)P3 in vivo. using 3-phosphoinositides were carried out in the presence of phos- PDK1 that is isolated from either control or insulin-stimu- pholipids containing 100 µM phosphatidylcholine and 100 µM phos- lated cells has similar activity towards GST–∆PH PKBα phatidylserine. Information regarding antibodies and other reagents as well as preparation of His–PDK1 used in this study is given in the Sup- [20,27]. Furthermore, experiments in vitro demonstrated plementary material. that despite binding to PtdIns(3,4,5)P3/PtdIns(3,4)P2, through its PH domain [22,41], these 3-phosphoinositides Buffer solutions Buffer A consists of 50 mM Tris-HCl pH 7.5, 1 mM EGTA, 1 mM have no influence on the rate at which PDK1 phosphory- EDTA, 1% (by mass) Triton X-100, 1 mM sodium orthovanadate, lates ∆PH PKBα, p70 S6 kinase or SGK; substrates that do 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 0.27 M sucrose, not interact with 3-phosphoinositides [3,17,18]. Impor- 1 µM microcystin-LR, 0.1% (by volume) β-mercaptoethanol and ‘com- tantly, the interaction with PIF confers on PDK1 the ability plete’ proteinase inhibitor cocktail (one tablet per 25 ml, Boehringer Mannheim). Buffer B consists of 50 mM Tris-HCl pH 7.5, 0.1 mM to be activated directly by PtdIns(3,4,5)P3/PtdIns(3,4)P2 in EGTA and 0.1% (by volume) β-mercaptoethanol. Buffer C consists of vitro (Figure 8). This does not happen if the PH domain of 50 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.27 M sucrose PDK1 is deleted (data not shown). and 0.1% (by volume) β-mercaptoethanol. Research Paper Acquisition of PDK2 activity by PDK1 Balendran et al. 403

Yeast two-hybrid screen 2. Shepherd PR, Withers DJ, Siddle K: Phosphoinositide 3-kinase: the Myc-tagged human PDK1, the kinase domain of PDK1 (residues key switch in insulin signalling. Biochem J 1988, 333:471-479. 1–404) and the PH domain of PDK1 (428–556) were subcloned into 3. Alessi DR, Downes CP: The role of PI 3-kinase in insulin action. 1436: EcoRI–SalI of the yeast pAS2-1 vector. A yeast two-hybrid screen was Biochim Biophys Acta 1998, 151-164. 4. Woscholski R, Parker PJ: Inositol lipid 5-phosphatases – traffic carried out using a human skeletal muscle library subcloned into the signals and signal traffic. Trends Biochem Sci 1997, 22:427-431. pGAD10 vector (Clontech) transformed into the yeast strain Y166. 5. Myers MP, Pass I, Batty IH, VanderKaay J, Stolarov JP, Hemmings BA, Yeast cells (4 × 106) were transformed with pGAD10 library constructs et al.: The lipid phosphatase activity of PTEN is critical for its and PDK1 constructs in the pAS2-1 vector. Further information on the tumor suppressor function. Proc Natl Acad Sci USA 1998, yeast two-hybrid screen is provided in the Supplementary material. The 95:13513-13518. expression of all GST fusion proteins in 293 cells was performed as 6. Maehama T, Dixon JE: The tumor suppressor, PTEN/MMAC1, described previously ([19] and see Supplementary material). dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem 1998, α 273:13375-13378 Measurement of PKB activation after incubation with GST–PIF 7. Kohn AD, Kovacina KS, Roth RA: Insulin stimulates the kinase This assay was carried out in two stages as described previously ([19], activity of Rac-pk, a pleckstrin-homology domain-containing and see Supplementary material). Briefly, in the first stage, GST–PKBα Ser/Thr kinase. EMBO J 1995, 14:4288-4295. was incubated with GST–PIF (or other indicated PDK1 proteins in the 8. 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