1 MRC Protein Phosphorylation Unit, MSI/WTB complex, University of Dundee, Dow Street, Dundee, DD1 5EH, UK
2 School of Life Sciences, MSI/WTB complex, University of Dundee, Dow Street, Dundee, DD1 5EH, UK
3 Division of Cell and Developmental Biology, MSI/WTB complex, University of Dundee, Dow Street, Dundee, DD1 5EH, UK
* Author for correspondence (e-mail: b.j.collins{at}dundee.ac.uk)
Accepted 2 August 2005
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Protein kinase, Docking sites, Akt, SGK, PKC, RSK, AGC kinase
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The structure of the catalytic domain of PDK1 revealed that the phospho-hydrophobic-motif-binding pocket was composed of a hydrophobic groove located next to a cluster of basic residues forming a phosphate groove (Biondi et al., 2002) (Fig. 1D-F). Mutagenesis of the hydrophobic groove prevented PDK1 from activating S6K, SGK and RSK in vitro, without affecting PKB activation (Biondi et al., 2001
; Frodin et al., 2002
). The crucial role that the hydrophobic groove of PDK1 played in vivo was emphasized in a knockin study in which disruption of this site abolished activation of S6K and RSK, but not PKB (Collins et al., 2003
). To date, the role of the phosphate groove of PDK1 has only been studied in vitro. Mutation of Arg131, located in the phosphate groove, abolished binding of PDK1 to phosphopeptides encompassing the hydrophobic motifs of S6K (Biondi et al., 2002
) and RSK (Frodin et al., 2002
), indicating that this site would play a key role in regulating PDK1 activity. The aim of this study was both to define the physiological role that the phosphate groove of PDK1 plays in enabling PDK1 to regulate the activation of its diverse substrates and to define its role in mouse development.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antibodies
The following antibodies were raised in sheep and affinity purified on the appropriate antigen: PDK1 whole protein (full-length human PDK1 protein), PDK1 mouse C-terminus (residues 540-559 of mouse PDK1, RKIQEVWRQQYQSNPDAAVQ), total GSK3 (residues 471-483 of rat GSK3
, QAPDATPTLTNSS), total GSK3ß (full-length human protein), total S6K1 (residues 25-44 of rat S6K1, AGVFDIDLDQPEDAGSEDEL), S6 phospho S235-P (residues 229-242 of human S6, AKRRRLpSSLRASTS), total RSK (residues 712-734 of human p90 RSK2, RNQSPVLEPVGRSTLAQRRGIKK), RSK phospho T360-P (Thr359 in human RSK1, residues 355-364 of rat RSK1, FTSRTpPRDSP), RSK phospho S381-P (Ser380 in human RSK1, residues 376-385 of rat RSK1, FRGFSpFVATG), GST-FOXO1a (full-length human protein), PKC
(residues 3-24 of rat PKC
, SRTDPKMDRSGGRVRLKAHYGG) and TSC2 (residues 1791-1814 of mouse TSC2, ATPTYETGQRKRLISSVDDFTEFV). The total PKB
antibody used to immunoprecipitate and immunoblot PKB
was a mouse monoclonal antibody raised against residues 1-149 of human PKB and was purchased from Upstate (05-591). The total GAPDH antibody was a mouse monoclonal antibody and was purchased from Calbiochem (CB1001). Mouse monoclonal antibodies recognizing GST were purchased from Sigma. The following antibodies were purchased from Cell Signalling Technology and the catalogue number indicated: PKB phospho T308-P (#9275), PKB phospho S473-P (#9271), GSK3
/GSK3ß phospho S21-P/S9-P (#9336), PKC
/
phospho T410-P/T403-P (#9378), S6K1 phospho T421-P/S424-P (#9204), S6K1 phospho T389-P (#9205), total S6 Protein (#2212), human RSK1 phospho T573-P (#9346), total ERK1/2 (#9102), ERK1/2 phospho T202/Y204-P (#9101), TSC2 phospho T1462-P (#3611), TSC2 phospho S939-P (#3615), FOXO1a/FOXO3a phospho T24-P/T32-P (#9464), FOXO1a phospho S256-P (#9461), total PRK2 (#2612), and PRK1/PRK2 phospho T778/T816-P (#2611). We found that the pan-PDK1 site antibody (#9379) recognized the phosphorylated T-loop of S6K in cell extracts. The following antibodies were purchased from Santa Cruz and the catalogue number indicated: RSK2 phospho S227-P (sc-12445-R), PKC
(Sc-208), PKCßII (Sc-210), PKC
(Sc-213), PKC
(Sc-211), PKC
(Sc-214) and total PRK1 (sc-7161). Secondary antibodies coupled to horseradish peroxidase (HRP) were from Pierce. Secondary antibodies coupled to IRDye800 flurophore for use with the LI-COR Odyssey infrared detection system were purchased from Rockland.
General methods and buffers
Restriction enzyme digests, DNA ligations, site-directed mutagenesis, PCR, Southern blotting and other recombinant DNA procedures were performed using standard protocols. DNA sequencing was performed by The Sequencing Service (School of Life Sciences, University of Dundee, Scotland; www.dnaseq.co.uk) using Applied Biosystems Big-Dye Ver 3.1 chemistry on an Applied Biosystems model 3730 automated capillary DNA sequencer. Lysis buffer was 50 mM Tris-HCl pH 7.5, 1 mM EGTA, 1 mM EDTA, 1% (by mass) Triton X-100, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 0.27 M sucrose, 0.1% (by vol) 2-mercaptoethanol and `Complete' proteinase inhibitor cocktail (one tablet per 50 ml). Buffer A was 50 mM Tris-HCl pH 7.5, 0.1 mM EGTA and 0.1% (by vol) 2-mercaptoethanol. Embryo membrane digestion buffer was 50 mM KCl, 10 mM Tris, pH 7.5, 2.5 mM MgCl2, 0.5% Tween 20 and 0.1 mg/ml proteinase K.
Construction of the R131M knockin targeting vector
A BAC clone containing the mouse genomic PDK1 sequence from a 129Sv mouse BAC library has been used to define the mouse intron/exon sequences of the kinase domain of PDK1 (Williams et al., 2000). This revealed that the kinase domain of PDK1 commences at exon 2 and Arg131 is located in exon 4. A pMC1-neo-pA cassette flanked by loxP sites (floxed; Gu et al., 1993
) was introduced at the 5' end of exon 3 (see Fig. 2A). The 5' homology arm was constructed with two fragments: a HindIII fragment of 3.3 kb containing exon 2 and a 1.7 kb HindIII-NdeI fragment from the mouse PDK1 BAC clone. A 3.8 kb NdeI-SacI fragment from the BAC clone containing exons 3 and 4 was used to construct the 3' homology region and the codon encoding for Arg131 (AGA) was altered by mutagenesis to encode Met (ATG). This mutation was previously shown to prevent PDK1 from interacting with a phosphopeptide encompassing the hydrophobic motif of RSK (Frodin et al., 2002
). Appropriate linkers were used to introduce an EcoRV restriction site at the HindIII site 3' of exon 4. The PGK-TK-pA cassette was included at the 3' end of the 3' arm for negative selection. Further details of the sequence of this targeting construct are available by request. The final knockin construct was purified on a caesium chloride gradient and was linearized using NotI before electroporation.
|
Microinjection of positively targeted ES cells into blastocysts
In order to obtain blastocysts for microinjection, CB6f1 (a hybrid of BALB/c and C57Bl/6) female mice were induced to superovulate by the injection of pregnant mare serum gonadotropin (PMSG), which has follicle-stimulating hormone activity. This was followed 48 hours later by the injection of human chorionic gonadotropin (HCG), which has lutenizing hormone (LH) activity, thus mimicking the LH surge that normally brings about ovulation. These females were then mated with C57Bl/6 males. Blastocysts were removed at 2.5 days post coitus (dpc) and cultured overnight. The following morning (3.5 dpc) each expanded blastocyst was injected with 10-15 PDK1131Mneo/+ ES cells. Around 40 blastocysts were injected on each round of microinjection. Following microinjection, the blastocysts were returned to culture medium for 2 hours, prior to transfer to CD1 pseudopregnant females. The offspring were born 17 days following re-implantation. These animals were chimaeras, having developed from both the injected ES cells and the recipient blastocyst. Subsequent matings were continued with C57Bl/6 mice. The Neomycin cassette was excised in the PDK1131Mneo/+ mice by mating with the Bal1 mouse line, which expresses the Cre recombinase enzyme in all tissues (Betz et al., 1996
). Neomycin excision was identified using the Primers P1 (5'-CTATGCTGTGTTACTTCTTGGAGCACAG) and P2 (5'-AATAGCCAGGGCTACACAGAGAAACCTTTC) as described in Fig. 2. PCR yields a product of 200 bp for the wild-type allele and 330 bp for the 131M allele in which the Neo has been excised (PDK1131M/+) (Fig. 2C).
Analysis of phenotype of PDK1131M/131M embryos
Heterozygous PDK1131M/+ mice were intercrossed and the day of plugging was designated E0.5. Pregnant females were sacrificed and embryos were dissected from the uterus in PBS. Embryos were then photographed using a Leica M275 microscope before being fixed overnight in 4% PFA in PBS, then stored in PBS with 0.02% sodium azide. Genotyping was performed on embryonic membranes that were incubated overnight in embryo membrane digestion buffer at 65°C and subjected to PCR with the P1 and P2 primers (Fig. 2A,C).
Generation and isolation of PDK1131M/131M-knockin ES cells
Female PDK1131M/+ mice were induced to superovulate by the injection of PMSG. This was followed 48 hours later by the injection of HCG. These mice were then mated with male PDK1131M/+ mice. Blastocysts were removed at 2.5 dpc and cultured on 24-well plates on a feeder layer of MEFs for 1-2 weeks to allow the ES cells to grow. Wells were trypsinized and 80% of the aliquot frozen in two batches while the remaining 20% was used to grow cells for DNA preparation. Cells were analysed by Southern blotting with the 3' probe and by PCR using P1 and P2. 20% of the cell lines were found to possess the PDK1131M/131M genotype.
Cell culture, stimulation and cell lysis
PDK1+/+, PDK1131M/131M and PDK1-/- ES cells were grown on gelatinized tissue culture plastic in KnockOut DMEM containing 10% KnockOut SR supplemented with 0.1 mM non-essential amino acids, antibiotics (100 units penicillin G, 100 µg/ml streptomycin), antimycotic (1 µg/ml Ciproxin Infusion), 2 mM L-glutamine, 0.1 mM ß-mercaptoethanol and 25 ng/ml murine leukaemia inhibitory factor. The ES cells were cultured to 80% confluence on 15 cm diameter dishes and incubated for 4 hours in KnockOut DMEM lacking serum. The cells were then stimulated with the indicated agonists as described in the figure legends. The cells were lysed in 0.4 ml of ice-cold lysis buffer and centrifuged at 4°C for 15 minutes at 13,000 g. The supernatants were aliquoted, frozen in liquid nitrogen and stored at -80°C until use. Protein concentrations were determined by the Bradford method using bovine serum albumin as a standard.
Immunoprecipitation and assay of protein kinases
500 µg ES cell lysate protein was used to immunoprecipitate and assay PKB, S6K1 and RSK isoforms. The lysates were incubated at 4°C for 1 hour on a shaking platform with 5 µg of each antibody coupled to 10 µl of protein G-Sepharose. The immunoprecipitates were washed twice with 1 ml of lysis buffer containing 0.5 M NaCl, and once with 1 ml of buffer A. The standard assay (50 µl) contained: washed Protein G-Sepharose immunoprecipitate, 50 mM Tris/HCl pH 7.5, 0.1 mM EGTA, 0.1% (by vol) 2-mercaptoethanol, 2.5 µM PKI (TTYADFIASGRTGRRNAIHD, peptide inhibitor of cyclic-AMP-dependent protein kinase), 10 mM magnesium acetate, 0.1 mM [
32P]ATP (
200 cpm/pmol) and Crosstide (GRPRTSSFAEG). For PKB and S6K assays, 100 µM Crosstide was used; for RSK assays, 30 µM Crosstide was used (Alessi et al., 1996
). The assays were carried out for 30 minutes at 30°C, the assay tubes being agitated continuously to keep the immunoprecipitate in suspension, then terminated and analysed as described previously (Alessi et al., 1995
). PDK1 was immunoprecipitated from 1 mg of ES cell lysate protein with the anti-mouse PDK1 C-terminal antibody and the immunoprecipitates were washed and assayed as above, except that T308tide (KTFCGTPEYLAPEVRR, 1 mM) was used as the substrate (Biondi et al., 2000
). In all assays, 1 mUnit of activity was that amount of enzyme that catalysed the phosphorylation of 1 pmol of substrate in 1 minute.
Expression of GST-PDK1 in 293 cells and GST-SGK1 in ES cells
GST-PDK1 and GST-PDK1(R131M) was expressed in 293 cells and affinity purified on glutathione-Sepaharose as described previously (Alessi et al., 1997). Constructs encoding the expression of GST-SGK1 or GST-SGK1(S422D) lacking the first 60 N-terminal residues have been described previously (Kobayashi and Cohen, 1999
). The 60 N-terminal amino acids of SGK encode polyubiquitination sites and, unless these are removed, SGK cannot be expressed at significant levels (Brickley et al., 2002
). ES cells were grown to 90% confluence on 15 cm tissue culture dishes in Dulbecco's modified Eagle's medium containing high glucose supplemented with 15% foetal calf serum, 0.1 mM non-essential amino acids, 2 mM L-glutamine, 0.1 mM 2-mercaptoethanol and 25 ng/ml murine leukaemia inhibitory factor. After washing cells twice in PBS, cells were then fed with 11 ml of DMEM (with no LIF, serum or antibiotics). 22 µg of GST-SGK1 ebg2T plasmid was premixed for 20 minutes with 25 µl Lipofectamine 2000 in 1 ml of optiMEM I and was added dropwise to each dish and the cells were then incubated for 5 hours. Media was then made up to contain 15% foetal calf serum, 0.1 mM non-essential amino acids, 2 mM L-glutamine, 0.1 mM 2-mercaptoethanol and 25 ng/ml murine leukaemia inhibitory factor. 24 hours post-transfection, the cells were stimulated as described in the figure legends and lysed in 0.4 ml lysis buffer and the lysates centrifuged at 13000 g for 15 minutes at 4°C. After immunobloting the lysates with anti-GST antibody to verify expression levels of SGK, the supernatants containing similar levels of SGK were incubated for 1 hour on a shaking platform with 20 µl of glutathione-Sepharose equilibrated previously in lysis buffer. The suspension was centrifuged for 2 minutes at 13000 g and the beads were washed twice with lysis buffer containing 0.5 M NaCl and once with buffer A. The purified SGK was either assayed for SGK1 activity employing the same assay and peptide substrate (100 µM Crosstide) as described above or immunoblotted with the anti-GST antibody after resuspension in SDS sample buffer.
Affinity purification of PDK1 on HM-PRK2 Sepharose
Streptavidin-Sepharose High Performance (0.5 ml) equilibrated in lysis buffer was incubated with 25 µg of Biotinylated HM-PRK2 peptide (Biotin-C6spacer: REPRILSEEEQEMFRDFDYIADWC) on a shaking platform for 30 minutes and the beads washed with lysis buffer to remove any unconjugated peptide. 0.5 mg of ES cell lysate protein was incubated with 10 µl of the conjugated HM-PRK2 peptide-Sepharose on a shaking platform for 1 hour at 4°C, which was washed three times with 1 ml of lysis buffer. The beads were resuspended in a volume of 15 µl of lysis buffer to which 5 µl of 4x SDS Sample Buffer was added. The samples were then immunoblotted for PDK1 as described below.
Immunoblotting
Unless stated otherwise, 25 µg of protein lysate in SDS Sample Buffer was subjected to SDS/polyacrylamide gel electrophoresis for immunoblotting. Exceptions were: ERK total, 10 µg; ERK-P, 5 µg; GAPDH, 5 µg; S6 total, 10 µg; and PDK1 total, 40 µg. Proteins were transferred to nitrocellulose. For phospho-specific antibody blots, the nitrocellulose membranes were immunoblotted at 4°C for 16 hours using the indicated antibodies (2 µg/ml for the sheep antibodies or 500-fold dilution for commercial antibodies) in the presence of 10 µg/ml of the de-phosphopeptide antigen used to raise the antibody for sheep-raised phospho-specific antibodies. For total blots, the nitrocellulose membranes were immunoblotted at 4°C for 16 hours using the indicated antibodies (1 µg/ml for the sheep and rabbit-raised antibodies and 1000-fold dilution for the mouse monoclonal PKB and GAPDH antibodies). PKC
was used at 2 µg/ml. The blots were incubated in 50 mM Tris/HCl pH 7.5, 0.15 M NaCl, 0.2% (by vol) Tween containing either 5% (by mass) skimmed milk for sheep-raised antibodies or 0.5-1% BSA (by mass) for rabbit and mouse-raised antibodies. Detection was performed using HRP-conjugated secondary antibodies and the enhanced chemiluminescence reagent or, for the PKC isoform and indicated PKB blots, IRDye800 flurophore-conjugated antibody and using the Odyssey infrared detection system (LI-COR Biosciences). Band intensity was quantitated using LI-COR software.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PDK1 is normally expressed in knockin ES cells
Employing two different PDK1 antibodies, we found that PDK1 is expressed at similar levels in PDK1131M/131M and PDK1+/+ ES cells (Fig. 3A). We also immunoprecipitated PDK1 from ES cell lysates and assayed its activity using the T308tide peptide substrate that does not interact with the phospho-hydrophobic-motif-binding pocket (Biondi et al., 2000). The activity of PDK1 isolated from the PDK1131M/131M-knockin ES cells was similar to that measured from PDK1+/+ wild-type cells. We also expressed recombinant wild-type GST-PDK1 and mutant GST-PDK1(R131M) and found that these displayed identical specific activities (Fig. 3B). In order to confirm disruption of the phosphate groove of PDK1 in the PDK1131M/131M-knockin ES cells, cell lysates were incubated with streptavidin-Sepharose conjugated to a biotinylated peptide that encompasses the hydrophobic motif of PRK2 that interacts strongly with the phospho-hydrophobic-motif-binding pocket of PDK1 (Balendran et al., 1999
; Biondi et al., 2000
). Using this approach, PDK1 could be affinity purified from wild-type PDK1+/+ ES cells, but not from PDK1131M/131M ES cells, confirming disruption of the phosphate groove in knockin cells (Fig. 3A, lower panel).
|
PKB is activated in knockin ES cells
In order to assess PKB activation, serum-starved PDK1+/+ and PDK1131M/131M ES cells were stimulated with IGF1 in the presence or absence of the PI 3-kinase inhibitor wortmannin. PKB
activity was assayed following its immunoprecipitation, and PKB
phosphorylation at Thr308 and Ser473 was measured by immunoblot analysis with phosphospecific antibodies. In both PDK1+/+ and PDK1131M/131M ES cells, IGF1 induced a marked activation and phosphorylation of Thr308 and Ser473 that was inhibited by wortmannin (Fig. 4, upper panel). The activation of PKB and phosphorylation of Thr308 was slightly reduced in IGF1-stimulated PDK1131M/131M ES cells. Quantitative analysis of Thr308 phosphorylation employing LI-COR infrared analysis in two additional experiments confirmed a slight reduction of Thr308 phosphorylation in the knockin cells (Fig. 4, lower panel). IGF1 induced the phosphorylation of the PKB substrates glycogen synthase kinase-3 (GSK3) and tuberous sclerosis complex-2 (TSC2), as well as the FOXO-1a forkhead transcription factor, in both PDK1+/+ and PDK1131M/131M ES cells, albeit to a slightly reduced extent than observed in wild-type cells (Fig. 4).
|
|
As phosphorylation of the hydrophobic motif of S6K is regulated by PKB, which is activated in PDK1131M/131M ES cells (Fig. 4), we were interested to explore whether the reduction of Thr389 phosphorylation of S6K in the PDK1131M/131M-knockin cells was the result of increased dephosphorylation of this residue, as was previously proposed to occur in the absence of T-loop phosphorylation (Collins et al., 2003). We therefore treated ES cell lines with the protein phosphatase inhibitor okadaic acid and found that it markedly stimulated S6K activity as well as phosphorylation of Thr389 in the IGF1-stimulated PDK1131M/131M cells, but not the PDK1-/- ES cells, which lack PKB activity (Williams et al., 2000
) (Fig. 5).
Reduced activation of SGK1 in knockin ES cells
As we have not previously been able to measure endogenous SGK activity in ES cells (Collins et al., 2003), we transfected PDK1+/+ and PDK1131M/131M ES cells with a construct encoding wild-type SGK1, and measured SGK1 activity as well as hydrophobic motif phosphorylation at Ser422, in unstimulated and IGF1-treated cells in the presence or absence or wortmannin (Fig. 6A). In PDK1+/+ ES cells, IGF1 increased activity approximately threefold without significantly enhancing the high basal level of Ser422 phosphorylation, similar to what has been observed before in ES cells (Collins et al., 2003
). In PDK1131M/131M ES cells, the basal SGK1 activity was nearly tenfold lower than control cells and IGF1 stimulated SGK1 activity to a level that was approximately fourfold lower than that observed in PDK1+/+ ES cells. Wortmannin blocked IGF1-induced SGK1 activation in both the control and knockin ES cells. It had been shown previously that an SGK1 mutant in which the hydrophobic residue is changed to Asp [SGK1(S422D)] is constitutively active when expressed in cells (Kobayashi and Cohen, 1999
) owing to its high affinity for the phospho-hydrophobic-motif-binding pocket of PDK1 (Biondi et al., 2001
). When expressed in PDK1131M/131M-knockin ES cells, the SGK1(S422D) mutant possessed more comparable activity to that observed in the wild-type cells (Fig. 6B).
|
Activation of RSK is reduced in knockin ES cells
RSK activity was measured after immunoprecipitation from serum-starved ES cells with an antibody that immunoprecipitates all RSK isoforms. In PDK1+/+ ES cells, RSK activity was stimulated approximately twofold by the phorbol ester TPA (Fig. 7), which activates RSK through the ERK mitogen-activated protein (MAP) kinase pathway. Treatment of ES cells with the PD184352 MEK inhibitor (Sebolt-Leopold et al., 1999) prevented ERK phosphorylation and RSK activation. In unstimulated PDK1131M/131M ES cells, RSK activity was considerably reduced, to a level similar to that observed for PDK1+/+ ES cells treated with PD184352 (Fig. 7). Stimulation of PDK1131M/131M ES cells with TPA increased RSK activity approximately threefold, whereas treatment with PD184352 reduced RSK activity to below basal levels. Reduced activation of RSK in the PDK1131M/131M ES cells is not a result of lack of TPA-induced ERK activation, as TPA stimulated ERK phosphorylation to the same extent as in PDK1+/+ ES cells. Moreover, phosphorylation of RSK at three ERK-dependent phosphorylation sites (Thr359, Thr573, Ser386) was not inhibited and was even moderately enhanced in the PDK1131M/131M-knockin ES cells (Fig. 7). Instead, the reduction of basal and TPA-stimulated RSK activity in the PDK1131M/131M ES cells correlated with lower phosphorylation of RSK at the T-loop site (Ser227), assessed with a phosphospecific antibody. Phosphorylation of GSK3
/GSK3ß, a physiological substrate of RSK (Frame and Cohen, 2001
), was also reduced in the PDK1131M/131M ES cells.
|
Levels of PKC isoforms in the PDK1131M/131M-knockin ES cell line
As outlined in the Introduction, phosphorylation of PKC isoforms by PDK1 plays an important role in stabilizing these enzymes. To study the importance of the phosphate groove of PDK1 in stabilizing PKC isoforms, we assessed the levels of these enzymes in the PDK1131M/131M ES cells. As a control, we also employed hydrophobic-groove-knockin ES cells (PDK1155E/155E) (Collins et al., 2003) and PDK1-knockout ES cells (PDK1-/-) (Williams et al., 2000
). The levels of PKC isoforms were assessed by quantitative immunoblot analysis employing a LI-COR Odyssey infrared imaging system. In the PDK1131M/131M ES cells the levels of conventional and novel PKC isoforms that require hydrophobic motif phosphorylation for activation (PKC
, PKCßII, PKC
, PKC
and PKC
) were more similar to those found in the PDK1+/+ cells than in the PDK1155E/155E and PDK1-/- cells, where the levels of these PKC isoforms were markedly reduced (Fig. 8A). The levels of atypical PKC isoforms (PKC
, PRK1 and PRK2) that possess an acidic residue at the hydrophobic motif rather than a phosphorylatable Ser/Thr are moderately reduced in the PDK1131M/131M ES cells, albeit to level lower than that observed in PDK1155E/155E and PDK1-/- cells. We also measured T-loop phosphorylation of PKC
, PRK1 and PRK2 and found that the phosphorylation was reduced to a lesser extent in the PDK1131M/131M ES cells compared with PDK1155E/155E and PDK1-/- cells (Fig. 8A).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
By contrast, PDK1131M/131M embryos survived almost to birth but nevertheless some overt defects in craniofacial development and limb posture are evident and are suggestive of defects in cranial neural crest development and in the functioning of the peripheral nervous system. These findings, together with lack of dorsal root ganglia reported in PDK1-/- and PtdIns(3,4,5)P3-binding-deficient PDK1-knockin embryos (McManus et al., 2004), suggest a role for PDK1 substrates in the formation and/or survival of neural-crest-derived tissues. All of these PDK1 mutant phenotypes display a reduction in forebrain size (although this is less evident in PDK1131M/131M embryos). This is a characteristic feature of `flat-top' mutant mice that have a mutation in mTOR that leads to a reduction in its kinase activity and hence phosphorylation of S6K (Hentges et al., 1999
; Hentges et al., 2001
). As attenuation or loss of S6K activation is observed in all PDK1 mutants, this comparison suggests that forebrain defects in these animals are attributable to reduced S6K activity.
The results of this study demonstrate that the phosphate groove of PDK1 plays an important role in enabling PDK1 to activate S6K, SGK and RSK maximally. Both the basal and stimulated activities of these enzymes were markedly reduced in the PDK1131M/131M-knockin ES cells (Figs 5, 6, 7). Importantly, agonists still induced a substantial activation of S6K, SGK and RSK in the knockin cells. This is in contrast to the PDK1155E/155E hydrophobic-groove-knockin ES cells, in which S6K and RSK are completely inactive and not stimulated by agonists (Collins et al., 2003). In addition to phosphorylation of the hydrophobic motif, activation of S6K is accompanied by phosphorylation of other residues including C-terminal Ser/Thr-Pro residues. Phosphorylation of these residues is regulated by mTOR and plays a role in enabling S6K to be activated (Isotani et al., 1999
). Indeed, mutation of these residues to Glu has been shown to facilitate activation of S6K by PDK1 in vitro (Pullen et al., 1998
) and activation of S6K in cells (Dennis et al., 1998
; Han et al., 1995
). PDK1 does not regulate the phosphorylation of two of these residues (Thr421 and Ser424) as, in PDK1-/- ES cells, IGF1 still induced normal phosphorylation of these residues (McManus et al., 2004
). Consistent with this, IGF1-induced phosphorylation of Thr421/Ser424 is not affected in PDK1131M/131M-knockin ES cells, and even increased under conditions in which phosphorylation of the T-loop and hydrophobic motif of S6K are markedly reduced (Fig. 5). It is possible that phosphorylation of the C-terminal Ser/Thr-Pro residues of S6K induces a conformational change that exposes the hydrophobic motif and enables PDK1 to bind S6K through its hydrophobic groove independently of the phosphate groove. Consistent with this observation, the PDK1(R131M) phosphate groove mutant (Frodin et al., 2002
), unlike the PDK1(L155E) mutant (Biondi et al., 2001
), was still capable of phosphorylating S6K in vitro, but to a lower extent than wild-type PDK1. It is also conceivable that phosphorylation of the hydrophobic motif itself could induce a conformational change, facilitating its direct interaction with the hydrophobic groove of PDK1. The finding that stimulation of the phosphorylation of the hydrophobic motif of S6K by treatment with the phosphatase inhibitor okadaic acid markedly enhanced S6K activity in the PDK1131M/131M-knockin cells (Fig. 5) supports this notion. Moreover, the finding that mutation of the hydrophobic motif phosphorylation site of SGK to an acidic residue also markedly enhanced SGK activity in PDK1131M/131M-knockin cells (Fig. 6B) indicates that phosphorylation of the hydrophobic motif promotes the interaction of SGK with PDK1 in a phosphate-groove-independent manner, probably mediated through the hydrophobic groove of PDK1. Similar arguments also apply to RSK, which is also phosphorylated at sites other than the hydrophobic motif upon activation (Dalby et al., 1998
; Frodin and Gammeltoft, 1999
).
Previous work has suggested that phosphorylation of PKC isoforms by PDK1 plays an important role in stabilizing these enzymes (Newton, 2002). Studies by Newton and colleagues (Gao et al., 2001
) showed that the hydrophobic motif of PKCßII is important for the activation of this enzyme by PDK1, indicating a role for the phospho-hydrophobic-motif-binding pocket of PDK1 in regulating PKC isoforms. Consistent with this, we have found that the levels of PKC isoforms were markedly decreased in PDK1155E/155E hydrophobic-groove-knockin ES cells (McManus et al., 2004
). As outlined in the Introduction, T-loop phosphorylation of PKC isoforms by PDK1 results in the hydrophobic motifs of these enzymes becoming autophosphorylated. If this model is correct, the mechanism by which PDK1 recognizes PKC isoforms must be distinct from that of S6K, SGK and RSK, in that PDK1-mediated activation of PKC isoforms will not be triggered by phosphorylation of the hydrophobic motif. It is likely that the binding of PKC isoforms to diacylglycerol/phorbol esters at membranes, together with Ca2+ binding, induces conformational changes that promote phosphorylation and hence activation by PDK1. The finding that the levels of PKC isoforms are not reduced in the PDK1131M/131M phosphate-groove-knockin ES cells, in contrast to PDK1155E/155E hydrophobic-groove-knockin ES cells, suggests that PDK1 docks to the unphosphorylated hydrophobic motif of PKC isoforms through its hydrophobic groove independently of the phosphate-binding pocket. A model for the activation of PKC isoforms by PDK1 based on these observations is presented in Fig. 8B.
In summary, our studies define the crucial role that the phosphate groove of PDK1 plays in enabling PDK1 to activate S6K, SGK and RSK maximally in vivo, but also provide evidence for a phosphate-groove-independent mechanism in regulating activation of these enzymes. Our results also provide further insight into the mechanism by which PDK1 regulates the phosphorylation of conventional and novel PKC isoforms, suggesting that the hydrophobic groove rather than the phosphate groove is a key determinant enabling PDK1 to phosphorylate these enzymes. This study also provides a further example of how knockin technology can be exploited to define the physiological importance of a domain on a signalling protein.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alessi, D. R., Cohen, P., Ashworth, A., Cowley, S., Leevers, S. J. and Marshall, C. J. (1995). Assay and expression of mitogen-activated protein kinase, MAP kinase kinase, and Raf. Methods Enzymol. 255, 279-290.[Medline]
Alessi, D. R., Caudwell, F. B., Andjelkovic, M., Hemmings, B. A. and Cohen, P. (1996). Molecular basis for the substrate specificity of protein kinase B; comparison with MAPKAP kinase-1 and p70 S6 kinase. FEBS Lett. 399, 333-338.[CrossRef][Medline]
Alessi, D. R., Deak, M., Casamayor, A., Caudwell, F. B., Morrice, N., Norman, D. G., Gaffney, P., Reese, C. B., MacDougall, C. N., Harbison, D. et al. (1997). 3-Phosphoinositide-dependent protein kinase-1 (PDK1): structural and functional homology with the Drosophila DSTPK61 kinase. Curr. Biol. 7, 776-789.[CrossRef][Medline]
Balendran, A., Casamayor, A., Deak, M., Paterson, A., Gaffney, P., Currie, R., Downes, C. P. and Alessi, D. R. (1999). PDK1 acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2. Curr. Biol. 9, 393-404.[CrossRef][Medline]
Betz, U. A., Vosshenrich, C. A., Rajewsky, K. and Muller, W. (1996). Bypass of lethality with mosaic mice generated by Cre-loxP-mediated recombination. Curr. Biol. 6, 1307-1316.[CrossRef][Medline]
Biondi, R. M., Cheung, P. C., Casamayor, A., Deak, M., Currie, R. A. and Alessi, D. R. (2000). Identification of a pocket in the PDK1 kinase domain that interacts with PIF and the C-terminal residues of PKA. EMBO J. 19, 979-988.
Biondi, R. M., Kieloch, A., Currie, R. A., Deak, M. and Alessi, D. R. (2001). The PIF-binding pocket in PDK1 is essential for activation of S6K and SGK, but not PKB. EMBO J. 20, 4380-4390.
Biondi, R. M., Komander, D., Thomas, C. C., Lizcano, J. M., Deak, M., Alessi, D. R. and Van Aalten, D. M. (2002). High resolution crystal structure of the human PDK1 catalytic domain defines the regulatory phosphopeptide docking site. EMBO J. 21, 4219-4228.
Brazil, D. P. and Hemmings, B. A. (2001). Ten years of protein kinase B signalling: a hard Akt to follow. Trends Biochem. Sci. 26, 657-664.[CrossRef][Medline]
Brickley, D. R., Mikosz, C. A., Hagan, C. R. and Conzen, S. D. (2002). Ubiquitin modification of serum and glucocorticoid-induced protein kinase-1 (SGK-1). J. Biol. Chem. 277, 43064-43070.
Collins, B. J., Deak, M., Arthur, J. S., Armit, L. J. and Alessi, D. R. (2003). In vivo role of the PIF-binding docking site of PDK1 defined by knockin mutation. EMBO J. 22, 4202-4211.
Dalby, K. N., Morrice, N., Caudwell, F. B., Avruch, J. and Cohen, P. (1998). Identification of regulatory phosphorylation sites in mitogen-activated protein kinase (MAPK)-activated protein kinase-1a/p90rsk that are inducible by MAPK. J. Biol. Chem. 273, 1496-1505.
Dennis, P. B., Pullen, N., Pearson, R. B., Kozma, S. C. and Thomas, G. (1998). Phosphorylation sites in the autoinhibitory domain participate in p70(s6k) activation loop phosphorylation. J. Biol. Chem. 273, 14845-14852.
Frame, S. and Cohen, P. (2001). GSK3 takes centre stage more than 20 years after its discovery. Biochem. J. 359, 1-16.[CrossRef][Medline]
Frodin, M. and Gammeltoft, S. (1999). Role and regulation of 90 kDa ribosomal S6 kinase (RSK) in signal transduction. Mol. Cell Endocrinol. 151, 65-77.[CrossRef][Medline]
Frodin, M., Jensen, C. J., Merienne, K. and Gammeltoft, S. (2000). A phosphoserine-regulated docking site in the protein kinase RSK2 that recruits and activates PDK1. EMBO J. 19, 2924-2934.
Frodin, M., Antal, T. L., Dummler, B. A., Jensen, C. J., Deak, M., Gammeltoft, S. and Biondi, R. M. (2002). A phosphoserine/threonine-binding pocket in AGC kinases and PDK1 mediates activation by hydrophobic motif phosphorylation. EMBO J. 21, 5396-5407.
Gao, T., Toker, A. and Newton, A. C. (2001). The carboxyl terminus of protein kinase c provides a switch to regulate its interaction with the phosphoinositide-dependent kinase, PDK-1. J. Biol. Chem. 276, 19588-19596.
Gu, H., Zou, Y. R. and Rajewsky, K. (1993). Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting. Cell 73, 1155-1164.[CrossRef][Medline]
Han, J. W., Pearson, R. B., Dennis, P. B. and Thomas, G. (1995). Rapamycin, wortmannin, and the methylxanthine SQ20006 inactivate p70s6k by inducing dephosphorylation of the same subset of sites. J. Biol. Chem. 270, 21396-21403.
Hentges, K., Thompson, K. and Peterson, A. (1999). The flat-top gene is required for the expansion and regionalization of the telencephalic primordium. Development 126, 1601-1609.
Hentges, K. E., Sirry, B., Gingeras, A. C., Sarbassov, D., Sonenberg, N., Sabatini, D. and Peterson, A. S. (2001). FRAP/mTOR is required for proliferation and patterning during embryonic development in the mouse. Proc. Natl. Acad. Sci. USA 98, 13796-13801.
Isotani, S., Hara, K., Tokunaga, C., Inoue, H., Avruch, J. and Yonezawa, K. (1999). Immunopurified mammalian target of rapamycin phosphorylates and activates p70 S6 kinase alpha in vitro. J. Biol. Chem. 274, 34493-34498.
Joyner, A. L. (1993). Gene Targeting, A Practical Approach. Oxford: IRL Press.
Kobayashi, T. and Cohen, P. (1999). Activation of serum- and glucocorticoid-regulated protein kinase by agonists that activate phosphatidylinositide 3-kinase is mediated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and PDK2. Biochem. J. 339, 319-328.[CrossRef][Medline]
Lang, F. and Cohen, P. (2001). Regulation. and physiological roles, of, serum- and glucocorticoid-induced protein kinase isoforms. Sci. STKE 108, RE17.
Lawlor, M. A., Mora, A., Ashby, P. R., Williams, M. R., Murray-Tait, V., Malone, L., Prescott, A. R., Lucocq, J. M. and Alessi, D. R. (2002). Essential role of PDK1 in regulating cell size and development in mice. EMBO J. 21, 3728-3738.
Leslie, N. R., Biondi, R. M. and Alessi, D. R. (2001). Phosphoinositide-regulated kinases and phosphoinositide phosphatases. Chem. Rev. 101, 2365-2380.[CrossRef][Medline]
Lizcano, J. M., Deak, M., Morrice, N., Kieloch, A., Hastie, C. J., Dong, L., Schutkowski, M., Reimer, U. and Alessi, D. R. (2002). Molecular basis for the substrate specificity of NIMA-related kinase-6 (NEK6). Evidence that NEK6 does not phosphorylate the hydrophobic motif of ribosomal S6 protein kinase and serum- and glucocorticoid-induced protein kinase in vivo. J. Biol. Chem. 277, 27839-27849.
McManus, E. J., Collins, B. J., Ashby, P. R., Prescott, A. R., Murray-Tait, V., Armit, L. J., Arthur, J. S. and Alessi, D. R. (2004). The in vivo role of PtdIns(3,4,5)P(3) binding to PDK1 PH domain defined by knockin mutation. EMBO J. 23, 2071-2082.
Mora, A., Komander, D., Van Aalten, D. M. and Alessi, D. R. (2004). PDK1, the master regulator of AGC kinase signal transduction. Semin. Cell Dev. Biol. 15, 161-170.[CrossRef][Medline]
Newton, A. C. (2002). Regulation of the ABC kinases by phosphorylation: protein kinase C as a paradigm. Biochem. J. 20, 361-371.[CrossRef]
Pullen, N., Dennis, P. B., Andjelkovic, M., Dufner, A., Kozma, S. C., Hemmings, B. A. and Thomas, G. (1998). Phosphorylation and activation of p70s6k by PDK1. Science 279, 707-710.
Scheid, M. P. and Woodgett, J. R. (2001). PKB/Akt: functional insights from genetic models. Nat. Rev. Mol. Cell. Biol. 2, 760-768.[CrossRef][Medline]
Scheid, M. P. and Woodgett, J. R. (2003). Unravelling the activation mechanisms of protein kinase B/Akt. FEBS Lett. 546, 108-112.[CrossRef][Medline]
Sebolt-Leopold, J. S., Dudley, D. T., Herrera, R., Van Becelaere, K., Wiland, A., Gowan, R. C., Tecle, H., Barrett, S. D., Bridges, A., Przybranowski, S. et al. (1999). Blockade of the MAP kinase pathway suppresses growth of colon tumors in vivo. Nat. Med. 5, 810-816.[CrossRef][Medline]
Shamji, A. F., Nghiem, P. and Schreiber, S. L. (2003). Integration of growth factor and nutrient signaling: implications for cancer biology. Mol. Cell 12, 271-280.[CrossRef][Medline]
Stewart, C. L., Schuetze, S., Vanek, M. and Wagner, E. F. (1987). Expression of retroviral vectors in transgenic mice obtained by embryo infection. EMBO J. 6, 383-388.[Abstract]
Volarevic, S. and Thomas, G. (2001). Role of S6 phosphorylation and S6 kinase in cell growth. Prog. Nucleic Acid Res. Mol. Biol. 65, 101-127.[Medline]
Williams, M. R., Arthur, J. S., Balendran, A., van der Kaay, J., Poli, V., Cohen, P. and Alessi, D. R. (2000). The role of 3-phosphoinositide-dependent protein kinase 1 in activating AGC kinases defined in embryonic stem cells. Curr. Biol. 10, 439-448.[CrossRef][Medline]
|