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First published online 11 October 2005
doi: 10.1242/jcs.02617

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In vivo role of the phosphate groove of PDK1 defined by knockin mutation

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

View larger version (59K): [in a new window]   Fig. 1. Regulation of AGC kinase by PDK1 and structure of the phospho-hydrophobic-motif-binding pocket. Model described in the Introduction by which activation of PKB (A), S6K (B) and RSK (C) by PDK1 in response to extracellular agonists is controlled. Abbreviations: PH, Pleckstrin-homology; HM, hydrophobic motif; PHM, phospho-hydrophobic motif; NTKD, N-terminal kinase domain of RSK; CTKD, C-terminal kinase domain of RSK. (D) Ribbon representation of the kinase domain of PDK1 based on the structure of the catalytic domain reported previously (Biondi et al., 2002). The residues on the N-terminal lobe making up the hydrophobic groove are shown in green, whereas the residues making up the phosphate groove are in orange. ATP is shown as a ball and stick model with phosphate ions highlighted in purple and oxygen atoms highlighted in red. Locations of the T-loop phosphorylated Ser241 is indicated (E). Residues forming the hydrophobic and phosphate grooves are illustrated. The PDK1 backbone is shown as a grey ribbon. Hydrogen bonds forming with the oxygen atoms (red) of the sulphate ion (green) that lies within the phosphate groove when PDK1 is crystallized (Biondi et al., 2002) are indicated as black dotted lines. (F) Surface representation of the hydrophobic and phosphate groove. Position of the Arg131 residue that is mutated in this study is shown in red.

View larger version (30K): [in a new window]   ES cell knockin strategy. (A) Diagram illustrating the targeting knockin construct, the 5' end of the gene for PDK1 and the allele modification generated. The position of the 3' probe used to genotype targeted knockin cells in B is shown. The position of the PCR primers used to genotype the Cre recombinase-mediated excision of the neomycin cassette is indicated by arrows. The position of Arg131/Met131 in exon 4 is represented by an asterisk. The position of the introduced novel EcoRV restriction site is marked. (+), wild-type allele; (131Mneo), targeted knockin allele with the neomycin cassette still present; (131M), targeted knockin allele with the neomycin cassette removed. (B) Genomic DNA purified from the indicated ES cell lines was digested with EcoRV, electrophoresed on a 1% agarose gel, transferred to nitrocellulose and the membrane incubated with the 32P-labelled 3' probe. The wild-type allele generates a 17 kb fragment whereas the targeted knockin allele generates a 7.2 kb fragment in this analysis. (C) Genomic DNA purified from the indicated ES cell lines or embryos was used as a template for PCR with the P1 and P2 primers. The wild-type allele (+) generates a 200 bp product, whereas a 330 bp product is obtained with the targeted allele in which the neomycin cassette is excised (131M). (D) Genomic DNA purified from the indicated ES cell lines was subjected to PCR using primers 5'-GCCTCCAAGGAGATCAGTACACAG and 5'-GGTAGTCGCAGGGCCTGTGCTG to generate a 460 bp product that encompasses the 131 mutation region on exon 4. The resultant PCR products were ligated into the pCR-Topo 2.1 vector, transformed into Escherichia coli and clones sequenced. The numbers of the wild type Arg131 and knockin Met131 sequences obtained for each cell line is indicated.

View larger version (27K): [in a new window]   Expression and activity of PDK1 in knockin ES cells. (A) The wild-type PDK1+/+, knockin PDK1131M/131M and knockout PDK1-/- ES cells (Williams et al., 2000) were cultured to 80% confluence and lysed. PDK1 was immunoprecipitated from the cell lysate and assayed with the T308tide peptide as described in the Materials and Methods. The results shown are the average specific activity ±s.e.m. of three separate dishes of cells with assays for each dish performed in duplicate. The cell lysates were also immunoblotted with PDK1 antibody 1 (raised against the C-terminal 20 residues of mouse PDK1) or PDK1 antibody 2 (raised against the recombinant human PDK1 protein). The lysates were also incubated with Sepharose conjugated to the HM-PRK2 peptide in order to affinity purify PDK1 and bound PDK1 visualized by immunoblot analysis as described in the Materials and Methods. Similar results were obtained in three separate experiments. It should be noted that PDK1, as observed in other cell lines, is detected as two bands on immunoblot analysis for reasons that are not yet known (Alessi et al., 1997; Balendran et al., 1999). (B) Equal amounts of recombinant wild-type GST-PDK1 and mutant GST-PDK1(R131M) were assayed with the T308tide peptide and levels of PDK1 present in each assay was assessed by Coomassie staining of a polyacrylamide gel. The assays were performed in triplicate and results presented as the average ±s.e.m.

View larger version (54K): [in a new window]   Activation of PKB and phosphorylation of PKB substrates in knockin cells. The wild-type and knockin ES cells were cultured to 80% confluence. Cells were deprived of serum for 4 hours and either left untreated or incubated in the presence or absence of 100 nM wortmannin for 10 minutes and then stimulated with 20 ng/ml IGF1 for 15 minutes. The cells were lysed and PKB immunoprecipitated and assayed. The results shown are the average specific activity ±s.e.m. of three separate dishes of cells with assays for each dish performed in duplicate. The ES cell lysates were also immunoblotted with the indicated antibodies. For the TSC2 and FOXO1a blots, the ES cells were treated as above except IGF1 stimulation was performed for 30 minutes. Cells were lysed and lysates immunoblotted with the indicated antibodies. Similar results were obtained in two separate experiments. In the lower panel, the results of two further experiments are shown in which quantitation of the immunoblots was performed using the LI-COR Odyssey infrared imaging system as described in the Materials and Methods. The results are plotted relative to the values of the IGF1-stimulated sample in PDK1+/+ cells, which is given a value of 1.0. Abbreviation: HM, hydrophobic motif.

View larger version (48K): [in a new window]   S6K1 activation is impaired in knockin cells. The indicated ES cells were cultured to 80% confluence. Cells were deprived of serum for 4 hours and either left untreated or incubated in the presence or absence of 500 nM okadaic acid for 30 minutes, 100 nM wortmannin for 10 minutes or in the presence or absence of 100 nM rapamycin for 30 minutes, and then stimulated with 20 ng/ml IGF1 for 30 minutes. The cells were lysed and S6K1 immunoprecipitated and assayed. The results shown are the average specific activity ±s.e.m. of two separate dishes of cells with assays for each dish performed in duplicate. The ES cell lysates were also immunoblotted with the indicated antibodies. Thr389 is the phosphorylated S6K hydrophobic motif residue. S6K phosphorylates the S6 protein (S6-P) at Ser235. Similar results were obtained in three separate experiments. Abbreviation: HM, hydrophobic motif.

View larger version (24K): [in a new window]   SGK1 activation is impaired in knockin cells. The indicated ES cells lines were transfected with a DNA construct encoding GST-SGK1 (A) or GST-SGK1(S422D) (B). At 24 hours post-transfection, the ES cells were deprived of serum for 5 hours, incubated in the presence or absence of 100 nM wortmannin for 10 minutes and then either left unstimulated or stimulated with 50 ng/ml IGF1 for 20 minutes. The cells were lysed, and GST-SGK1 was affinity purified from the cell lysate on glutathione-Sepharose and assayed. The results shown are the average ±s.e.m. for three dishes of cells each assayed in duplicate. The purified GST-SGK1 was immunoblotted with the anti-GST antibody (SGK1-Total) to ensure that similar amounts of enzyme were assayed for each condition as well as with the S6K T389 phosphospecific antibody that also crossreacts with the phosphorylated hydrophobic motif of SGK (Lizcano et al., 2002). Similar results were obtained in two separate experiments. Abbreviations: HM, hydrophobic motif.

View larger version (55K): [in a new window]   RSK isoform activation is impaired in knockin cells. The indicated ES cells were cultured to 80% confluence. Cells were deprived of serum for 4 hours and were either left untreated or incubated in the presence or absence of 2 µM PD 184352 for 30 minutes and then either left unstimulated or stimulated with 0.4 µg/ml TPA for 15 minutes. The cells were lysed and RSK isoforms immunoprecipitated with an antibody that recognizes all isoforms and assayed. The results shown are the average specific activity ±s.e.m. of three separate dishes of cells with assays for each dish performed in duplicate. The ES cell lysates were also immunoblotted with the indicated antibodies. Ser227 (numbering based on human RSK2) is phosphorylated by PDK1, Thr359 and Thr573 (numbering based on human RSK1) are phosphorylated by ERK and Ser386 (numbering based on human RSK2) is the hydrophobic motif (HM) phosphorylation residue and is phosphorylated by the C-terminal RSK kinase domain. The RSK2 Ser386-P blot was performed on RSK2 immunoprecipitated from 0.5 mg of ES cell lysate. Similar results were obtained in two separate experiments. Abbreviations: NTKD, N-terminal kinase domain of RSK; CTKD, C-terminal kinase domain of RSK.

View larger version (37K): [in a new window]   Immunoblotting of PKC isoforms in ES cells. (A) The indicated wild-type, knockin and knockout PDK1 ES cells lines were cultured to 80% confluence, washed twice in PBS and lysed. Lysates were subjected to SDS polyacrylamide gel electrophoresis and immunoblotted with the indicated antibodies. Quantitation of the immunoblots, indicated immediately below each blot, was performed using the LI-COR Odyssey infrared imaging system as described in the Materials and Methods. The value of expression of each PKC isoform detected in the control PDK1+/+ cells is taken as 1.0 and the expression of the isoforms in other cells is expressed relative to this. As a loading control, the cell lysates were immunoblotted for glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Similar results were obtained in two independent experiments. (B) Diagram illustrating a potential mechanism of activation for the conventional and novel PKC isoforms. Allosteric modification of inactive PKC by diacylglycerol and/or Ca2+ and membrane localization allows PDK1 to dock with the hydrophobic motif of PKC via the PDK1 hydrophobic groove, independently of the PDK1 phosphate groove. Once docked, PDK1 then phosphorylates the T-loop of PKC. This enables PKC isoforms to autophosphorylate their hydrophobic motif and become active.

View larger version (43K): [in a new window]   Phenotypes of PDK1131M/131M embryos. (A) Representative PDK1131M/+ or PDK1131M/131M embryos at the indicated embryonic stage were dissected in PBS and imaged on a Leica M275 microscope and wholemount photographs were taken. In all images, embryos are shown at the same magnification. Abbreviations: EP, eye pigment; CT, cranial tissue, SLJ, short lower jaw; SUL, slack upper limb. Bar, 2560 µM. (B) PDK1+/131M mice were crossed and the resulting proportion of progeny at each stage is indicated. Genotyping was performed as described in the Materials and Methods.