In vivo role of the PIF-binding docking site of PDK1 defined by knock-in mutation - PubMed (original) (raw)
In vivo role of the PIF-binding docking site of PDK1 defined by knock-in mutation
Barry J Collins et al. EMBO J. 2003.
Abstract
PKB/Akt, S6K, SGK and RSK are mediators of responses triggered by insulin and growth factors and are activated following phosphorylation by 3-phosphoinositide-dependent protein kinase-1 (PDK1). To investigate the importance of a substrate-docking site in the kinase domain of PDK1 termed the 'PIF-pocket', we generated embryonic stem (ES) cells in which both copies of the PDK1 gene were altered by knock-in mutation to express a form of PDK1 retaining catalytic activity, in which the PIF-pocket site was disrupted. The knock-in ES cells were viable, mutant PDK1 was expressed at normal levels and insulin-like growth factor 1 induced normal activation of PKB and phosphorylation of the PKB substrates GSK3 and FKHR. In contrast, S6K, RSK and SGK were not activated, nor were physiological substrates of S6K and RSK phosphorylated. These experiments establish the importance of the PIF-pocket in governing the activation of S6K, RSK, SGK, but not PKB, in vivo. They also illustrate the power of knock-in technology to probe the physiological roles of docking interactions in regulating the specificity of signal transduction pathways.
Figures
Fig. 1. ES cell knock-in strategy. (A) Diagram illustrating the targeting knock-in construct, the 5′ end of the PDK1 gene and the allele modification generated. The black boxes represent exons and the black triangles loxP sites. The position of the 3′ probe used to genotype targeted knock-in cells in (B) is shown. The positions of the PCR primers used to genotype the Cre recombinase-mediated excision of the neomycin cassette are indicated by arrows. The position of Leu155/Glu155 in exon 4 is represented by an asterisk. The position of the novel _Eco_RV restriction site is marked. + = wild-type allele; 155Eneo = the targeted knock-in allele with the neomycin cassette still present; 155E = the targeted knock-in allele with the neomycin cassette removed. (B) Genomic DNA purified from the indicated ES cell lines was digested with _Eco_RV, 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 knock-in allele generates a 7.2 kb fragment in this analysis. (C) Genomic DNA purified from the indicated ES cell lines 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 (155E). (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 155 mutation region on exon 4. The resultant PCR products were ligated into the pCR-Topo 2.1 vector, transformed into E.coli and clones sequenced. The numbers of the wild-type Leu155 and knock-in Glu155 sequences obtained for each cell line are indicated.
Fig. 2. Expression and activity of PDK1 in knock-in ES cells. The indicated ES cells were cultured to 80% confluence and lysed. PDK1 was immunoprecipitated from the cell lysate and assayed with the indicated peptide as described in Materials and methods. The results shown are the average ± SEM of three separate dishes of cells with each assay 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 recombinant human PDK1 protein). The lysates were also incubated with Sepharose conjugated to PIF to affinity purify PDK1 as described in Materials and methods. The washed resin was then immunoblotted for PDK1 using PDK1 antibody 1. Similar results were obtained in two separate experiments. It should be noted that PDK1 in ES cells, as observed in other cell lines, is detected as two bands on immunoblot analysis (Balendran et al., 1999a; Williams et al., 2000).
Fig. 3. Activation of PKBα in PDK1155E/155E knock-in cells. The indicated ES cells were deprived of serum for 4 h, incubated in the presence or absence of 100 nM wortmannin for 10 min and then either left unstimulated or stimulated with 20 ng/ml IGF1 for 15 min. The cells were lysed, and PKBα was immunoprecipitated and assayed. The results shown are the average ± SEM for three dishes of cells each assayed in duplicate. The ES cell lysates were also immunoblotted with the indicated antibodies. Similar results were obtained in four separate experiments.
Fig. 4. S6K1 is not activated in PDK1155E/155E knock-in cells. (A) The indicated ES cells were deprived of serum for 4 h, incubated in the presence or absence of 100 nM rapamycin or 500 nM okadaic acid for 30 min, and then either left unstimulated or stimulated with 20 ng/ml IGF1 for 30 min. The cells were lysed, and S6K1 was immunoprecipitated and assayed. The results shown are the average ± SEM for three dishes of cells each assayed in duplicate. The ES cell lysates were also immunoblotted with the indicated antibodies. Thr389 is the hydrophobic motif residue. (B) The indicated ES cells lines were transfected with a DNA construct encoding the expression of wild-type GST–S6K. At 24 h post-transfection, the ES cells were deprived of serum for 4 h, incubated in the presence or absence of 100 nM rapamycin for 30 min and then either left unstimulated or stimulated with 20 ng/ml IGF1 for 30 min. The cells were lysed, and GST–S6K was affinity purified from the cell lysate on glutathione–Sepharose and immunoblotted with the indicated antibodies. Thr252 is the T-loop residue phosphorylated by PDK1. Similar results were obtained in two separate experiments.
Fig. 5. RSK isoforms are not activated in PDK1155E/155E knock-in cells. The indicated ES cells were deprived of serum for 4 h, incubated in the presence or absence of 2 µM PD 184352 for 1 h and then either left unstimulated or stimulated with 0.4 µg/ml TPA for 15 min. The cells were lysed, and RSK isoforms were immunoprecipitated with an antibody that recognizes all isoforms and assayed. The results shown are the average ± SEM for two dishes of cells each assayed in triplicate. The ES cell lysates were also immunoblotted with the indicated antibodies. Ser227 is phosphorylated by PDK1, Thr360 and Thr573 are phosphorylated by ERK, and Ser380 is phosphorylated by the C-terminal kinase domain of RSK (CT-KD). Similar results were obtained in two separate experiments.
Fig. 6. SGK1 is inactive in PDK1155E/155E knock-in cells. (A) The indicated ES cells lines were transfected with a DNA construct encoding GST–SGK1. At 44 h post-transfection, the ES cells were deprived of serum for 4 h, incubated in the presence or absence of 100 nM wortmannin for 10 min and then either left unstimulated or stimulated with 20 ng/ml IGF1 for 20 min. 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 ± SEM for three dishes of cells each assayed in triplicate. 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 a phospho-antibody recognizing Ser422, the hydrophobic motif. (B) As (A), except that the indicated ES cells lines were transfected with a construct encoding expression of GST–SGK1[S422D]. Similar results were obtained in two separate experiments.
Fig. 7. Phosphorylation of FKHR at Thr24, Ser256 and Ser319 in PDK1155E/155E knock-in cells. The indicated ES cell lines were deprived of serum for 4 h, incubated in the presence or absence of 100 nM wortmannin for 10 min and then either left unstimulated or stimulated with 20 ng/ml IGF1 for 30 min. The cells were lysed, and FKHR was immunoprecipitated and immunoblotted with the indicated antibodies. For the blotting of the Ser319 site, two different batches of wild-type PDK1+/+ ES cell lines were employed that consistently gave marginally different responses to IGF1. Similar results were obtained in two separate experiments in which each stimulation was performed in triplicate.
Fig. 8. Summary of the mechanism of activation of PKB, S6K and SGK by PDK1. PKB is activated following its recruitment to the plasma membrane where it is phosphorylated at Thr308 by PDK1 and at Ser473 by a distinct unknown hydrophobic motif kinase, termed PDK2. The mutual binding of PKB and PDK1 through their PH domains co-localizes PDK1 and PKB. Once PKB is phosphorylated at Thr308, a hydrophobic motif-binding site is formed in the catalytic domain, resulting in the intramolecular binding of the PKB hydrophobic motif phosphorylated at Ser473 to this site. This is the step that leads to the maximal activation of PKB. In contrast, for S6K and SGK, it is the phosphorylation of these enzymes at their hydrophobic motif that enables PDK1 to interact through its PIF-pocket and hence phosphorylate the T-loop of these substrates. PI-3-kinase regulates the phosphorylation of S6K and SGK1 at their hydrophobic motif. Phosphorylation of the T-loop of S6K and SGK is predicted to promote the formation of a binding site within the kinase domain of these enzymes, for their own phosphorylated hydrophobic motif, leading to activation. We propose that this protects dephosphorylation of the hydrophobic motif by protein phosphatases. In PDK1155E/155E cells, S6K is not phosphorylated at its T-loop and therefore the hydrophobic motif phosphorylation site will remain exposed and is thus more likely to be dephosphorylated.
References
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