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PMID: 12912918 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

In vivo role of the PIF-binding docking site of PDK1 defined by knock-in mutation.

The EMBO journal ·Vol. 22 ·No. 16 ·2003-08-15 ·Pages 4202-11

Collins BJ, Deak M, Arthur JS, Armit LJ, Alessi DR

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.

MeSH Terms
3-Phosphoinositide-Dependent Protein Kinases Adaptor Proteins, Signal Transducing Alleles Binding Sites Carrier Proteins/metabolism DNA-Binding Proteins/metabolism Enzyme Activation/drug effects Forkhead Box Protein O1 Forkhead Transcription Factors Humans Insulin-Like Growth Factor I/pharmacology Intracellular Signaling Peptides and Proteins Models, Biological Mutation Neoplasm Proteins Phosphorylation Protein Binding Protein Serine-Threonine Kinases/chemistry,genetics,metabolism Protein Structure, Tertiary Proto-Oncogene Proteins Proto-Oncogene Proteins c-akt Stem Cells/metabolism Substrate Specificity Transcription Factors/metabolism
Chemicals
Adaptor Proteins, Signal Transducing Carrier Proteins DNA-Binding Proteins FOXO1 protein, human FRAT1 protein, human Forkhead Box Protein O1 Forkhead Transcription Factors Intracellular Signaling Peptides and Proteins Neoplasm Proteins Proto-Oncogene Proteins Transcription Factors Insulin-Like Growth Factor I 3-Phosphoinositide-Dependent Protein Kinases AKT1 protein, human PDPK1 protein, human Protein Serine-Threonine Kinases Proto-Oncogene Proteins c-akt
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Collins Barry J
MRC Protein Phosphorylation Unit, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, UK. b.j.collins@dundee.ac.uk
Deak Maria
Arthur J Simon C
Armit Laura J
Alessi Dario R
References (47)
47 references, click to expand
  1. Regulation and physiological roles of serum- and glucocorticoid-induced protein kinase isoforms.
    Sci STKE. 2001 Nov 13;2001(108):re17 PMID: 11707620
  2. Ten years of protein kinase B signalling: a hard Akt to follow.
    Trends Biochem Sci. 2001 Nov;26(11):657-64 PMID: 11701324
  3. Two novel phosphorylation sites on FKHR that are critical for its nuclear exclusion.
    EMBO J. 2002 May 1;21(9):2263-71 PMID: 11980723
  4. Molecular mechanism for the regulation of protein kinase B/Akt by hydrophobic motif phosphorylation.
    Mol Cell. 2002 Jun;9(6):1227-40 PMID: 12086620
  5. Essential role of PDK1 in regulating cell size and development in mice.
    EMBO J. 2002 Jul 15;21(14):3728-38 PMID: 12110585
  6. mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery.
    Cell. 2002 Jul 26;110(2):163-75 PMID: 12150925
  7. Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action.
    Cell. 2002 Jul 26;110(2):177-89 PMID: 12150926
  8. Multiple phosphoinositide 3-kinase-dependent steps in activation of protein kinase B.
    Mol Cell Biol. 2002 Sep;22(17):6247-60 PMID: 12167717
  9. High resolution crystal structure of the human PDK1 catalytic domain defines the regulatory phosphopeptide docking site.
    EMBO J. 2002 Aug 15;21(16):4219-28 PMID: 12169624
  10. TSC1-TSC2: a complex tale of PKB-mediated S6K regulation.
    Nat Cell Biol. 2002 Sep;4(9):E214-6 PMID: 12205484
  11. A phosphoserine/threonine-binding pocket in AGC kinases and PDK1 mediates activation by hydrophobic motif phosphorylation.
    EMBO J. 2002 Oct 15;21(20):5396-407 PMID: 12374740
  12. Crystal structure of an activated Akt/protein kinase B ternary complex with GSK3-peptide and AMP-PNP.
    Nat Struct Biol. 2002 Dec;9(12):940-4 PMID: 12434148
  13. Modulation of insulin-stimulated degradation of human insulin receptor substrate-1 by Serine 312 phosphorylation.
    J Biol Chem. 2003 Mar 7;278(10):8199-211 PMID: 12510059
  14. The many forks in FOXO's road.
    Sci STKE. 2003 Mar 4;2003(172):RE5 PMID: 12621150
  15. Signalling specificity of Ser/Thr protein kinases through docking-site-mediated interactions.
    Biochem J. 2003 May 15;372(Pt 1):1-13 PMID: 12600273
  16. Insulin-induced Drosophila S6 kinase activation requires phosphoinositide 3-kinase and protein kinase B.
    Biochem J. 2003 Sep 1;374(Pt 2):297-306 PMID: 12841848
  17. Identification of regulatory phosphorylation sites in mitogen-activated protein kinase (MAPK)-activated protein kinase-1a/p90rsk that are inducible by MAPK.
    J Biol Chem. 1998 Jan 16;273(3):1496-505 PMID: 9430688
  18. Regulation of the p70 S6 kinase by phosphorylation in vivo. Analysis using site-specific anti-phosphopeptide antibodies.
    J Biol Chem. 1998 Jun 26;273(26):16621-9 PMID: 9632736
  19. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor.
    Cell. 1999 Mar 19;96(6):857-68 PMID: 10102273
  20. 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. 1999 Apr 15;339 ( Pt 2):319-28 PMID: 10191262
  21. PDK1 acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2.
    Curr Biol. 1999 Apr 22;9(8):393-404 PMID: 10226025
  22. Protein modification: docking sites for kinases.
    Curr Biol. 1999 May 6;9(9):R329-31 PMID: 10322109
  23. Serum and glucocorticoid-inducible kinase (SGK) is a target of the PI 3-kinase-stimulated signaling pathway.
    EMBO J. 1999 Jun 1;18(11):3024-33 PMID: 10357815
  24. Phosphorylation of the transcription factor forkhead family member FKHR by protein kinase B.
    J Biol Chem. 1999 Jun 11;274(24):17179-83 PMID: 10358075
  25. Blockade of the MAP kinase pathway suppresses growth of colon tumors in vivo.
    Nat Med. 1999 Jul;5(7):810-6 PMID: 10395327
  26. Role and regulation of 90 kDa ribosomal S6 kinase (RSK) in signal transduction.
    Mol Cell Endocrinol. 1999 May 25;151(1-2):65-77 PMID: 10411321
  27. Mammalian TOR controls one of two kinase pathways acting upon nPKCdelta and nPKCepsilon.
    J Biol Chem. 1999 Dec 3;274(49):34758-64 PMID: 10574945
  28. Evidence that 3-phosphoinositide-dependent protein kinase-1 mediates phosphorylation of p70 S6 kinase in vivo at Thr-412 as well as Thr-252.
    J Biol Chem. 1999 Dec 24;274(52):37400-6 PMID: 10601311
  29. Identification of a pocket in the PDK1 kinase domain that interacts with PIF and the C-terminal residues of PKA.
    EMBO J. 2000 Mar 1;19(5):979-88 PMID: 10698939
  30. The role of 3-phosphoinositide-dependent protein kinase 1 in activating AGC kinases defined in embryonic stem cells.
    Curr Biol. 2000 Apr 20;10(8):439-48 PMID: 10801415
  31. A phosphoserine-regulated docking site in the protein kinase RSK2 that recruits and activates PDK1.
    EMBO J. 2000 Jun 15;19(12):2924-34 PMID: 10856237
  32. Role of S6 phosphorylation and S6 kinase in cell growth.
    Prog Nucleic Acid Res Mol Biol. 2001;65:101-27 PMID: 11008486
  33. Cellular signaling: pivoting around PDK-1.
    Cell. 2000 Oct 13;103(2):185-8 PMID: 11057891
  34. Protein kinase SGK mediates survival signals by phosphorylating the forkhead transcription factor FKHRL1 (FOXO3a).
    Mol Cell Biol. 2001 Feb;21(3):952-65 PMID: 11154281
  35. Identification of a docking groove on ERK and p38 MAP kinases that regulates the specificity of docking interactions.
    EMBO J. 2001 Feb 1;20(3):466-79 PMID: 11157753
  36. Discovery of PDK1, one of the missing links in insulin signal transduction. Colworth Medal Lecture.
    Biochem Soc Trans. 2001 May;29(Pt 2):1-14 PMID: 11356119
  37. Phosphorylation of the protein kinase mutated in Peutz-Jeghers cancer syndrome, LKB1/STK11, at Ser431 by p90(RSK) and cAMP-dependent protein kinase, but not its farnesylation at Cys(433), is essential for LKB1 to suppress cell vrowth.
    J Biol Chem. 2001 Jun 1;276(22):19469-82 PMID: 11297520
  38. The target of rapamycin (TOR) proteins.
    Proc Natl Acad Sci U S A. 2001 Jun 19;98(13):7037-44 PMID: 11416184
  39. Two splice variants of protein kinase B gamma have different regulatory capacity depending on the presence or absence of the regulatory phosphorylation site serine 472 in the carboxyl-terminal hydrophobic domain.
    J Biol Chem. 2001 Aug 3;276(31):29550-8 PMID: 11387345
  40. Regulation of elongation factor 2 kinase by p90(RSK1) and p70 S6 kinase.
    EMBO J. 2001 Aug 15;20(16):4370-9 PMID: 11500364
  41. The PIF-binding pocket in PDK1 is essential for activation of S6K and SGK, but not PKB.
    EMBO J. 2001 Aug 15;20(16):4380-90 PMID: 11500365
  42. GSK3 takes centre stage more than 20 years after its discovery.
    Biochem J. 2001 Oct 1;359(Pt 1):1-16 PMID: 11563964
  43. The p70 S6 kinase integrates nutrient and growth signals to control translational capacity.
    Prog Mol Subcell Biol. 2001;26:115-54 PMID: 11575164
  44. PKB/AKT: functional insights from genetic models.
    Nat Rev Mol Cell Biol. 2001 Oct;2(10):760-8 PMID: 11584303
  45. Carboxyl-terminal modulator protein (CTMP), a negative regulator of PKB/Akt and v-Akt at the plasma membrane.
    Science. 2001 Oct 12;294(5541):374-80 PMID: 11598301
  46. PKB/Akt: a key mediator of cell proliferation, survival and insulin responses?
    J Cell Sci. 2001 Aug;114(Pt 16):2903-10 PMID: 11686294
  47. Phosphoinositide-regulated kinases and phosphoinositide phosphatases.
    Chem Rev. 2001 Aug;101(8):2365-80 PMID: 11749378
Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
DOI
10.1093/emboj/cdg407
Email
b.j.collins@dundee.ac.uk
Published
2003-08-15
Pages
4202-11
Language
English
Region
England
NLM ID
8208664
PMCID
PMC175797
Subset
IM
Grants
Wellcome Trust · 065629 · United Kingdom
External Links
PubMed source DOI: 10.1093/emboj/cdg407 PMC Full Text View journal details
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