Regulation of 3-Phosphoinositide–Dependent Protein Kinase 1 Activity by Homodimerization in Live Cells

Sci. Signal., 26 October 2010
Vol. 3, Issue 145, p. ra78
DOI: 10.1126/scisignal.2000738

Regulation of 3-Phosphoinositide–Dependent Protein Kinase 1 Activity by Homodimerization in Live Cells

  1. Thomas A. Masters1,2,*,
  2. Véronique Calleja1,*,
  3. Daven A. Armoogum2,
  4. Richard J. Marsh2,
  5. Christopher J. Applebee1,
  6. Michel Laguerre3,
  7. Angus J. Bain2,, and
  8. Banafshé Larijani1,
  1. 1Cell Biophysics Laboratory, Cancer Research UK, Lincoln’s Inn Fields Laboratories, London Research Institute, London WC2A 3LY, UK.
  2. 2Ultrafast Laser Spectroscopy Group, Department of Physics and Astronomy, University College London, London WC1E 6BT, UK.
  3. 3IECB-CBMN-UMR 5248-CNRS, Institut Européen de Chimie et Biologie, University of Bordeaux, Pessac 33607, France.
  1. To whom correspondence should be addressed. E-mail: banafshe.larijani{at}cancer.org.uk (B.L.); a.bain{at}ucl.ac.uk (A.J.B.)
  • * These authors contributed equally to this work.

Abstract

3-Phosphoinositide–dependent kinase 1 (PDK1) plays a central role in regulating the activity of protein kinases that are essential for signaling; however, how PDK1 itself is regulated is largely unknown. We found that homodimerization of PDK1 is a spatially and temporally regulated mechanism for controlling PDK1 activity. We used Förster resonance energy transfer monitored by fluorescence lifetime imaging microscopy to observe PDK1 homodimerization in live cells. A pleckstrin homology (PH) domain–dependent, basal dimeric association of PDK1 was increased upon cell stimulation with growth factors; this association was prevented by a phosphatidylinositol 3-kinase inhibitor and by a mutation in, or a complete deletion of, the PH domain of PDK1. The distinct spatial distribution of PDK1 homodimers relative to that of heterodimers of PDK1 and protein kinase B (PKB), and the ability of monomeric mutants of PDK1 to phosphorylate PKB, suggested that the monomer was the active conformation. Mutation of the autophosphorylation residue threonine-513 to glutamate, which was predicted to destabilize the homodimer interface, enhanced the interaction between PDK1 and PKB and the activity of PKB. Through in vitro, time-resolved fluorescence intensity and anisotropy measurements, combined with existing crystal structures and computational molecular modeling, we determined the geometrical arrangement of the PDK1 homodimer. With this approach, we calculated the size of the population of PDK1 dimers in cells. This description of a previously uncharacterized regulatory mechanism for the activation of PDK1 offers possibilities for controlling PDK1 activity therapeutically.

Citation:

T. A. Masters, V. Calleja, D. A. Armoogum, R. J. Marsh, C. J. Applebee, M. Laguerre, A. J. Bain, and B. Larijani, Regulation of 3-Phosphoinositide–Dependent Protein Kinase 1 Activity by Homodimerization in Live Cells. Sci. Signal. 3, ra78 (2010).

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