Regulation of 3-Phosphoinositide–Dependent Protein Kinase 1 Activity by Homodimerization in Live Cells
Sticking Together
The “master kinase” phosphoinositide-dependent kinase 1 (PDK1) plays a central role in such processes as cellular proliferation and survival and has a wide range of targets, including protein kinase B (PKB). PDK1 is downstream of phosphatidylinositol 3-kinase (PI3K), and the generation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) triggers the translocation of PDK1 and PKB to the plasma membrane, where PDK1 phosphorylates PKB. Although the mechanisms by which PDK1 activates its substrates are well studied, less is known about how the activity of PDK1 is regulated. Masters et al. used a combination of Förster resonance energy transfer (FRET)–based analysis of fluorescently tagged proteins in live cells, as well as computational modeling, to show that a subset of cytosolic PDK1 exists in a homodimeric form. Disruption of the homodimeric interface increased the association between PDK1 and PKB, and this and other evidence suggested that monomeric—rather than dimeric—PDK1 was the active form. Together, these data suggest that homodimerization of PDK1 regulates its activity.
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.
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Supplementary Material
Summary
Methods
Fig. S1. Coimmunoprecipitation of myc-PDK1 with GFP-myc-PDK1 from COS-7 cells.
Fig. S2. In vitro phosphorylation of the PDKtide peptide by recombinant tagged PDK1.
Fig. S3. Analysis of sensitized acceptor fluorescence.
Fig. S4. Molecular modeling of the PDK1 homodimer.
Fig. S5. Example of the calculation of pixel enrichment.
Fig. S6. Probabilities that a PDK1 dimer can be detected by hetero-FRET.
Reference
Resources
File (3_ra78_sm.pdf)
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Information & Authors
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Published In
Science Signaling
Volume 3 | Issue 145
October 2010
October 2010
Copyright
Copyright © 2010, American Association for the Advancement of Science.
Submission history
Received: 13 November 2009
Accepted: 8 October 2010
Acknowledgments
Acknowledgments: We are grateful to P. J. Parker for critical reading of the manuscript and for scientific discussions. We are indebted to S. Kisakye-Nambozo, A. Borg, R. George, and S. Kjaer for help with protein expression and purification. Funding: This research project was funded by Cancer Research UK core funding and Engineering and Physical Science Council (EPSRC) grant EP/C536134/1. T.A.M. gratefully acknowledges the EPSRC doctoral training centre PhD programme at the University College London Centre for Mathematics and Physics in the Life Sciences and Experimental Biology (CoMPLEX) for financial support. Author contributions: A.J.B. and B.L. directed the research; T.A.M., V.C., and B.L. designed and performed the FLIM experiments; T.A.M. and V.C. designed the molecular biology tools; T.A.M. and V.C. performed the biochemical experiments; C.J.A. and B.L. developed the two-photon FLIM apparatus; A.J.B. designed the in vitro experiments; T.A.M., D.A.A., and R.J.M. planned and performed the in vitro experiments; T.A.M., R.J.M., and A.J.B. analyzed the in vitro data; M.L. performed the molecular modeling; T.A.M., V.C., D.A.A., R.J.M., M.L., A.J.B., and B.L. were involved in scientific discussions; and T.A.M., V.C., A.J.B., and B.L. wrote the manuscript. Competing interests: The authors declare that they have no competing interests.
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