Role of T-loop phosphorylation in PDK1 activation, stability, and substrate binding

David Komander, Gursant Kular, Maria Deak, Dario R. Alessi, Daan M. F. van Aalten

Research output: Contribution to journalArticle

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Abstract

3-Phosphoinositide-dependent protein kinase-1 (PDK1) phosphorylates the T-loop of several AGC (cAMP-dependent, cGMP-dependent, protein kinase C) family protein kinases, resulting in their activation. Previous structural studies have revealed that the aC-helix, located in the small lobe of the kinase domain of PDK1, is a key regulatory element, as it links a substrate interacting site termed the hydrophobic motif (HM) pocket with the phosphorylated Ser-241 in the T-loop. In this study we have demonstrated by mutational analysis that interactions between the phosphorylated Ser-241 and the aC-helix are not required for PDK1 activity or substrate binding through the HM-pocket but are necessary for PDK1 to be activated or stabilized by a peptide that binds to this site. The structure of an inactive T-loop mutant of PDK1, in which Ser-241 is changed to Ala, was also determined. This structure, together with surface plasmon resonance binding studies, demonstrates that the PDK1(S241A)-inactive mutant possesses an intact HM-pocket as well as an ordered aC-helix. These findings reveal that the integrity of the aC-helix and HM-pocket in PDK1 is not regulated by T-loop phosphorylation.
Original languageEnglish
Pages (from-to)18797-18802
Number of pages6
JournalJournal of Biological Chemistry
Volume280
Issue number19
DOIs
Publication statusPublished - 13 May 2005

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3-Phosphoinositide-Dependent Protein Kinases
Phosphorylation
Chemical activation
Substrates
Cyclic GMP-Dependent Protein Kinases
Surface Plasmon Resonance
Surface plasmon resonance
Protein Kinases
Protein Kinase C
Phosphotransferases
Peptides

Cite this

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abstract = "3-Phosphoinositide-dependent protein kinase-1 (PDK1) phosphorylates the T-loop of several AGC (cAMP-dependent, cGMP-dependent, protein kinase C) family protein kinases, resulting in their activation. Previous structural studies have revealed that the aC-helix, located in the small lobe of the kinase domain of PDK1, is a key regulatory element, as it links a substrate interacting site termed the hydrophobic motif (HM) pocket with the phosphorylated Ser-241 in the T-loop. In this study we have demonstrated by mutational analysis that interactions between the phosphorylated Ser-241 and the aC-helix are not required for PDK1 activity or substrate binding through the HM-pocket but are necessary for PDK1 to be activated or stabilized by a peptide that binds to this site. The structure of an inactive T-loop mutant of PDK1, in which Ser-241 is changed to Ala, was also determined. This structure, together with surface plasmon resonance binding studies, demonstrates that the PDK1(S241A)-inactive mutant possesses an intact HM-pocket as well as an ordered aC-helix. These findings reveal that the integrity of the aC-helix and HM-pocket in PDK1 is not regulated by T-loop phosphorylation.",
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Role of T-loop phosphorylation in PDK1 activation, stability, and substrate binding. / Komander, David; Kular, Gursant; Deak, Maria; Alessi, Dario R.; van Aalten, Daan M. F.

In: Journal of Biological Chemistry, Vol. 280, No. 19, 13.05.2005, p. 18797-18802.

Research output: Contribution to journalArticle

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