Keywords

PDK1, Trans-autophosphorylation, PIP3, PH Domain, Hydrophobic Motif (HM), Switch-like Activation, AGC Kinase, Cell Signaling, Structural Activation

Reference

DOI: 10.1038/s41467-022-29368-4

Abstract

PDK1 is a ‘master’ kinase that activates numerous AGC kinases like Akt, PKC, S6K. This study elucidates PIP3-driven trans-autophosphorylation of PDK1 via a linker-swapped dimer, uncovering the role of PH domain-mediated autoinhibition and positive cooperativity in PIP3 binding, which together define a switch-like activation mechanism of PDK1.

Pre-knowledge

  • PDK1 phosphorylates ~23 kinases (Akt, PKC, Sgk, S6K, RSK).
  • Activation site: Ser241 (activation loop).
  • PDK1 consists of an N-terminal kinase domain (51–359) and a C-terminal PH domain that binds PIP3.
  • PDK1 lacks canonical C-terminal tail (HM) seen in other AGC kinases.
  • Activation thought to involve membrane-localized dimerization and trans-autophosphorylation.
  • αG helix is essential in protein-protein interactions and is part of the PDK1 dimer interface.

Key Mechanistic Insights

1. PDK1 Forms a PIP3-Mediated Face-to-Face Dimer for Trans-Autophosphorylation

  • Monomeric in solution, but dimerizes upon membrane PIP3 binding.
  • Y288-centered αG helix crucial for dimer interface; Y288 mutations (A/E) abolish autophosphorylation but not conformation.
  • Artificial dimerization restores phosphorylation, confirming dimer-dependent autoactivation.

Membrane recruitment drives specific dimerization critical for activation.

2. Hydrophobic Motif (HM)-like Sequence in Linker Promotes Dimerization and Activation

  • Linker between kinase and PH domain (359-389) contains Phe-Gly-Cys-Met (383-386) — a functional HM-like motif engaging hydrophobic pocket.
  • PIFtide (PRK2 HM peptide) occupancy in PDK1’s hydrophobic pocket promotes trans-autophosphorylation.
  • First time PDK1 identified to encode its own HM within the flexible linker.

3. Linker-Swapped Dimer Mechanism Mediated by the HM

  • Modeled PDK1 dimer shows 29.4 Å distance between C-terminal of one kinase and HM of the other.
  • Dimer stabilized via HM-pocket interaction across protomers — linker-swap mechanism.
  • Crosslinking-MS confirms model: extensive intermolecular crosslinks between N and C termini of two protomers.
  • Artificial HM fusion (PIFtide via (GS)4 linker) accelerates autophosphorylation, supporting linker-swap dimer hypothesis.

Linker-swap dimerization as a new regulatory mechanism in AGC kinases.

4. PH Domain Autoinhibition and Relief by PIP3 Binding

  • PH domain hinders kinase domain association and trans-autophosphorylation.
  • PDK1_FL autophosphorylates slower than truncated forms.
  • PH domain blocks catalytic cleft, substrate binding helix (αD), and αG dimerization surface.
  • PIP3 binding to PH domain disrupts autoinhibition, enabling dimer formation.
  • SAXS & HDX-MS data suggest PH domain occludes the kinase dimerization interface until PIP3 binding.

PH domain acts as a molecular brake lifted upon PIP3 binding.

5. Switch-like Activation Driven by PIP3 Cooperativity

  • PIP3-containing liposomes increase PDK1 autoP rate 5-fold.
  • Cooperative binding: PDK1 shows switch-like behavior, with positive cooperativity in PIP3 binding.
  • PDK1_FL binds to PIP3-liposomes with higher affinity when dimerized, indicating activation depends on membrane context.
  • Liposome binding assays and HDX-MS support that membrane-bound PDK1 adopts an activation-competent state.

PIP3-driven dimerization and activation represents a fail-safe mechanism to prevent cytosolic PDK1 activity.

Experimental Strategies

  • Intact MS: precise phospho-state monitoring (1h & O/N time points).
  • SEC-MALS & MW ratio: confirming monomeric status in solution.
  • Crosslinking-MS (XL-MS): mapping intermolecular interactions in dimer and monomer.
  • HDX-MS: probing structural dynamics in presence/absence of PH domain, PIFtide, and PIP3.
  • Liposome pelleting assay: quantifying PIP3 binding affinity.
  • Mutagenesis (Y288A/E, S241A, linker variants) to dissect functional motifs and mechanisms.

Take-home Concept

  • PDK1 is autoinhibited via its PH domain until membrane recruitment.
  • PIP3 drives cooperative dimerization, enabling trans-autophosphorylation of activation loop (S241).
  • Linker region encodes a functional HM, engaging hydrophobic pocket of opposite protomer — essential for autoactivation.
  • After activation, PDK1 dimers dissociate to act on downstream kinases (e.g., Akt, PKC).
  • A dynamic balance of inhibition and activation through precise structural mechanisms — regulated by membrane lipid context.

RD’s Final Thoughts

  • PDK1 as a minimal but master regulator — beautifully employs a built-in switch combining dimerization, autoP, and membrane sensing.
  • Mechanism of face-to-face dimerization via αG helix + linker-swap HM could apply to other AGC kinases.
  • Lipid-mediated signaling meets kinase autoactivation — reminds me of CaMKII’s regulated assembly/disassembly, but here via PIP3.
  • Opens thoughts for designing synthetic regulatory switches in kinases, coupling localization and activation.

Masterpiece of kinase regulation via dimer and membrane code.