Keywords

Protein Kinase, Autophosphorylation, Activation Loop, Kinase Dimerization, Biophysics, Signal Transduction

Reference

DOI: https://doi.org/10.7554/eLife.88210

Abstract

Protein kinases are central to eukaryotic signaling via ATP-dependent phosphorylation of protein substrates. Tight regulation of kinase activity ensures accurate signaling. A key regulatory mechanism is activation loop autophosphorylation, but how this occurs (cis vs. trans) remains controversial.
Classically, trans-autophosphorylation involves dimerization of kinase domains, but recent discoveries suggest possible cis-autophosphorylation mechanisms. This review critically examines biochemical, kinetic, and structural evidence for kinase dimerization and autophosphorylation, proposing a framework to evaluate kinase autoregulation and implications for cellular signaling.

Notes

1. Anatomy of a Protein Kinase

  • Glycine loop (156-161; 4ekk): organizes γ-phosphate for phosphoryl transfer.
  • αD helix (233-242; 4ekk) & Activation loop (294-306; 4ekk): substrate binding and recognition.
  • αG helix (352-363; 4ekk): mediates protein-protein interactions (e.g., dimerization, trans-inhibition, substrate recognition).
  • Active conformation requires hydrophobic spines (C-spine & R-spine) and a conserved salt bridge (β3-lysine & αC-glutamate).
  • Activation loop: site of phosphorylation, highest divergence, and flexibility.

2. Kinase Regulation Concepts

  • Inactive and active states modulated by cis (intramolecular) and trans (intermolecular) regulatory mechanisms.
  • Activation loop phosphorylation is pivotal — but mechanism (cis vs. trans) remains debated.
  • Steric occlusion: substrate site blocked in cis (e.g., CaMKK, PKA) or trans (e.g., DAPK dimers).
  • Allostery: distal interactions modulating activity — in both cis and trans.
  • Regulation via disorder-to-order transitions in activation loops is a key feature in many kinases (e.g., PKB).

3. Kinase Inhibition and Activation

Inhibition

  • Activation loop occlusion: prevents substrate and ATP binding (e.g., RTKs, Src).
  • Steric block by inhibitory elements (cis: C-terminal helix in CaMKK; trans: DAPK dimer).
  • 14-3-3 proteins interacting with B-raf C-terminal to prevent activation.

Activation

  • C-terminal extensions in AGC kinases stabilize active conformations via cis-interactions.
  • Aurora kinases and others regulated by C-terminal motifs and activation loop phosphorylation.
  • Activation loop phosphorylation in trans: dimerization enables cross-phosphorylation (e.g., PDK1 on PIP3 membranes).
  • Potential for cis-autophosphorylation: proposed in PKD but controversial.

4. Cis vs. Trans Autophosphorylation Mechanisms

  • Trans-autophosphorylation: bimolecular, concentration-dependent, often via face-to-face dimerization.
  • Cis-autophosphorylation: unimolecular, concentration-independent, occurring within the same molecule.
  • The mechanism remains enigmatic as phosphorylation requires kinase activity prior to its own activation loop phosphorylation.

5. Interpreting Biochemical Evidence

Techniques:

  • Intact Mass Spectrometry: precise, but lacks site specificity.
  • Tandem MS: site-specific, but peptide ionization and cleavage issues.
  • Radionuclide assays and phospho-specific Western blots: semi-quantitative, limited specificity.

Interpretation:

  • Concentration-dependent activity → supports trans-mechanism but non-definitive.


Figure: a pic from the paper for clarifying. (Click to enlarge)

  • Mutational analysis of dimer interfaces can suggest dimerization roles but has pitfalls due to overlapping cis/trans interfaces.
  • Multiple converging evidence streams required to distinguish cis and trans clearly.

6. Kinetic Models of Autophosphorylation

  • Cis: unimolecular, linear product formation, concentration-independent.
  • Trans: bimolecular, concentration-dependent, requires partner interaction.
  • Dimerization scenarios: transient (weak, signal-induced) vs. constitutive dimers (stable).

7. Structural Evidence and Its Ambiguities

  • Crystal structures show dimerization but may reflect lattice artifacts driven by activation loop exchange.
  • Activation loop docking in trans can promote crystal growth, but doesn’t confirm physiological relevance.
  • Interpretation of crystal dimers requires solution validation (e.g., crosslinking, NMR, SAXS).
  • Example: Chk2 activation loop exchange forming crystal lattice dimers — but physiological relevance unclear.

8. The Enigma of Activation Loop Autophosphorylation

  • Logical problem: kinase needs activity to phosphorylate its own loop before it becomes active.
  • Autophosphorylation mechanistically distinct from substrate phosphorylation — supported by evidence that canonical substrate motif mutations don’t affect autophosphorylation (e.g., PKD).
  • Autophosphorylation motifs often non-canonical — not phosphorylated as peptides in trans.
  • Physiological constraints: low cellular kinase concentrations (nM), crowded environments — challenging for purely diffusion-limited trans autophosphorylation.
  • Autophosphorylation may require scaffolds, localization, or induced dimerization to occur efficiently in cells.

RD’s Thoughts and Learnings

  • Strong conceptual framework but mechanistic gaps remain for cis vs. trans activation loop phosphorylation.
  • Structural ambiguity of activation loop dimers in crystal structures is a real issue — needs careful biochemical and biophysical validation.
  • Exciting but perplexing: kinase needs to be active to autophosphorylate — yet phosphorylation often activates the kinase. A chicken-or-egg paradox in molecular biology.
  • Important reminder that in vitro biochemistry must be interpreted with caution, especially regarding physiological relevance of dimerization and activation mechanisms.
  • Potential insights for kinase drug design: targeting dimer interfaces, activation loops, or allosteric regulatory sites.
  • Possible link to disease: misregulated activation loop phosphorylation could lead to pathological kinase activation (e.g., cancer).

Take-home Messages

  • Protein kinase activation loop autophosphorylation is central to kinase regulation, but mechanistic details remain under debate (cis vs. trans).
  • Kinase dimerization is a key element in many models, but structural and biochemical data need careful interpretation.
  • Activation loop phosphorylation motifs differ from substrate motifs, hinting at distinct mechanisms.
  • The paradox of needing activity to autophosphorylate raises fundamental questions about kinase biology.
  • RD finds this review essential to understand the state-of-the-art on kinase regulation — but many open questions remain for future research.

RD’s final thought: This review maps the landscape — but solving the kinase activation puzzle will take more creative experiments.