Keywords

Kinase, Activation, Mechanisms, Conformational Plasticity, Pseudosubstrate, Cell Signaling, Cell

Reference

DOI: 10.1016/s0092-8674(02)00741-9

Abstract

Protein kinases play essential roles in eukaryotic signaling pathways, regulating growth and development.
Despite highly conserved catalytic cores, kinases utilize diverse mechanisms for activation and inhibition.
The active state adopts a conserved conformation, but inactive states exhibit remarkable plasticity, enabling regulation via domain interactions, phosphorylation, or pseudosubstrate binding.
Structural studies reveal how conformational flexibility underlies kinase function and regulation, with implications for drug targeting, such as Gleevec’s stabilization of inactive Abl kinase.

Notes

1. General Summary

  • Protein kinases are critical regulatory enzymes, making up ~2% of eukaryotic genomes.
  • Switching between active/inactive states underpins precise control of cellular processes.
  • The catalytic domain consists of a two-lobed structure:
    • N-lobe: binds ATP.
    • C-lobe: catalyzes phosphotransfer.
  • ATP binds in the cleft between lobes, stabilized by the P-loop (Glycine loop).
  • Activation loop phosphorylation triggers conformational changes necessary for catalysis.

2. Structural Insights

  • Active State:
    • Activation loop is phosphorylated, adopting an open conformation to accommodate substrate.
    • αC helix forms an ion pair (Glu91-Lys72) crucial for ATP positioning.
    • Catalytic residues (Asp166, Lys72) properly aligned for phosphotransfer.
  • Inactive States:
    • Diverse conformations depending on interaction with regulatory domains (e.g., SH2/SH3 in Src).
    • Helix αC repositioned, disrupting the ion pair and misaligning catalytic residues.
    • Activation loop can block substrate binding or be trapped in an alternative conformation.
  • Plasticity enables regulation and targeting by inhibitors (e.g., Gleevec binds inactive Abl but not Src).

3. Mechanisms of Regulation

  • Phosphorylation-Dependent Activation:
    • e.g., ERK2, Insulin receptor kinase (IRK) — phosphorylation opens activation loop, stabilizing active conformation.
  • Helix αC Repositioning:
    • Essential for activation loop and ATP positioning.
    • e.g., CDKs require cyclins to reposition αC and activation loop for activation.
  • Pseudosubstrate Inhibition:
    • Intra pseudosubstrate: Autoinhibitory sequences mimic substrates, block active site (e.g., twitchin, PAK).
    • Inter pseudosubstrate: Regulatory proteins provide inhibitory segments (e.g., Rho family GTPases activate PAK by displacing pseudosubstrate).
  • Alternative Inhibitory Segments:
    • e.g., EphB2, TβR-I, use N-terminal inhibitory motifs to prevent catalysis until phosphorylated.

4. Functional Implications

  • Conformational plasticity allows fine-tuned regulation of kinase activity, responding to signals, partners, or inhibitors.
  • Diverse inactive conformations provide multiple points for therapeutic targeting, as seen with Gleevec in Abl.
  • αC helix and activation loop form a dynamic regulatory unit, modulating access and alignment of catalytic residues.
  • Glycine loop (P-loop) and P+1 loop are critical structural motifs coordinating ATP and substrate, embedded in the activation segment.

5. RD’s Thoughts and Takeaways

  • 🧡 I LOVE THIS PAPER 🧡
  • The discussion on αC helix and activation loop interdependence is highly insightful, clarifying many kinase mechanisms.
  • The role of pseudosubstrate autoinhibition shows how kinases can self-regulate and be externally controlled, essential for signal specificity.
  • Gleevec example illustrates the value of targeting inactive conformations—key lesson for kinase drug discovery.
  • Plasticity in inactive states reflects evolutionary flexibility of kinases, crucial for complex signaling networks.
  • RD also now sees Glycine loop = P loop, important for ATP binding; P+1 loop in activation segment — critical for positioning.

Take-home Messages

  • Kinase conformational plasticity underlies precise signaling regulation.
  • Activation loops and αC helix are central hubs of conformational control.
  • Pseudosubstrate mechanisms provide self-inhibition and fast activation.
  • Therapeutic targeting of inactive states (e.g., Gleevec) exploits plasticity for specificity.
  • Kinase regulation integrates structural adaptability with functional demands, making them versatile yet tightly controlled signaling nodes.