Keywords

Autophosphorylation, Kinase Activation, PKA, PKC, MLCK, RTK, Src, Rhodopsin Kinase, Mos, Signal Transduction, Post-translational Modification, Enzyme Regulation

Reference

DOI: 10.1007/BF01076757

Abstract Summary

  • Autophosphorylation is a widespread regulatory mechanism among protein kinases, influencing their catalytic activity, protein interactions, and subcellular localization.
  • Occurs both in cis (intramolecular) and in trans (intermolecular), often following regulatory ligand binding.
  • Influences substrate affinity, enzyme stability, and downstream signal specificity.

Key Points

1. PKA (cAMP-dependent Protein Kinase)

  • Exists as a tetramer (R2C2); R = regulatory subunit, C = catalytic subunit.
  • Autophosphorylation of RII subunits (Ser-95/Ser-112) reduces affinity for C, facilitating activation upon cAMP binding.
  • C subunit autophosphorylation sites:
    • Thr-197 (near active site, essential for high substrate affinity, enhances activity).
    • Ser-338, Ser-10 (possible modulatory roles).
  • R autophosphorylation inhibits PP-1, possibly prolonging PKA effects.

2. Phosphorylase Kinase

  • First kinase identified with autophosphorylation capacity.
  • Subunit composition: α, β, γ, δ; δ = calmodulin.
  • Autophosphorylation of α and β subunits enhances activity.
  • Requires Ca²⁺, and troponin C can stimulate it.
  • Physiological significance remains unclear.

3. MLCK (Myosin Light Chain Kinase)

  • Ca²⁺/calmodulin-dependent autophosphorylation at Ser-160, Ser-234.
  • Does not affect kinetics but increases sensitivity to inhibition by phosphorylated MLC.
  • Amino-terminal region likely influences substrate interaction.

4. PKC (Protein Kinase C)

  • Autophosphorylation at multiple sites (Ser-16, Thr-17, Thr-314, Thr-324, Thr-634, Thr-641) enhances activity.
  • Increases phorbol ester affinity, reduces Ca²⁺ sensitivity.
  • Influences substrate specificity, regulation, and downregulation.

5. Mos (MAPK Pathway Regulator)

  • Autophosphorylation critical during GVBD (germinal vesicle breakdown).
  • Stabilizes Mos for metaphase II arrest, prevents ubiquitin-mediated degradation.
  • Possibly initiated by another kinase but sustained via autoP (esp. Ser-3).

6. Rhodopsin Kinase (RK/GRK1)

  • Phosphorylates photoactivated rhodopsin (Rho)*.
  • Autophosphorylation at Ser-488, Thr-489:
    • Decreases affinity for phosphorylated Rho*.
    • Facilitates enzyme-substrate complex dissociation via electrostatic repulsion.

7. Receptor Tyrosine Kinases (RTKs)

  • Example: Insulin Receptor (IR):
    • Autophosphorylation of Tyr-1146, Tyr-1150, Tyr-1151 enhances kinase activity.
    • Enables insulin-independent activity.
    • Tyr-1316, Tyr-1322 act as negative regulators.
  • RTKs cluster into 3 groups: EGFR, IR (tetrameric), PDGFR.

8. Src Family Kinases

  • Positive regulation: AutoP near catalytic site enhances activity.
  • Negative regulation: AutoP in C-terminal tail inhibits activity.
  • Family members: c-Src, c-Yes, Fyn, c-Fgr, Lck, Hck, Lyn, Blk.

Functional Effects of Autophosphorylation

  1. Catalytic activity enhancement.
  2. Altered sensitivity or specificity for allosteric effectors.
  3. Protection or targeting for proteolysis.
  4. Membrane association modulation.
  5. Endocytosis regulation.
  6. Mediating protein-protein interactions.

“Autophosphorylation impacts virtually all aspects of kinase function."

Evolutionary Perspectives

  • Two models:
    1. Autophosphorylation domains evolved from unrelated phosphorylation sites (possibly as competitive inhibitors).
    2. Post-fusion evolution: Autophosphorylation sites evolved after regulatory and catalytic domains combined, allowing opportunistic regulation.
  • The diversity of autoP sites suggests second pathway is more likely.

Discussion Highlights

  • Common themes:
    • Triggered by regulatory ligands.
    • Multiple sites phosphorylated, often following kinase domain specificity.
    • Enhances ligand and substrate affinity, prolonging activation.
    • Tissue-specific variations modulated by phosphatases.
  • Philosophical thought:
    • “It is an error in logic to assume that autophosphorylation has relevance in all instances, but equally flawed to dismiss its importance where functional effects are yet unobserved.”
  • AutoP as a unifying and flexible mechanism of kinase regulation—context-dependent, tissue-dependent, dynamic.

Future Questions

  • How do specific autoP patterns affect cell-specific signaling and disease states?
  • Can engineered autoP sites be used to modulate kinase behavior for therapeutic purposes?
  • How does autophosphorylation crosstalk with other PTMs (e.g., ubiquitination, acetylation)?

RD’s Takeaways

  • Love the breadth of autoP effects—from enzyme activity to degradation control!
  • The dual role in activating and inhibiting kinases (e.g., Src) is fascinating—context matters!
  • Insightful for kinase signaling pathway models—adds complexity beyond linear cascades.