Keywords

CDPK, Groundnut, Autophosphorylation, Calcium Signaling, Kinase Activation, Protein Phosphorylation

Reference

DOI: 10.1104/pp.120.3.859

Abstract

GnCDPK, a Ca2+-dependent protein kinase (CDPK) from groundnut (Arachis hypogea), undergoes autophosphorylation on threonine residues in a Ca2+-dependent, calmodulin-independent manner.
This autophosphorylation is required for its activation, as prior autophosphorylation eliminates a characteristic lag phase in exogenous substrate phosphorylation.
Interestingly, autophosphorylation enhances substrate phosphorylation under low ATP conditions but does not affect Ca2+ sensitivity or ATP affinity.
Autophosphorylation therefore primes GnCDPK for efficient substrate phosphorylation, without altering its calcium dependency.

Notes

1. Experimental Framework and Approach

  • GnCDPK purified from dry groundnut seeds, previously characterized for MLCpep (chicken smooth muscle myosin light chain peptide) phosphorylation.
  • Autophosphorylation analyzed under various conditions:
    • Ca2+ dependence (K₀.₅ = 0.5 μM).
    • Mg2+ requirement (5-10 mM).
    • Calmodulin (CaM) independence.
  • Used 32P labeling to detect phosphorylation levels and stoichiometry.

2. Cool Findings

  • GnCDPK autophosphorylates in a Ca2+-dependent but calmodulin-independent manner, inhibited by EGTA.
  • Autophosphorylation occurs primarily on Thr residues, while MLCpep phosphorylation favors Ser.
  • Autophosphorylation reaches saturation within 2 minutes, with a stoichiometry of ~0.2 mol phosphate/mol protein — similar to soybean CDPK.
  • Distinct ATP affinities:
    • Autophosphorylation Km = 100 nM.
    • Exogenous substrate phosphorylation Km = 10 μM — 100x lower affinity for ATP during substrate phosphorylation!
  • Prior autophosphorylation eliminates the lag observed in substrate phosphorylation under low ATP conditions.
  • Lag phase in substrate phosphorylation:
    • Without prior autophosphorylation, a 1-2 min lag is observed at low ATP (5 μM).
    • Pre-incubation with ATP (1 μM, 2-10 min) removes lag, boosting initial substrate phosphorylation rate.

3. Inspiration and Analytical Approaches

  • To determine intra- vs. inter-molecular autophosphorylation:
    • Linear relationship between rate of autophosphorylation and enzyme concentration implies intramolecular (cis) autophosphorylation.
    • Van’t Hoff plot (log velocity vs. log enzyme concentration).
  • Phospho-amino acid analysis: Confirmed pThr for autoP, pSer for substrate P.
  • 32P incorporation measurement via liquid scintillation counting — (RD: could adopt this technique too!).

4. Key Mechanistic Insights

  • Autophosphorylation primes GnCDPK, enhancing its substrate phosphorylation without altering Ca2+ dependency or ATP affinity.
  • Pre-phosphorylated GnCDPK shows a sharp increase in MLCpep phosphorylation; non-autophosphorylated enzyme displays a delayed and reduced response.
  • Autophosphorylation does NOT influence Ca2+ sensitivity — phosphorylation of MLCpep remains Ca2+-dependent in both phosphorylated and unphosphorylated forms.
  • Autophosphorylation enhances affinity for MLCpep (Km 50 μM vs. 250 μM for non-P form), but not for ATP (Km ~10 μM both).
  • Nonhydrolyzable ATP analogs fail to substitute for ATP in priming GnCDPK, indicating true phosphorylation is required, not just ATP binding.

5. Broader Implications and Comparative Notes

  • “Autophosphorylation is a common regulatory mechanism that can adjust kinase activity or modulate dependence on activators."
  • In GnCDPK, autophosphorylation does not modulate Ca2+ sensitivity, but enhances readiness for substrate phosphorylation, possibly via conformational shifts.
  • Comparison to other systems:
    • Soybean CDPK: Similar low stoichiometry of autoP.
    • CaMKII: Autophosphorylation modulates Ca2+ dependence — not observed in GnCDPK, suggesting distinct regulatory strategies.
    • Wingbean CDPK: AutoP is Ca2+-independent, unlike GnCDPK.
  • Model: Autophosphorylation might induce cooperative interactions among phosphorylated and unphosphorylated kinase molecules, as seen in CaMK systems.

6. RD’s Reflections and Inspiration

  • Lag phase concept is fascinating: implies structural adjustment required before efficient substrate phosphorylation.
  • Different ATP affinities for autoP and substrate P — possible conformational gating mechanism?
  • Stoichiometry ~0.2 mol/mol hints at partial activation — intriguing in context of CDPK complex formation or multimerization.
  • Cross-kingdom comparison (e.g., CaMK, other CDPKs) valuable for understanding evolutionary tuning of autoP function.
  • Potential application to study phosphorylation “priming” in other plant-specific kinases (e.g., receptor kinases or CPKs in immune signaling).

Take-home Messages

  • GnCDPK autophosphorylates on Thr residues in a Ca2+-dependent, calmodulin-independent manner.
  • Autophosphorylation primes the kinase for efficient substrate phosphorylation, eliminating lag under low ATP.
  • Enhances substrate affinity (Km drop) but does not alter ATP affinity or Ca2+ sensitivity.
  • Partial autophosphorylation (0.2 mol/mol) suggests complex regulation and potential cooperative behavior.
  • Autophosphorylation is a preparatory step ensuring rapid and effective phosphorylation of downstream substrates under physiological constraints.
  • Overall, GnCDPK demonstrates how autophosphorylation refines enzymatic responsiveness, a feature that may apply broadly to plant kinase regulation.