Keywords

CDPK, CaM, EF-hand, Soybean, Calcium-binding kinase, Evolution, Actin-regulation

Reference

DOI: 10.1126/science.1852075

Abstract

This foundational study reports the purification and cloning of a novel calcium-dependent protein kinase (CDPK) from soybean. Unlike classical CaM-dependent kinases, CDPK directly binds calcium via a C-terminal calmodulin-like domain (CaM-LD) containing four EF-hands, functioning without external calmodulin (CaM) or phospholipids. CDPK defines a new family of calcium-regulated protein kinases, featuring a fused kinase and CaM-like regulatory domain, potentially evolved from a gene fusion event.

Notes

1. Structural and Evolutionary Significance of CDPK

  • First discovery of a Ca2+-binding kinase that doesn’t need CaM.
  • Single polypeptide contains:
    • N-terminal kinase domain (homologous to CaMK family).
    • C-terminal CaM-like domain (CLD) with four EF-hands for calcium binding.
  • Predicted MW: ~57 kDa.

Key evolutionary insight: CDPK likely arose from fusion of two pre-existing genes: (1) a CaM-dependent kinase and (2) a CaM-like Ca2+-binding protein — a fused, self-contained Ca2+ sensor-effector module.

2. Functional Model and Activation Mechanism

  • Pseudosubstrate/autoinhibitory domain proposed:
    • Rich in basic residues, possibly mimicking substrate to block kinase active site in the absence of Ca2+.
  • Direct Ca2+ binding to CLD relieves autoinhibition, activating the kinase.
  • Avoids slower CaM diffusion, enabling rapid Ca2+ response.

Speed advantage: CDPK may respond faster to Ca2+ transients than CaM-dependent kinases, as activation doesn’t require external CaM recruitment.

3. EF-Hand and Calcium Binding Domain Insights

  • EF-hand: 12 amino acid helix-loop-helix motif, oxygen-containing ligands coordinate Ca2+.
  • Spaced EF-hands in CDPK resemble those in calpain, suggesting evolutionary flexibility in calcium-sensing domains.
  • CDPK = second known kinase family with an embedded CaM-like domain (after calpain proteases in animal cells).

Evolutionary note: The fusion of a kinase domain and CaM-like domain in CDPK represents a unique, tightly coupled sensing-acting unit — an evolutionary “shortcut” for calcium signaling.

4. Functional Connection to Actin and Cytoplasmic Streaming

  • Cytoplasmic Ca2+ flux is critical for actin-dependent cytoplasmic streaming.
  • Later studies confirm soybean CDPK-α colocalizes with actin, suggesting CDPK regulates actin dynamics in response to calcium.

Possible role: CDPK might be a key link between Ca2+ signals and actin cytoskeleton remodeling — hinting at roles in cell motility, growth, and morphogenesis.

5. Pseudosubstrate Regulation Concept

  • The basic-rich pseudosubstrate may sit in the kinase’s active site to block ATP/substrate access until displaced upon Ca2+ binding.
  • Possible mechanism for activation:
  1. Ca2+ binds CLD, changing its conformation.
  2. CLD “pulls” pseudosubstrate out of the kinase domain.
  3. Active site freed, enabling kinase function.

Connection to autoP hypothesis: Post-activation, autophosphorylation of activation loop might further stabilize active conformation, providing a second regulatory layer.

6. Evolutionary Fusion and Diversification

  • The fusion model implies modular assembly of functional proteins:
    • Evolution glues together sensor (CaM-like) + effector (kinase) domains to create autonomous calcium sensors.
  • CDPK represents a prototype for plant-specific calcium regulation, distinct from animal pathways.
  • Parallel example: calpain (Ca2+-dependent protease) also has a fused CaM-like domain, though structurally distinct (lower identity).

Protein evolution principle: Domain fusion allows evolution to create new regulatory proteins rapidly by combining pre-existing functional modules.

Take-home Messages

  • CDPK is the first identified kinase that binds Ca2+ directly via its own fused CaM-like domain, independent of calmodulin.
  • EF-hand-rich CaM-like domain enables direct, fast response to calcium signals.
  • Pseudosubstrate region proposed to block kinase domain until Ca2+ relieves inhibition.
  • The fusion of sensor-effector domains is a key evolutionary innovation for autonomous regulation.
  • CDPK likely plays a role in Ca2+-regulated actin cytoskeletal dynamics.

Final Thought

CDPKs represent an elegant evolutionary solution to intracellular calcium sensing — compact, efficient, and specialized, merging signal detection and execution in a single molecule.