Keywords

CDPK, N-terminal domain, Tobacco, RSG, Substrate recognition, Kinase engineering, Calcium signaling

Reference

DOI: 10.1105/tpc.109.073577

Abstract

This study identifies the variable N-terminal domain of tobacco CDPK1 as a crucial determinant for substrate recognition, particularly for the transcription factor REPRESSION OF SHOOT GROWTH (RSG). Remarkably, by transferring this N-terminal domain to another CDPK, the substrate specificity of the kinase can be altered, indicating a powerful strategy for reprogramming CDPK specificity and rewiring signaling pathways. This discovery reveals that CDPK substrate selection is not confined to the catalytic domain but critically involves a variable N-terminal domain, opening new avenues in kinase engineering.

Notes

1. N-terminal Domain Drives Substrate Recognition

  • CDPK1 binds RSG in a Ca2+-dependent manner, but the N-terminal domain is required for this specific recognition.
  • Deletion of the N-terminal domain abolishes RSG binding, although kinase activity remains intact when using casein (control substrate).

The N-terminal domain separates substrate recognition from catalytic activity—providing a “plug-and-play” module for target selection.

2. Pinpointing Critical Residues in the N-domain

  • Through truncation and mutational analyses, Arg10 (R10) was identified as essential for RSG binding and phosphorylation.
  • R10A mutant: retains normal kinase activity (using casein), but fails to phosphorylate RSG and loses in vivo function.

First demonstration that a single N-terminal residue can dictate specific substrate recognition in CDPKs.

3. Engineering CDPK Substrate Specificity via Chimeras

  • Chimeric CDPKs combining:
    • N-terminal domain of NtCDPK1 and
    • Catalytic domain of AtCPK9 (Arabidopsis)
      → gained the ability to recognize and phosphorylate RSG, following the identity of the N-domain.

Proof of concept: swapping N-terminal domains rewires kinase signaling toward specific substrates.

4. Experimental Highlights: Verifying Kinase Activity and Specificity

  • Casein phosphorylation assays confirmed that catalytic activity was unaffected by N-domain mutations (R10A).
  • Synthetic peptides of RSG phosphorylation site (S114) used to validate specificity.
  • Anti-pS114 antibodies confirmed in vitro and in vivo phosphorylation by WT and chimeric CDPKs, but not by R10A mutants.

Elegant setup to disentangle “general kinase function” from “specific target recognition”.

5. Substrate Specificity Beyond Active Site: A Modular View

  • Traditional models emphasize substrate recognition via:

    1. Catalytic cleft recognition.
    2. Docking motifs (linear motifs on substrates).
    3. Subcellular localization, scaffolds, expression patterns.

  • CDPKs lack scaffold proteins and accessory subunits, making this N-terminal domain-based substrate selection especially notable.

N-terminal domains act as pseudo-scaffold-like elements embedded in the kinase, dictating specificity “in cis”.

6. General Implications for CDPK and Kinase Engineering

  • Chimeric CDPKs demonstrate programmable kinase-substrate recognition, a strategy not fully explored in plants before.
  • The 81% similarity of CDPK1 KD to CaMK suggests that adding a variable N-terminal domain to other kinases (e.g., mammalian) could tune substrate specificity.

Possibility of extending N-terminal guided substrate recognition to other kinase families, beyond CDPKs.

7. Interesting Functional Insight on RSG-CDPK1 Interaction

  • The interaction between RSG and CDPK1 N-domain exposes S114 for phosphorylation.
  • N-terminal domain increases local substrate concentration near the active site, boosting phosphorylation efficiency.
  • ~3000-fold effective local concentration increase within a 10-nm radius from the kinase active site — biophysical underpinning of N-domain contribution.

N-terminal domain acts like a “substrate trap”, focusing kinase activity onto specific targets.

8. Broader Perspective on Substrate Specificity

  • Substrate selection involves multi-layered mechanisms:
    1. Active site–substrate sequence compatibility.
    2. Docking motifs.
    3. Subcellular localization.
    4. Expression timing.
    5. Scaffold proteins.
  • For CDPK, lacking scaffolds and targeting domains, N-terminal domain emerges as the key selector.

CDPKs thus represent a model where substrate specificity is “hardwired” into the protein, offering insights for synthetic biology.

Final Thought

“If we can swap N-terminal domains to rewire CDPK substrate specificity, could we extend this idea to reprogram other kinases, building synthetic signaling pathways tailored to specific needs?"