Tobacco Calcium-Dependent Protein Kinases Are Differentially Phosphorylated In Vivo as Part of a Kinase Cascade That Regulates Stress Response

Keywords

CDPK, NtCDPK2, NtCDPK3, Intra/Inter-Phosphorylation, Membrane Localization, Stress Signaling, Calcium-Dependent Protein Kinase

Reference

DOI: 10.1074/jbc.M109.052126

Abstract

NtCDPK2 and NtCDPK3, two calcium-dependent protein kinases (CDPKs) from tobacco, are differentially phosphorylated in vivo in response to biotic and abiotic stress, primarily within their variable N-terminal regions.
Despite high sequence similarity (91%), distinct phosphorylation patterns emerge, with NtCDPK2 Thr65 being an intra-molecular autophosphorylation site, while Ser40 is phosphorylated by an upstream kinase.
Membrane localization is essential for these phosphorylation events, and domain-swap experiments reveal that the N-terminus dictates phosphorylation specificity.
Additionally, a truncated active form of NtCDPK2 (VK2) induces cell death, linking phosphorylation and localization to CDPK signaling outcomes.

Notes

1. In Vivo Phosphorylation Patterns and Kinetics

• Stress induces rapid phosphorylation of both NtCDPK2 and NtCDPK3:
  • NtCDPK2: Ser40 (inter-molecular) and Thr65 (intra-molecular autophosphorylation), detected within 2 min.
  • NtCDPK3: Ser54 and a PEST domain site (both stress-induced, inter-molecular phosphorylation).
• Electrophoretic mobility shifts reflect these phosphorylation events — two distinct shifts per kinase.
• Dephosphorylation occurs within 2–4 hours, and shifts are phosphatase-sensitive (cantharidin inhibition).

2. Dissection of Intra- and Inter-Molecular Phosphorylation

• Intra-molecular autophosphorylation:
  • Thr65 (NtCDPK2) is confirmed as an autoP-site via in vitro kinase assay and absence in kinase-inactive mutants.
• Inter-molecular phosphorylation:
  • Ser40 (NtCDPK2) and Ser54 (NtCDPK3) remain phosphorylated in kinase-dead mutants, indicating action by an upstream kinase.
  • Not replicated in vitro — supporting an in vivo kinase cascade mechanism.

3. N-Terminal Specificity and Domain Swapping

• Phosphorylation patterns are encoded in the N-terminal regions, not the kinase domain:
  • N-terminal swap experiments between NtCDPK2 and NtCDPK3 reproduce the donor’s phosphorylation pattern.
• Mutating Ser40 and Thr65 in NtCDPK2 or Ser54 in NtCDPK3 directly abolishes specific mobility shifts in the hybrids.
• PEST domains contribute to additional phosphorylation-dependent shifts — deletion of PEST regions alters patterns.

4. Membrane Localization Requirement

• Myristoylation (G2A) and palmitoylation (C5S) mutants of NtCDPK2 lose membrane association and fail to phosphorylate Ser40.
• Similar effects seen for NtCDPK3 G2A mutant, affecting PEST phosphorylation.
• Correct membrane localization is essential for phosphorylation by upstream kinases.

5. Functional Consequences: Cell Death and Gain-of-Function Phenotypes

• Truncated NtCDPK2 (VK2) lacking regulatory domains induces cell death, indicating constitutive activity.
• Phosphorylation of Ser40 and Thr65 occurs without stimulation in VK2, mirroring constitutive active state.
• This effect depends on membrane localization — disrupted by G2A/C5S mutations.
• Parallels with other CDPKs (e.g., StCDPK5) where truncated forms or localization mutants affect ROS production and stress signaling.

6. Comparison of NtCDPK2 and NtCDPK3 Mechanisms

7. Broader Implications for CDPK Regulation

• Three-step CDPK regulation:
  1. Ca2+ binding → relieves auto-inhibition.
  2. Auto/Inter phosphorylation.
  3. Membrane localization enables full activation and phosphorylation by upstream kinases.
• Phosphorylation may adjust Ca2+ sensitivity, subcellular localization, or protein-protein interactions.
• N-terminal P-sites as determinants of specificity and functional outcome, independent of kinase domain sequence.

8. RD’s Takeaways and Inspiration

• N-terminal-driven regulation adds a fascinating layer to CDPK specificity.
• Strong parallel to CaMK and other animal kinases where phosphorylation tunes sensitivity, localization, and signaling.
• Auto- and inter-molecular phosphorylation events must be carefully dissected in other CDPK studies — this paper sets a great methodological example.
• Membrane association as a checkpoint — crucial for understanding CDPK activation in plant immunity and signaling.
• Love the “necessary but not sufficient” view on phosphorylation — aligns well with broader kinase regulation concepts.
• Exciting connection to ROS production pathways and membrane-localized immune responses.

Take-home Messages

• NtCDPK2 and NtCDPK3 are phosphorylated in vivo in a stress-dependent, N-terminal-specific manner.
• Intra- and inter-molecular phosphorylation events dictate kinase behavior — with Ser40 (NtCDPK2) and Ser54 (NtCDPK3) as upstream kinase targets.
• Membrane localization is critical for phosphorylation and function — governed by N-terminal myristoylation/palmitoylation.
• Phosphorylation can induce constitutive kinase activity and trigger cell death, highlighting its functional importance.
• N-terminal domains encode the “identity” and functional outcome of CDPK phosphorylation, independent of the catalytic domain.
• This study shapes a new paradigm for CDPK function — as dynamic signaling hubs regulated by calcium, phosphorylation, and membrane association.