Identification of a new motif for CDPK phosphorylation in vitro that suggests ACC synthase may be a CDPK substrate

Cinta Hernández Sebastià, Shane C. Hardin, Steven D. Clouse, Joseph J. Kieber, Steven C. Huber

Research output: Contribution to journalArticlepeer-review


1-Amino-cyclopropane-1-carboxylate synthase (ACS) catalyzes the rate-determining step in the biosynthesis of the plant hormone ethylene, and there is evidence for regulation of stability of the protein by reversible protein phosphorylation. The site of phosphorylation of the tomato enzyme, LeACS2, was recently reported to be Ser460, but the requisite protein kinase has not been identified. In the present study, a synthetic peptide based on the known regulatory phosphorylation site (KKNNLRLS460FSKRMY) in LeACS2 was found to be readily phosphorylated in vitro by several calcium-dependent protein kinases (CDPKs), but not a plant SNF1-related protein kinase or the kinase domain of the receptor-like kinase, BRI1, involved in brassinosteroid signaling. Studies with variants of the LeACS2-Ser460 peptide establish a fundamentally new phosphorylation motif that is broadly targeted by CDPKs: φ-1-[ST]0+1-X-Basic +3-Basic+4, where φ is a hydrophobic residue. Database analysis using the new motif predicts a number of novel phosphorylation sites in plant proteins. Finally, we also demonstrate that CDPKs and SnRK1s do not recognize motifs presented in the reverse order, indicating that side chain interactions alone are not sufficient for substrate recognition.

Original languageEnglish (US)
Pages (from-to)81-91
Number of pages11
JournalArchives of Biochemistry and Biophysics
Issue number1
StatePublished - Aug 1 2004


  • 1-Amino-cyclopropane-1-carboxylate synthase
  • Calcium-dependent protein kinase
  • Ethylene biosynthesis
  • Phosphorylation motif
  • Phosphorylation site prediction
  • Synthetic peptide phosphorylation

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology


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