Poster: Protein modification

Abs # 803: Identification of a New Motif for CDPK phosphorylation in vitro

Presenter:	Huber, Steven C., schuber1@life.uiuc.edu
Authors	Sebastia, Cinta H. (A)   Hardin, Shane C. (A)   Clouse, Steven D. (B)   Kieber, Joseph J. (C)   Huber, Steven C. (A)
Affiliations:	(A): USDA/ARS, Univ of Illinois, Dept of Plant Biology (B): NC State University, Dept. of Horticultural Sciences (C): University of North Carolina, Biology Department

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. Phosphorylation of the LeACS2-Ser460 peptide by CDPKs was surprising because the sequence lacks a basic residue at P-3/P-4 (relative to the phosphorylated Ser at position P) and a hydrophobic group at P-5, both of which are considered essential recognition elements in the classic motif targeted by CDPKs. We demonstrate that phosphorylation of the LeACS2-Ser460 peptide is most dependent on basic residues at P+3/P+4 and hydrophobic residues at P-1 and P+1. The results establish a fundamentally new phosphorylation motif that is broadly targeted by CDPKs: Φ-_-1-[ST]₀-Φ₊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.