TY - JOUR
T1 - Identification of a new motif for CDPK phosphorylation in vitro that suggests ACC synthase may be a CDPK substrate
AU - Sebastià, Cinta Hernández
AU - Hardin, Shane C.
AU - Clouse, Steven D.
AU - Kieber, Joseph J.
AU - Huber, Steven C.
N1 - Funding Information:
This work was supported in part by the US Department of Energy (Grant DE-AI05-2003ER to S.C.H.) and the US Department of Agriculture (Grant 2003-02631 to J.J.K.). C.H.S. was the recipient of a postdoctoral fellowship from the Ministerio de Educacion, Cultura y Deporte, Madrid, Spain.
PY - 2004/8/1
Y1 - 2004/8/1
N2 - 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-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.
AB - 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-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.
KW - 1-Amino-cyclopropane-1-carboxylate synthase
KW - Calcium-dependent protein kinase
KW - Ethylene biosynthesis
KW - Phosphorylation motif
KW - Phosphorylation site prediction
KW - Synthetic peptide phosphorylation
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U2 - 10.1016/j.abb.2004.04.025
DO - 10.1016/j.abb.2004.04.025
M3 - Article
C2 - 15234272
AN - SCOPUS:3042554224
SN - 0003-9861
VL - 428
SP - 81
EP - 91
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 1
ER -