Identification of a new motif for CDPK phosphorylation in vitro that suggests ACC synthase may be a CDPK substrate.
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: phi -1-[ST]0- phi +1-X-Basic+3-Basic+4, where phi 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.
Hernández Sebastià
Cinta
C
US Department of Agriculture, Agricultural Research Service, and Department of Plant Biology and Crop Science, University of Illinois, 1201 W. Gregory Drive, Urbana, IL 61801-3838, USA.
Hardin
Shane C
SC
Clouse
Steven D
SD
Kieber
Joseph J
JJ
Huber
Steven C
SC
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D004789:Enzyme Activation; D008190:Lyases / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D003313:Zea mays / Q000201:enzymology*
D010940:Plant Proteins; D011494:Protein Kinases; D008190:Lyases; C021773:1-aminocyclopropanecarboxylate synthase