title: Identification of a novel phosphorylation motif for CDPKs: phosphorylation of synthetic peptides lacking basic residues at P-3/P-4.
abstract: The Ca(2+)-dependent protein kinases (CDPKs) are members of a large subfamily of protein kinases in plants that have been implicated in the control of numerous aspects of plant growth and development. One known substrate of the CDPKs is the ER-located ACA2 calcium pump, which is regulated by phosphorylation of Ser(45). In the present study, a synthetic peptide based on the known regulatory phosphorylation site (RRFRFTANLS(45)KRYEA) was efficiently phosphorylated in vitro by CDPKs but not a plant SNF1-related protein kinase. Phosphorylation of the Ser(45)-ACA2 peptide was surprising because the sequence lacks basic residues at P-3/P-4 (relative to the phosphorylated Ser at position P) that are considered to be essential recognition elements for CDPKs. We demonstrate that phosphorylation of the Ser(45)-ACA2 peptide is dependent on the cluster of basic residues found N-terminal (P-6 to P-9) as well as C-terminal (P + 1/P + 2) to the phosphorylated Ser. The results establish a new general phosphorylation motif for CDPKs: [Basic-Basic-X-Basic]-phi-X(4)-S/T-X-Basic (where phi is a hydrophobic residue). The motif predicts a number of new phosphorylation sites in plant proteins. Evidence is presented that the novel motif may explain the phosphorylation by CDPKs of Ser271 in the aquaporin PM28A.
authors.affiliation: Department of Biological Sciences, Zhejiang University, Huajiachi Campus, Hangzhou, Zhejiang, 310029, China.
authors.forename: J Z
authors.initials: JZ
authors.lastname: Huang
authors.forename: S C
authors.initials: SC
authors.lastname: Hardin
authors.forename: S C
authors.initials: SC
authors.lastname: Huber
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D020346:Aquaporins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D010940:Plant Proteins; D011494:Protein Kinases / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D018724:Spinacia oleracea / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D020346:Aquaporins; C100932:PM28A protein, Spinacia oleracea; D010455:Peptides; D010940:Plant Proteins; D012694:Serine; D011494:Protein Kinases; D000252:Calcium-Transporting ATPases