Phosphorylation of synthetic peptides by a CDPK and plant SNF1-related protein kinase. Influence of proline and basic amino acid residues at selected positions.
Spinach (Spinacia oleracea L.) leaf sucrose-phosphate synthase (SPS) can be inactivated by phosphorylation of Ser-158 by calmodulin-like domain protein kinases (CDPKs) or SNF1-related protein kinases (SnRK1) in vitro. While the phosphorylation site sequence is relatively conserved, most of the deduced sequences of SPS from dicot species surrounding the Ser-158 regulatory phosphorylation site contain a Pro residue at P-4 (where P is the phosphorylated Ser); spinach is the exception and contains an Arg at P-4. We show that a Pro at P-4 selectively inhibits phosphorylation of the peptide by a CDPK relative to a SnRK1. The presence of a Pro at P-4, by allowing a tight turn in the peptide substrate, may interfere with proper binding of residues at P-5 and beyond. Both kinases had greater activity with peptides having basic residues at P-6 and P+5 (in addition to the known requirement for an Arg at P-3/P-4), and when the residue at P-6 was a His, the pH optimum for phosphorylation of the peptide was acid shifted. The results are used to predict proteins that may be selectively phosphorylated by SnRK1s (as opposed to CDPKs), such as SPS in dicot species, or may be phosphorylated in a pH-dependent manner.
Huang
J Z
JZ
Department of Biological Sciences, Zhejiang University, Hangzhou, Zhejiang, 310029 People's Republic of China. jzhuang@zjh.edu.cn
Huber
S C
SC
D000595:Amino Acid Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D018515:Plant Leaves / Q000201:enzymology; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D018724:Spinacia oleracea / Q000201:enzymology; D013379:Substrate Specificity
D010455:Peptides; C071570:SNF1-related protein kinases; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases