Journal Article

Phosphorylation of Synthetic Peptides by a CDPK and Plant SNF1-Related Protein Kinase. Influence of Proline and Basic Amino Acid Residues at Selected Positions

Jian-Zhong Huang and Steven C. Huber

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 42, issue 10, pages 1079-1087
Published in print October 2001 | ISSN: 0032-0781
Published online October 2001 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pce137
Phosphorylation of Synthetic Peptides by a CDPK and Plant SNF1-Related Protein Kinase. Influence of Proline and Basic Amino Acid Residues at Selected Positions

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  • Biochemistry
  • Molecular and Cell Biology
  • Plant Sciences and Forestry

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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.

Keywords: Key words: CDPKs — Phosphorylation motif — Protein kinase specificity — SNF1-related protein kinase — Spinacia oleracea.; Abbreviations: CDPK, calmodulin-like domain protein kinase, also known as calmodulin-domain protein kinase or calcium-dependent protein kinase; DTT, dithiothreitol; EGTA, ethyleneglycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid; FPLC, fast protein liquid chromatography; MOPS, 3-(N-morpholino)-propanesulfonic acid; PEG, polyethylene glycol; PMSF, phenylmethylsulfonyl fluoride; SNF1, sucrose non-fermenting 1 (protein kinase) in S. cerevisiae; SnRK1, SNF1-related protein kinase; SPS, sucrose-phosphate synthase.

Journal Article.  6674 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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