Site-specific phosphorylation of L-form starch phosphorylase by the protein kinase activity from sweet potato roots

Guang Huar Young, Han Min Chen, Chi Tsai Lin, Kuang Ching Tseng, Jiann Shing Wu, Rong Huay Juang*

*Corresponding author for this work

Research output: Contribution to journalJournal Article peer-review

26 Scopus citations

Abstract

A 78-amino acid insertion (L78) is found in the low-affinity type (L-form) of starch phosphorylase (L-SP, EC 2.4.1.1). This insertion blocks the starch-binding site on the L-SP molecule, and it decreases the binding affinity of L-SP toward starch. The computational analysis of the amino acid sequence on L78 predicts several phosphorylation sites at its Ser residues. Indeed, from the immunoblotting results using antibodies against phosphoamino acids, we observed that the purified L-SP from mature sweet potato (Ipomoea batatas) roots is phosphorylated. This observation led us to the detection of a protein kinase activity in the protein fraction of the crude extract from the sweet potato roots. The kinase was partially purified by liquid chromatography, and its native molecular mass was estimated as 338 kDa. An expressed peptide (L78P) containing the essential part of L78 was intensively phosphorylated by the kinase. However, H-SP (the high-affinity isomer of SP lacking the L78 insertion) and the proteolytic modified L-SP, which lost its L78 fragment, could not be phosphorylated. Furthermore, using L78P mutants by site-directed mutagenesis at Ser residues on L78, we demonstrate that only one Ser residue on L78 is phosphorylated by the kinase. These results imply that this kinase is specific to L-SP, or more precisely, to the L78 insertion. The in vitro phosphorylated L-SP shows higher sensitivity to proteolytic modification, but has no change in its kinetic parameters.

Original languageEnglish
Pages (from-to)468-478
Number of pages11
JournalPlanta
Volume223
Issue number3
DOIs
StatePublished - 02 2006
Externally publishedYes

Keywords

  • Ipomoea
  • Protein kinase
  • Protein phosphorylation
  • Starch phosphorylase
  • Sweet potato roots

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