Ion binding properties of the dehydrin ERD14 are dependent upon phosphorylation.
The ERD14 protein (early response to dehydration) is a member of the dehydrin family of proteins which accumulate in response to dehydration-related environmental stresses. Here we show the Arabidopsis dehydrin, ERD14, possesses ion binding properties. ERD14 is an in vitro substrate of casein kinase II; the phosphorylation resulting both in a shift in apparent molecular mass on SDS-PAGE gels and increased calcium binding activity. The phosphorylated protein bound significantly more calcium than the nonphosphorylated protein, with a dissociation constant of 120 microm and 2.86 mol of calcium bound per mol of protein. ERD14 is phosphorylated by extracts of cold-treated tissues, suggesting that the phosphorylation status of this protein might be modulated by cold-regulated kinases or phosphatases. Calcium binding properties of ERD14 purified from Arabidopsis extracts were comparable with phosphorylated Escherichia coli-expressed ERD14. Approximately 2 mol of phosphate were incorporated per mol of ERD14, indicating a minimum of two phosphorylation sites. Western blot analyses confirmed that threonine and serine are possible phosphorylation sites on ERD14. Utilizing matrix assisted laser desorption ionization-time of flight/mass spectrometry we identified five phosphorylated peptides that were present in both in vivo and in vitro phosphorylated ERD14. Our results suggest that the polyserine (S) domain is most likely the site of phosphorylation in ERD14 responsible for the activation of calcium binding.
Alsheikh
Muath K
MK
Department of Biology, Indiana University, Purdue University Indianapolis, Indianapolis, Indiana 46202-5132, USA.
Heyen
Bruce J
BJ
Randall
Stephen K
SK
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000378:metabolism* / Q000502:physiology; D001665:Binding Sites; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002412:Cations; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D007477:Ions; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors
D029681:Arabidopsis Proteins; D002412:Cations; C089006:ERD14 protein, Arabidopsis; D007477:Ions; D002118:Calcium