ACA8 is a plasma membrane-localized isoform of calmodulin (CaM)-regulated Ca2+-ATPase of Arabidopsis thaliana. Several phosphopeptides corresponding to portions of the regulatory N-terminus of ACA8 have been identified in phospho-proteomic studies. To mimic phosphorylation of the ACA8 N-terminus, each of the serines found to be phosphorylated in those studies (Ser19, Ser22, Ser27, Ser29, Ser57, and Ser99) has been mutated to aspartate. Mutants have been expressed in Saccharomyces cerevisiae and characterized: mutants S19D and S57D-and to a lesser extent also mutants S22D and S27D-are deregulated, as shown by their low activation by CaM and by tryptic cleavage of the N-terminus. The His-tagged N-termini of wild-type and mutant ACA8 (6His-M-1-I-116) were expressed in Escherichia coli, affinity-purified, and used to analyse the kinetics of CaM binding by surface plasmon resonance. All the analysed mutations affect the kinetics of interaction with CaM to some extent: in most cases, the altered kinetics result in marginal changes in affinity, with the exception of mutants S57D (K-D similar to 10-fold higher than wild-type ACA8) and S99D (K-D about half that of wild-type ACA8). The ACA8 N-terminus is phosphorylated in vitro by two isoforms of A. thaliana calcium-dependent protein kinase (CPK1 and CPK16); phosphorylation of mutant 6His-M-1-I-116 peptides shows that CPK16 is able to phosphorylate the ACA8 N-terminus at Ser19 and at Ser22. The possible physiological implications of the subtle modulation of ACA8 activity by phosphorylation of its N-terminus are discussed.
|Titolo:||Phosphorylation of serine residues in the N-terminus modulates the activity of ACA8, a plasma membrane Ca2+-ATPase of Arabidopsis thaliana|
|Parole Chiave:||Arabidopsis thaliana; Ca 2+-ATPase; calcium-dependent protein kinase; calmodulin; phosphorylation; plasma membrane|
|Settore Scientifico Disciplinare:||Settore BIO/04 - Fisiologia Vegetale|
|Data di pubblicazione:||2012|
|Digital Object Identifier (DOI):||10.1093/jxb/err346|
|Appare nelle tipologie:||01 - Articolo su periodico|