PHOSPHO-REGULATION OF ACA8, A PLASMA MEMBRANE CA2+-ATPASE OF ARABIDOPSIS THALIANA

ACA8 is a plasma membrane-localized isoform of calmodulin (CaM)-regulated Ca2+-ATPase of Arabidopsis thaliana. Phospho-proteomic studies identified several phosphopeptides corresponding to portions of its regulatory N-terminus. Each of the Ser found to be phosphorylated in those studies (S19, S22, S27, S29, S57, and S99) has been mutated to Asp, to mimic phosphorylation of the ACA8 N-terminus, and to Ala to prevent phosphorylation. Mutants have been expressed in Saccharomyces cerevisiae and characterized: as shown by the low activation by CaM, mutants S19D, S57D, S22D and S27D are deregulated. Moreover, the low response to CaM of ACA8 mutants S22A, S27A, and S29A points the relevance of these serine residues per se in determining the amplitude of the response of ACA8 to CaM. To analyse the effect of S to D mutation on the kinetic of CaM binding, His-tagged N-termini of wild-type and mutant ACA8 (6His-1M-I116) were expressed in Escherichia coli, affinity-purified and used in surface plasmon resonance experiments. 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 (KD 10-fold higher than wild-type ACA8) and S99D (KD about half that of wild-type ACA8). Since S19 is in a consensus motive for calcium-dependent protein kinases (CDPKs) the ACA8 N-terminus has been subjected to in vitro phosphorylation assays with two isoforms of A. thaliana CDPKs: CDPK1, that phosphorylates ACA2 (an endoplasmic reticulum localised isoform of A. thaliana ACA) and CDPK16, a plasma membrane localised isoform of CDPK. Results show that both kinases are able to phosphorylate ACA8 N-terminus, but CDPK16 with higher extent. Phosphorylation of mutant 6His-1M-I116 peptides mapped CDPK16 phosphorylation site at S19 and at S22. Furthermore, we identified by two-hybrid screening two isoforms of CBL-interacting protein kinases (CIPKs) as putative interactors of ACA8 N-terminus region: CIPK9 and CIPK14. BiFC analysis in Nicotiana benthamiana confirmed the two-hybrid results, showing that interaction between ACA8 full length and CIPK9 or CIPK14 occurs in planta at the plasma membrane. Moreover, phosphorylation assay demonstrate that both kinases phosphorylate ACA8 N-terminus in vitro. Implications of these results are discussed.