The eluate was diluted 5-fold with GST binding buffer containing 50mM TRIS-HCl pH 7

The eluate was diluted 5-fold with GST binding buffer containing 50mM TRIS-HCl pH 7.4, 150mM NaCl, 10mM EDTA, 1mM dithiothreitol (DTT), and 0.4 % Triton X-100, and incubated for 30min with 2ml of glutathioneSepharose 4B (GE Healthcare Bio-Science AB, Sweden, catalogue no. thalianacalcium-dependent protein kinase (CPK1 and CPK16); phosphorylation of mutant 6His-1M-I116peptides 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. Keywords:Arabidopsis thaliana, Ca2+-ATPase, calcium-dependent protein kinase, calmodulin, plasma DC42 membrane, phosphorylation == Introduction == Cytosolic calcium is a key element in the transduction of a variety of endogenous and environmental signals in plant cells. An increasing amount of evidence indicates that signal specificity is encoded by the SBI-477 amplitude, frequency, and time extension of cytosolic Ca2+waves, which in turn depend on the activity of Ca2+channelswhich when open flood the cytosol with Ca2+from the apoplast and/or intracellular storesand of active Ca2+transporterswhich extrude Ca2+to the apoplast or sequester it in intracellular stores. Fine-tuning of the Ca2+transport systems in response to different signals is thus a crucial feature of Ca2+-mediated signal transduction (Sanderset al., 2002;Boursiac and Harper, 2007;McAinsh SBI-477 and Pittman, 2009;Das and Pandey, 2010;Doddet al., 2010;Bonza and De Michelis, 2011;Pittmanet al., 2011). In plant cells, Ca2+extrusion from the cytoplasm is accomplished either through tonoplast-localized Ca2+H+antiporters powered by a proton-motive force, or through Ca2+pumps powered by ATP hydrolysis, localized both at the plasma membrane (PM) and at intracellular membranes. PM Ca2+pumps are likely to play a crucial role in re-establishing the low basal Ca2+concentration especially after its increase due to opening of PM Ca2+channels. Indeed, the SBI-477 available evidence, albeit fragmentary, demonstrates their involvement in fundamental processes such as development, hormonal regulation, and response to biotic and abiotic stresses; however, their physiological role and the mechanisms underlying their regulation in response to specific signals have not been ascertained yet (Boursiac and Harper, 2007;Bonza and De Michelis, 2011;Pittmanet al., 2011). PM Ca2+pumps are calmodulin (CaM)-regulated Ca2+-ATPases, belonging to the P-type ATPase superfamily: three isoforms of CaM-regulated Ca2+-ATPase, all belonging to the same cluster, have been identified as PM-localized pumps inArabidopsis thaliana: among these, the best characterized at the biochemical level is ACA8, a widely expressed isoform found in all plant organs (Bonza and De Michelis, 2011;Pittmanet al., 2011). ACA8, like other plant isoforms of CaM-regulated Ca2+-ATPases, SBI-477 has an extended cytosolic N-terminal domain containing an autoinhibitory domain partially overlapping the CaM-binding site: CaM binding suppresses the autoinhibitory action of the N-terminal domain and determines both the increase ofVmaxand the decrease of theK0.5for free Ca2+(Bonza and De Michelis, 2011;Pittmanet al., 2011). ACA8 is also regulated by acidic phospholipids such as phosphatidylserine or phosphatidylinositol-4P, which activate the pump via two distinct mechanisms, involving their binding to different sites: acidic phospholipids binding to a site in the protein N-terminus, overlapping the autoinhibitory and CaM-binding domain, stimulates ACA8 activity similar to CaM or to cleavage of the N-terminus, while their binding to a second, as yet unidentified, site further stimulates ACA8 activity by lowering itsK0.5for free Ca2+(Meneghelliet al., 2008). CaM-regulated Ca2+-ATPases can also be modulated by phosphorylation. In the pumps of animal cells, which have the regulatory domain localized at the extended C-terminus, the C-terminal portion is the target of phosphorylation by different protein kinases that phosphorylate different amino acids in different isoforms: each phosphorylation event has a peculiar effect on the pump activity (Enyediet al., 1996,1997;Penniston and Enyedi, 1998;Vermaet al., 1999). In plants, it has been shown thatin vitrophosphorylation of a serine residue just downstream the CaM-binding site of ACA2anA. thalianaisoform of the endoplasmic reticulumby a calcium-dependent protein kinase (CDPK) severely inhibits CaM-stimulated enzyme activity, without disrupting CaM binding (Hwanget al., 2000). Also the N-terminus of BCA1an isoform of CaM-regulated Ca2+-ATPase of the tonoplast ofBrassica oleraceacan be phosphorylatedin vitroby protein kinase C at two serine residues, one within the.