title: Phosphorylation of serine residues in the N-terminus modulates the activity of ACA8, a plasma membrane Ca2+-ATPase of Arabidopsis thaliana.
abstract: ACA8 is a plasma membrane-localized isoform of calmodulin (CaM)-regulated Ca(2+)-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-(1)M-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) ≈ 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-(1)M-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.
authors.affiliation: Dipartimento di Biologia L. Gorini, Università degli Studi di Milano, Istituto di Biofisica del CNR, Sezione di Milano, via G. Celoria 26, 20133 Milano, Italy.
authors.forename: Sonia
authors.initials: S
authors.lastname: Giacometti
authors.forename: Claudia Adriana
authors.initials: CA
authors.lastname: Marrano
authors.forename: Maria Cristina
authors.initials: MC
authors.lastname: Bonza
authors.forename: Laura
authors.initials: L
authors.lastname: Luoni
authors.forename: Margherita
authors.initials: M
authors.lastname: Limonta
authors.forename: Maria Ida
authors.initials: MI
authors.lastname: De Michelis
mesh_terms: D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D002147:Calmodulin; D012694:Serine; C508150:Aca8 protein, Arabidopsis; D000252:Calcium-Transporting ATPases
references.citation: Plant Physiol. 2009 Jun;150(2):889-903
references.pmid: 19376835
references.citation: Planta. 2004 Mar;218(5):814-23
references.pmid: 14648121
references.citation: J Proteomics. 2009 Apr 13;72(3):439-51
references.pmid: 19245862
references.citation: Mol Cell Proteomics. 2007 Jul;6(7):1198-214
references.pmid: 17317660
references.citation: Plant Physiol. 2000 Aug;123(4):1495-506
references.pmid: 10938365
references.citation: Plant Physiol. 2000 Feb;122(2):517-26
references.pmid: 10677444
references.citation: Plant Physiol. 2002 Jun;129(2):469-85
references.pmid: 12068094
references.citation: J Biol Chem. 2009 Nov 6;284(45):30881-8
references.pmid: 19740735
references.citation: J Cell Biol. 1994 Feb;124(3):351-63
references.pmid: 7507493
references.citation: Plant J. 2007 Sep;51(5):931-40
references.pmid: 17651370
references.citation: Annu Rev Plant Biol. 2004;55:263-88
references.pmid: 15377221
references.citation: J Membr Biol. 1998 Sep 15;165(2):101-9
references.pmid: 9744998
references.citation: Mol Cell Proteomics. 2007 Oct;6(10):1711-26
references.pmid: 17586839
references.citation: FEBS Lett. 2004 Sep 10;574(1-3):20-4
references.pmid: 15358533
references.citation: Plant Biol (Stuttg). 2011 May;13(3):421-30
references.pmid: 21489092
references.citation: Nature. 1970 Aug 15;227(5259):680-5
references.pmid: 5432063
references.citation: J Biol Chem. 2006 Jan 13;281(2):1058-65
references.pmid: 16267044
references.citation: Mol Cell Proteomics. 2003 Nov;2(11):1234-43
references.pmid: 14506206
references.citation: Plant Cell. 2004 Sep;16(9):2394-405
references.pmid: 15308754
references.citation: Proc Natl Acad Sci U S A. 2000 May 23;97(11):6224-9
references.pmid: 10823962
references.citation: J Biol Chem. 1997 Oct 31;272(44):27525-8
references.pmid: 9346883
references.citation: Plant Cell. 2002;14 Suppl:S401-17
references.pmid: 12045291
references.citation: Mol Syst Biol. 2008;4:193
references.pmid: 18463617
references.citation: J Biol Chem. 1996 Dec 13;271(50):32461-7
references.pmid: 8943312
references.citation: Plant Physiol. 1993 Oct;103(2):385-390
references.pmid: 12231945
references.citation: Plant J. 2010 Jul 1;63(1):1-17
references.pmid: 20374526
references.citation: J Biol Chem. 1999 Jan 1;274(1):527-31
references.pmid: 9867875
references.citation: J Bioenerg Biomembr. 2007 Dec;39(5-6):409-14
references.pmid: 18000746
references.citation: Plant Physiol. 2003 Aug;132(4):1840-8
references.pmid: 12913141
references.citation: Mol Membr Biol. 2008 Sep;25(6-7):539-46
references.pmid: 18988067
references.citation: Annu Rev Plant Biol. 2010;61:593-620
references.pmid: 20192754
references.citation: Biochemistry. 1994 Jun 14;33(23):7267-77
references.pmid: 8003490
references.citation: Proteomics. 2008 Sep;8(17):3536-47
references.pmid: 18686298
references.citation: Biochemistry. 2000 Apr 11;39(14):4004-11
references.pmid: 10747788
references.citation: FEBS Lett. 2010 Dec 1;584(23):4783-8
references.pmid: 21073872
references.citation: Plant Physiol. 2010 Jul;153(3):1161-74
references.pmid: 20466843
references.citation: New Phytol. 2009 Jan;181(2):275-294
references.pmid: 19121028
references.citation: Curr Genomics. 2010 Mar;11(1):2-13
references.pmid: 20808518