title: Characterization of phospho-(tyrosine)-mimetic calmodulin mutants.
abstract: Calmodulin (CaM) phosphorylated at different serine/threonine and tyrosine residues is known to exert differential regulatory effects on a variety of CaM-binding enzymes as compared to non-phosphorylated CaM. In this report we describe the preparation and characterization of a series of phospho-(Y)-mimetic CaM mutants in which either one or the two tyrosine residues present in CaM (Y99 and Y138) were substituted to aspartic acid or glutamic acid. It was expected that the negative charge of the respective carboxyl group of these amino acids mimics the negative charge of phosphate and reproduce the effects that distinct phospho-(Y)-CaM species may have on target proteins. We describe some physicochemical properties of these CaM mutants as compared to wild type CaM, after their expression in Escherichia coli and purification to homogeneity, including: i) changes in their electrophoretic mobility in the absence and presence of Ca2+; ii) ultraviolet (UV) light absorption spectra, far- and near-UV circular dichroism data; iii) thermal stability in the absence and presence of Ca2+; and iv) Tb3+-emitted fluorescence upon tyrosine excitation. We also describe some biochemical properties of these CaM mutants, such as their differential phosphorylation by the tyrosine kinase c-Src, and their action as compared to wild type CaM, on the activity of two CaM-dependent enzymes: cyclic nucleotide phosphodiesterase 1 (PDE1) and endothelial nitric oxide synthase (eNOS) assayed in vitro.
authors.affiliation: Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Madrid, Spain.
authors.forename: Silviya R
authors.initials: SR
authors.lastname: Stateva
authors.affiliation: Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Madrid, Spain; Universidad Central de Venezuela, Facultad de Ciencias, Instituto de Biología Experimental, Caracas, Venezuela.
authors.forename: Valentina
authors.initials: V
authors.lastname: Salas
authors.affiliation: Universidad Central de Venezuela, Facultad de Ciencias, Instituto de Biología Experimental, Caracas, Venezuela; Instituto de Estudios Avanzados (IDEA), Caracas, Venezuela.
authors.forename: Gustavo
authors.initials: G
authors.lastname: Benaim
authors.affiliation: Instituto de Química Física Rocasolano, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
authors.forename: Margarita
authors.initials: M
authors.lastname: Menéndez
authors.affiliation: Instituto de Química Física Rocasolano, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
authors.forename: Dolores
authors.initials: D
authors.lastname: Solís
authors.affiliation: Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Madrid, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Villalobo
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002417:Cattle; D055598:Chemical Phenomena; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1 / Q000378:metabolism; D009154:Mutation; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D019000:Phosphotyrosine / Q000737:chemistry*; D055550:Protein Stability; D051381:Rats; D013696:Temperature
chemical_list: D002147:Calmodulin; D019000:Phosphotyrosine; D052250:Nitric Oxide Synthase Type III; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1
references.citation: Biochemistry. 2001 Aug 14;40(32):9605-17
references.pmid: 11583160
references.citation: Methods Enzymol. 1990;182:50-68
references.pmid: 2314256
references.citation: Protein Expr Purif. 1999 Aug;16(3):388-95
references.pmid: 10425159
references.citation: Biochemistry. 1997 Feb 25;36(8):2017-24
references.pmid: 9047299
references.citation: Nature. 1970 Aug 15;227(5259):680-5
references.pmid: 5432063
references.citation: Biochim Biophys Acta. 2014 Feb;1843(2):398-435
references.pmid: 24188867
references.citation: Physiol Rev. 1999 Jul;79(3):661-82
references.pmid: 10390515
references.citation: J Biol Chem. 1986 Aug 5;261(22):10429-38
references.pmid: 3525545
references.citation: Biochemistry. 2012 May 1;51(17):3651-61
references.pmid: 22486744
references.citation: FEBS Lett. 1983 Mar 7;153(1):169-73
references.pmid: 6825857
references.citation: Biochim Biophys Acta. 2005 Aug 10;1751(2):119-39
references.pmid: 16027053
references.citation: Anal Biochem. 2001 Sep 15;296(2):179-87
references.pmid: 11554713
references.citation: Eur J Biochem. 1999 Jun;262(3):790-802
references.pmid: 10411641
references.citation: Circulation. 2013 Mar 5;127(9):1009-17
references.pmid: 23388215
references.citation: Biochemistry. 1981 Jul 21;20(15):4407-14
references.pmid: 7284330
references.citation: Acta Crystallogr B. 2002 Jun;58(Pt 3 Pt 1):380-8
references.pmid: 12037359
references.citation: Proc Natl Acad Sci U S A. 1986 Jun;83(12):4190-3
references.pmid: 2424020
references.citation: Biochem J. 1986 Sep 1;238(2):485-90
references.pmid: 3800949
references.citation: Eur J Biochem. 2003 Feb;270(3):404-14
references.pmid: 12542690
references.citation: Eur J Biochem. 1994 Sep 15;224(3):909-16
references.pmid: 7925415
references.citation: J Biol Chem. 1982 Feb 25;257(4):1845-54
references.pmid: 6276400
references.citation: PLoS Comput Biol. 2011 Jul;7(7):e1002114
references.pmid: 21829336
references.citation: Biochem Biophys Res Commun. 1983 Feb 28;111(1):301-5
references.pmid: 6830594
references.citation: FEBS Lett. 2002 Feb 20;513(1):107-13
references.pmid: 11911888
references.citation: Biochim Biophys Acta. 1980 Jun 26;623(2):257-70
references.pmid: 7397213
references.citation: Cell. 2002 Mar 22;108(6):739-42
references.pmid: 11955428
references.citation: Biochemistry. 1999 Sep 21;38(38):12266-79
references.pmid: 10493794
references.citation: Biochem J. 1994 May 1;299 ( Pt 3):863-8
references.pmid: 8192677
references.citation: Eur J Biochem. 2002 Aug;269(15):3619-31
references.pmid: 12153558
references.citation: Trends Cell Biol. 2000 Aug;10(8):322-8
references.pmid: 10884684
references.citation: Biochemistry. 1992 Sep 1;31(34):7779-86
references.pmid: 1510964
references.citation: J Biol Chem. 1977 Jun 25;252(12):4108-17
references.pmid: 193856
references.citation: J Biol Chem. 2012 May 25;287(22):18173-81
references.pmid: 22493455
references.citation: Protein Expr Purif. 2005 Jun;41(2):384-92
references.pmid: 15866726
references.citation: Physiol Rev. 2011 Apr;91(2):651-90
references.pmid: 21527734
references.citation: Clin Exp Allergy. 2004 Feb;34(2):170-6
references.pmid: 14987293
references.citation: Protein Expr Purif. 1998 Feb;12(1):25-8
references.pmid: 9473453
references.citation: Arch Biochem Biophys. 1994 Nov 15;315(1):119-26
references.pmid: 7526800
references.citation: Nucleic Acids Res. 1979 Nov 24;7(6):1513-23
references.pmid: 388356
references.citation: Annu Rev Biochem. 1989;58:951-98
references.pmid: 2673026
references.citation: J Am Heart Assoc. 2014 Jun 23;3(3):e000996
references.pmid: 24958779