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Título : Characterisation of tyrosine-phosphorylation-defective calmodulin mutants
Autor : Salas, Valentina
Sánchez-Torres, Juan
Cusidó-Hita, David M.
García-Marchan, Yael
Sojo, Felipe
Benaím, Gustavo
Villalobo, Antonio
Palabras clave : Calmodulin mutants
Calmodulin phosphorylation
c-Src
Epidermal growth factor receptor
phenylalanine
Escherichia coli
Fecha de publicación : 2005
Editorial : Protein Expression and PuriWcation
Citación : Vol. 41;No. 2 pp 384–392
Resumen : Using site-directed mutagenesis, we have produced three calmodulin (CaM) mutants in which one or the two tyrosine residues of native CaM were substituted by phenylalanine. The three variants, denoted CaM(Y99F), CaM(Y138F), and CaM(Y99F/Y138F), were highly expressed in transformed Escherichia coli BL21(DE3)pLysS and puriWed in high yield. The three CaM mutants were able to activate the cyclic nucleotide phosphodiesterase and the plasma membrane Ca2+-ATPase, and present the characteristic Ca2+- induced electrophoretic mobility shift of native CaM. CaM(Y138F) and CaM(Y99F/Y138F), however, showed a slightly higher electrophoretic mobility than CaM(Y99F) or wild type CaM. The molar extinction coeYcient of native CaM at 276 nm decreases 50% in CaM(Y99F) and CaM(Y138F), while the 276nm peak disappears in CaM(Y99F/Y138F). Terbium Xuorescence studies with the diVerent CaM mutants indicate that Y99 (but not Y138) closely interacts with Ca2+ in the III Ca2+-binding domain. The epidermal growth factor receptor (EGFR) and the non-receptor tyrosine kinase c-Src phosphorylate CaM(Y99F) and CaM(Y138F) at a lesser extent than wild type CaM, while they fail to phosphorylate CaM(Y99F/Y138F) as expected. All resulting phospho-(Y)CaM species present the characteristic Ca2+-induced electrophoretic mobility shift observed in non-phosphorylated CaM. Quantitative analysis of the diVerent phospho-(Y)CaM species suggests that the relative phosphorylation of Y99 and Y138 in wild type CaM by both the EGFR and c-Src is diVerent than the respective phosphorylation of either Y99 in CaM(Y138F) or Y138 in CaM(Y99F).
Descripción : Corresponding author. Fax: +58 212 903 5118. E-mail address: gbenaim@reacciun.ve (G. Benaim). These authors contributed equally to this work. Abbreviations used: CaM, calmodulin; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; EGTA, [ethylenebis (oxyethylenenitrilo)] tetraacetic acid; Hepes, 4-(2-hydroxyethyl)-1 piperazineethanesulfonic acid; IPTG, isopropyl-1-thio- -D-galactopiranoside; Jak2, Janus kinase 2; P-CaM, phosphorylated calmodulin; PCR, polymerase chain reaction; PMSF, phenylmethylsulfonyl Xuoride; PTB, phosphotyrosine binding; PVDS, polyvinylidene diXuoride; SDS–PAGE, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate; SH2, Src non-catalytic homology 2; Tris, tris(hydroxymethyl)-aminomethane. 1046-5928/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2005.01.004.
URI : http://hdl.handle.net/123456789/15700
ISSN : 1096-0279 (Electronic)
1046-5928 (Linking)
1046-5928 (Print)
doi:10.1016/j.pep.2005.01.004
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