Characterisation of tyrosine-phosphorylation-defective calmodulin mutants.
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 purified in high yield. The three CaM mutants were able to activate the cyclic nucleotide phosphodiesterase and the plasma membrane Ca(2+)-ATPase, and present the characteristic Ca(2+)-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 coefficient of native CaM at 276 nm decreases 50% in CaM(Y99F) and CaM(Y138F), while the 276nm peak disappears in CaM(Y99F/Y138F). Terbium fluorescence studies with the different CaM mutants indicate that Y99 (but not Y138) closely interacts with Ca(2+) in the III Ca(2+)-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 Ca(2+)-induced electrophoretic mobility shift observed in non-phosphorylated CaM. Quantitative analysis of the different 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 different than the respective phosphorylation of either Y99 in CaM(Y138F) or Y138 in CaM(Y99F).
Salas
Valentina
V
Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Arturo Duperier 4, E-28029 Madrid, Spain.
Sánchez-Torres
Juan
J
Cusidó-Hita
David M
DM
García-Marchan
Yael
Y
Sojo
Felipe
F
Benaim
Gustavo
G
Villalobo
Antonio
A
D019943:Amino Acid Substitution; D000818:Animals; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
D002147:Calmodulin; D014443:Tyrosine; D010649:Phenylalanine