Characterization of tescalcin, a novel EF-hand protein with a single call-binding site: Metal-binding properties, localization in tissues and cells, and effect on calcineurin

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The tescalcin gene is preferentially expressed during mouse testis differentiation. Here, we demonstrate that this gene encodes a 24 kDa Ca2+- and Mg2+-binding protein with one consensus EF-hand and three additional domains with EF-hand homology. Equilibrium dialysis with Ca-45(2+) revealed that recombinant tescalcin binds approximately one Ca2+ ion at physiological concentrations (pCa 4.5). The intrinsic tryptophan fluorescence of tescalcin was significantly reduced by Ca2+, indicative of a conformational change. The apparent K-d for Ca2+ was 0.8 muM. A point mutation in the consensus EF-hand (D123A) abolished Ca-45(2+) binding and prevented the fluorescence quenching, demonstrating that the consensus EF-hand alone mediates the Ca2+-induced conformational change. Tescalcin also binds Mg2+ (K-d 73 muM), resulting in a much smaller fluorescence decrease. In the presence of 1 mM Mg2+, tescalcin's Ca2+ affinity is shifted to 3.5 muM. These results illustrate that tescalcin should bind Mg2+ constitutively in a quiescent cell, replacing it with Ca2+ during stimulation. We also show that tescalcin is most abundant in adult mouse heart, brain, and stomach, as well as in HeLa and HL-60 cells. Immunofluorescence microscopy revealed that tescalcin is present in the cytoplasm and nucleus, with concentration in membrane ruffles and lamellipodia in the presence of serum, where it colocalizes with the small guanosine triphosphatase Rac-1. Tescalcin shares sequence and functional homology with calcineurin-B homologous protein (CHP), and we found that tescalcin, like CHP, can inhibit the phosphatase activity of calcineurin A. Hence, tescalcin is a novel calcineurin B-like protein that binds a single Ca2+ ion.
Publisher
AMER CHEMICAL SOC
Issue Date
2003-12
Language
English
Article Type
Article
Keywords

MYOFIBRILLAR ADENOSINE-TRIPHOSPHATASE; CA2+-BINDING PROTEIN; NA+/H+ EXCHANGER; HOMOLOGOUS PROTEIN; CALCIUM SENSOR; PHOSPHATASE-ACTIVITY; N-MYRISTOYLATION; CALMODULIN; CA2+; TROPONIN

Citation

BIOCHEMISTRY, v.42, no.49, pp.14553 - 14565

ISSN
0006-2960
DOI
10.1021/bi034870f
URI
http://hdl.handle.net/10203/240867
Appears in Collection
MSE-Journal Papers(저널논문)
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