Characterization of tescalcin, a novel EF-hand protein with a single Ca2+-binding site: metal-binding properties, localization in tissues and cells, and effect on calcineurin.
The tescalcin gene is preferentially expressed during mouse testis differentiation. Here, we demonstrate that this gene encodes a 24 kDa Ca(2+)- and Mg(2+)-binding protein with one consensus EF-hand and three additional domains with EF-hand homology. Equilibrium dialysis with (45)Ca(2+) revealed that recombinant tescalcin binds approximately one Ca(2+) ion at physiological concentrations (pCa 4.5). The intrinsic tryptophan fluorescence of tescalcin was significantly reduced by Ca(2+), indicative of a conformational change. The apparent K(d) for Ca(2+) was 0.8 microM. A point mutation in the consensus EF-hand (D123A) abolished (45)Ca(2+) binding and prevented the fluorescence quenching, demonstrating that the consensus EF-hand alone mediates the Ca(2+)-induced conformational change. Tescalcin also binds Mg(2+) (K(d) 73 microM), resulting in a much smaller fluorescence decrease. In the presence of 1 mM Mg(2+), tescalcin's Ca(2+) affinity is shifted to 3.5 microM. These results illustrate that tescalcin should bind Mg(2+) constitutively in a quiescent cell, replacing it with Ca(2+) 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 Ca(2+) ion.
Gutierrez-Ford
Christina
C
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida 33136, USA.
Levay
Konstantin
K
Gomes
Aldrin V
AV
Perera
Erasmo M
EM
Som
Tapan
T
Kim
You-Me
YM
Benovic
Jeffrey L
JL
Berkovitz
Gary D
GD
Slepak
Vladlen Z
VZ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D019703:Calcineurin / Q000378:metabolism*; D065095:Calcineurin Inhibitors; D002118:Calcium / Q000037:antagonists & inhibitors / Q000737:chemistry*; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D002413:Cations, Divalent / Q000737:chemistry; D002942:Circular Dichroism; D006224:Cricetinae; D003956:Dialysis; D020832:EF Hand Motifs / Q000235:genetics; D004789:Enzyme Activation; D018922:HL-60 Cells; D006367:HeLa Cells; D006801:Humans; D007106:Immune Sera / Q000096:biosynthesis / Q000302:isolation & purification; D020014:K562 Cells; D008274:Magnesium / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D009928:Organ Specificity / Q000235:genetics; D016716:PC12 Cells; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D020539:Sequence Analysis, Protein; D013050:Spectrometry, Fluorescence; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry
D065095:Calcineurin Inhibitors; D002135:Calcium-Binding Proteins; D002413:Cations, Divalent; D007106:Immune Sera; D012333:RNA, Messenger; D011994:Recombinant Proteins; C486009:TESC protein, human; C420458:Tesc protein, mouse; D014364:Tryptophan; D019703:Calcineurin; D008274:Magnesium; D002118:Calcium