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

Abstract

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.

The Etv1/Er81 transcription factor orchestrates activity-dependent gene regulation in the terminal maturation program of cerebellar granule cells

“…The testican gene turned out to be expressed mainly in stellate cells of the cerebellum. We selected 14 genes from the previous study and three additional TTXsuppressive genes which were identified in this study and expressed in the mature cerebellum (10)(11)(12). PCR analysis confirmed that the expression of all 17 genes was inhibited by TTX to different degrees, ranging from 15-85% ( Fig.…”

Fine Mapping of Collagen-Induced Arthritis Quantitative Trait Loci in an Advanced Intercross Line

“…Tesc encodes a putative EF-hand Ca 2ϩ -binding protein. It shares sequence and functional homology with calcineurin-B homologous protein (CHP), which can inhibit the phosphatase activity of calcineurin A, which regulates the NFAT nuclear translocation and activation (53,54).…”

Regulation of Rice NADPH Oxidase by Binding of Rac GTPase to Its N-Terminal Extension

“…To further study if Ca 2þ binding by the EF-hand motifs of the N-terminal region of rbohB is involved in Rac1-rbohB interaction, we mutated the conserved EF-hand motifs of Os rbohB, substituting the Asp residues at positions 242 and 286, generating the mutants OsrbohB D242A (ef1) and D286A (ef2) (Gutierrez-Ford et al, 2003). Mutations in these conserved Asp residues, which participate in Ca 2þ coordination, are known to disrupt Ca 2þ binding (see Supplemental Figure 3 online).…”