The Ca(2+)-binding domains in non-muscle type alpha-actinin: biochemical and genetic analysis.

Abstract

Abstract. Dictyostelium c~-actinin is a Ca:+-regulated F-actin cross-linking protein. To test the inhibitory function of the two EF hands, point mutations were introduced into either one or both Ca2+-binding sites. After mutations, the two EF hands were distinguishable with respect to their regulatory activities. Inactivation of EF hand I abolished completely the F-actin cross-linking activity of Dictyostelium discoideum o~-actinin but Ca 2+ binding by EF hand H was still observed in a *sCa 2+ overlay assay. In contrast, after mutation of EF hand II the molecule was still active and inhibited by Ca2+; however, ~500-fold more Ca 2+ was necessary for inhibition and 45Ca2+ binding could not be detected in the overlay assay. These data indicate that EF hand I has a low affinity for Ca 2+ and EF hand II a high affinity, implying a regulatory function of EF hand I in the inhibition of F-actin cross-linking activity. Biochemical data is presented which allows us to distinguish two functions of the EF hand domains in D. discoideum tx-actinin: (a) at the level of the EFhands, the Ca2+-binding affinity of EF hand I was increased by EF hand II in a cooperative manner, and (b) at the level of the two subunits, the EF hands acted as an on/off switch for actin-binding in the neighboring subunit. To corroborate in vitro observations in an in vivo system we tried to rescue the abnormal phenotype of a mutant (Witke, W., M. Schleicher, A. A. Noegel. 1992. Cell. 68:53-62) by introducing the mutated t~-actinin cDNAs. In agreement with the biochemical data, only the molecule modified in EF hand II could rescue the abnormal phenotype. Considering the fact that the active construct is "always on" because it requires nonphysiological, high Ca ~+ concentrations for inactivation, it is interesting to note that an unregulated ot-actinin was able to rescue the mutant phenotype.

Dictyostelium discoideum contains a family of calmodulin‐related EF‐hand proteins that are developmentally regulated

“…This regulation is mediated by two perfect EF-hand regions in the C-terminus of the subunits [4,11]. The concentrations of the small acidic Ca2+.binding proteins c, almodulin [12] and calfumirin [13] are also essentially unaltered during growth and development.…”

“…Many of the effects of Ca 2+ are exerted via proteins containing EF-hand domains for Ca 2+ binding as a common structural motif [I,2], Proteins like a-actinin or spectrin harbor intramolecular EF-regions as regulatory domains [3,4], others act either as monomeric proteins (e.g. calmodulin, SI00) that transiently interact with their targets or as permanently bound regulatory subunits [5][6][7].…”

The C‐Terminal Domain of α‐Spectrin is Structurally Related to Calmodulin

“…Binding of calcium to the EF-hands might therefore regulate binding to F-actin. This could explain the regulation of actin binding by calcium in several non-muscle a-actinins Witke et al, 1993).…”

The Cytoskeletal/Non-muscle Isoform of α-Actinin Is Phosphorylated on Its Actin-binding Domain by the Focal Adhesion Kinase

“…Calcium binding to the EF hand modules in ␣-actinin decreases the interaction between ␣-actinin and actin (26,27). Fukami et al (28,29) have shown that the skeletal muscle isoform of ␣-actinin binds phosphatidylinositol 4,5-bisphosphate and that the actin gelating activity of the protein was enhanced by the phospholipid.…”