The formation, disassembly, and organization of actin filaments in the cell are accompanied by numerous actin-binding proteins (ABPs). One group of ABPs are crosslinkers of actin filaments, which includes α-actinin. In vertebrates, four isoforms of α-actinin are known. Among these, two isoforms (2 and 3) are mainly localized in muscle cells, while the other two (1 and 4) are present in all cell types. All isoforms form antiparallel dimers, with one actin-binding domain (ABD) at each end of the dimer. In addition to the ABD, each α-actinin molecule contains a central rod domain and a calmodulin-like domain (CaMD), composed of two tandem EF-hand motifs (EF1–2 and EF3–4). One of the differences between muscle and non-muscle isoforms is that non-muscle α-actinins are regulated by Ca$^{2+}$, whereas the activity of muscle α-actinins is calcium-independent. Besides calcium, non-muscle α-actinins are also regulated by cleavage by the protease calpain, by phosphatidylinositol intermediates, and by phosphorylation of tyrosine residues. In addition to these modes of regulation, it was recently discovered that mechanical stress also affects the function of α-actinin, and an additional level of regulation is provided by the formation of heterodimers of α-actinin-1 and -4. In this study, we aimed to characterize new potential mechanisms for regulating the function of non-muscle forms of α-actinins. The first objective was to contribute to the characterization of the propensity for heterodimer formation between α-actinin-1 and -4. To this end, we aimed to express and isolate α-actinin-1/4 heterodimers via expression from a bicistronic RNA. We were partially successful, as the use of this method did result in heterodimer formation, but we were unable to fully purify them from the mixture, which also contained homodimers of the individual α-actinins. The second objective was to investigate additional potential sites for post-translational modifications and their impact on α-actinin function regulation. We first examined proteomic databases to identify which amino acid residues in α-actinin-1 are most likely to be phosphorylated in cells. We then examined the locations of these residues within the α-actinin-1 homodimer. Based on this, we hypothesized that phosphorylation of residue S348 – due to its location near the linker region between EF1–2 and EF3–4 – could influence calcium binding and, consequently, α-actinin’s ability to crosslink actin filaments. By introducing phosphomimetic mutations (S348E and S348D) at this site and performing isothermal titration calorimetry (ITC), we found that phosphorylation at this site likely does not affect calcium binding, as the calcium-binding affinity of the mutated dimers was similar to that of wild-type half-dimers. However, protein precipitation occurred during ITC measurements, so these results are not entirely conclusive.
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