Subcellular distribution of thymosin beta4.

The localization of Oregon Green cadaverine-labeled thymosin beta(4), its fragments, and variants was investigated in cytoplasm-depleted A431 cells and in microinjected cells without and with fixation. The studied thymosin beta(4) variants included substitutions of the lysine residues within the basic cluster (14-KSKLKK-19) and the actin-binding motif (17-LKKTETQ-23). In contrast to Oregon Green cadaverine, none of the variants or fragments of thymosin beta(4) could pass the intact nuclear pore of cytoplasm-depleted cells and were hence excluded from the nucleus. However, an equal distribution of all thymosin beta(4) variants was observed in living cells. The nuclear localization is neither dependent on the actin-binding ability of thymosin beta(4) nor on its basic lysine cluster. The equal distribution of the beta-thymosins, the ability of the fragments thymosin beta(4)(1-26) and beta(4)(27-43) to enter the nucleus in intact cells immediately after injection, and their exclusion from cytoplasm-depleted nuclei make it unlikely that they are transported by a single transport protein. A passive but regulated diffusion could explain the described ability of thymosin beta(4) to shuttle into the nucleus.

Influence of the N terminus and the actin-binding motif of thymosin beta4 on its interaction with G-actin.

Thymosin beta(4) binds G-actin in a 1:1 ratio and prevents its aggregation to F-actin by sequestration. Substitution or modification of single amino acid residues within the N-terminal sequence 1 to 22 of thymosin beta(4) alters its interaction with G-actin. We generated thymosin beta(4) variants with amino acid substitutions within the N-terminal alpha-helix and the putative actin-binding motif. None of the E. coli-generated thymosin beta(4) variants was modified or acetylated at its N terminus. The stability of the complex of G-actin with nonacetylated thymosin beta(4) or beta(4)(A7V) is higher than the one with naturally occurring thymosin beta(4), which is always acetylated. The complex of G-actin with nonacetylated thymosin beta(4)(A7V,K18,19A) and beta(4)(K14,16,18,19A) is 15 times less stable compared to the complex with thymosin beta(4). The G-actin sequestering activities of all thymosin beta(4) variants correspond to their complex stabilities with G-actin, except for nonacetylated thymosin beta(4)(A7V), where it is attenuated. Thymosin beta(4)(Delta17-23) missing the putative actin-binding motif shows no interaction with G-actin.