C-terminal variations in beta-thymosin family members specify functional differences in actin-binding properties

Mammalian cells express several isoforms of beta-thymosin, a major actin monomer sequestering factor, including thymosins beta4, beta10, and beta15. Differences in actin-binding properties of different beta-thymosin family members have not been investigated. We find that thymosin beta15 binds actin with a 2.4-fold higher affinity than does thymosin beta4. Mutational analysis was performed to determine the amino acid differences in thymosin beta15 that specify its increased actin-affinity. Previous work with thymosin beta4 identified an alpha-helical domain, as well as a conserved central motif, as crucial for actin binding. Mutational analysis confirms that these domains are also vital for actin binding in thymosin beta15, but that differences in these domains are not responsible for the variation in actin-binding properties between thymosins beta4 and beta15. Truncation of the unique C-terminal residues in thymosin beta15 inhibits actin binding, suggesting that this domain also has an important role in mediating actin-binding affinity. Replacement of the 10 C-terminal amino acids of thymosin beta15 with those of thymosin beta4 did, however, reduce the actin-binding affinity of the hybrid relative to thymosin beta15. Similarly, replacement of the thymosin beta4 C-terminal amino acids with those of thymosin beta15 led to increased actin binding. We conclude that functional differences between closely related beta-thymosin family members are, in part, specified by the C-terminal variability between these isoforms. Such differences may have consequences for situations where beta-thymosins are differentially expressed as in embryonic development and in cancer.