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 mo nomer sequestering factor, including thymosins beta 4, beta 10, and beta 15 . Differences in actin-binding properties of different beta-thymosin family members have not been investigated. We find that thymosin beta 15 binds ac tin with a 2.4-fold higher affinity than does thymosin beta 4. Mutational a nalysis was performed to determine the amino acid differences in thymosin b eta 15 that specify its increased actin-affinity. Previous work with thymos in beta 4 identified an alpha-helical domain, as well as a conserved centra l motif, as crucial for actin binding. Mutational analysis confirms that th ese domains are also vital for actin binding in thymosin beta 15, but that differences in these domains are not responsible for the variation in actin -binding properties between thymosins beta 4 and beta 15. Truncation of the unique C-terminal residues in thymosin beta 15 inhibits actin binding, sug gesting that this domain also has an important role in mediating actin-bind ing affinity. Replacement of the 10 C-terminal amino acids of thymosin beta 15 with those of thymosin beta 4 did, however, reduce the actin-binding af finity of the hybrid relative to thymosin beta 15. Similarly, replacement o f the thymosin beta 4 C-terminal amino acids with those of thymosin beta 15 led to increased actin binding. We conclude that functional differences be tween 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 ex pressed as in embryonic development and in cancer. (C) 2000 Wiley-Liss, Inc .