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C‐terminal variations in β‐thymosin family members specify functional differences in actin‐binding properties

Mammalian cells express several isoforms of β‐thymosin, a major actin monomer sequestering factor, including thymosins β4, β10, and β15. Differences in actin‐binding properties of different β‐thymosin family members have not been investigated. We find that thymosin β15 binds actin with a 2.4‐fold higher affinity than does thymosin β4. Mutational analysis was performed to determine the amino acid differences in thymosin β15 that specify its increased actin‐affinity. Previous work with thymosin β4 identified an α‐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 β15, but that differences in these domains are not responsible for the variation in actin‐binding properties between thymosins β4 and β15. Truncation of the unique C‐terminal residues in thymosin β15 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 β15 with those of thymosin β4 did, however, reduce the actin‐binding affinity of the hybrid relative to thymosin β15. Similarly, replacement of the thymosin β4 C‐terminal amino acids with those of thymosin β15 led to increased actin binding. We conclude that functional differences between closely related β‐thymosin family members are, in part, specified by the C‐terminal variability between these isoforms. Such differences may have consequences for situations where β‐thymosins are differentially expressed as in embryonic development and in cancer. J. Cell. Biochem. 77:277–287, 2000. © 2000 Wiley‐Liss, Inc.