Abstract
Our laboratory has previously identified a cDNA clone encoding the small heat shock protein HspB3. A hybridoma that produces a monoclonal antibody specific to HspB3 was also generated. By using Western blot analysis, HspB3 could be detected in rat skeletal muscle and most abundantly in the heart. Further screening human heart cDNA library using HspB3 as a bait with the yeast two-hybrid system . We have identified that HspB3 is capable of interacting with actin. The goal of this project is to identify the domains required for the interaction between these two proteins. Through serial deletion of these two genes followed by yeast two hybrid analysis, we have identified the region between amino acid position 25-36 of HspB3 is critical for interacting with actin. In actin, amino acid region 322-337, which is near the C-terminus is necessary for its interaction with HspB3. To confirm the interaction between HspB3 and actin in vivo, confocal microcopy was used. When a wild type HspB3 and a truncated mutant of HspB3 (HspB3/△25~36aa) were over-expressed in RD (Human embryonal rhabdomyosarcoma) cell line, the wild type of HspB3 could be co-localized with actin, but the truncated HspB3 could not . Moreover, unlike the wild type HspB3 that did not affect cell morphology, overexpression of truncated HspB3 in RD cells resulted in loss of cell membrane integrity. We concluded that HspB3 is a true actin binding protein. Our results have provided basic information concerning the function role of the small heat shock protein HspB3.