Poster abstracts

Poster number 52 submitted by Christopher Schwebach

Characterizing a Novel Method of Human Plastin Regulation

Christopher S. Schwebach (Department of Chemistry and Biochemistry, Ohio State), Jonathan C. Wright (Department of Chemistry and Biochemistry, Ohio State), Elena Kudryashova (Department of Chemistry and Biochemistry, Ohio State)

Abstract:
The actin cytoskeleton is a vast network intricately regulated by numerous actin binding proteins. Plastins are one family of such proteins that non-covalently crosslink actin into bundles and contribute to cellular processes including cell migration and invasion. Bundling is achieved upon binding of two actin binding domains (ABD1 and ABD2) to actin filaments. Our recent findings show that the ABDs are not functionally equal. ABD1 acts as the primary, unregulated and constitutively interacting, but low affinity (~3uM) domain. In contrast, we found using fluorescence anisotropy (FA) that ABD2 alone binds actin with an affinity of ~15nM, i.e., much higher than those of full-length plastin or ABD1 alone. By pyrene-actin polymerization and TIRF microscopy we found that ABD2 is also able to nucleate actin, a property not previously described for full-length plastins. These results imply inhibition of ABD2 in the full protein context. Next, we found that ABD1 and 2 bind each other with high affinity (~20 nM) when expressed in trans and that ABD1 inhibits ABD2’s ability to nucleate actin filaments. Mutations that increase PLS association with actin in yeast also lead to dramatically enhanced bundling capabilities when introduced to human plastins. Interestingly, these mutations also decrease the thermal stability of full-length plastin to the level of ABD1 and ABD2 suggesting weaker association between the domains. Importantly, this same shift in thermal stability is seen in wild-type plastin in the presence of actin. These data support a novel mode of plastin regulation where ABD1 allosterically inhibits ABD2. Upon ABD1-actin binding, ABD2 inhibition is released allowing bundling. Understanding these complex mechanisms will lead to targeted therapies treating cancer metastases, which are known to be stimulated by ectopic expression of PLS2.

Keywords: Actin, Biochemistry, Plastin