Regulation of the formin INF2 by actin monomers and calcium/calmodulin.

In response to increased intracellular calcium, the formin INF2 polymerizes 20-30% of the total cellular actin pool within 30 s, suggesting robust regulation. INF2 regulation requires an autoinhibitory interaction between the N-terminal diaphanous inhibitory domain (DID) and the C-terminal diaphanous autoregulatory domain (DAD). DID mutations are dominantly linked to two human diseases and constitutively activate INF2. However, DAD binding to actin monomers competes with DID binding, disrupting regulation. Here, we use a novel cell-free assay for the detailed investigation of INF2 regulation. Contrary to our previous findings, INF2 inhibition does not require CAP proteins but does require actin "buffering" by monomer-binding proteins such as profilin or thymosin. INF2 is activated by calcium-bound calmodulin (CALM) through CALM binding to the N terminus. In addition, the N terminus plays an important role in INF2 regulation beyond CALM binding. These findings support a role of actin monomer-binding proteins not only in regulating overall actin dynamics but also in specific regulation of an actin polymerization factor.