title: Dissecting requirements for auto-inhibition of actin nucleation by the formin, mDia1.
abstract: The mammalian formin, mDia1, is an actin nucleation factor. Experiments in cells and in vitro show that the N-terminal region potently inhibits nucleation by the formin homology 2 (FH2) domain-containing C terminus and that RhoA binding to the N terminus partially relieves this inhibition. Cellular experiments suggest that potent inhibition depends upon the presence of the diaphanous auto-regulatory domain (DAD) C-terminal to FH2. In this study, we examine in detail the N-terminal and C-terminal regions required for this inhibition and for RhoA relief. Limited proteolysis of an N-terminal construct from residues 1-548 identifies two stable truncations: 129-548 and 129-369. Analytical ultracentrifugation suggests that 1-548 and 129-548 are dimers, whereas 129-369 is monomeric. All three N-terminal constructs inhibit nucleation by the full C terminus. Although inhibition by 1-548 is partially relieved by RhoA, inhibition by 129-548 or 129-369 is RhoA-resistant. At the C terminus, DAD deletion does not affect nucleation but decreases inhibitory potency of 1-548 by 20,000-fold. Synthetic DAD peptide binds both 1-548 and 129-548 with similar affinity and partially relieves nucleation inhibition. C-terminal constructs are stable dimers. Our conclusions are as follows: 1) DAD is an affinity-enhancing motif for auto-inhibition; 2) an N-terminal domain spanning residues 129-369 (called DID for diaphanous inhibitory domain) is sufficient for auto-inhibition; 3) a dimerization region C-terminal to DID increases the inhibitory ability of DID; and 4) DID alone is not sufficient for RhoA relief of auto-inhibition, suggesting that sequences N-terminal to DID are important to RhoA binding. An additional finding is that FH2 domain-containing constructs of mDia1 and mDia2 lose >75% nucleation activity upon freeze-thaw.
authors.affiliation: Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
authors.forename: Fang
authors.initials: F
authors.lastname: Li
authors.forename: Henry N
authors.initials: HN
authors.lastname: Higgs
mesh_terms: D000199:Actins / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000502:physiology*; D000080865:Formins; D051379:Mice; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D000199:Actins; D002352:Carrier Proteins; C491464:Diap1 protein, mouse; D000080865:Formins; D020742:rhoA GTP-Binding Protein