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.
Highlights
Mammals possess 15 formin genes, in seven distinct phylogenetic groups [15]
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
Summary
DNA Constructs—Constructs of mouse mDia (accession number U96963) were generated by reverse transcription-PCR and cloned into pGEX-KT, as described previously [9]. C-terminal constructs (748 –1255, 748 –1203, 748 –1175, and mDia2-(521–1171)) were purified by fast protein liquid chromatography on a SourceQ15 5/5 or 10/10 column (Amersham Biosciences), concentrated on SP Sepharose Fast Flow (Amersham Biosciences), and dialyzed into the following buffer: 2 mM NaPO4, pH 7.0, 150 mM NaCl, 0.1 mM EGTA, 0.5 mM DTT. Residual inhibitor was inactivated by the addition of dithiothreitol (Calbiochem) to 10 mM from a 1 M stock in water (freshly made) and further incubation for 15 min on ice. The major proteolytic fragment from each digestion was isolated by fast protein liquid chromatography on a SourceQ15 5/5 column (Amersham Biosciences) and dialyzed in the same buffer used for mDia 548 storage (described above), frozen in Ͻ50-ml aliquots in liquid nitrogen, and stored at Ϫ70 °C. SЈ is calculated by the equation SЈ ϭ (S ϫ Mt)/(fmax Ϫ fmin), where S is raw slope in arbitrary units/s, Mt is M concentration of total polymerizable monomer in M, and fmax and fmin are fluorescence of fully polymerized and unpolymerized actin respectively, in arbitrary units
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