Abstract
The actin cytoskeleton is a dynamic array of filaments that undergoes rapid remodeling to drive many cellular processes. An essential feature of filament remodeling is the spatio-temporal regulation of actin filament nucleation. One family of actin filament nucleators, the Diaphanous-related formins, is activated by the binding of small G-proteins such as RhoA. However, RhoA only partially activates formins, suggesting that additional factors are required to fully activate the formin. Here we identify one such factor, IQ motif containing GTPase activating protein-1 (IQGAP1), which enhances RhoA-mediated activation of the Diaphanous-related formin (DIAPH1) and targets DIAPH1 to the plasma membrane. We find that the inhibitory intramolecular interaction within DIAPH1 is disrupted by the sequential binding of RhoA and IQGAP1. Binding of RhoA and IQGAP1 robustly stimulates DIAPH1-mediated actin filament nucleation in vitro In contrast, the actin capping protein Flightless-I, in conjunction with RhoA, only weakly stimulates DIAPH1 activity. IQGAP1, but not Flightless-I, is required to recruit DIAPH1 to the plasma membrane where actin filaments are generated. These results indicate that IQGAP1 enhances RhoA-mediated activation of DIAPH1 in vivo Collectively these data support a model where the combined action of RhoA and an enhancer ensures the spatio-temporal regulation of actin nucleation to stimulate robust and localized actin filament production in vivo.
Highlights
The actin cytoskeleton is a dynamic array of filaments that undergoes rapid remodeling to drive many cellular processes
Using DIAPH1 as a model formin, we examined the role of Fli-I, which interacts with the Diaphanous autoregulatory domain (DAD), and IQ motif containing GTPase activating protein-1 (IQGAP1), which interacts with the DAD interacting domain (DID) and targets DIAPH1 to the leading edge of migrating cells [18], on formin-dependent actin polymerization activity
A recombinant IQGAP1-Diaphanous-binding region (DBR) domain fused to glutathione S-transferase (GST-IQGAP1-DBR) was added to purified DIAPH1 N- or C-terminal fragments fused to His6 or maltose-binding protein (MBP), respectively, and interactions were determined by pulldown assays and Western blotting (Fig. 1)
Summary
The scaffold-protein IQGAP1 enhances and spatially restricts the actin-nucleating activity of Diaphanous-related formin 1 (DIAPH1). One family of actin filament nucleators, the Diaphanous-related formins, is activated by the binding of small G-proteins such as RhoA. IQGAP1, but not Flightless-I, is required to recruit DIAPH1 to the plasma membrane where actin filaments are generated These results indicate that IQGAP1 enhances RhoA-mediated activation of DIAPH1 in vivo. In the case of the formins DAAM1 and DIAPH1, Fli-I binding enhances RhoA-mediated activation of the actin filament nucleation activity in vitro [12]. The scaffold protein IQGAP1, a DID interacting partner, was required to target DIAPH1 to the plasma membrane [18] Taken together, these data suggest that DID and DAD interactors have the potential to regulate small G-protein–mediated activation and targeting of formins. Using DIAPH1 as a model formin, we examined the role of Fli-I, which interacts with the DAD, and IQGAP1, which interacts with the DID and targets DIAPH1 to the leading edge of migrating cells [18], on formin-dependent actin polymerization activity
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