Activation of Arp2/3 Complex: Addition of the First Subunit of the New Filament by a WASP Protein Triggers Rapid ATP Hydrolysis on Arp2

In response to activation by WASP-family proteins, the Arp2/3 complex nucleates new actin filaments from the sides of preexisting filaments. The Arp2/3-activating (VCA) region of WASP-family proteins binds both the Arp2/3 complex and an actin monomer and the Arp2 and Arp3 subunits of the Arp2/3 complex bind ATP. We show that Arp2 hydrolyzes ATP rapidly—with no detectable lag—upon nucleation of a new actin filament. Filamentous actin and VCA together do not stimulate ATP hydrolysis on the Arp2/3 complex, nor do monomeric and filamentous actin in the absence of VCA. Actin monomers bound to the marine macrolide Latrunculin B do not polymerize, but in the presence of phalloidin-stabilized actin filaments and VCA, they stimulate rapid ATP hydrolysis on Arp2. These data suggest that ATP hydrolysis on the Arp2/3 complex is stimulated by interaction with a single actin monomer and that the interaction is coordinated by VCA. We show that capping of filament pointed ends by the Arp2/3 complex (which occurs even in the absence of VCA) also stimulates rapid ATP hydrolysis on Arp2, identifying the actin monomer that stimulates ATP hydrolysis as the first monomer at the pointed end of the daughter filament. We conclude that WASP-family VCA domains activate the Arp2/3 complex by driving its interaction with a single conventional actin monomer to form an Arp2–Arp3–actin nucleus. This actin monomer becomes the first monomer of the new daughter filament.

[1]  R. Mullins,et al.  Arp2/3 complex requires hydrolyzable ATP for nucleation of new actin filaments , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[2]  T D Pollard,et al.  Sequences, structural models, and cellular localization of the actin- related proteins Arp2 and Arp3 from Acanthamoeba , 1995, The Journal of cell biology.

[3]  T. Pollard,et al.  Direct demonstration of actin filament annealing in vitro , 1988, The Journal of cell biology.

[4]  T D Pollard,et al.  Molecular mechanisms controlling actin filament dynamics in nonmuscle cells. , 2000, Annual review of biophysics and biomolecular structure.

[5]  L A Selden,et al.  Impact of profilin on actin-bound nucleotide exchange and actin polymerization dynamics. , 1999, Biochemistry.

[6]  M. Welch,et al.  Reconstitution of human Arp2/3 complex reveals critical roles of individual subunits in complex structure and activity. , 2001, Molecular cell.

[7]  E. Andrianantoandro,et al.  Kinetic mechanism of end-to-end annealing of actin filaments. , 2001, Journal of molecular biology.

[8]  T. Pollard,et al.  Direct observation of dendritic actin filament networks nucleated by Arp2/3 complex and WASP/Scar proteins , 2000, Nature.

[9]  R. Mullins,et al.  Different WASP family proteins stimulate different Arp2/3 complex-dependent actin-nucleating activities , 2001, Current Biology.

[10]  T. Pollard,et al.  The Arp2/3 complex nucleates actin filament branches from the sides of pre-existing filaments , 2001, Nature Cell Biology.

[11]  P. Rubenstein,et al.  The Effects of Severely Decreased Hydrophobicity in a Subdomain 3/4 Loop on the Dynamics and Stability of Yeast G-actin* , 1997, The Journal of Biological Chemistry.

[12]  T. Pollard,et al.  Hydrolysis of ATP by polymerized actin depends on the bound divalent cation but not profilin. , 2002, Biochemistry.

[13]  T. Pollard,et al.  A conserved amphipathic helix in WASP/Scar proteins is essential for activation of Arp2/3 complex , 2003, Nature Structural Biology.

[14]  Sam T. Pickering.  A DIRECT DEMONSTRATION , 1909 .

[15]  Thomas D. Pollard,et al.  Interaction of WASP/Scar proteins with actin and vertebrate Arp2/3 complex , 2000, Nature Cell Biology.

[16]  R. Mullins,et al.  Actin assembly mediated by Arp2/3 complex and WASP family proteins. , 2000, Methods in enzymology.

[17]  E. Shacter,et al.  Organic extraction of Pi with isobutanol/toluene. , 1984, Analytical biochemistry.

[18]  T. Pollard,et al.  Interactions of ADF/cofilin, Arp2/3 complex, capping protein and profilin in remodeling of branched actin filament networks , 2000, Current Biology.

[19]  T D Pollard,et al.  The interaction of Arp2/3 complex with actin: nucleation, high affinity pointed end capping, and formation of branching networks of filaments. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[20]  T D Pollard,et al.  Direct real-time observation of actin filament branching mediated by Arp2/3 complex using total internal reflection fluorescence microscopy , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[21]  T. Pollard,et al.  Mechanism of Interaction of Acanthamoeba Actophorin (ADF/Cofilin) with Actin Filaments* , 1999, The Journal of Biological Chemistry.

[22]  T. Pollard,et al.  Crystal Structure of Arp2/3 Complex , 2001, Science.

[23]  M. Carlier,et al.  ATP hydrolysis on actin-related protein 2/3 complex causes debranching of dendritic actin arrays , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[24]  T. Pollard,et al.  Scar, a WASp-related protein, activates nucleation of actin filaments by the Arp2/3 complex. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[25]  Thomas D. Pollard,et al.  Mechanism of action of cytochalasin B on actin , 1980, Cell.

[26]  K C Holmes,et al.  Refinement of the F-actin model against X-ray fiber diffraction data by the use of a directed mutation algorithm. , 1993, Journal of molecular biology.

[27]  M. Kirschner,et al.  The Interaction between N-WASP and the Arp2/3 Complex Links Cdc42-Dependent Signals to Actin Assembly , 1999, Cell.

[28]  T. Mitchison,et al.  Interaction of human Arp2/3 complex and the Listeria monocytogenes ActA protein in actin filament nucleation. , 1998, Science.

[29]  T. Pollard,et al.  Inhibition of the Arp2/3 complex-nucleated actin polymerization and branch formation by tropomyosin , 2001, Current Biology.

[30]  M. Carlier,et al.  Continuous monitoring of Pi release following nucleotide hydrolysis in actin or tubulin assembly using 2-amino-6-mercapto-7-methylpurine ribonucleoside and purine-nucleoside phosphorylase as an enzyme-linked assay. , 1996, Biochemistry.