Phosphorylation of the Arp2 subunit relieves auto-inhibitory interactions for Arp2/3 complex activation.

Actin filament assembly by the actin-related protein (Arp) 2/3 complex is necessary to build many cellular structures, including lamellipodia at the leading edge of motile cells and phagocytic cups, and to move endosomes and intracellular pathogens. The crucial role of the Arp2/3 complex in cellular processes requires precise spatiotemporal regulation of its activity. While binding of nucleation-promoting factors (NPFs) has long been considered essential to Arp2/3 complex activity, we recently showed that phosphorylation of the Arp2 subunit is also necessary for Arp2/3 complex activation. Using molecular dynamics simulations and biochemical assays with recombinant Arp2/3 complex, we now show how phosphorylation of Arp2 induces conformational changes permitting activation. The simulations suggest that phosphorylation causes reorientation of Arp2 relative to Arp3 by destabilizing a network of salt-bridge interactions at the interface of the Arp2, Arp3, and ARPC4 subunits. Simulations also suggest a gain-of-function ARPC4 mutant that we show experimentally to have substantial activity in the absence of NPFs. We propose a model in which a network of auto-inhibitory salt-bridge interactions holds the Arp2 subunit in an inactive orientation. These auto-inhibitory interactions are destabilized upon phosphorylation of Arp2, allowing Arp2 to reorient to an activation-competent state

Phosphorylation of the Arp2/3 complex is necessary to nucleate actin filaments

The actin-related protein 2/3 (Arp2/3) complex is the primary nucleator of new actin filaments in most crawling cells. Nucleation-promoting factors (NPFs) of the Wiskott-Aldrich syndrome protein (WASP)/Scar family are the currently recognized activators of the Arp2/3 complex. We now report that the Arp2/3 complex must be phosphorylated on either threonine or tyrosine residues to be activated by NPFs. Phosphorylation of the Arp2/3 complex is not necessary to bind NPFs or the sides of actin filaments but is critical for binding the pointed end of actin filaments and nucleating actin filaments. Mass spectrometry revealed phosphorylated Thr237 and Thr238 in Arp2, which are evolutionarily conserved residues. In cells, phosphorylation of only the Arp2 subunit increases in response to growth factors, and alanine substitutions of Arp2 T237 and T238 or Y202 inhibits membrane protrusion. These findings reveal an additional level of regulation of actin filament assembly independent of WASP proteins, and show that phosphorylation of the Arp2/3 complex provides a logical “or gate” capable integrating diverse upstream signals

The Nck-interacting kinase NIK increases Arp2/3 complex activity by phosphorylating the Arp2 subunit

The nucleating activity of the Arp2/3 complex promotes the assembly of branched actin filaments that drive plasma membrane protrusion in migrating cells. Arp2/3 complex binding to nucleation-promoting factors of the WASP and WAVE families was previously thought to be sufficient to increase nucleating activity. However, phosphorylation of the Arp2 subunit was recently shown to be necessary for Arp2/3 complex activity. We show in mammary carcinoma cells that mutant Arp2 lacking phosphorylation assembled with endogenous subunits and dominantly suppressed actin filament assembly and membrane protrusion. We also report that Nck-interacting kinase (NIK), a MAP4K4, binds and directly phosphorylates the Arp2 subunit, which increases the nucleating activity of the Arp2/3 complex. In cells, NIK kinase activity was necessary for increased Arp2 phosphorylation and plasma membrane protrusion in response to epidermal growth factor. NIK is the first kinase shown to phosphorylate and increase the activity of the Arp2/3 complex, and our findings suggest that it integrates growth factor regulation of actin filament dynamics

Human β-actin does not support <i>A</i>. <i>fumigatus</i> viability.

<p>(A) Hyphal plugs from the wild type and <i>HsactB</i> heterokaryon strains grown for 48 hrs at 37°C on GMM. Cores of agar containing hyphae taken from the colony periphery of previous cultures where transferred to new GMM agar plates for culture. (B) Hyphae of the <i>HsactB</i> heterokaryon growing on GMM agar plates after 48 hrs at 37°C. Blunted hyphal tips (black arrowheads) that regularly lysed (white arrowheads) were noted. Scale bar = 50 μm (C) Conidia harvested from each cultures represented in Panel A were inoculated onto hygromycin selective agar and cultured for 48 hrs at 37°C. Note the lack of germination of the <i>HsactB</i> heterokaryon strain under selection, indicating inability of human actin to support <i>A</i>. <i>fumigatus</i> viability.</p

F-actin stabilization alters actin dynamics and cell wall construction in <i>A</i>. <i>fumigatus</i>.

<p>Conidia of the WT and <i>Scact1</i> strains were inoculated onto cover slips submerged in GMM and incubated for 24 hr at 37°C. Adherent hyphae were subsequently treated with jasplakinolide (50 μg/ml) for 2 hours at 37°C. Cultures were fixed and immunostained with an anti-actin antibody (red) either alone (A and D) or in combination with Hoechst (blue) to detect nuclear position (B and E). White, block arrows indicate areas of actin structure accumulation. Note normal polarization of the cytoskeleton to the hyphal tip in the WT strain in the presence of jasplakinolide (A and B) and the disorganization of aggregated actin into clumps in the jasplakinolide-treated <i>Scact1</i> strain (D and E). Small white arrowheads denote nuclei (B and E). To detect changes in cell wall deposition, calcofluor white staining was performed on unfixed samples treated with 50 μg/ml jasplakinolide (C and F). White arrowheads denote areas of aberrant cell wall deposition in the jasplakinolide-treated <i>Scact1</i> strain (C and F). Scale bar = 50 μm.</p

Differential Support of <i>Aspergillus fumigatus</i> Morphogenesis by Yeast and Human Actins

<div><p>The actin cytoskeleton is highly conserved among eukaryotes and is essential for cellular processes regulating growth and differentiation. In fungi, filamentous actin (F-actin) orchestrates hyphal tip structure and extension via organization of exocytic and endocytic processes at the hyphal tip. Although highly conserved, there are key differences among actins of fungal species as well as between mammalian and fungal actins. For example, the F-actin stabilizing molecules, phalloidin and jasplakinolide, bind to actin structures in yeast and human cells, whereas phalloidin does not bind actin structures of Aspergillus. These discrepancies suggest structural differences between Aspergillus actin filaments and those of human and yeast cells. Additionally, fungal actin kinetics are much faster than those of humans, displaying 5-fold faster nucleation and 40-fold faster nucleotide exchange rates. Limited published studies suggest that these faster actin kinetics are required for normal growth and morphogenesis of yeast cells. In the current work, we show that replacement of Aspergillus actin with yeast actin generates a morphologically normal strain, suggesting that Aspergillus actin kinetics are similar to those of yeast. In contrast to wild type A. fumigatus, F-actin in this strain binds phalloidin, and pharmacological stabilization of these actin structures with jasplakinolide inhibits germination and alters morphogenesis in a dose-dependent manner. We also show that human β-actin cannot support Aspergillus viability, even though the amino acid sequences of human and Aspergillus actins are 89.3% identical. Our findings show that minor differences in actin protein sequence account for loss of phalloidin and jasplakinolide sensitivity in Aspergillus species.</p></div

Yeast actin supports growth and viability of <i>A</i>. <i>fumigatus</i>.

<p>(A) Western blot analysis of actin protein expression levels in the wild type (WT) and <i>S</i>. <i>cerevisiae ACT1</i> expressing strains (<i>Scact1</i>). Total protein lysates from 24 hr submerged cultures were separated by SDS-PAGE and detected with an anti-actin antibody (~42 kDa band). Coomassie stained total protein is shown as a loading control. (B) Representative cultures comparing growth and colony morphology of the WT and <i>Scact1</i> strains. Conidia were point inoculated onto the center of each GMM agar plate and incubated for up to 5 days at 37°C. (C) Quantification of radial outgrowth (colony diameter) over 120 hrs post inoculation growth. Data represents the average of three experiments ± standard deviation.</p

The <i>Scact1</i> strain is sensitive to the F-actin stabilizing agent, jasplakinolide.

<p>(A) Jasplakinolide treatment inhibits <i>Scact1</i> germination in a dose dependent manner. Conidia from the WT and <i>Scact1</i> strains were cultured in the presence of increasing doses of jasplakinolide and scored for germ tube formation after 12 hrs incubation at 37°C. Data are presented as the average of three experiments ± standard deviation. (B) Jasplakinolide inhibits growth and morphogenesis of the <i>Scact1</i> but not WT strain. Conidia were inoculated into a multi-well plate containing liquid GMM and ascending concentrations of jasplakinolide and incubated for 24 hrs at 37°C. Effects on WT and <i>Scact1</i> growth at 50 μg/ml jasplakinolide are shown. Jasplakinolide treatment was associated with decreased growth, increased branching and swollen hyphal tips (white arrowhead, enlarged panel to right).</p

Actin structures of <i>Scact1</i> stain with rhodamine-phalloidin.

<p>Staining of <i>Scact1</i> hyphae with rhodamine-phalloidin detected actin patches and rings. Actin patches were arranged as a sub-apical actin collar (A), as previously identified in filamentous fungi, and also found positioned along the hyphal cortex (B). Actin rings (C) were detected at sites of newly forming septa. Staining of the WT strain with rhodamine-phalloidin produced no detectable fluorescent signal (data not shown). Scale bar = 50 μm.</p

Phosphorylation of the Arp2 subunit relieves auto-inhibitory interactions for Arp2/3 complex activation.

Actin filament assembly by the actin-related protein (Arp) 2/3 complex is necessary to build many cellular structures, including lamellipodia at the leading edge of motile cells and phagocytic cups, and to move endosomes and intracellular pathogens. The crucial role of the Arp2/3 complex in cellular processes requires precise spatiotemporal regulation of its activity. While binding of nucleation-promoting factors (NPFs) has long been considered essential to Arp2/3 complex activity, we recently showed that phosphorylation of the Arp2 subunit is also necessary for Arp2/3 complex activation. Using molecular dynamics simulations and biochemical assays with recombinant Arp2/3 complex, we now show how phosphorylation of Arp2 induces conformational changes permitting activation. The simulations suggest that phosphorylation causes reorientation of Arp2 relative to Arp3 by destabilizing a network of salt-bridge interactions at the interface of the Arp2, Arp3, and ARPC4 subunits. Simulations also suggest a gain-of-function ARPC4 mutant that we show experimentally to have substantial activity in the absence of NPFs. We propose a model in which a network of auto-inhibitory salt-bridge interactions holds the Arp2 subunit in an inactive orientation. These auto-inhibitory interactions are destabilized upon phosphorylation of Arp2, allowing Arp2 to reorient to an activation-competent state