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.

Protrusion of the lamellipod in the crawling sperm of Ascaris is tightly coupled to the localized vectorial assembly and bundling of the major sperm protein cytoskeleton. In cell-free extracts of sperm, vesicles derived from the leading edge membrane reconstitute protrusion by directing the assembly of columnar meshworks of major sperm protein filaments that push the vesicle forward as they elongate. Treatment with proteases or a tyrosine phosphatase abolished vesicle activity, suggesting the involvement of a membrane phosphoprotein. Fractionation of vesicle proteins by sequential detergent lysis, size exclusion chromatography and immunoprecipitation with antiphosphotyrosine antibody identified a 48 kDa integral membrane phosphoprotein as the only sperm membrane component required to nucleate major sperm protein polymerization under physiological conditions. Immunolabeling assays showed that this protein is distributed uniformly in the sperm plasma membrane, but that its active phosphorylated form is located only at sites of major sperm protein polymerization at the leading edge. Because this protein specifies sites of cytoskeletal assembly, we have named it major sperm protein polymerization organizing protein (MPOP). The phosphorylation of MPOP is pH sensitive and appears to require a soluble tyrosine kinase. Comparison of the activity of MPOP to that of analogous membrane proteins in actin-based systems emphasizes the importance of precise transmission of information from the membrane to the cytoskeleton in amoeboid cell motility.

Legionella pneumophila, the etiological agent of legionellosis, replicates within phagocytic cells. Crucial to biogenesis of the replicative vacuole is the Dot/Icm type 4 secretion system, which translocates a large number of effectors into the host cell cytosol. Among them is LegK2, a protein kinase that plays a key role in Legionella infection. Here, we identified the actin nucleator ARP2/3 complex as a target of LegK2. LegK2 phosphorylates the ARPC1B and ARP3 subunits of the ARP2/3 complex. LegK2-dependent ARP2/3 phosphorylation triggers global actin cytoskeleton remodeling in cells, and it impairs actin tail formation by Listeria monocytogenes, a well-known ARP2/3-dependent process. During infection, LegK2 is addressed to the Legionella-containing vacuole surface and inhibits actin polymerization on the phagosome, as revealed by legK2 gene inactivation. Consequently, LegK2 prevents late endosome/lysosome association with the phagosome and finally contributes to remodeling of the bacterium-containing phagosome into a replicative niche. The inhibition of actin polymerization by LegK2 and its effect on endosome trafficking are ARP2/3 dependent since it can be phenocopied by a specific chemical inhibitor of the ARP2/3 complex. Thus, LegK2-ARP2/3 interplay highlights an original mechanism of bacterial virulence with an unexpected role in local actin remodeling that allows bacteria to control vesicle trafficking in order to escape host defenses.

Arp2 phosphorylation by the kinase NIK increases the actin-nucleating activity of the Arp2/3 complex and is necessary for plasma membrane protrusion in response to EGF.