The current study focuses on the molecular mechanisms responsible for actin assembly on a defined membrane surface: the phagosome. Mature phagosomes were surrounded by filamentous actin in vivo in two different cell types. Fluorescence microscopy was used to study in vitro actin nucleation/polymerization (assembly) on the surface of phagosomes isolated from J774 mouse macrophages. In order to prevent non-specific actin polymerization during the assay, fluorescent G-actin was mixed with thymosin beta4. The cytoplasmic side of phagosomes induced de novo assembly and barbed end growth of actin filaments. This activity varied cyclically with the maturation state of phagosomes, both in vivo and in vitro. Peripheral membrane proteins are crucial components of this actin assembly machinery, and we demonstrate a role for ezrin and/or moesin in this process. We propose that this actin assembly process facilitates phagosome/endosome aggregation prior to membrane fusion.

Particle ingestion by phagocytosis results from sequential rearrangements of the actin cytoskeleton and overlying membrane. To assemble a chronology of molecular events during phagosome formation and to examine the contributions of phosphoinositide 3-kinase (PI 3-kinase) to these dynamics, a method was developed for synchronizing Fc␥ receptor-mediated phagocytosis by murine macrophages. Erythrocytes opsonized with complement component C3bi were bound to macrophages at 37°C, a condition that does not favor particle phagocytosis. Addition of soluble anti-erythrocyte IgG resulted in rapid opsonization of the bound erythrocytes, followed by their immediate internalization via phagocytosis. Cellular content of F-actin, as measured by binding of rhodamine-phalloidin, increased transiently during phagocytosis, and this increase was not diminished by inhibitors of PI 3-kinase. Immunofluorescence localization of myosins in macrophages fixed at various times during phagocytosis indicated that myosins II and IXb were concentrated in early phagosomes, myosin IC increased later, and myosin V appeared after phagosome closure. Other cytoskeletal proteins showed similar variations in the timing of their appearance in phagosomes. The PI 3-kinase inhibitor wortmannin did not change the dynamics of PI 3-kinase or ezrin localization but prevented the loss of PAK1 from phagosomes. These results suggest that PI 3-kinase deactivates PAK1, and that this may be needed for phagosome closure. INTRODUCTIONPhagocytosis occurs by the extension of plasma membrane around an extracellular particle, followed by internalization of the particle into a membrane-bounded intracellular vesicle, the phagosome. In macrophages, different cell surface receptors stimulate different kinds of phagocytic response (Aderem and Underhill, 1999). Macrophage Fc␥ receptors mediate phagocytosis of IgG-coated particles. The complement receptor CR3 binds particles opsonized with C3bi, but requires additional activation with phorbol-12-myristate-13-acetate, fibronectin, or other signals to mediate phagocytosis (Wright and Griffin, 1985).Ligation of Fc receptors initiates an intracellular signaling cascade that impinges ultimately on the actin cytoskeleton (May and Machesky, 2001). Fc␥ receptor-mediated phagocytosis can be considered a morphogenetic process, in which the actin cytoskeleton is reorganized into a cup-shaped, cell surface protrusion that constricts at its outer margin to form an enclosure (Swanson and Baer, 1995). Consistent with such a mechanism, this lab and others have experimentally distinguished two component activities of phagocytosis: pseudopod extension and phagosome closure (Araki et al., 1996;Crowley et al., 1997;Lowry et al., 1998). Pseudopod extension appears to be mediated by localized, oriented, actin polymerization beneath the plasma membrane. The mechanism of phagosome closure is not known, but apparently entails contractile activities (Swanson et al., 1999), as well as membrane insertion and vesicular trafficking (Cox et al., 1999;Bajno et...

The Src homology 2 (SH2) domain-containing protein SH2-B␤ binds to and is a substrate of the growth hormone (GH) and cytokine receptor-associated tyrosine kinase JAK2. SH2-B␤ also binds, via its SH2 domain, to multiple activated growth factor receptor tyrosine kinases. We have previously implicated SH2-B␤ in GH and platelet-derived growth factor regulation of the actin cytoskeleton. We extend these findings by establishing a potentiating effect of SH2-B␤ on GH-dependent cell motility and defining regions of SH2-B␤ required for this potentiation. Time-lapse video microscopy, phagokinetic, and/or wounding assays demonstrate reduced movement of cells overexpressing SH2-B␤ lacking an intact SH2 domain because of a point mutation or a C-terminal truncation. An N-terminal proline-rich domain (amino acids 85-106) of SH2-B␤ is required for inhibition of cellular motility by SH2 domain-deficient mutants. Co-immunoprecipitation experiments indicate that Rac binds to this domain. GH is shown to activate endogenous Rac, and dominant negative mutants of SH2-B␤ are shown to inhibit membrane ruffling induced by constitutively active Rac. These findings suggest that SH2-B␤ is an adapter protein that facilitates actin rearrangement and cellular motility by recruiting Rac and potentially Rac-regulating, Rac effector, or other actinregulating proteins to activated cytokine (e.g. GH) and growth factor receptors.Cell migration is critical for many vital biological functions, including embryonic development, the inflammatory immune response, wound repair, tumor formation and metastasis, and tissue remodeling and growth. The actin cytoskeleton provides both the protrusive and contractile forces required for cell migration via a combination of actin polymerization and depolymerization, actin filament cross-linking, and the interaction of myosin-based motors with actin filaments (1). The complexity of cell motility and the fact that it is regulated by many hormones, cytokines, and growth factors, including growth hormone (GH)

p21-Activated serine-threonine kinase (PAK1) is implicated in breast cancer. We have shown previously that PAK1 is tyrosyl phosphorylated by prolactin (PRL)-activated Janus tyrosine kinase (JAK2). Although a role for both PRL and PAK1 in breast cancer is widely acknowledged, the mechanism remains poorly understood. In the present study, PRL-activated PAK1 stimulates the invasion of TMX2-28 human breast cancer cells through Matrigel. Three-dimensional (3D) collagen IV stimulates the secretion of the matrix proteases, metalloproteinase (MMP)-1 and -3 that is further enhanced by the PRL-dependent tyrosyl phosphorylation of PAK1. 3D collagen IV also stimulates the expression and secretion of MMP-2, but in contrast to MMP-1 and -3, PRL/PAK1 signaling down-regulates MMP-2 expression and secretion. In contrast, MMP-9 expression and secretion are stimulated by 3D collagen I, not collagen IV, and are not affected by PRL but are down-regulated by PAK1. MMP-1 and -3 are required and MMP-2 contributes to PRL-dependent invasion. ERK1/2 signaling appears to be required for the enhanced expression and secretion of MMP-1 and -3 and enhanced PRL-dependent invasion. p38 MAPK and c-Jun N-terminal kinase 1/2 pathways participate in production of MMP-1 and -3 as well as in PRL/PAK1-dependent cell invasion. Together, these data illustrate the complex interaction between the substratum and PRL/PAK1 signaling in human breast cancer cells and suggest a pivotal role for PRL-dependent PAK1 tyrosyl phosphorylation in MMP secretion.

The Src homology-2 (SH2) domain-containing protein SH2-B␤ is a substrate of the growth hormone (GH) receptor-associated tyrosine kinase JAK2. Here we tested whether SH2-B␤ is involved in GH regulation of the actin cytoskeleton. Based on cell fractionation and confocal microscopy, we find SH2-B␤ present at the plasma membrane and in the cytosol. SH2-B␤ colocalized with filamentous actin in GH and platelet-derived growth factor (PDGF)-induced membrane ruffles. To test if SH2-B␤ is required for actin reorganization, we transiently overexpressed wild-type or mutant SH2-B␤ in 3T3-F442A cells and assayed for GH-and PDGF-induced membrane ruffling and fluid phase pinocytosis. Overexpression of wild-type SH2-B␤ enhanced ruffling and pinocytosis produced by submaximal GH but not submaximal PDGF. Point mutant SH2-B␤ (R555E) and truncation mutant ⌬C555, both lacking a functional SH2 domain, inhibited membrane ruffling and pinocytosis induced by GH and PDGF. Mutant ⌬N504, which possesses a functional SH2 domain and enhances JAK2 kinase activity in overexpression systems, also inhibited GH-stimulated membrane ruffling. ⌬N504 failed to inhibit GH-induced nuclear localization of Stat5B, indicating JAK2 is active in these cells. Taken together, these results show that SH2-B␤ is required for GH-induced actin reorganization by a mechanism discrete from the action of SH2-B␤ as a stimulator of JAK2 kinase activity.