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P21-activated kinase 4 (PAK4) is a Cdc42 effector protein thought to regulate cell adhesion disassembly in a kinase-dependent manner. We found that PAK4 expression is significantly higher in high-grade human breast cancer patient samples, whereas depletion of PAK4 modifies cell adhesion dynamics of breast cancer cells. Surprisingly, systematic analysis of PAK4 functionality revealed that PAK4-driven adhesion turnover is neither dependent on Cdc42 binding nor kinase activity. Rather, reduced expression of PAK4 leads to a concomitant loss of RhoU expression. We report that RhoU is targeted for ubiquitination by the Rab40A-Cullin 5 complex and demonstrate that PAK4 protects RhoU from ubiquitination in a kinase-independent manner. Overexpression of RhoU rescues the PAK4 depletion phenotype, whereas loss of RhoU expression reduces cell adhesion turnover and migration. These data support a new kinase-independent mechanism for PAK4 function, where an important role of PAK4 in cellular adhesions is to stabilize RhoU protein levels. Thus, PAK4 and RhoU cooperate to drive adhesion turnover and promote cell migration. [ABSTRACT FROM AUTHOR]

Abstract: P21-activated kinase 4 (PAK4) is a member of the p21-activated kinase (PAK) family. Historically much of the attention has been directed towards founding family member PAK1 but the focus is now shifting towards PAK4. It is a pluripotent serine/threonine kinase traditionally recognised as a downstream effector of the Rho-family GTPases. However, emerging research over the last few years has revealed that this kinase is much more than that. New findings have shed light on the molecular mechanism of PAK4 activation and how this kinase is critical for early development. Moreover, the number of PAK4 substrates and binding partners is rapidly expanding highlighting the increasing amount of cellular functions controlled by PAK4. We propose that PAK4 should be considered a signalling integrator regulating numerous fundamental cellular processes, including actin cytoskeletal dynamics, cell morphology and motility, cell survival, embryonic development, immune defence and oncogenic transformation. This review will outline our current understanding of PAK4 biology. [Copyright &y& Elsevier]

Phagocytosis is the force-dependent complex cellular process by which immune cells engulf particles. Although there has been considerable progress in understanding ligand-receptor-induced actin polymerisation in pushing the membrane around the particle, significantly less is known about how localised contractile activities regulate cup closure in coordination with the actin cytoskeleton. Herein, we show that the unconventional class-I myosin, myosin 1G (Myo1G) is localised at phagocytic cups following Fcc-receptor (FcγR) ligation in macrophages. This progressive recruitment is dependent on the activity of phosphoinositide 3-kinase and is particularly important for engulfment of large particles. Furthermore, point mutations in the conserved pleckstrin homology-like domain of Myo1G abolishes the localisation of the motor protein at phagocytic cups and inhibits engulfment downstream of FccR. Binding of Myo1G to both F-actin and phospholipids might enable cells to transport phospholipids towards the leading edge of cups and to facilitate localised contraction for cup closure. [ABSTRACT FROM AUTHOR]

The adaptor protein Nck has been shown to link receptor ligation to actin-based signalling in a diverse range of cellular events, such as changes in cell morphology and motility. It has also been implicated in phagocytosis. However, its molecular role in controlling actin remodelling associated with phagocytic uptake remains to be clarified. Here, we show that Nck, which is recruited to phagocytic cups, is required for Fcγ receptor (FcγR)- but not complement receptor 3 (CR3)-induced phagocytosis. Nck recruitment in response to FcγR ligation is mediated by the phosphorylation of tyrosine 282 and 298 in the ITAMmotif in the cytoplasmic tail of the receptor. In the absence of FcγR phosphorylation, there is also no recruitment of N-WASP or Cdc42 to phagocytic cups. Nck promotes FcγR-mediated phagocytosis by recruiting N-WASP to phagocytic cups. Efficient phagocytosis, however, only occurs, if the CRIB domain of N-WASP can also interact with Cdc42. Our observations demonstrate that Nck and Cdc42 collaborate to stimulate N-WASP-dependent FcγR-mediated phagocytosis. [ABSTRACT FROM AUTHOR]

Background: Phagocytosis is the fundamental cellular process by which eukaryotic cells bind and engulf particles by their cell membrane. Particle engulfment involves particle recognition by cell-surface receptors, signaling and remodeling of the actin cytoskeleton to guide the membrane around the particle in a zipper-like fashion. Despite the signaling complexity, phagocytosis also depends strongly on biophysical parameters, such as particle shape, and the need for actin-driven force generation remains poorly understood. Results: Here, we propose a novel, three-dimensional and stochastic biophysical model of phagocytosis, and study the engulfment of particles of various sizes and shapes, including spiral and rod-shaped particles reminiscent of bacteria. Highly curved shapes are not taken up, in line with recent experimental results. Furthermore, we surprisingly find that even without actin-driven force generation, engulfment proceeds in a large regime of parameter values, albeit more slowly and with highly variable phagocytic cups. We experimentally confirm these predictions using fibroblasts, transfected with immunoreceptor FcγRIIa for engulfment of immunoglobulin G-opsonized particles. Specifically, we compare the wild-type receptor with a mutant receptor, unable to signal to the actin cytoskeleton. Based on the reconstruction of phagocytic cups from imaging data, we indeed show that cells are able to engulf small particles even without support from biological actin-driven processes. Conclusions: This suggests that biochemical pathways render the evolutionary ancient process of phagocytic highly robust, allowing cells to engulf even very large particles. The particle-shape dependence of phagocytosis makes a systematic investigation of host-pathogen interactions and an efficient design of a vehicle for drug delivery possible. [ABSTRACT FROM AUTHOR]