Regulation of macropinocytosis by p21-activated kinase-1.
Journal - Molecular biology of the cell (UNITED STATES )
The process of macropinocytosis is an essential aspect of normal cell function, contributing to both growth and motile processes of cells. p21-activated kinases (PAKs) are targets for activated Rac and Cdc42 guanosine 5'-triphosphatases and have been shown to regulate the actin-myosin cytoskeleton. In fibroblasts PAK1 localizes to areas of membrane ruffling, as well as to amiloride-sensitive pinocytic vesicles. Expression of a PAK1 kinase autoinhibitory domain blocked both platelet-derived growth factor- and RacQ61L-stimulated uptake of 70-kDa dextran particles, whereas an inactive version of this domain did not, indicating that PAK kinase activity is required for normal growth factor-induced macropinocytosis. The mechanisms by which PAK modulate macropinocytosis were examined in NIH3T3 cell lines expressing various PAK1 constructs under the control of a tetracycline-responsive transactivator. Cells expressing PAK1 (H83,86L), a mutant that dramatically stimulates formation of dorsal membrane ruffles, exhibited increased macropinocytic uptake of 70-kDa dextran particles in the absence of additional stimulation. This effect was not antagonized by coexpression of dominant-negative Rac1-T17N. In the presence of platelet-derived growth factor, both PAK1 (H83,86L) and a highly kinase active PAK1 (T423E) mutant dramatically enhanced the uptake of 70-kDa dextran. Neither wild-type PAK1 nor vector controls exhibited enhanced macropinocytosis, nor did PAK1 (H83,86L) affect clathrin-dependent endocytic mechanisms. Active versions of PAK1 enhanced both growth factor-stimulated 70-kDa dextran uptake and efflux, suggesting that PAK1 activity modulated pinocytic vesicle cycling. These data indicate that PAK1 plays an important regulatory role in the process of macropinocytosis, perhaps related to the requirement for PAK in directed cell motility.
|ISSN : ||1059-1524|
|Mesh Heading : ||3T3 Cells Amino Acid Substitution Animals Biological Transport Dextrans Genetic Vectors Mice Mutagenesis, Site-Directed Pinocytosis Platelet-Derived Growth Factor Protein-Serine-Threonine Kinases Recombinant Proteins Trans-Activators Transfection p21-Activated Kinases rac1 GTP-Binding Protein drug effects pharmacokinetics drug effects pharmacology genetics metabolism metabolism metabolism|
|Mesh Heading Relevant : ||physiology metabolism|
Localization of p21-activated kinase 1 (PAK1) to pseudopodia, membrane ruffles, and phagocytic cups in activated human neutrophils.
Journal - Journal of leukocyte biology (UNITED STATES )
Leukocyte chemoattractants are known to stimulate signaling pathways that involve Rho family GTPases. Direct evidence for the regulation of the leukocyte cytoskeleton by Rho GTPases and their effector targets is limited. The p21-activated kinases (PAKs) are specific targets of activated GTP-bound Rac and Cdc42, and have been proposed as regulators of chemoattractant-driven actin cytoskeletal changes in fibroblasts. PAK1 colocalizes with F-actin to cortical actin structures in stimulated fibroblasts, and activated PAK1 mutants induce membrane ruffling and polarized cytoskeletal rearrangements. We investigated whether PAK1 was associated with remodeling of the actin cytoskeleton in activated human neutrophils. We monitored the redistribution of PAK1 and F-actin into the actin cytoskeleton after stimulation of human neutrophils with the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) or the particulate stimulus, opsonized zymosan (OZ). PAK1 exhibited a similar distribution as F-actin in fMLP-stimulated leukocytes, localizing in membrane ruffles and to lamellipodia at the leading edge of polarized cells. Addition of OZ induced phagocytic uptake of this particulate stimulus, and PAK1 re-localized to the F-actin-rich pseudopodia and phagocytic cups associated with this process. Once the OZ was internalized, there was little PAK1 localized around the ingested particles, suggesting that PAK1 may be regulating the cytoskeletal extensions and events required for engulfment of bacteria, but not the subsequent steps of internalization. Localization of PAK1 and F-actin in cytoskeletal structures was abolished by the actin polymerization inhibitor cytochalasin D and the phosphatidylinositol 3-kinase inhibitor wortmannin. Our data suggest that PAK1 may regulate a subset of cytoskeletal dynamics initiated by chemoattractant and phagocytic stimuli in human neutrophils.
|ISSN : ||0741-5400|
|Mesh Heading : ||1-Phosphatidylinositol 3-Kinase Actins Adult Androstadienes Cell Cycle Proteins Cell Polarity Complement C3b Cytochalasin D Cytoskeleton Enzyme Inhibitors GTP-Binding Proteins Humans Microscopy, Confocal Microscopy, Fluorescence N-Formylmethionine Leucyl-Phenylalanine Neutrophils Opsonin Proteins Protein-Serine-Threonine Kinases Pseudopodia Zymosan cdc42 GTP-Binding Protein p21-Activated Kinases rac GTP-Binding Proteins antagonists & inhibitors physiology ultrastructure pharmacology physiology drug effects physiology pharmacology drug effects ultrastructure pharmacology physiology pharmacology drug effects ultrastructure pharmacology drug effects physiology pharmacology|
|Mesh Heading Relevant : ||Chemotaxis, Leukocyte Phagocytosis chemistry enzymology analysis enzymology|
Rho GTPases and leukocyte cytoskeletal regulation.
Journal - Current opinion in hematology (UNITED STATES )
The Rho GTPases (Rho, Rac, and Cdc42) regulate assembly of the actin cytoskeleton in many cells, including leukocytes. Recent work in identifying the protein targets of these GTPases is providing greater insight into the mechanisms used by cells to control cytoskeletal dynamics for a variety of purposes.
|ISSN : ||1065-6251|
|Mesh Heading : ||Animals Cytoskeleton GTP Phosphohydrolases Humans Leukocytes Membrane Proteins rhoB GTP-Binding Protein ultrastructure|
|Mesh Heading Relevant : ||GTP-Binding Proteins physiology physiology physiology physiology physiology|
Localization of p21-activated kinase 1 (PAK1) to pinocytic vesicles and cortical actin structures in stimulated cells.
Journal - The Journal of cell biology (UNITED STATES )
The mechanisms through which the small GTPases Rac1 and Cdc42 regulate the formation of membrane ruffles, lamellipodia, and filopodia are currently unknown. The p21-activated kinases (PAKs) are direct targets of active Rac and Cdc42 which can induce the assembly of polarized cytoskeletal structures when expressed in fibroblasts, suggesting that they may play a role in mediating the effects of these GTPases on cytoskeletal dynamics. We have examined the subcellular localization of endogenous PAK1 in fibroblast cell lines using specific PAK1 antibodies. PAK1 is detected in submembranous vesicles in both unstimulated and stimulated fibroblasts that colocalize with a marker for fluid-phase uptake. In cells stimulated with PDGF, in v-Src-transformed fibroblasts, and in wounded cells, PAK1 redistributed into dorsal and membrane ruffles and into the edges of lamellipodia, where it colocalizes with polymerized actin. PAK1 was also colocalized with F-actin in membrane ruffles extended as a response to constitutive activation of Rac1. PAK1 appears to precede F-actin in translocating to cytoskeletal structures formed at the cell periphery. The association of PAK1 with the actin cytoskeleton is prevented by the actin filament-disrupting agent cytochalasin D and by the phosphatidylinositol 3-kinase inhibitor wortmannin. Co-immunoprecipitation experiments demonstrate an in vivo interaction of PAK1 with filamentous (F)-actin in stimulated cells. Microinjection of a constitutively active PAK1 mutant into Rat-1 fibroblasts overexpressing the insulin receptor (HIRcB cells) induced the formation of F-actin- and PAK1-containing structures reminiscent of dorsal ruffles. These data indicate a close correlation between the subcellular distribution of endogenous PAK1 and the formation of Rac/Cdc42-dependent cytoskeletal structures and support an active role for PAK1 in regulating cortical actin rearrangements.
|ISSN : ||0021-9525|
|Mesh Heading : ||3T3 Cells Actins Animals Cell Membrane Cell Transformation, Viral Cytoplasmic Granules Cytoskeleton Genes, src Mice Mutation Pinocytosis Platelet-Derived Growth Factor Protein-Serine-Threonine Kinases Pseudopodia p21-Activated Kinases drug effects enzymology ultrastructure chemistry metabolism chemistry chemistry physiology physiology pharmacology genetics metabolism chemistry|
|Mesh Heading Relevant : ||chemistry enzymology physiology analysis|