Regulation of Macropinocytosis by p21-activated Kinase-1

Abstract

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 5Ј-triphosphatases and have been shown to regulate the actin-myosin cytoskeleton. In fibroblasts PAK1 localizes to areas of membrane ruffling, as well as to amiloridesensitive 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 plateletderived 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. Introductionp21-activated kinases (PAKs 1, 2, 3) are a group of 62-68-kDa serine/threonine kinases originally identified as targets of the small guanosine 5Ј-triphosphatases (GTPases) Rac and Cdc42 (Manser et al., 1994;Knaus and Bokoch, 1998). GTP-bound forms of Rac and Cdc42 stimulate the activity of PAKs by binding to a specific N-terminal p21-binding site (Manser et al., 1994;Burbelo et al., 1995;, thereby relieving the influence of an adjacent kinase autoinhibitory domain (Zhao et al., 1998;Zenke et al., 1999). PAKs have been implicated as effectors in several Racand Cdc42-regulated signaling pathways, including regulation of the phagocyte NADPH oxidase , p38 and C-jun N-terminal kinase activation (Zhang et al., 1995;Bagrodia et al., 1995;Tang et al., 1997), and modulation of the actin cytoskeleton (Dharmawardhane et al., 1997;Manser et al., 1997;Daniels et al., 1998).Introduction of a constitutively active PAK1 (H83,86L) N-terminal mutant into a variety of adherent cells induces the formation of cortical actin structures similar to those known to be regulated by Rac and Cdc42, including membrane ruffles and lamellipodia, filopodia, and focal complexes (Dharmawardhane et al., 1997;M...

Involvement of a Rac1-Dependent Macropinocytosis Pathway in Plasmid DNA Delivery by Electrotransfection

“…Similar results have also been observed in previous studies. 67,96 Therefore, data in our study could only lead to a conclusion that Rac1 was required for electroporation-induced macropinocytosis. Future studies should be directed to address the issue on how Rac1 activation mediates macropinocytosis.…”

“…62 It has also been reported that two of the Rho family GTPases, cell division control protein 42 (Cdc42) and Rac1, can regulate cell motility, [63][64][65] phagocytosis, 66 and macropinocytosis. 67 Therefore, we hypothesized that electrotransfection could lead to actin remodeling and enhance macropinocytosis through activation of Cdc42 and Rac1. To test this hypothesis, we performed experiments with two pharmacological inhibitors, ML141 68 and EHT1864, 69 that targeted Cdc42 and Rac1, respectively.…”

EMT, the cytoskeleton, and cancer cell invasion

“…DCRs are enriched with actin assembly proteins, such as Arp2/3, WASP and cortactin, suggesting that DCR formation requires actin assembly. As mediators of growth factor signaling, RhoGTPases, like Rac1 or Cdc42, are required for DCRs formation as well [155]. Other reports indicate the involvement of protein kinase A (PKA) and the cytoplasmic tyrosine kinase Abl in DCR formation [156].…”

Regulation of endocytic traffic by Rho GTPases

“…Macropinocytosis bears comparison with Type I phagocytosis -it is stimulated by type I PtdIns4P 5-kinases and also involves Rac-dependent recruitment of the PAK1 kinase [39]. Importantly, PAK1 activation is both necessary and sufficient for macropinocytosis [40], although determining the mechanistic basis of PAK function is still a key research goal.…”