Regulation of macropinocytosis by p21-activated kinase-1

The process of macropinocytosis is an essential aspect of normal cell funct ion, contributing to both growth and motile processes of cells, p21-activat ed kinases (PAKs) are targets for activated Pac and Cdc42 guanosine 5'-trip hosphatases and have been shown to regulate the actin-myosin cytoskeleton. Ln fibroblasts PAK1 localizes to areas of membrane ruffling, as well as to amiloride-sensitive pinocytic vesicles. Expression of a PAK1 kinase autoinh ibitory domain blocked both platelet-derived growth factor- and RacQ61L-sti mulated uptake of 70-kDa dextran particles, whereas an inactive version of this domain did not, indicating that PAK kinase activity is required for no rmal growth factor-induced macropinocytosis. The mechanisms by which PAK mo dulate macropinocytosis were examined in NIH3T3 cell lines expressing vario us PAK1 constructs under the control of a tetracycline-responsive transacti vator. Cells expressing PAK1 (H83,86L), a mutant that dramatically stimulat es formation of dorsal membrane ruffles, exhibited increased macropinocytic uptake of 70-kDa dextran particles in the absence of additional stimulatio n. This effect was not antagonized by coexpression of dominant-negative Rac 1-T17N. In the presence of platelet-derived growth factor, both PAK1 (H83,8 6L) and a highly kinase active PAK1 (T423E) mutant dramatically enhanced th e uptake of 70-kDa dextran. Neither wild-type PAK1 nor vector controls exhi bited enhanced macropinocytosis, nor did PAK1 (H83,86L) affect clathrin-dep endent endocytic mechanisms. Active versions of PAK1 enhanced both growth f actor-stimulated 70-kDa dextran uptake and efflux, suggesting that PAK1 act ivity modulated pinocytic vesicle cycling. These data indicate that PAK1 pl ays an important regulatory role in the process of macropinocytosis, perhap s related to the requirement for PAK in directed cell motility.