Stimulation of
growth factor signaling has been implicated in the development of invasive phenotypes and the activation of
p21-activated kinase (Pak1) in human
breast cancer cells (Adam, L., Vadlamudi, R., Kondapaka, S. B., Chernoff, J., Mendelsohn, J., and Kumar, R. (1998) J. Biol. Chem. 273, 28238-28246; Adam, L., Vadlamudi, R., Mandal, M., Chernoff, J., and Kumar, R. (2000) J. Biol. Chem. 275, 12041-12050). To study the role of Pak1 in the regulation of motility and growth of breast epithelial cells, we developed human epithelial MCF-7 clones that overexpressed the
kinase-active T423E Pak1 mutant under an inducible
tetracycline promoter or that stably expressed the
kinase-active H83L,H86L Pak1 mutant, which is deficient in
small GTPase binding sites. The expression of both T423E and H83L,H86L Pak1 mutants in breast epithelial cells was accompanied by increased cell motility without any apparent effect on the growth rate of cells. The T423E Pak1 mutant was primarily localized to filopodia, and the H83L,H86L Pak1 mutant was primarily localized to ruffles. Cells expressing T423E Pak1 exhibited a regulatable stimulation of
mitogen-activated protein kinase and
Jun N-terminal kinase activities. The expression of
kinase-active Pak1 mutants significantly stimulated anchorage-independent growth of cells in soft
agar in a preferential
mitogen-activated protein kinase-sensitive manner. In addition, regulatable expression of
kinase-active Pak1 resulted in an abnormal organization of mitotic spindles characterized by appearance of multiple spindle orientations. We also provide evidence to suggest a close correlation between the status of
Pak1 kinase activity and base-line invasiveness of human
breast cancer cells and
breast tumor grades. This study is the first demonstration of Pak1 regulation of anchorage-independent growth, potential Pak1 regulation of invasiveness, and abnormal organization of mitotic spindles of human epithelial
breast cancer cells.