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J. Cell Biol.,
Volume 145, Number 4, May 17, 1999 837-849
Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111
The p21 (Cdc42/Rac) activated kinase Pak1
regulates cell morphology and polarity in most, if not
all, eukaryotic cells. We and others have established
that Pak's effects on these parameters are mediated by
changes in the organization of cortical actin. Because
cell motility requires polarized rearrangements of the actin/myosin cytoskeleton, we examined the role of
Pak1 in regulating cell movement. We established
clonal tetracycline-regulated NIH-3T3 cell lines that inducibly express either wild-type Pak1, a kinase-dead, or
constitutively-active forms of this enzyme, and examined the morphology, F-actin organization, and motility
of these cells. Expression of any of these forms of Pak1
induced dramatic changes in actin organization which
were not inhibited by coexpression of a dominant-negative form of Rac1. Cells inducibly expressing wild-type
or constitutively-active Pak1 had large, polarized lamellipodia at the leading edge, were more motile than their
normal counterparts when plated on a fibronectin-coated surface, and displayed enhanced directional
movement in response to an immobilized collagen gradient. In contrast, cells expressing a kinase-dead form
of Pak1 projected multiple lamellipodia emerging from
different parts of the cell simultaneously. These cells,
though highly motile, displayed reduced persistence of
movement when plated on a fibronectin-coated surface and had defects in directed motility toward immobilized collagen. Expression of constitutively activated
Pak1 was accompanied by increased myosin light chain
(MLC) phosphorylation, whereas expression of kinase-dead Pak1 had no effect on MLC. These results suggest that Pak1 affects the phosphorylation state of MLC,
thus linking this kinase to a molecule that directly affects cell movement.
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