The serine/threonine p21-activated kinase (PAK) is an effector for Rac and
Cdc42, but its role in regulating cytoskeletal organization has been contro
versial,To address this issue, we investigated the role of PAI( in migratio
n of microvascular endothelial cells. We found that a dominant negative (DN
) mutant of PAK significantly inhibited cell migration and increased stress
fibers and focal adhesions, The DN effect mapped to the most NH2-terminal
proline-rich SH3-binding sequence. Observation of a green fluorescent prote
in-tagged alpha-actinin construct in living cells revealed that the DN cons
truct had no effect on membrane ruffling, but dramatically inhibited stress
fiber and focal contact motility and turnover. Constitutively active PAK i
nhibited migration equally well and also increased stress fibers and focal
adhesions, but had a somewhat weaker effect on their dynamics. In contrast
to their similar effects on motility, DN PAK decreased cell contractility,
whereas active PAK increased contractility. Active PAK also increased myosi
n light chain (MLC) phosphorylation, as indicated by staining with an antib
ody to phosphorylated MLC, whereas DN PAK had little effect, despite the in
crease in actin stress fibers. These results demonstrate that although PAK
is not required for extension of lamellipodia, it has substantial effects o
n cell adhesion and contraction. These data suggest a model in which PAK pl
ays a role coordinating the formation of new adhesions at the leading edge
with contraction and detachment at the trailing edge.