A role for p21-activated kinase in endothelial cell migration.
The serine/threonine p21-activated kinase (PAK) is an effector for Rac and Cdc42, but its role in regulating cytoskeletal organization has been controversial. To address this issue, we investigated the role of PAK in migration 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 NH(2)-terminal proline-rich SH3-binding sequence. Observation of a green fluorescent protein-tagged alpha-actinin construct in living cells revealed that the DN construct had no effect on membrane ruffling, but dramatically inhibited stress fiber and focal contact motility and turnover. Constitutively active PAK inhibited 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 myosin light chain (MLC) phosphorylation, as indicated by staining with an antibody to phosphorylated MLC, whereas DN PAK had little effect, despite the increase in actin stress fibers. These results demonstrate that although PAK is not required for extension of lamellipodia, it has substantial effects on cell adhesion and contraction. These data suggest a model in which PAK plays a role coordinating the formation of new adhesions at the leading edge with contraction and detachment at the trailing edge.
Kiosses
W B
WB
Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Daniels
R H
RH
Otey
C
C
Bokoch
G M
GM
Schwartz
M A
MA
D000185:Actinin / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002448:Cell Adhesion; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology*; D004730:Endothelium, Vascular / Q000502:physiology*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008833:Microcirculation; D018715:Microscopy, Video; D008954:Models, Biological; D018994:Myosin Light Chains / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D016596:Vinculin / Q000378:metabolism; D054462:p21-Activated Kinases; D018909:src Homology Domains
D008164:Luminescent Proteins; D018994:Myosin Light Chains; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000185:Actinin; D016596:Vinculin; D049452:Green Fluorescent Proteins; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases