A Role for P21-Activated Kinase in Endothelial Cell Migration

Abstract

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 in-creased stress fibers and focal adhesions. The DN effect mapped to the most NH2-terminal proline-rich SH3-binding sequence. Observation of a green fluorescent protein-tagged α-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.

Activation of Cdc42, Rac, PAK, and Rho-Kinase in Response to Hepatocyte Growth Factor Differentially Regulates Epithelial Cell Colony Spreading and Dissociation

“…In agreement with our data, Kiosses et al (1999) demonstrated that the amino-terminal proline-rich domain of Pak, and not the Rac/Cdc42 interaction domain, was required for the inhibition of cell migration by a dominant negative Pak protein.…”

Polarization and Movement of Keratocytes: A Multiscale Modelling Approach

“…Experimental observations further increase the relevance of such questions: Fashena et al (2002) showed that keratocyte-like movements occur in MCF7 breast adenocarcinoma cells overexpressing HEF1 (a cas-family protein), with the formation of a large leading lamellipod, enhanced ruffling and pronounced trailing edge. Similar observations were made in human microvascular endothelial cell line, HMEC-1 (Kiosses et al, 1999;Fischer et al, 2003). More surprisingly, Asano et al (2004) reported that the deletion of a single gene, amiB, from Dictyostelium cells, transforms this typically amoeboid cell to a crescent-shaped keratocyte-like cell with persistent movement and higher velocity.…”

Filamin B Plays a Key Role in Vascular Endothelial Growth Factor-induced Endothelial Cell Motility through Its Interaction with Rac-1 and Vav-2

“…These defects could be explained by an alteration of Vav-2-Rac-1-PAK signaling. In endothelial cells, PAK activity is necessary for migration, and it is up-regulated by the formation of new adhesions; in accordance with this, overexpression of a PAK dominant negative form inhibits cell migration (45). However, transfection of a constitutively active PAK mutant was also detrimental for migration because PAK inactivation is required for detachment and tail retraction.…”