Human p21-activated kinase (Pak1) regulates actin organization in mammalian cells.
BACKGROUND
The Rho family GTPases Cdc42, Rac1 and RhoA regulate the reorganization of the actin cytoskeleton induced by extracellular signals such as growth factors. In mammalian cells, Cdc42 regulates the formation of filopodia, whereas Rac regulates lamellipodia formation and membrane ruffling, and RhoA regulates the formation of stress fibers. Recently, the serine/threonine protein kinase p65(pak) autophosphorylates, thereby increasing its catalytic activity towards exogenous substrates. This kinase is therefore a candidate effector for the changes in cell shape induced by growth factors.


RESULTS
Here, we report that the microinjection of activated Pak1 protein into quiescent Swiss 3T3 cells induces the rapid formation of polarized filopodia and membrane ruffles. The prolonged overexpression of Pak1 amino-terminal mutants that are unable to bind Cdc42 or Rac1 results in the accumulation of filamentous actin in large, polarized membrane ruffles and the formation of vinculin-containing focal complexes within these structures. This phenotype resembles that seen in motile fibroblasts. The amino-terminal Pak1 mutant displays enhanced binding to the adaptor protein Nck, which contains three Src-homology 3 (SH3) domains. Mutation of a proline residue within a conserved SH3-binding region at the amino terminus of Pak1 interferes with SH3-protein binding and alters the effects of Pak1 on the cytoskeleton.


CONCLUSIONS
These results indicate that Pak1, acting through a protein that contains an SH3 domain, regulates the structure of the actin cytoskeleton in mammalian cells, and may serve as an effector for Cdc42 and/or Rac1 in promoting cell motility.
Sells
M A
MA
Fox Chase Cancer Centre, 7701 Burholme Avenue, Philadelphia, Pennsylvania 19111, USA.
Knaus
U G
UG
Bagrodia
S
S
Ambrose
D M
DM
Bokoch
G M
GM
Chernoff
J
J
D016475:3T3 Cells / Q000187:drug effects / Q000648:ultrastructure; D008841:Actin Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure*; D000199:Actins / Q000378:metabolism* / Q000648:ultrastructure; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D019556:COS Cells; D018797:Cell Cycle Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000648:ultrastructure; D002465:Cell Movement; D003599:Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure; D019204:GTP-Binding Proteins / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D051379:Mice; D008845:Microinjections; D008954:Models, Biological; D015513:Oncogene Proteins / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000494:pharmacology / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016596:Vinculin / Q000378:metabolism; D020846:cdc42 GTP-Binding Protein, Saccharomyces cerevisiae; D054462:p21-Activated Kinases; D020744:rac GTP-Binding Proteins; D018909:src Homology Domains
D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; C078332:Nck protein; D015513:Oncogene Proteins; D011993:Recombinant Fusion Proteins; D016596:Vinculin; D005982:Glutathione Transferase; C515903:PAK1 protein, human; C515905:Pak1 protein, mouse; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D019204:GTP-Binding Proteins; D020846:cdc42 GTP-Binding Protein, Saccharomyces cerevisiae; D020744:rac GTP-Binding Proteins