Temporal and spatial distribution of activated Pak1 in fibroblasts.
p21-activated kinases (Paks) are effectors of the small GTPases Cdc42 and Rac, and are thought to mediate some of the cytoskeletal and transcriptional activities of these proteins. To localize activated Pak1 in cells, we developed an antibody directed against a phosphopeptide that is contained within the activation loop of Pak1. This antibody specifically recognizes the activated form of Pak1. Immunofluorescence analysis of NIH-3T3 cells coexpressing activated Cdc42 or Rac1 plus wild-type Pak1 shows that activated Pak1 accumulates at sites of focal adhesion, throughout filopodia and within the body and edges of lamellipodia. Platelet-derived growth factor stimulation of NIH-3T3 cells shows a pattern of Pak1 activation similar to that observed with Rac1. During closure of a fibroblast monolayer wound, Pak1 is rapidly activated and localizes to the leading edge of motile cells, then gradually tapers off as the wound closes. The activation of Pak1 by wounding is blocked by inhibitors of phosphatidylinositol 3-kinase, and Src family kinases, but not by an inhibitor of the epidermal growth factor receptor. These findings indicate that activated Pak1, and by extension, probably activated Cdc42 or Rac, accumulates at sites of cortical actin remodeling in motile fibroblasts.
Sells
M A
MA
Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
Pfaff
A
A
Chernoff
J
J
D016475:3T3 Cells; D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D002457:Cell Extracts / Q000276:immunology; D002465:Cell Movement; D004789:Enzyme Activation / Q000187:drug effects; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000502:physiology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D022001:Focal Adhesions / Q000187:drug effects / Q000378:metabolism; D007106:Immune Sera / Q000096:biosynthesis / Q000276:immunology; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011554:Pseudopodia / Q000187:drug effects / Q000378:metabolism; D016415:Sequence Alignment; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D014945:Wound Healing; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism; D054462:p21-Activated Kinases; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
D000199:Actins; D002457:Cell Extracts; D066246:ErbB Receptors; D007106:Immune Sera; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010982:Platelet-Derived Growth Factor; D017346:Protein Serine-Threonine Kinases; D020764:cdc42 GTP-Binding Protein; D054462:p21-Activated Kinases; D020830:rac1 GTP-Binding Protein; C515905:Pak1 protein, mouse