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Temporal and spatial distribution of activated Pak1 in fibroblasts.

Sells MA, Pfaff A, Chernoff J - J. Cell Biol. (2000)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.

ABSTRACT
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.

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Colocalization of activated Pak1 and paxillin at the leading edge of motile fibroblasts. A monolayer of wild-type Pak1 expressing NIH-3T3 S2-6 cells were wounded as in the legend to Fig. 6. The leading edge of the wounded monolayer is depicted. Cells were stained with (A) antiphospho-Pak1 (red); (B) antipaxillin (blue); and (C) phalloidin (green). A merged image (D) is also shown. Bar, 25 μm.
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Figure 7: Colocalization of activated Pak1 and paxillin at the leading edge of motile fibroblasts. A monolayer of wild-type Pak1 expressing NIH-3T3 S2-6 cells were wounded as in the legend to Fig. 6. The leading edge of the wounded monolayer is depicted. Cells were stained with (A) antiphospho-Pak1 (red); (B) antipaxillin (blue); and (C) phalloidin (green). A merged image (D) is also shown. Bar, 25 μm.

Mentions: In cells closest to the wound edge, activated Pak1 accumulates in leading edge lamellipodia that extend into the wounded region (Fig. 7 A). A substantial portion of the activated fraction of Pak1 also colocalizes with paxillin, a marker of focal adhesions (Fig. 7B and Fig. D). Thus, in a wounded monolayer model, Pak1 is activated in cells flanking the wound edge and accumulates in actin-based structures that extend into the wounded region.


Temporal and spatial distribution of activated Pak1 in fibroblasts.

Sells MA, Pfaff A, Chernoff J - J. Cell Biol. (2000)

Colocalization of activated Pak1 and paxillin at the leading edge of motile fibroblasts. A monolayer of wild-type Pak1 expressing NIH-3T3 S2-6 cells were wounded as in the legend to Fig. 6. The leading edge of the wounded monolayer is depicted. Cells were stained with (A) antiphospho-Pak1 (red); (B) antipaxillin (blue); and (C) phalloidin (green). A merged image (D) is also shown. Bar, 25 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2150672&req=5

Figure 7: Colocalization of activated Pak1 and paxillin at the leading edge of motile fibroblasts. A monolayer of wild-type Pak1 expressing NIH-3T3 S2-6 cells were wounded as in the legend to Fig. 6. The leading edge of the wounded monolayer is depicted. Cells were stained with (A) antiphospho-Pak1 (red); (B) antipaxillin (blue); and (C) phalloidin (green). A merged image (D) is also shown. Bar, 25 μm.
Mentions: In cells closest to the wound edge, activated Pak1 accumulates in leading edge lamellipodia that extend into the wounded region (Fig. 7 A). A substantial portion of the activated fraction of Pak1 also colocalizes with paxillin, a marker of focal adhesions (Fig. 7B and Fig. D). Thus, in a wounded monolayer model, Pak1 is activated in cells flanking the wound edge and accumulates in actin-based structures that extend into the wounded region.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus