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The Cdc42 Effector Kinase PAK4 Localizes to Cell-Cell Junctions and Contributes to Establishing Cell Polarity.

Selamat W, Tay PL, Baskaran Y, Manser E - PLoS ONE (2015)

Bottom Line: The serine/threonine kinase PAK4 is a Cdc42 effector whose role is not well understood; overexpression of PAK4 has been associated with some cancers, and there are reports that correlate kinase level with increased cell migration in vitro.In U2OS osteosarcoma and MCF-7 breast cancer cell lines, PAK4 depletion did not affect collective cell migration, but affected cell polarization.We also confirm β-catenin as a target for PAK4 in these cells.

View Article: PubMed Central - PubMed

Affiliation: small G-protein Signaling and Kinases (sGSK) Group, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.

ABSTRACT
The serine/threonine kinase PAK4 is a Cdc42 effector whose role is not well understood; overexpression of PAK4 has been associated with some cancers, and there are reports that correlate kinase level with increased cell migration in vitro. Here we report that PAK4 is primarily associated with cell-cell junctions in all the cell lines we tested, and fails to accumulate at focal adhesions or at the leading edge of migrating cells. In U2OS osteosarcoma and MCF-7 breast cancer cell lines, PAK4 depletion did not affect collective cell migration, but affected cell polarization. By contrast, Cdc42 depletion (as reported by many studies) caused a strong defect in junctional assembly in multiple cells lines. We also report that the depletion of PAK4 protein or treatment of cells with the PAK4 inhibitor PF-3758309 can lead to defects in centrosome reorientation (polarization) after cell monolayer wounding. These experiments are consistent with PAK4 forming part of a conserved cell-cell junctional polarity Cdc42 complex. We also confirm β-catenin as a target for PAK4 in these cells. Treatment of cells with PF-3758309 caused inhibition of β-catenin Ser-675 phosphorylation, which is located predominantly at cell-cell junctions.

No MeSH data available.


Related in: MedlinePlus

PAK4 loss does not affect collective migration rates of U2OS or MCF-7 cells.A) (Left panel) U2OS or MCF-7 cells were transfected with siRNA directed to PAK4 or Cdc42 as indicated. The cell lysates (30 μg per lane) were probed for expression of PAK4, Cdc42 or tubulin. (Right panel) U2OS cells were transfected with siRNA directed to Rac1 as indicated and the lysates were probed for expression of Rac1 or tubulin. B) Low power images of the same area of the monolayer scratch wound are shown before and after 4h cell migration into the gap. The wound-edge is represented in yellow and red corresponding to the start and end of imaging respectively. C) Bar chart depicting the area covered over 4h after the scratch was applied by either the U2OS or MCF-7 cells, with standard error of mean. The area was calculated using ImageJ software. *P value < 0.05, **P value < 0.005. Scale bar: 50μm.
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pone.0129634.g003: PAK4 loss does not affect collective migration rates of U2OS or MCF-7 cells.A) (Left panel) U2OS or MCF-7 cells were transfected with siRNA directed to PAK4 or Cdc42 as indicated. The cell lysates (30 μg per lane) were probed for expression of PAK4, Cdc42 or tubulin. (Right panel) U2OS cells were transfected with siRNA directed to Rac1 as indicated and the lysates were probed for expression of Rac1 or tubulin. B) Low power images of the same area of the monolayer scratch wound are shown before and after 4h cell migration into the gap. The wound-edge is represented in yellow and red corresponding to the start and end of imaging respectively. C) Bar chart depicting the area covered over 4h after the scratch was applied by either the U2OS or MCF-7 cells, with standard error of mean. The area was calculated using ImageJ software. *P value < 0.05, **P value < 0.005. Scale bar: 50μm.

Mentions: We tested the effects of PAK4 knockdown in MCF-7 (adenocarcinoma) or U2OS (osteosarcoma) on collective cell migration. The efficacy of knockdown in both cell types was similar (~90% as determined by band signal intensity analysis using ImageJ (Fig 3A). Typical images of leading edge U2OS cells after a monolayer scratch are illustrated in Fig 3B; the position of the monolayer edge at the start and 4h later are marked in yellow and red respectively. The area covered by the migrated cells over this 4h period determined in different regions was averaged over three independent experiments (Fig 3C). Under these conditions, neither U2OS nor MCF-7 cell migration was significantly affected by PAK4 loss. By contrast, knockdown of either Cdc42 or Rac1 caused a significant reduction in migratory rates. We conclude that the role of PAK4 in collective cell migration is cell type and/or growth factor dependent. The U2OS cells treated with PAK4 siRNA also showed no overall changes in the distribution of F-actin or myosin IIB (S1 Fig).


The Cdc42 Effector Kinase PAK4 Localizes to Cell-Cell Junctions and Contributes to Establishing Cell Polarity.

Selamat W, Tay PL, Baskaran Y, Manser E - PLoS ONE (2015)

PAK4 loss does not affect collective migration rates of U2OS or MCF-7 cells.A) (Left panel) U2OS or MCF-7 cells were transfected with siRNA directed to PAK4 or Cdc42 as indicated. The cell lysates (30 μg per lane) were probed for expression of PAK4, Cdc42 or tubulin. (Right panel) U2OS cells were transfected with siRNA directed to Rac1 as indicated and the lysates were probed for expression of Rac1 or tubulin. B) Low power images of the same area of the monolayer scratch wound are shown before and after 4h cell migration into the gap. The wound-edge is represented in yellow and red corresponding to the start and end of imaging respectively. C) Bar chart depicting the area covered over 4h after the scratch was applied by either the U2OS or MCF-7 cells, with standard error of mean. The area was calculated using ImageJ software. *P value < 0.05, **P value < 0.005. Scale bar: 50μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4466050&req=5

pone.0129634.g003: PAK4 loss does not affect collective migration rates of U2OS or MCF-7 cells.A) (Left panel) U2OS or MCF-7 cells were transfected with siRNA directed to PAK4 or Cdc42 as indicated. The cell lysates (30 μg per lane) were probed for expression of PAK4, Cdc42 or tubulin. (Right panel) U2OS cells were transfected with siRNA directed to Rac1 as indicated and the lysates were probed for expression of Rac1 or tubulin. B) Low power images of the same area of the monolayer scratch wound are shown before and after 4h cell migration into the gap. The wound-edge is represented in yellow and red corresponding to the start and end of imaging respectively. C) Bar chart depicting the area covered over 4h after the scratch was applied by either the U2OS or MCF-7 cells, with standard error of mean. The area was calculated using ImageJ software. *P value < 0.05, **P value < 0.005. Scale bar: 50μm.
Mentions: We tested the effects of PAK4 knockdown in MCF-7 (adenocarcinoma) or U2OS (osteosarcoma) on collective cell migration. The efficacy of knockdown in both cell types was similar (~90% as determined by band signal intensity analysis using ImageJ (Fig 3A). Typical images of leading edge U2OS cells after a monolayer scratch are illustrated in Fig 3B; the position of the monolayer edge at the start and 4h later are marked in yellow and red respectively. The area covered by the migrated cells over this 4h period determined in different regions was averaged over three independent experiments (Fig 3C). Under these conditions, neither U2OS nor MCF-7 cell migration was significantly affected by PAK4 loss. By contrast, knockdown of either Cdc42 or Rac1 caused a significant reduction in migratory rates. We conclude that the role of PAK4 in collective cell migration is cell type and/or growth factor dependent. The U2OS cells treated with PAK4 siRNA also showed no overall changes in the distribution of F-actin or myosin IIB (S1 Fig).

Bottom Line: The serine/threonine kinase PAK4 is a Cdc42 effector whose role is not well understood; overexpression of PAK4 has been associated with some cancers, and there are reports that correlate kinase level with increased cell migration in vitro.In U2OS osteosarcoma and MCF-7 breast cancer cell lines, PAK4 depletion did not affect collective cell migration, but affected cell polarization.We also confirm β-catenin as a target for PAK4 in these cells.

View Article: PubMed Central - PubMed

Affiliation: small G-protein Signaling and Kinases (sGSK) Group, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.

ABSTRACT
The serine/threonine kinase PAK4 is a Cdc42 effector whose role is not well understood; overexpression of PAK4 has been associated with some cancers, and there are reports that correlate kinase level with increased cell migration in vitro. Here we report that PAK4 is primarily associated with cell-cell junctions in all the cell lines we tested, and fails to accumulate at focal adhesions or at the leading edge of migrating cells. In U2OS osteosarcoma and MCF-7 breast cancer cell lines, PAK4 depletion did not affect collective cell migration, but affected cell polarization. By contrast, Cdc42 depletion (as reported by many studies) caused a strong defect in junctional assembly in multiple cells lines. We also report that the depletion of PAK4 protein or treatment of cells with the PAK4 inhibitor PF-3758309 can lead to defects in centrosome reorientation (polarization) after cell monolayer wounding. These experiments are consistent with PAK4 forming part of a conserved cell-cell junctional polarity Cdc42 complex. We also confirm β-catenin as a target for PAK4 in these cells. Treatment of cells with PF-3758309 caused inhibition of β-catenin Ser-675 phosphorylation, which is located predominantly at cell-cell junctions.

No MeSH data available.


Related in: MedlinePlus