Limits...
p21-Activated kinase (PAK) regulates cytoskeletal reorganization and directional migration in human neutrophils.

Itakura A, Aslan JE, Kusanto BT, Phillips KG, Porter JE, Newton PK, Nan X, Insall RH, Chernoff J, McCarty OJ - PLoS ONE (2013)

Bottom Line: In this study, we characterized the role of p21-activated kinase (PAK) downstream of Rho GTPases in cytoskeletal remodeling and chemotactic processes of human neutrophils.We found that PAK activation occurred upon stimulation of neutrophils with f-Met-Leu-Phe (fMLP), and PAK accumulated at the actin-rich leading edge of stimulated neutrophils, suggesting a role for PAK in Rac-dependent actin remodeling.Treatment with the pharmacological PAK inhibitor, PF3758309, abrogated the integrity of RhoA-mediated actomyosin contractility and surface adhesion.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Oregon Health and Science University, Portland, Oregon, United States of America.

ABSTRACT
Neutrophils serve as a first line of defense in innate immunity owing in part to their ability to rapidly migrate towards chemotactic factors derived from invading pathogens. As a migratory function, neutrophil chemotaxis is regulated by the Rho family of small GTPases. However, the mechanisms by which Rho GTPases orchestrate cytoskeletal dynamics in migrating neutrophils remain ill-defined. In this study, we characterized the role of p21-activated kinase (PAK) downstream of Rho GTPases in cytoskeletal remodeling and chemotactic processes of human neutrophils. We found that PAK activation occurred upon stimulation of neutrophils with f-Met-Leu-Phe (fMLP), and PAK accumulated at the actin-rich leading edge of stimulated neutrophils, suggesting a role for PAK in Rac-dependent actin remodeling. Treatment with the pharmacological PAK inhibitor, PF3758309, abrogated the integrity of RhoA-mediated actomyosin contractility and surface adhesion. Moreover, inhibition of PAK activity impaired neutrophil morphological polarization and directional migration under a gradient of fMLP, and was associated with dysregulated Ca(2+) signaling. These results suggest that PAK serves as an important effector of Rho-family GTPases in neutrophil cytoskeletal reorganization, and plays a key role in driving efficient directional migration of human neutrophils.

Show MeSH

Related in: MedlinePlus

PAK2 localizes to the leading edge of activated neutrophils.Replicate samples of total neutrophil (PMN) cell lysates (50 µg per lane) were analyzed for (A) the expression of PAK1 (68 kD), PAK2 (61 kD), PAK3 (65 kD) and PAK4 (72 kD) or (D) phosphorylation of PAK1/2 Thr432/402 at indicated time points after the addition of fMLP (10 nM) by Western blot. Human neutrophils adherent on fibronectin surfaces were treated in the presence or absence of fMLP (10 nM) for 3 min and stained for (B) PAK1, PAK2 or PAK4 (green) and F-actin (red), (E) phospho-PAK2 Ser20 (green) and Rac1-GTP (red), (F) phospho-PAK2 Ser20 (green) and Cdc42-GTP (red), or (G) phospho-PAK1/2 Thr423/402 (green) and F-actin (red). In selected experiments, neutrophils were pretreated with vehicle (0.1% DMSO), EHT1864 (Rac1/2 inhibitor, 50 µM) or wortmannin (PI3K inhibitor, 100 nM). Results are quantified from at least 60 cells and presented as the mean percentage±SEM of (C) neutrophils displaying PAK immunofluorescence at actin-rich leading edge, or (H) neutrophils displaying phospho-PAK1/2 immunofluorescence in the fields of view. Representative images obtained from 3 independent experiments are shown. * P<0.05, compared to the basal level. Scale ba = 10 µm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3760889&req=5

pone-0073063-g001: PAK2 localizes to the leading edge of activated neutrophils.Replicate samples of total neutrophil (PMN) cell lysates (50 µg per lane) were analyzed for (A) the expression of PAK1 (68 kD), PAK2 (61 kD), PAK3 (65 kD) and PAK4 (72 kD) or (D) phosphorylation of PAK1/2 Thr432/402 at indicated time points after the addition of fMLP (10 nM) by Western blot. Human neutrophils adherent on fibronectin surfaces were treated in the presence or absence of fMLP (10 nM) for 3 min and stained for (B) PAK1, PAK2 or PAK4 (green) and F-actin (red), (E) phospho-PAK2 Ser20 (green) and Rac1-GTP (red), (F) phospho-PAK2 Ser20 (green) and Cdc42-GTP (red), or (G) phospho-PAK1/2 Thr423/402 (green) and F-actin (red). In selected experiments, neutrophils were pretreated with vehicle (0.1% DMSO), EHT1864 (Rac1/2 inhibitor, 50 µM) or wortmannin (PI3K inhibitor, 100 nM). Results are quantified from at least 60 cells and presented as the mean percentage±SEM of (C) neutrophils displaying PAK immunofluorescence at actin-rich leading edge, or (H) neutrophils displaying phospho-PAK1/2 immunofluorescence in the fields of view. Representative images obtained from 3 independent experiments are shown. * P<0.05, compared to the basal level. Scale ba = 10 µm.

Mentions: The PAK isoforms described in humans (PAK1–6) display a wide range of tissue distribution. PAK2 and PAK4 are ubiquitously expressed, while PAK1, PAK3, PAK5 and PAK6 expression are specific for tissues such as brain (PAK1, PAK3, PAK5 and PAK6) and spleen (PAK1) [20], [21]. Previous studies of the PAK pathways in neutrophils have relied on biochemical assays to monitor PAK1 and PAK2 activation [4], [5]. However, little is known about the expression and activity of PAK isoforms in regulating neutrophil cytoskeletal reorganization and migration. To characterize the expression of PAK isoforms in human neutrophils, whole human neutrophil lysates were separated by SDS-PAGE and examined for PAK expression by Western blot using PAK isoform-specific antibodies. As shown in Fig. 1A, neutrophils express detectable levels of PAK1, PAK2 and PAK4. Next, to examine PAK isoform expression and subcellular localization, PAKs were studied by immunofluorescence microscopy of fixed neutrophils. PAK1, PAK2 and PAK4 were all detected in the cytosol of unstimulated neutrophils (Fig. 1B). Upon the stimulation with the bacteria-derived peptide fMLP, PAK2 colocalized with the actin-rich leading edge of neutrophils, whereas PAK1 and PAK4 localization was excluded from leading edge and primarily found in the cytosol and/or the back of stimulated cells (Fig. 1B and 1C).


p21-Activated kinase (PAK) regulates cytoskeletal reorganization and directional migration in human neutrophils.

Itakura A, Aslan JE, Kusanto BT, Phillips KG, Porter JE, Newton PK, Nan X, Insall RH, Chernoff J, McCarty OJ - PLoS ONE (2013)

PAK2 localizes to the leading edge of activated neutrophils.Replicate samples of total neutrophil (PMN) cell lysates (50 µg per lane) were analyzed for (A) the expression of PAK1 (68 kD), PAK2 (61 kD), PAK3 (65 kD) and PAK4 (72 kD) or (D) phosphorylation of PAK1/2 Thr432/402 at indicated time points after the addition of fMLP (10 nM) by Western blot. Human neutrophils adherent on fibronectin surfaces were treated in the presence or absence of fMLP (10 nM) for 3 min and stained for (B) PAK1, PAK2 or PAK4 (green) and F-actin (red), (E) phospho-PAK2 Ser20 (green) and Rac1-GTP (red), (F) phospho-PAK2 Ser20 (green) and Cdc42-GTP (red), or (G) phospho-PAK1/2 Thr423/402 (green) and F-actin (red). In selected experiments, neutrophils were pretreated with vehicle (0.1% DMSO), EHT1864 (Rac1/2 inhibitor, 50 µM) or wortmannin (PI3K inhibitor, 100 nM). Results are quantified from at least 60 cells and presented as the mean percentage±SEM of (C) neutrophils displaying PAK immunofluorescence at actin-rich leading edge, or (H) neutrophils displaying phospho-PAK1/2 immunofluorescence in the fields of view. Representative images obtained from 3 independent experiments are shown. * P<0.05, compared to the basal level. Scale ba = 10 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3760889&req=5

pone-0073063-g001: PAK2 localizes to the leading edge of activated neutrophils.Replicate samples of total neutrophil (PMN) cell lysates (50 µg per lane) were analyzed for (A) the expression of PAK1 (68 kD), PAK2 (61 kD), PAK3 (65 kD) and PAK4 (72 kD) or (D) phosphorylation of PAK1/2 Thr432/402 at indicated time points after the addition of fMLP (10 nM) by Western blot. Human neutrophils adherent on fibronectin surfaces were treated in the presence or absence of fMLP (10 nM) for 3 min and stained for (B) PAK1, PAK2 or PAK4 (green) and F-actin (red), (E) phospho-PAK2 Ser20 (green) and Rac1-GTP (red), (F) phospho-PAK2 Ser20 (green) and Cdc42-GTP (red), or (G) phospho-PAK1/2 Thr423/402 (green) and F-actin (red). In selected experiments, neutrophils were pretreated with vehicle (0.1% DMSO), EHT1864 (Rac1/2 inhibitor, 50 µM) or wortmannin (PI3K inhibitor, 100 nM). Results are quantified from at least 60 cells and presented as the mean percentage±SEM of (C) neutrophils displaying PAK immunofluorescence at actin-rich leading edge, or (H) neutrophils displaying phospho-PAK1/2 immunofluorescence in the fields of view. Representative images obtained from 3 independent experiments are shown. * P<0.05, compared to the basal level. Scale ba = 10 µm.
Mentions: The PAK isoforms described in humans (PAK1–6) display a wide range of tissue distribution. PAK2 and PAK4 are ubiquitously expressed, while PAK1, PAK3, PAK5 and PAK6 expression are specific for tissues such as brain (PAK1, PAK3, PAK5 and PAK6) and spleen (PAK1) [20], [21]. Previous studies of the PAK pathways in neutrophils have relied on biochemical assays to monitor PAK1 and PAK2 activation [4], [5]. However, little is known about the expression and activity of PAK isoforms in regulating neutrophil cytoskeletal reorganization and migration. To characterize the expression of PAK isoforms in human neutrophils, whole human neutrophil lysates were separated by SDS-PAGE and examined for PAK expression by Western blot using PAK isoform-specific antibodies. As shown in Fig. 1A, neutrophils express detectable levels of PAK1, PAK2 and PAK4. Next, to examine PAK isoform expression and subcellular localization, PAKs were studied by immunofluorescence microscopy of fixed neutrophils. PAK1, PAK2 and PAK4 were all detected in the cytosol of unstimulated neutrophils (Fig. 1B). Upon the stimulation with the bacteria-derived peptide fMLP, PAK2 colocalized with the actin-rich leading edge of neutrophils, whereas PAK1 and PAK4 localization was excluded from leading edge and primarily found in the cytosol and/or the back of stimulated cells (Fig. 1B and 1C).

Bottom Line: In this study, we characterized the role of p21-activated kinase (PAK) downstream of Rho GTPases in cytoskeletal remodeling and chemotactic processes of human neutrophils.We found that PAK activation occurred upon stimulation of neutrophils with f-Met-Leu-Phe (fMLP), and PAK accumulated at the actin-rich leading edge of stimulated neutrophils, suggesting a role for PAK in Rac-dependent actin remodeling.Treatment with the pharmacological PAK inhibitor, PF3758309, abrogated the integrity of RhoA-mediated actomyosin contractility and surface adhesion.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Oregon Health and Science University, Portland, Oregon, United States of America.

ABSTRACT
Neutrophils serve as a first line of defense in innate immunity owing in part to their ability to rapidly migrate towards chemotactic factors derived from invading pathogens. As a migratory function, neutrophil chemotaxis is regulated by the Rho family of small GTPases. However, the mechanisms by which Rho GTPases orchestrate cytoskeletal dynamics in migrating neutrophils remain ill-defined. In this study, we characterized the role of p21-activated kinase (PAK) downstream of Rho GTPases in cytoskeletal remodeling and chemotactic processes of human neutrophils. We found that PAK activation occurred upon stimulation of neutrophils with f-Met-Leu-Phe (fMLP), and PAK accumulated at the actin-rich leading edge of stimulated neutrophils, suggesting a role for PAK in Rac-dependent actin remodeling. Treatment with the pharmacological PAK inhibitor, PF3758309, abrogated the integrity of RhoA-mediated actomyosin contractility and surface adhesion. Moreover, inhibition of PAK activity impaired neutrophil morphological polarization and directional migration under a gradient of fMLP, and was associated with dysregulated Ca(2+) signaling. These results suggest that PAK serves as an important effector of Rho-family GTPases in neutrophil cytoskeletal reorganization, and plays a key role in driving efficient directional migration of human neutrophils.

Show MeSH
Related in: MedlinePlus