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Role of the PI3K regulatory subunit in the control of actin organization and cell migration.

Jiménez C, Portela RA, Mellado M, Rodríguez-Frade JM, Collard J, Serrano A, Martínez-A C, Avila J, Carrera AC - J. Cell Biol. (2000)

Bottom Line: We demonstrate that PDGF activates Cdc42 and its downstream effector N-WASP to mediate filopodia formation, actin stress fiber disassembly, and a reduction in focal adhesion complexes.Induction of the Cdc42 pathway is independent of phosphoinositide 3-kinase (PI3K) enzymatic activity, but it is dependent on the p85alpha regulatory subunit of PI3K.Finally, data are provided showing that activation of this pathway is required for PDGF-induced cell migration on collagen.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Oncology, Centro Nacional de Biotecnología, Madrid, Spain.

ABSTRACT
Cell migration represents an important cellular response that utilizes cytoskeletal reorganization as its driving force. Here, we describe a new signaling cascade linking PDGF receptor stimulation to actin rearrangements and cell migration. We demonstrate that PDGF activates Cdc42 and its downstream effector N-WASP to mediate filopodia formation, actin stress fiber disassembly, and a reduction in focal adhesion complexes. Induction of the Cdc42 pathway is independent of phosphoinositide 3-kinase (PI3K) enzymatic activity, but it is dependent on the p85alpha regulatory subunit of PI3K. Finally, data are provided showing that activation of this pathway is required for PDGF-induced cell migration on collagen. These observations show the essential role of the PI3K regulatory subunit p85alpha in controlling PDGF receptor-induced cytoskeletal changes and cell migration, illustrating a novel signaling pathway that links receptor stimulation at the cell membrane with actin dynamics.

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Increased p85α-PI3K regulatory subunit expression induces a decrease in actin stress fibers and adhesion complexes. (A) N-myc-p110-CAAX stable NIH-3T3 cells were prepared as detailed in Materials and Methods. Lysates of selected clones (200 μg/sample) were immunoprecipitated with anti-myc Ab, and the associated PI3K activity was tested in vitro as in Fig. 3 A. Two representative N-myc-p110-CAAX–positive clones (lanes 2 and 3) are shown. (B) NIH-3T3 stable transfectants expressing p110-CAAX, HA-p85α, or HA-p65PI3K (indicated) were incubated in 0.1% serum for 16 h. Cells were subsequently fixed and stained for filamentous actin using FITC-conjugated phalloidin as in Fig. 1. (C) Cells were treated as in B and stained for paxillin distribution by indirect immunofluorescence as in Fig. 1. (D) NIH-3T3 cells preincubated in medium containing 0.1% serum were microinjected with different cDNAs (0.2 mg/ml) encoding HA-p65PI3K, HA-p85α, or HA-p65PI3K plus cDNA encoding N-myc-wt-p110. Cells were incubated for 3 h and subsequently fixed and stained simultaneously with FITC-phalloidin and anti-HA Ab. Phalloidin staining is illustrated; microinjected HA-positive cells are indicated by an arrow. In the bottom right panel, HA staining is shown for the p85α sample. (E) NIH-3T3 cells were transfected with cDNA encoding either SH3Bcr mutant or p50α, and cells were incubated and stained as in Fig. 2. Quantitation of fluorescence intensity of F-actin (in arbitrary units) in the indicated cross-sections is shown beneath the SH3Bcr and p50 panels. The figure illustrates one representative experiment of four performed with similar results. Bars, 100 μm.
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Figure 4: Increased p85α-PI3K regulatory subunit expression induces a decrease in actin stress fibers and adhesion complexes. (A) N-myc-p110-CAAX stable NIH-3T3 cells were prepared as detailed in Materials and Methods. Lysates of selected clones (200 μg/sample) were immunoprecipitated with anti-myc Ab, and the associated PI3K activity was tested in vitro as in Fig. 3 A. Two representative N-myc-p110-CAAX–positive clones (lanes 2 and 3) are shown. (B) NIH-3T3 stable transfectants expressing p110-CAAX, HA-p85α, or HA-p65PI3K (indicated) were incubated in 0.1% serum for 16 h. Cells were subsequently fixed and stained for filamentous actin using FITC-conjugated phalloidin as in Fig. 1. (C) Cells were treated as in B and stained for paxillin distribution by indirect immunofluorescence as in Fig. 1. (D) NIH-3T3 cells preincubated in medium containing 0.1% serum were microinjected with different cDNAs (0.2 mg/ml) encoding HA-p65PI3K, HA-p85α, or HA-p65PI3K plus cDNA encoding N-myc-wt-p110. Cells were incubated for 3 h and subsequently fixed and stained simultaneously with FITC-phalloidin and anti-HA Ab. Phalloidin staining is illustrated; microinjected HA-positive cells are indicated by an arrow. In the bottom right panel, HA staining is shown for the p85α sample. (E) NIH-3T3 cells were transfected with cDNA encoding either SH3Bcr mutant or p50α, and cells were incubated and stained as in Fig. 2. Quantitation of fluorescence intensity of F-actin (in arbitrary units) in the indicated cross-sections is shown beneath the SH3Bcr and p50 panels. The figure illustrates one representative experiment of four performed with similar results. Bars, 100 μm.

Mentions: PDGF treatment induces activation and translocation of p85/p110 PI3K to its receptor, an essential event for Rac-mediated lamellipodium formation (Klippel et al. 1992; Wennström et al. 1994a,Wennström et al. 1994b). The molecular basis for Cdc42 activation after PDGF-R stimulation nonetheless remains unclear. We previously noticed that cells expressing p85α or its truncation product p65PI3K (Jiménez, et al. 1998) exhibited filopodium-like extensions and contained decreased numbers of actin stress fibers (our unpublished observations). Thus, we decided to study the ability of PI3K regulatory subunit to mediate actin cytoskeleton changes in greater detail. We have compared the phenotypes of cells expressing p110-CAAX (a constitutive active mutant of the p110 catalytic subunit; Jiménez et al. 1998), the p85α regulatory subunit, or p65PI3K (a p85 mutant that enhances p110 activation; Jiménez et al. 1998). p85α and p65PI3K stable cell lines have been described previously and express transfected proteins at levels similar to that of endogenous p85α (Jiménez et al. 1998); p110-CAAX cell lines, described here, were prepared similarly (Fig. 4 A).


Role of the PI3K regulatory subunit in the control of actin organization and cell migration.

Jiménez C, Portela RA, Mellado M, Rodríguez-Frade JM, Collard J, Serrano A, Martínez-A C, Avila J, Carrera AC - J. Cell Biol. (2000)

Increased p85α-PI3K regulatory subunit expression induces a decrease in actin stress fibers and adhesion complexes. (A) N-myc-p110-CAAX stable NIH-3T3 cells were prepared as detailed in Materials and Methods. Lysates of selected clones (200 μg/sample) were immunoprecipitated with anti-myc Ab, and the associated PI3K activity was tested in vitro as in Fig. 3 A. Two representative N-myc-p110-CAAX–positive clones (lanes 2 and 3) are shown. (B) NIH-3T3 stable transfectants expressing p110-CAAX, HA-p85α, or HA-p65PI3K (indicated) were incubated in 0.1% serum for 16 h. Cells were subsequently fixed and stained for filamentous actin using FITC-conjugated phalloidin as in Fig. 1. (C) Cells were treated as in B and stained for paxillin distribution by indirect immunofluorescence as in Fig. 1. (D) NIH-3T3 cells preincubated in medium containing 0.1% serum were microinjected with different cDNAs (0.2 mg/ml) encoding HA-p65PI3K, HA-p85α, or HA-p65PI3K plus cDNA encoding N-myc-wt-p110. Cells were incubated for 3 h and subsequently fixed and stained simultaneously with FITC-phalloidin and anti-HA Ab. Phalloidin staining is illustrated; microinjected HA-positive cells are indicated by an arrow. In the bottom right panel, HA staining is shown for the p85α sample. (E) NIH-3T3 cells were transfected with cDNA encoding either SH3Bcr mutant or p50α, and cells were incubated and stained as in Fig. 2. Quantitation of fluorescence intensity of F-actin (in arbitrary units) in the indicated cross-sections is shown beneath the SH3Bcr and p50 panels. The figure illustrates one representative experiment of four performed with similar results. Bars, 100 μm.
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Figure 4: Increased p85α-PI3K regulatory subunit expression induces a decrease in actin stress fibers and adhesion complexes. (A) N-myc-p110-CAAX stable NIH-3T3 cells were prepared as detailed in Materials and Methods. Lysates of selected clones (200 μg/sample) were immunoprecipitated with anti-myc Ab, and the associated PI3K activity was tested in vitro as in Fig. 3 A. Two representative N-myc-p110-CAAX–positive clones (lanes 2 and 3) are shown. (B) NIH-3T3 stable transfectants expressing p110-CAAX, HA-p85α, or HA-p65PI3K (indicated) were incubated in 0.1% serum for 16 h. Cells were subsequently fixed and stained for filamentous actin using FITC-conjugated phalloidin as in Fig. 1. (C) Cells were treated as in B and stained for paxillin distribution by indirect immunofluorescence as in Fig. 1. (D) NIH-3T3 cells preincubated in medium containing 0.1% serum were microinjected with different cDNAs (0.2 mg/ml) encoding HA-p65PI3K, HA-p85α, or HA-p65PI3K plus cDNA encoding N-myc-wt-p110. Cells were incubated for 3 h and subsequently fixed and stained simultaneously with FITC-phalloidin and anti-HA Ab. Phalloidin staining is illustrated; microinjected HA-positive cells are indicated by an arrow. In the bottom right panel, HA staining is shown for the p85α sample. (E) NIH-3T3 cells were transfected with cDNA encoding either SH3Bcr mutant or p50α, and cells were incubated and stained as in Fig. 2. Quantitation of fluorescence intensity of F-actin (in arbitrary units) in the indicated cross-sections is shown beneath the SH3Bcr and p50 panels. The figure illustrates one representative experiment of four performed with similar results. Bars, 100 μm.
Mentions: PDGF treatment induces activation and translocation of p85/p110 PI3K to its receptor, an essential event for Rac-mediated lamellipodium formation (Klippel et al. 1992; Wennström et al. 1994a,Wennström et al. 1994b). The molecular basis for Cdc42 activation after PDGF-R stimulation nonetheless remains unclear. We previously noticed that cells expressing p85α or its truncation product p65PI3K (Jiménez, et al. 1998) exhibited filopodium-like extensions and contained decreased numbers of actin stress fibers (our unpublished observations). Thus, we decided to study the ability of PI3K regulatory subunit to mediate actin cytoskeleton changes in greater detail. We have compared the phenotypes of cells expressing p110-CAAX (a constitutive active mutant of the p110 catalytic subunit; Jiménez et al. 1998), the p85α regulatory subunit, or p65PI3K (a p85 mutant that enhances p110 activation; Jiménez et al. 1998). p85α and p65PI3K stable cell lines have been described previously and express transfected proteins at levels similar to that of endogenous p85α (Jiménez et al. 1998); p110-CAAX cell lines, described here, were prepared similarly (Fig. 4 A).

Bottom Line: We demonstrate that PDGF activates Cdc42 and its downstream effector N-WASP to mediate filopodia formation, actin stress fiber disassembly, and a reduction in focal adhesion complexes.Induction of the Cdc42 pathway is independent of phosphoinositide 3-kinase (PI3K) enzymatic activity, but it is dependent on the p85alpha regulatory subunit of PI3K.Finally, data are provided showing that activation of this pathway is required for PDGF-induced cell migration on collagen.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Oncology, Centro Nacional de Biotecnología, Madrid, Spain.

ABSTRACT
Cell migration represents an important cellular response that utilizes cytoskeletal reorganization as its driving force. Here, we describe a new signaling cascade linking PDGF receptor stimulation to actin rearrangements and cell migration. We demonstrate that PDGF activates Cdc42 and its downstream effector N-WASP to mediate filopodia formation, actin stress fiber disassembly, and a reduction in focal adhesion complexes. Induction of the Cdc42 pathway is independent of phosphoinositide 3-kinase (PI3K) enzymatic activity, but it is dependent on the p85alpha regulatory subunit of PI3K. Finally, data are provided showing that activation of this pathway is required for PDGF-induced cell migration on collagen. These observations show the essential role of the PI3K regulatory subunit p85alpha in controlling PDGF receptor-induced cytoskeletal changes and cell migration, illustrating a novel signaling pathway that links receptor stimulation at the cell membrane with actin dynamics.

Show MeSH
Related in: MedlinePlus