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Activated R-ras, Rac1, PI 3-kinase and PKCepsilon can each restore cell spreading inhibited by isolated integrin beta1 cytoplasmic domains.

Berrier AL, Mastrangelo AM, Downward J, Ginsberg M, LaFlamme SE - J. Cell Biol. (2000)

Bottom Line: In contrast, L61Rac1 and myr-PKCstraightepsilon each increased cell spreading independent of PI 3-kinase activity.Additionally, the dominant-negative mutant of Rac1, N17Rac1, abrogated cell spreading and inhibited the ability of p110alpha-CAAX and myr-PKCstraightepsilon to increase cell spreading.These studies suggest that R-Ras, PI 3-kinase, Rac1 and PKCepsilon require the function of integrin beta cytoplasmic domains to regulate cell spreading and that Rac1 is downstream of PI 3-kinase and PKCepsilon in a pathway involving integrin beta cytoplasmic domain function in cell spreading.

View Article: PubMed Central - PubMed

Affiliation: Center for Cell Biology and Cancer Research, Albany Medical College, Albany, New York 12208, USA.

ABSTRACT
Attachment of many cell types to extracellular matrix proteins triggers cell spreading, a process that strengthens cell adhesion and is a prerequisite for many adhesion-dependent processes including cell migration, survival, and proliferation. Cell spreading requires integrins with intact beta cytoplasmic domains, presumably to connect integrins with the actin cytoskeleton and to activate signaling pathways that promote cell spreading. Several signaling proteins are known to regulate cell spreading, including R-Ras, PI 3-kinase, PKCepsilon and Rac1; however, it is not known whether they do so through a mechanism involving integrin beta cytoplasmic domains. To study the mechanisms whereby cell spreading is regulated by integrin beta cytoplasmic domains, we inhibited cell spreading on collagen I or fibrinogen by expressing tac-beta1, a dominant-negative inhibitor of integrin function, and examined whether cell spreading could be restored by the coexpression of either V38R-Ras, p110alpha-CAAX, myr-PKCepsilon, or L61Rac1. Each of these activated signaling proteins was able to restore cell spreading as assayed by an increase in the area of cells expressing tac-beta1. R-Ras and Rac1 rescued cell spreading in a GTP-dependent manner, whereas PKCstraightepsilon required an intact kinase domain. Importantly, each of these signaling proteins required intact beta cytoplasmic domains on the integrins mediating adhesion in order to restore cell spreading. In addition, the rescue of cell spreading by V38R-Ras was inhibited by LY294002, suggesting that PI 3-kinase activity is required for V38R-Ras to restore cell spreading. In contrast, L61Rac1 and myr-PKCstraightepsilon each increased cell spreading independent of PI 3-kinase activity. Additionally, the dominant-negative mutant of Rac1, N17Rac1, abrogated cell spreading and inhibited the ability of p110alpha-CAAX and myr-PKCstraightepsilon to increase cell spreading. These studies suggest that R-Ras, PI 3-kinase, Rac1 and PKCepsilon require the function of integrin beta cytoplasmic domains to regulate cell spreading and that Rac1 is downstream of PI 3-kinase and PKCepsilon in a pathway involving integrin beta cytoplasmic domain function in cell spreading.

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The morphology of cotransfected cells adherent to collagen I. (A) The morphology of cells expressing tac or tac-β1 alone or cells coexpressing tac-β1 and either V38R-Ras, L61Rac1, or myr-PKCε. Shown is tac epitope expression (FITC fluorescence) for representative cells from the quantitative experiment shown in Fig. 1. Also included is tac-β1 expression of a representative cell from the cotransfection of tac-β1 and p110α-CAAX. Fluorescence images were obtained with Spot software and composites were generated in adobe photoshop. Scale bar: 10 μm. (B) Coexpression of either V38R-Ras, L61Rac1, or myr-PKCε in addition to rescuing cell spreading also restores the localization of tac-β1 to the focal contact. Fibroblasts were cotransfected with tac-β1 and either V38R-Ras, L61Rac1, or myr-PKCε, and cells adherent to collagen were costained for tac and signaling protein expression as described previously. The tac-FITC staining (right) and corresponding interference reflection pattern (left) of coexpressing cells is shown. Scale bar: 10 μm.
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Figure 4: The morphology of cotransfected cells adherent to collagen I. (A) The morphology of cells expressing tac or tac-β1 alone or cells coexpressing tac-β1 and either V38R-Ras, L61Rac1, or myr-PKCε. Shown is tac epitope expression (FITC fluorescence) for representative cells from the quantitative experiment shown in Fig. 1. Also included is tac-β1 expression of a representative cell from the cotransfection of tac-β1 and p110α-CAAX. Fluorescence images were obtained with Spot software and composites were generated in adobe photoshop. Scale bar: 10 μm. (B) Coexpression of either V38R-Ras, L61Rac1, or myr-PKCε in addition to rescuing cell spreading also restores the localization of tac-β1 to the focal contact. Fibroblasts were cotransfected with tac-β1 and either V38R-Ras, L61Rac1, or myr-PKCε, and cells adherent to collagen were costained for tac and signaling protein expression as described previously. The tac-FITC staining (right) and corresponding interference reflection pattern (left) of coexpressing cells is shown. Scale bar: 10 μm.

Mentions: We next examined the morphology of cells coexpressing tac-β1 and each of the signaling proteins. As expected, cells expressing tac had a normal fibroblast morphology, whereas cells expressing tac-β1 were not spread (Fig. 4 A). Cells expressing tac-β1 and either p110α-CAAX or myr-PKCε were spread and had many membrane projections (Fig. 4 A). In some instances, tac-β1 was observed in focal adhesions of myr-PKCε–expressing cells (Fig. 4 B). The morphology of cells coexpressing tac-β1 and either p110α-caax or myr-pkcε was similar to many of the untransfected cells or the cells expressing tac alone, indicating that this is a common morphology for fibroblasts in our assay. Many of the cells coexpressing tac-β1 and V38R-Ras exhibited large polarized lamellipodia (Fig. 4 A) with prominent focal adhesion staining of tac-β1 (Fig. 4 B). Cells coexpressing tac-β1 and L61Rac1 were symmetrically spread and exhibited membrane ruffling (Fig. 4 A) and tac-β1 localization in focal adhesions at the cell periphery (Fig. 4 B). Thus, expression of activated forms of PI 3-kinase, PKCε, R-Ras, and Rac1 increased cell spreading through distinct cell morphologies. Interestingly, cells expressing L61Rac1 alone or V38R-Ras alone had cell morphologies very similar to cells coexpressing tac-β1, which tended to have smaller cell areas (Fig. 4 A and 5). Cells expressing either myr-PKCε or p110α-CAAX alone had cell morphologies similar to untransfected cells or cells transfected with tac alone (Fig. 5).


Activated R-ras, Rac1, PI 3-kinase and PKCepsilon can each restore cell spreading inhibited by isolated integrin beta1 cytoplasmic domains.

Berrier AL, Mastrangelo AM, Downward J, Ginsberg M, LaFlamme SE - J. Cell Biol. (2000)

The morphology of cotransfected cells adherent to collagen I. (A) The morphology of cells expressing tac or tac-β1 alone or cells coexpressing tac-β1 and either V38R-Ras, L61Rac1, or myr-PKCε. Shown is tac epitope expression (FITC fluorescence) for representative cells from the quantitative experiment shown in Fig. 1. Also included is tac-β1 expression of a representative cell from the cotransfection of tac-β1 and p110α-CAAX. Fluorescence images were obtained with Spot software and composites were generated in adobe photoshop. Scale bar: 10 μm. (B) Coexpression of either V38R-Ras, L61Rac1, or myr-PKCε in addition to rescuing cell spreading also restores the localization of tac-β1 to the focal contact. Fibroblasts were cotransfected with tac-β1 and either V38R-Ras, L61Rac1, or myr-PKCε, and cells adherent to collagen were costained for tac and signaling protein expression as described previously. The tac-FITC staining (right) and corresponding interference reflection pattern (left) of coexpressing cells is shown. Scale bar: 10 μm.
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Figure 4: The morphology of cotransfected cells adherent to collagen I. (A) The morphology of cells expressing tac or tac-β1 alone or cells coexpressing tac-β1 and either V38R-Ras, L61Rac1, or myr-PKCε. Shown is tac epitope expression (FITC fluorescence) for representative cells from the quantitative experiment shown in Fig. 1. Also included is tac-β1 expression of a representative cell from the cotransfection of tac-β1 and p110α-CAAX. Fluorescence images were obtained with Spot software and composites were generated in adobe photoshop. Scale bar: 10 μm. (B) Coexpression of either V38R-Ras, L61Rac1, or myr-PKCε in addition to rescuing cell spreading also restores the localization of tac-β1 to the focal contact. Fibroblasts were cotransfected with tac-β1 and either V38R-Ras, L61Rac1, or myr-PKCε, and cells adherent to collagen were costained for tac and signaling protein expression as described previously. The tac-FITC staining (right) and corresponding interference reflection pattern (left) of coexpressing cells is shown. Scale bar: 10 μm.
Mentions: We next examined the morphology of cells coexpressing tac-β1 and each of the signaling proteins. As expected, cells expressing tac had a normal fibroblast morphology, whereas cells expressing tac-β1 were not spread (Fig. 4 A). Cells expressing tac-β1 and either p110α-CAAX or myr-PKCε were spread and had many membrane projections (Fig. 4 A). In some instances, tac-β1 was observed in focal adhesions of myr-PKCε–expressing cells (Fig. 4 B). The morphology of cells coexpressing tac-β1 and either p110α-caax or myr-pkcε was similar to many of the untransfected cells or the cells expressing tac alone, indicating that this is a common morphology for fibroblasts in our assay. Many of the cells coexpressing tac-β1 and V38R-Ras exhibited large polarized lamellipodia (Fig. 4 A) with prominent focal adhesion staining of tac-β1 (Fig. 4 B). Cells coexpressing tac-β1 and L61Rac1 were symmetrically spread and exhibited membrane ruffling (Fig. 4 A) and tac-β1 localization in focal adhesions at the cell periphery (Fig. 4 B). Thus, expression of activated forms of PI 3-kinase, PKCε, R-Ras, and Rac1 increased cell spreading through distinct cell morphologies. Interestingly, cells expressing L61Rac1 alone or V38R-Ras alone had cell morphologies very similar to cells coexpressing tac-β1, which tended to have smaller cell areas (Fig. 4 A and 5). Cells expressing either myr-PKCε or p110α-CAAX alone had cell morphologies similar to untransfected cells or cells transfected with tac alone (Fig. 5).

Bottom Line: In contrast, L61Rac1 and myr-PKCstraightepsilon each increased cell spreading independent of PI 3-kinase activity.Additionally, the dominant-negative mutant of Rac1, N17Rac1, abrogated cell spreading and inhibited the ability of p110alpha-CAAX and myr-PKCstraightepsilon to increase cell spreading.These studies suggest that R-Ras, PI 3-kinase, Rac1 and PKCepsilon require the function of integrin beta cytoplasmic domains to regulate cell spreading and that Rac1 is downstream of PI 3-kinase and PKCepsilon in a pathway involving integrin beta cytoplasmic domain function in cell spreading.

View Article: PubMed Central - PubMed

Affiliation: Center for Cell Biology and Cancer Research, Albany Medical College, Albany, New York 12208, USA.

ABSTRACT
Attachment of many cell types to extracellular matrix proteins triggers cell spreading, a process that strengthens cell adhesion and is a prerequisite for many adhesion-dependent processes including cell migration, survival, and proliferation. Cell spreading requires integrins with intact beta cytoplasmic domains, presumably to connect integrins with the actin cytoskeleton and to activate signaling pathways that promote cell spreading. Several signaling proteins are known to regulate cell spreading, including R-Ras, PI 3-kinase, PKCepsilon and Rac1; however, it is not known whether they do so through a mechanism involving integrin beta cytoplasmic domains. To study the mechanisms whereby cell spreading is regulated by integrin beta cytoplasmic domains, we inhibited cell spreading on collagen I or fibrinogen by expressing tac-beta1, a dominant-negative inhibitor of integrin function, and examined whether cell spreading could be restored by the coexpression of either V38R-Ras, p110alpha-CAAX, myr-PKCepsilon, or L61Rac1. Each of these activated signaling proteins was able to restore cell spreading as assayed by an increase in the area of cells expressing tac-beta1. R-Ras and Rac1 rescued cell spreading in a GTP-dependent manner, whereas PKCstraightepsilon required an intact kinase domain. Importantly, each of these signaling proteins required intact beta cytoplasmic domains on the integrins mediating adhesion in order to restore cell spreading. In addition, the rescue of cell spreading by V38R-Ras was inhibited by LY294002, suggesting that PI 3-kinase activity is required for V38R-Ras to restore cell spreading. In contrast, L61Rac1 and myr-PKCstraightepsilon each increased cell spreading independent of PI 3-kinase activity. Additionally, the dominant-negative mutant of Rac1, N17Rac1, abrogated cell spreading and inhibited the ability of p110alpha-CAAX and myr-PKCstraightepsilon to increase cell spreading. These studies suggest that R-Ras, PI 3-kinase, Rac1 and PKCepsilon require the function of integrin beta cytoplasmic domains to regulate cell spreading and that Rac1 is downstream of PI 3-kinase and PKCepsilon in a pathway involving integrin beta cytoplasmic domain function in cell spreading.

Show MeSH
Related in: MedlinePlus