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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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Coexpression of either myr-PKCε, V38R-Ras, L61Rac1, or p110α-CAAX with tac-β1 can restore cell spreading on collagen I. (A) Diagram of the control tac receptor containing the extracellular and transmembrane domains of the small (tac) subunit of the human interleukin-2 receptor and the tac-β1 chimera containing the same domains of the interleukin-2 receptor fused to the human integrin β1A cytoplasmic domain. (B) Fibroblasts were transfected with tac (a) or tac-β1 (b), or cotransfected with tac-β1 and myr-PKCε-Flag (c and e) or tac-β1 and myc-V38R-Ras (d and f). Cell area for 100 randomly sampled positively transfected cells is plotted as a function of either tac epitope expression (a–d), myr-PKCε-Flag expression (e) for the same cells shown in c, or myc-V38R-Ras expression (f) for the same cells shown in d. The x axis is a linear scale of cell area from 0 to 1,600 μm2, the y axis is a linear scale of either FITC fluorescence (tac epitope expression) units defined by Image Pro-Plus from 0 to 2.4 × 104 (a–d) or rhodamine fluorescence (Flag or myc epitope expression) from 0 to 1.2 × 105 (e and f). (C) Fibroblasts were transfected with tac (a) or tac-β1 (b), or cotransfected with tac-β1 and myc-L61Rac1 (c and d). Cell area for 90 randomly sampled cells expressing tac, tac-β1, or coexpressing tac-β1 and myc-L61Rac1 is plotted as a function of tac epitope expression (a–c) or myc epitope expression (d) for the cells shown in c. The x axis is a linear scale of cell area from 0 to 2,400 μm2, the y axis is a linear scale of FITC fluorescence (tac epitope expression) from 0 to 2.4 × 104 (a–c) or rhodamine fluorescence (myc epitope expression) from 0 to 1.2 × 105 (d). (D) Fibroblasts transfected with the control tac receptor (a) or tac-β1 (b) or tac-β1 and p110α-CAAX (c) were analyzed for cell-surface expression of the tac epitope and cell area, as described in Materials and Methods. Cell area for 98 randomly sampled positively transfected cells is plotted as a function of tac epitope expression. The x axis is a linear scale of cell area from 0 to 2,400 μm2 and the y axis is a linear scale of FITC fluorescence (tac epitope expression) from 0 to 3.2 × 104 (a) or 0 to 1.6 × 104 (b and c). (B–D) The vertical line positioned at a cell area of 560 μm2 indicates the separation of spread (right) and not spread (left) cells. It is important to note that our spreading assays primarily analyze cells expressing moderate to low levels of tac-β1, since cell attachment to collagen I is inhibited by the expression of high levels of tac-β1 (data not shown). This observation is consistent with our recent studies showing that high levels of tac-β1 inhibit cell attachment to fibronectin (Mastrangelo et al. 1999a). The range of FITC fluorescence represents the range of tac-β1 expression detected in the adherent transfected cells. These experiments were performed three times and similar results were obtained.
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Figure 1: Coexpression of either myr-PKCε, V38R-Ras, L61Rac1, or p110α-CAAX with tac-β1 can restore cell spreading on collagen I. (A) Diagram of the control tac receptor containing the extracellular and transmembrane domains of the small (tac) subunit of the human interleukin-2 receptor and the tac-β1 chimera containing the same domains of the interleukin-2 receptor fused to the human integrin β1A cytoplasmic domain. (B) Fibroblasts were transfected with tac (a) or tac-β1 (b), or cotransfected with tac-β1 and myr-PKCε-Flag (c and e) or tac-β1 and myc-V38R-Ras (d and f). Cell area for 100 randomly sampled positively transfected cells is plotted as a function of either tac epitope expression (a–d), myr-PKCε-Flag expression (e) for the same cells shown in c, or myc-V38R-Ras expression (f) for the same cells shown in d. The x axis is a linear scale of cell area from 0 to 1,600 μm2, the y axis is a linear scale of either FITC fluorescence (tac epitope expression) units defined by Image Pro-Plus from 0 to 2.4 × 104 (a–d) or rhodamine fluorescence (Flag or myc epitope expression) from 0 to 1.2 × 105 (e and f). (C) Fibroblasts were transfected with tac (a) or tac-β1 (b), or cotransfected with tac-β1 and myc-L61Rac1 (c and d). Cell area for 90 randomly sampled cells expressing tac, tac-β1, or coexpressing tac-β1 and myc-L61Rac1 is plotted as a function of tac epitope expression (a–c) or myc epitope expression (d) for the cells shown in c. The x axis is a linear scale of cell area from 0 to 2,400 μm2, the y axis is a linear scale of FITC fluorescence (tac epitope expression) from 0 to 2.4 × 104 (a–c) or rhodamine fluorescence (myc epitope expression) from 0 to 1.2 × 105 (d). (D) Fibroblasts transfected with the control tac receptor (a) or tac-β1 (b) or tac-β1 and p110α-CAAX (c) were analyzed for cell-surface expression of the tac epitope and cell area, as described in Materials and Methods. Cell area for 98 randomly sampled positively transfected cells is plotted as a function of tac epitope expression. The x axis is a linear scale of cell area from 0 to 2,400 μm2 and the y axis is a linear scale of FITC fluorescence (tac epitope expression) from 0 to 3.2 × 104 (a) or 0 to 1.6 × 104 (b and c). (B–D) The vertical line positioned at a cell area of 560 μm2 indicates the separation of spread (right) and not spread (left) cells. It is important to note that our spreading assays primarily analyze cells expressing moderate to low levels of tac-β1, since cell attachment to collagen I is inhibited by the expression of high levels of tac-β1 (data not shown). This observation is consistent with our recent studies showing that high levels of tac-β1 inhibit cell attachment to fibronectin (Mastrangelo et al. 1999a). The range of FITC fluorescence represents the range of tac-β1 expression detected in the adherent transfected cells. These experiments were performed three times and similar results were obtained.

Mentions: In agreement with previous studies (LaFlamme et al. 1994), we found that the majority of the tac-β1 positive cells had cell areas <560 μm2, indicating that they were inhibited in cell spreading (Fig. 1 B). In contrast, the majority of cells expressing the control tac receptor had cell areas >560 μm2, indicating that they were not inhibited in spreading (Fig. 1 B). When we tested the ability of activated R-Ras (V38R-Ras) and membrane-anchored PKCε (myr-PKCε) to restore tac-β1–inhibited cell spreading, we found that the coexpression of tac-β1 with either V38R-Ras or myr-PKCε resulted in an increase in cell area as compared with cells expressing tac-β1 alone (Fig. 1 B). Quantitation of the spreading assay revealed that 35% of cells coexpressing myr-PKCε and 52% of cells coexpressing V38R-Ras were spread, compared with 12% of cells expressing tac-β1 alone. Coexpression of constitutively active Rac1 (L61Rac1) also increased spreading of tac-β1–expressing cells (Fig. 1 C). Of the cells coexpressing tac-β1 and L61Rac1, 86% were spread, compared with 13% expressing tac-β1 alone. Cotransfection of the membrane-anchored catalytic subunit of PI 3-kinase also increased the area of tac-β1–expressing cells (Fig. 1 D). When we quantitated the spreading of cells cotransfected with tac-β1 and p110α-CAAX as a function of tac epitope expression only (Fig. 1 D), we found that 59% of the tac-β1 and p110α-CAAX cotransfected cells were spread compared with only 9% of the cells transfected with tac-β1 alone. In this instance, we were unable to quantitate p110α-CAAX expression by immunofluorescence. However, we have found in our assays that ∼80% of transfected cells coexpress cotransfected plasmids (data not shown). Thus, as an additional control, we analyzed the percentage of transfected cells that spread as a function of tac epitope expression in four separate experiments and found that 57 ± 9% of the cells cotransfected with tac-β1 and p110α-CAAX were spread compared with 12 ± 5% of the cells transfected with tac-β1 alone (quantitation is the mean ± SD).


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)

Coexpression of either myr-PKCε, V38R-Ras, L61Rac1, or p110α-CAAX with tac-β1 can restore cell spreading on collagen I. (A) Diagram of the control tac receptor containing the extracellular and transmembrane domains of the small (tac) subunit of the human interleukin-2 receptor and the tac-β1 chimera containing the same domains of the interleukin-2 receptor fused to the human integrin β1A cytoplasmic domain. (B) Fibroblasts were transfected with tac (a) or tac-β1 (b), or cotransfected with tac-β1 and myr-PKCε-Flag (c and e) or tac-β1 and myc-V38R-Ras (d and f). Cell area for 100 randomly sampled positively transfected cells is plotted as a function of either tac epitope expression (a–d), myr-PKCε-Flag expression (e) for the same cells shown in c, or myc-V38R-Ras expression (f) for the same cells shown in d. The x axis is a linear scale of cell area from 0 to 1,600 μm2, the y axis is a linear scale of either FITC fluorescence (tac epitope expression) units defined by Image Pro-Plus from 0 to 2.4 × 104 (a–d) or rhodamine fluorescence (Flag or myc epitope expression) from 0 to 1.2 × 105 (e and f). (C) Fibroblasts were transfected with tac (a) or tac-β1 (b), or cotransfected with tac-β1 and myc-L61Rac1 (c and d). Cell area for 90 randomly sampled cells expressing tac, tac-β1, or coexpressing tac-β1 and myc-L61Rac1 is plotted as a function of tac epitope expression (a–c) or myc epitope expression (d) for the cells shown in c. The x axis is a linear scale of cell area from 0 to 2,400 μm2, the y axis is a linear scale of FITC fluorescence (tac epitope expression) from 0 to 2.4 × 104 (a–c) or rhodamine fluorescence (myc epitope expression) from 0 to 1.2 × 105 (d). (D) Fibroblasts transfected with the control tac receptor (a) or tac-β1 (b) or tac-β1 and p110α-CAAX (c) were analyzed for cell-surface expression of the tac epitope and cell area, as described in Materials and Methods. Cell area for 98 randomly sampled positively transfected cells is plotted as a function of tac epitope expression. The x axis is a linear scale of cell area from 0 to 2,400 μm2 and the y axis is a linear scale of FITC fluorescence (tac epitope expression) from 0 to 3.2 × 104 (a) or 0 to 1.6 × 104 (b and c). (B–D) The vertical line positioned at a cell area of 560 μm2 indicates the separation of spread (right) and not spread (left) cells. It is important to note that our spreading assays primarily analyze cells expressing moderate to low levels of tac-β1, since cell attachment to collagen I is inhibited by the expression of high levels of tac-β1 (data not shown). This observation is consistent with our recent studies showing that high levels of tac-β1 inhibit cell attachment to fibronectin (Mastrangelo et al. 1999a). The range of FITC fluorescence represents the range of tac-β1 expression detected in the adherent transfected cells. These experiments were performed three times and similar results were obtained.
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Figure 1: Coexpression of either myr-PKCε, V38R-Ras, L61Rac1, or p110α-CAAX with tac-β1 can restore cell spreading on collagen I. (A) Diagram of the control tac receptor containing the extracellular and transmembrane domains of the small (tac) subunit of the human interleukin-2 receptor and the tac-β1 chimera containing the same domains of the interleukin-2 receptor fused to the human integrin β1A cytoplasmic domain. (B) Fibroblasts were transfected with tac (a) or tac-β1 (b), or cotransfected with tac-β1 and myr-PKCε-Flag (c and e) or tac-β1 and myc-V38R-Ras (d and f). Cell area for 100 randomly sampled positively transfected cells is plotted as a function of either tac epitope expression (a–d), myr-PKCε-Flag expression (e) for the same cells shown in c, or myc-V38R-Ras expression (f) for the same cells shown in d. The x axis is a linear scale of cell area from 0 to 1,600 μm2, the y axis is a linear scale of either FITC fluorescence (tac epitope expression) units defined by Image Pro-Plus from 0 to 2.4 × 104 (a–d) or rhodamine fluorescence (Flag or myc epitope expression) from 0 to 1.2 × 105 (e and f). (C) Fibroblasts were transfected with tac (a) or tac-β1 (b), or cotransfected with tac-β1 and myc-L61Rac1 (c and d). Cell area for 90 randomly sampled cells expressing tac, tac-β1, or coexpressing tac-β1 and myc-L61Rac1 is plotted as a function of tac epitope expression (a–c) or myc epitope expression (d) for the cells shown in c. The x axis is a linear scale of cell area from 0 to 2,400 μm2, the y axis is a linear scale of FITC fluorescence (tac epitope expression) from 0 to 2.4 × 104 (a–c) or rhodamine fluorescence (myc epitope expression) from 0 to 1.2 × 105 (d). (D) Fibroblasts transfected with the control tac receptor (a) or tac-β1 (b) or tac-β1 and p110α-CAAX (c) were analyzed for cell-surface expression of the tac epitope and cell area, as described in Materials and Methods. Cell area for 98 randomly sampled positively transfected cells is plotted as a function of tac epitope expression. The x axis is a linear scale of cell area from 0 to 2,400 μm2 and the y axis is a linear scale of FITC fluorescence (tac epitope expression) from 0 to 3.2 × 104 (a) or 0 to 1.6 × 104 (b and c). (B–D) The vertical line positioned at a cell area of 560 μm2 indicates the separation of spread (right) and not spread (left) cells. It is important to note that our spreading assays primarily analyze cells expressing moderate to low levels of tac-β1, since cell attachment to collagen I is inhibited by the expression of high levels of tac-β1 (data not shown). This observation is consistent with our recent studies showing that high levels of tac-β1 inhibit cell attachment to fibronectin (Mastrangelo et al. 1999a). The range of FITC fluorescence represents the range of tac-β1 expression detected in the adherent transfected cells. These experiments were performed three times and similar results were obtained.
Mentions: In agreement with previous studies (LaFlamme et al. 1994), we found that the majority of the tac-β1 positive cells had cell areas <560 μm2, indicating that they were inhibited in cell spreading (Fig. 1 B). In contrast, the majority of cells expressing the control tac receptor had cell areas >560 μm2, indicating that they were not inhibited in spreading (Fig. 1 B). When we tested the ability of activated R-Ras (V38R-Ras) and membrane-anchored PKCε (myr-PKCε) to restore tac-β1–inhibited cell spreading, we found that the coexpression of tac-β1 with either V38R-Ras or myr-PKCε resulted in an increase in cell area as compared with cells expressing tac-β1 alone (Fig. 1 B). Quantitation of the spreading assay revealed that 35% of cells coexpressing myr-PKCε and 52% of cells coexpressing V38R-Ras were spread, compared with 12% of cells expressing tac-β1 alone. Coexpression of constitutively active Rac1 (L61Rac1) also increased spreading of tac-β1–expressing cells (Fig. 1 C). Of the cells coexpressing tac-β1 and L61Rac1, 86% were spread, compared with 13% expressing tac-β1 alone. Cotransfection of the membrane-anchored catalytic subunit of PI 3-kinase also increased the area of tac-β1–expressing cells (Fig. 1 D). When we quantitated the spreading of cells cotransfected with tac-β1 and p110α-CAAX as a function of tac epitope expression only (Fig. 1 D), we found that 59% of the tac-β1 and p110α-CAAX cotransfected cells were spread compared with only 9% of the cells transfected with tac-β1 alone. In this instance, we were unable to quantitate p110α-CAAX expression by immunofluorescence. However, we have found in our assays that ∼80% of transfected cells coexpress cotransfected plasmids (data not shown). Thus, as an additional control, we analyzed the percentage of transfected cells that spread as a function of tac epitope expression in four separate experiments and found that 57 ± 9% of the cells cotransfected with tac-β1 and p110α-CAAX were spread compared with 12 ± 5% of the cells transfected with tac-β1 alone (quantitation is the mean ± SD).

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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Related in: MedlinePlus