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Rac mediates cytoskeletal rearrangements and increased cell motility induced by urokinase-type plasminogen activator receptor binding to vitronectin.

Kjøller L, Hall A - J. Cell Biol. (2001)

Bottom Line: In addition, a fourfold increase in the level of activated Rac is induced by uPAR expression.We conclude that uPAR interacts with VN both to initiate a p130Cas/Rac-dependent signaling pathway leading to actin reorganization and increased cell motility and to act as an adhesion receptor required for these responses.This mechanism may play a role in uPAR-mediated regulation of cell motility at sites where VN and uPAR are co-expressed, such as malignant tumors.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory for Molecular Cell Biology, Cancer Research Campaign Oncogene and Signal Transduction Group, University College London, United Kingdom.

ABSTRACT
The urokinase-type plasminogen activator receptor (uPAR) is involved in the regulation of cell motility in a variety of cell types. We show here that expression of human uPAR in growing murine fibroblasts leads to a dramatic reorganization of the actin cytoskeleton. uPAR expression induces multiple rapidly advancing protrusions that resemble the leading edge of migrating cells. The cytoskeletal changes are independent of uPA and activation of the RGD-binding activity of integrins but require uPAR binding to vitronectin (VN). The actin reorganization is blocked by coexpression of dominant negative versions of either Rac (N17Rac) or p130Cas, but not by inhibitors of Cdc42 or Rho, and is accompanied by a Rac-dependent increase in cell motility. In addition, a fourfold increase in the level of activated Rac is induced by uPAR expression. We conclude that uPAR interacts with VN both to initiate a p130Cas/Rac-dependent signaling pathway leading to actin reorganization and increased cell motility and to act as an adhesion receptor required for these responses. This mechanism may play a role in uPAR-mediated regulation of cell motility at sites where VN and uPAR are co-expressed, such as malignant tumors.

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Effect of uPAR expression on Rac activity in growing NIH 3T3 cells. (A) NIH 3T3 cells were transfected by electroporation with pRC/CMV-uPAR and/or pRK5–myc-wtRac as indicated. PRK5-myc transfection was used as a control. The levels of total cellular Rac and GST–PAK–CRIB precipitated active Rac were determined by SDS-PAGE on 15% gels followed by immunoblotting for Rac. Results shown for endogenous Rac activation are for cells lysed 4 h after transfection. Results shown for cotransfected wtRac activation are for cells lysed 16 h after transfection. (B) The level of activated Rac from cells lysed 16 h after transfection with pRK5–myc-wtRac or pRK5–myc-wtRac and pRC/CMV-uPAR was quantified by density scanning using NIH Image software. The ratio of activated to total Rac was determined and results are expressed relative to the Rac activity in cells transfected with pRK5–myc-wtRac. Results are mean ± SD from three independent experiments.
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Figure 7: Effect of uPAR expression on Rac activity in growing NIH 3T3 cells. (A) NIH 3T3 cells were transfected by electroporation with pRC/CMV-uPAR and/or pRK5–myc-wtRac as indicated. PRK5-myc transfection was used as a control. The levels of total cellular Rac and GST–PAK–CRIB precipitated active Rac were determined by SDS-PAGE on 15% gels followed by immunoblotting for Rac. Results shown for endogenous Rac activation are for cells lysed 4 h after transfection. Results shown for cotransfected wtRac activation are for cells lysed 16 h after transfection. (B) The level of activated Rac from cells lysed 16 h after transfection with pRK5–myc-wtRac or pRK5–myc-wtRac and pRC/CMV-uPAR was quantified by density scanning using NIH Image software. The ratio of activated to total Rac was determined and results are expressed relative to the Rac activity in cells transfected with pRK5–myc-wtRac. Results are mean ± SD from three independent experiments.

Mentions: To investigate whether Rac activation could also be detected in growing cells where a strong protrusive activity was induced by uPAR, the level of GTP-bound Rac was determined. Growing NIH 3T3 cells were mock-transfected, transfected with uPAR, or cotransfected with uPAR and wild-type Rac. Activated Rac was precipitated from cell lysates by the Rac-binding domain of p21-activated kinase (PAK) fused to GST (Sander et al. 1998). As seen in Fig. 7 A, an increase in the activity of both endogenous Rac and cotransfected wild-type Rac could be detected in response to uPAR. In cells transfected with wild-type Rac, uPAR expression induced a fourfold activation of Rac when lysates were analyzed 16 h after transfection (Fig. 7 B). Assays performed 4, 8, and 16 h after transfection yielded similar results. We conclude that uPAR induces Rac activation.


Rac mediates cytoskeletal rearrangements and increased cell motility induced by urokinase-type plasminogen activator receptor binding to vitronectin.

Kjøller L, Hall A - J. Cell Biol. (2001)

Effect of uPAR expression on Rac activity in growing NIH 3T3 cells. (A) NIH 3T3 cells were transfected by electroporation with pRC/CMV-uPAR and/or pRK5–myc-wtRac as indicated. PRK5-myc transfection was used as a control. The levels of total cellular Rac and GST–PAK–CRIB precipitated active Rac were determined by SDS-PAGE on 15% gels followed by immunoblotting for Rac. Results shown for endogenous Rac activation are for cells lysed 4 h after transfection. Results shown for cotransfected wtRac activation are for cells lysed 16 h after transfection. (B) The level of activated Rac from cells lysed 16 h after transfection with pRK5–myc-wtRac or pRK5–myc-wtRac and pRC/CMV-uPAR was quantified by density scanning using NIH Image software. The ratio of activated to total Rac was determined and results are expressed relative to the Rac activity in cells transfected with pRK5–myc-wtRac. Results are mean ± SD from three independent experiments.
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Related In: Results  -  Collection

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Figure 7: Effect of uPAR expression on Rac activity in growing NIH 3T3 cells. (A) NIH 3T3 cells were transfected by electroporation with pRC/CMV-uPAR and/or pRK5–myc-wtRac as indicated. PRK5-myc transfection was used as a control. The levels of total cellular Rac and GST–PAK–CRIB precipitated active Rac were determined by SDS-PAGE on 15% gels followed by immunoblotting for Rac. Results shown for endogenous Rac activation are for cells lysed 4 h after transfection. Results shown for cotransfected wtRac activation are for cells lysed 16 h after transfection. (B) The level of activated Rac from cells lysed 16 h after transfection with pRK5–myc-wtRac or pRK5–myc-wtRac and pRC/CMV-uPAR was quantified by density scanning using NIH Image software. The ratio of activated to total Rac was determined and results are expressed relative to the Rac activity in cells transfected with pRK5–myc-wtRac. Results are mean ± SD from three independent experiments.
Mentions: To investigate whether Rac activation could also be detected in growing cells where a strong protrusive activity was induced by uPAR, the level of GTP-bound Rac was determined. Growing NIH 3T3 cells were mock-transfected, transfected with uPAR, or cotransfected with uPAR and wild-type Rac. Activated Rac was precipitated from cell lysates by the Rac-binding domain of p21-activated kinase (PAK) fused to GST (Sander et al. 1998). As seen in Fig. 7 A, an increase in the activity of both endogenous Rac and cotransfected wild-type Rac could be detected in response to uPAR. In cells transfected with wild-type Rac, uPAR expression induced a fourfold activation of Rac when lysates were analyzed 16 h after transfection (Fig. 7 B). Assays performed 4, 8, and 16 h after transfection yielded similar results. We conclude that uPAR induces Rac activation.

Bottom Line: In addition, a fourfold increase in the level of activated Rac is induced by uPAR expression.We conclude that uPAR interacts with VN both to initiate a p130Cas/Rac-dependent signaling pathway leading to actin reorganization and increased cell motility and to act as an adhesion receptor required for these responses.This mechanism may play a role in uPAR-mediated regulation of cell motility at sites where VN and uPAR are co-expressed, such as malignant tumors.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory for Molecular Cell Biology, Cancer Research Campaign Oncogene and Signal Transduction Group, University College London, United Kingdom.

ABSTRACT
The urokinase-type plasminogen activator receptor (uPAR) is involved in the regulation of cell motility in a variety of cell types. We show here that expression of human uPAR in growing murine fibroblasts leads to a dramatic reorganization of the actin cytoskeleton. uPAR expression induces multiple rapidly advancing protrusions that resemble the leading edge of migrating cells. The cytoskeletal changes are independent of uPA and activation of the RGD-binding activity of integrins but require uPAR binding to vitronectin (VN). The actin reorganization is blocked by coexpression of dominant negative versions of either Rac (N17Rac) or p130Cas, but not by inhibitors of Cdc42 or Rho, and is accompanied by a Rac-dependent increase in cell motility. In addition, a fourfold increase in the level of activated Rac is induced by uPAR expression. We conclude that uPAR interacts with VN both to initiate a p130Cas/Rac-dependent signaling pathway leading to actin reorganization and increased cell motility and to act as an adhesion receptor required for these responses. This mechanism may play a role in uPAR-mediated regulation of cell motility at sites where VN and uPAR are co-expressed, such as malignant tumors.

Show MeSH
Related in: MedlinePlus