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Alpha(v)beta3 integrin expression up-regulates cdc2, which modulates cell migration.

Manes T, Zheng DQ, Tognin S, Woodard AS, Marchisio PC, Languino LR - J. Cell Biol. (2003)

Bottom Line: We describe here a novel pathway that modulates cell migration and that is activated by alphavbeta3 and, as downstream effector, by cdc2 (cdk1).Third, cdc2 inhibitors reduce cell migration without affecting cell adhesion.These results show that cdc2 is a downstream effector of the alphavbeta3 integrin, and that it promotes cell migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Yale University School of Medicine, New Haven, CT 06510, USA.

ABSTRACT
The alphavbeta3 integrin has been shown to promote cell migration through activation of intracellular signaling pathways. We describe here a novel pathway that modulates cell migration and that is activated by alphavbeta3 and, as downstream effector, by cdc2 (cdk1). We report that alphavbeta3 expression in LNCaP (beta3-LNCaP) prostate cancer cells causes increased cdc2 mRNA levels as evaluated by gene expression analysis, and increased cdc2 protein and kinase activity levels. We provide three lines of evidence that increased levels of cdc2 contribute to a motile phenotype on integrin ligands in different cell types. First, increased levels of cdc2 correlate with more motile phenotypes of cancer cells. Second, ectopic expression of cdc2 increases cell migration, whereas expression of dominant-negative cdc2 inhibits migration. Third, cdc2 inhibitors reduce cell migration without affecting cell adhesion. We also show that cdc2 increases cell migration via specific association with cyclin B2, and we unravel a novel pathway of cell motility that involves, downstream of cdc2, caldesmon. cdc2 and caldesmon are shown here to localize in membrane ruffles in motile cells. These results show that cdc2 is a downstream effector of the alphavbeta3 integrin, and that it promotes cell migration.

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Expression of cyclin B2 increases cell migration. (A) β3-LNCaP (β3-2) and HeLa cells cotransfected with pCMVβgal and pcDNA-3 (vector), pCMXcyclin A (A), pCMVcyclin B1 (B1), or pCMVcyclin B2 (B2) were processed 24 h after transfection, as described in Figs. 3 and 4. The mean and SEM of 10 random fields is shown. (B) Ectopically expressed cyclin B2 forms active kinase complex. Immunocomplexes precipitated from HeLa and cyclin B2–transfected HeLa RIPA extracts using nonimmune rabbit serum (n.i.), or rabbit polyclonal antibody to cyclin B2 (B2) or a c-myc agarose-conjugated rabbit polyclonal antibody (c-myc) were used in kinase assays using histone H1 as a substrate. (C) Cyclin B2– cells migrate poorly on FN. For migration assays (top left panel), 15,000, 30,000, or 60,000 cyclin B2– (B2 −/−) and wt (B2 +/+) MEFs were seeded in serum-free medium on 5 μg/ml FN-coated transwell insert filters. After 4 h, cells were fixed and stained with crystal violet and the cells on the top and bottom of the filter were counted. The mean and SEM of 10 random fields is shown. For proliferation assays (top right panel), 10,000 cells were seeded in serum-free medium on 5 μg/ml FN-coated 96-well plates in the presence of 1 μCi [3H]thymidine per well. After 4 h, cells were processed to determine [3H]thymidine incorporation, as described in Materials and methods. For adhesion assays (bottom panels), 50,000 cells were seeded for 2 h in serum-free medium in a 96-well plate coated with increasing concentrations of FN as described in Materials and methods.
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fig5: Expression of cyclin B2 increases cell migration. (A) β3-LNCaP (β3-2) and HeLa cells cotransfected with pCMVβgal and pcDNA-3 (vector), pCMXcyclin A (A), pCMVcyclin B1 (B1), or pCMVcyclin B2 (B2) were processed 24 h after transfection, as described in Figs. 3 and 4. The mean and SEM of 10 random fields is shown. (B) Ectopically expressed cyclin B2 forms active kinase complex. Immunocomplexes precipitated from HeLa and cyclin B2–transfected HeLa RIPA extracts using nonimmune rabbit serum (n.i.), or rabbit polyclonal antibody to cyclin B2 (B2) or a c-myc agarose-conjugated rabbit polyclonal antibody (c-myc) were used in kinase assays using histone H1 as a substrate. (C) Cyclin B2– cells migrate poorly on FN. For migration assays (top left panel), 15,000, 30,000, or 60,000 cyclin B2– (B2 −/−) and wt (B2 +/+) MEFs were seeded in serum-free medium on 5 μg/ml FN-coated transwell insert filters. After 4 h, cells were fixed and stained with crystal violet and the cells on the top and bottom of the filter were counted. The mean and SEM of 10 random fields is shown. For proliferation assays (top right panel), 10,000 cells were seeded in serum-free medium on 5 μg/ml FN-coated 96-well plates in the presence of 1 μCi [3H]thymidine per well. After 4 h, cells were processed to determine [3H]thymidine incorporation, as described in Materials and methods. For adhesion assays (bottom panels), 50,000 cells were seeded for 2 h in serum-free medium in a 96-well plate coated with increasing concentrations of FN as described in Materials and methods.

Mentions: Mammalian cdc2 is known to associate with cyclins A, B1, and B2 (for review see Kohn, 1999). Ectopic expression of cyclin B2, but not cyclin A or cyclin B1, increased β3-LNCaP and HeLa cell migration on FN (Fig. 5 A), without affecting cell adhesion to this substrate (unpublished data). The ectopically transfected cyclin B2 was able to form active kinase complex, as shown by immunoprecipitation kinase assays of nontransfected versus cyclin B2-transfected HeLa cells using a cyclin B2 antibody, as well as by immunoprecipitation kinase assay of cyclin B2 transfected HeLa cells using a c-myc antibody (Fig. 5 B).


Alpha(v)beta3 integrin expression up-regulates cdc2, which modulates cell migration.

Manes T, Zheng DQ, Tognin S, Woodard AS, Marchisio PC, Languino LR - J. Cell Biol. (2003)

Expression of cyclin B2 increases cell migration. (A) β3-LNCaP (β3-2) and HeLa cells cotransfected with pCMVβgal and pcDNA-3 (vector), pCMXcyclin A (A), pCMVcyclin B1 (B1), or pCMVcyclin B2 (B2) were processed 24 h after transfection, as described in Figs. 3 and 4. The mean and SEM of 10 random fields is shown. (B) Ectopically expressed cyclin B2 forms active kinase complex. Immunocomplexes precipitated from HeLa and cyclin B2–transfected HeLa RIPA extracts using nonimmune rabbit serum (n.i.), or rabbit polyclonal antibody to cyclin B2 (B2) or a c-myc agarose-conjugated rabbit polyclonal antibody (c-myc) were used in kinase assays using histone H1 as a substrate. (C) Cyclin B2– cells migrate poorly on FN. For migration assays (top left panel), 15,000, 30,000, or 60,000 cyclin B2– (B2 −/−) and wt (B2 +/+) MEFs were seeded in serum-free medium on 5 μg/ml FN-coated transwell insert filters. After 4 h, cells were fixed and stained with crystal violet and the cells on the top and bottom of the filter were counted. The mean and SEM of 10 random fields is shown. For proliferation assays (top right panel), 10,000 cells were seeded in serum-free medium on 5 μg/ml FN-coated 96-well plates in the presence of 1 μCi [3H]thymidine per well. After 4 h, cells were processed to determine [3H]thymidine incorporation, as described in Materials and methods. For adhesion assays (bottom panels), 50,000 cells were seeded for 2 h in serum-free medium in a 96-well plate coated with increasing concentrations of FN as described in Materials and methods.
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fig5: Expression of cyclin B2 increases cell migration. (A) β3-LNCaP (β3-2) and HeLa cells cotransfected with pCMVβgal and pcDNA-3 (vector), pCMXcyclin A (A), pCMVcyclin B1 (B1), or pCMVcyclin B2 (B2) were processed 24 h after transfection, as described in Figs. 3 and 4. The mean and SEM of 10 random fields is shown. (B) Ectopically expressed cyclin B2 forms active kinase complex. Immunocomplexes precipitated from HeLa and cyclin B2–transfected HeLa RIPA extracts using nonimmune rabbit serum (n.i.), or rabbit polyclonal antibody to cyclin B2 (B2) or a c-myc agarose-conjugated rabbit polyclonal antibody (c-myc) were used in kinase assays using histone H1 as a substrate. (C) Cyclin B2– cells migrate poorly on FN. For migration assays (top left panel), 15,000, 30,000, or 60,000 cyclin B2– (B2 −/−) and wt (B2 +/+) MEFs were seeded in serum-free medium on 5 μg/ml FN-coated transwell insert filters. After 4 h, cells were fixed and stained with crystal violet and the cells on the top and bottom of the filter were counted. The mean and SEM of 10 random fields is shown. For proliferation assays (top right panel), 10,000 cells were seeded in serum-free medium on 5 μg/ml FN-coated 96-well plates in the presence of 1 μCi [3H]thymidine per well. After 4 h, cells were processed to determine [3H]thymidine incorporation, as described in Materials and methods. For adhesion assays (bottom panels), 50,000 cells were seeded for 2 h in serum-free medium in a 96-well plate coated with increasing concentrations of FN as described in Materials and methods.
Mentions: Mammalian cdc2 is known to associate with cyclins A, B1, and B2 (for review see Kohn, 1999). Ectopic expression of cyclin B2, but not cyclin A or cyclin B1, increased β3-LNCaP and HeLa cell migration on FN (Fig. 5 A), without affecting cell adhesion to this substrate (unpublished data). The ectopically transfected cyclin B2 was able to form active kinase complex, as shown by immunoprecipitation kinase assays of nontransfected versus cyclin B2-transfected HeLa cells using a cyclin B2 antibody, as well as by immunoprecipitation kinase assay of cyclin B2 transfected HeLa cells using a c-myc antibody (Fig. 5 B).

Bottom Line: We describe here a novel pathway that modulates cell migration and that is activated by alphavbeta3 and, as downstream effector, by cdc2 (cdk1).Third, cdc2 inhibitors reduce cell migration without affecting cell adhesion.These results show that cdc2 is a downstream effector of the alphavbeta3 integrin, and that it promotes cell migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Yale University School of Medicine, New Haven, CT 06510, USA.

ABSTRACT
The alphavbeta3 integrin has been shown to promote cell migration through activation of intracellular signaling pathways. We describe here a novel pathway that modulates cell migration and that is activated by alphavbeta3 and, as downstream effector, by cdc2 (cdk1). We report that alphavbeta3 expression in LNCaP (beta3-LNCaP) prostate cancer cells causes increased cdc2 mRNA levels as evaluated by gene expression analysis, and increased cdc2 protein and kinase activity levels. We provide three lines of evidence that increased levels of cdc2 contribute to a motile phenotype on integrin ligands in different cell types. First, increased levels of cdc2 correlate with more motile phenotypes of cancer cells. Second, ectopic expression of cdc2 increases cell migration, whereas expression of dominant-negative cdc2 inhibits migration. Third, cdc2 inhibitors reduce cell migration without affecting cell adhesion. We also show that cdc2 increases cell migration via specific association with cyclin B2, and we unravel a novel pathway of cell motility that involves, downstream of cdc2, caldesmon. cdc2 and caldesmon are shown here to localize in membrane ruffles in motile cells. These results show that cdc2 is a downstream effector of the alphavbeta3 integrin, and that it promotes cell migration.

Show MeSH
Related in: MedlinePlus