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Rho GTPase-independent regulation of mitotic progression by the RhoGEF Net1.

Menon S, Oh W, Carr HS, Frost JA - Mol. Biol. Cell (2013)

Bottom Line: Accordingly, inhibition of Net1 expression results in spindle assembly checkpoint activation.The ability of Net1 to control mitosis is independent of RhoA or RhoB activation, as knockdown of either GTPase does not phenocopy effects of Net1 knockdown on nuclear morphology, and effects of Net1 knockdown are effectively rescued by expression of catalytically inactive Net1.These results identify Net1 as a novel regulator of mitosis and indicate that altered expression of Net1, as occurs in human cancers, may adversely affect genomic stability.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, TX 77008, USA.

ABSTRACT
Neuroepithelial transforming gene 1 (Net1) is a RhoA-subfamily-specific guanine nucleotide exchange factor that is overexpressed in multiple human cancers and is required for proliferation. Molecular mechanisms underlying its role in cell proliferation are unknown. Here we show that overexpression or knockdown of Net1 causes mitotic defects. Net1 is required for chromosome congression during metaphase and generation of stable kinetochore microtubule attachments. Accordingly, inhibition of Net1 expression results in spindle assembly checkpoint activation. The ability of Net1 to control mitosis is independent of RhoA or RhoB activation, as knockdown of either GTPase does not phenocopy effects of Net1 knockdown on nuclear morphology, and effects of Net1 knockdown are effectively rescued by expression of catalytically inactive Net1. We also observe that Net1 expression is required for centrosomal activation of p21-activated kinase and its downstream kinase Aurora A, which are critical regulators of centrosome maturation and spindle assembly. These results identify Net1 as a novel regulator of mitosis and indicate that altered expression of Net1, as occurs in human cancers, may adversely affect genomic stability.

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Expression of wild-type or catalytically inactive Net1 rescues nuclear morphology in Net1-depleted cells. HeLa cells were transfected with control (A) or Net1-specific (B) siRNAs. One day later the cells were transfected with plasmids expressing siRNA-resistant, HA-epitope-tagged wild-type or catalytically inactive Net1 (L321E). Two days later the cells were fixed and stained for HA-epitope expression (green), α-tubulin (red), CREST (purple), and DNA (blue). Representative micrographs. Bar, 10 μm. (C) Quantification of aberrant nuclear morphology. Average of three independent experiments. Errors are SEM. Statistical significance compared with control values was determined by Student's t test; *p < 0.05; **p < 0.01; ***p < 0.001. (D) Assessment of the activation state of wild-type and catalytically inactive Net1. HeLa cells were transfected with HA-epitope-tagged wild-type Net1 or Net1 L321E. Two days later the cells were lysed and tested for interaction with GST or GST-A17RhoA in pull-down assays. Left, Western blots for HA-Net1 proteins and GST in the glutathione-agarose pull downs. Right, Western blots of HA-Net1 proteins and GAPDH in cell lysates. A representative experiment from three independent experiments. (E) Quantification of Net1 activity assays. Statistical significance was determined by Student's t test; ***p < 0.001.
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Figure 7: Expression of wild-type or catalytically inactive Net1 rescues nuclear morphology in Net1-depleted cells. HeLa cells were transfected with control (A) or Net1-specific (B) siRNAs. One day later the cells were transfected with plasmids expressing siRNA-resistant, HA-epitope-tagged wild-type or catalytically inactive Net1 (L321E). Two days later the cells were fixed and stained for HA-epitope expression (green), α-tubulin (red), CREST (purple), and DNA (blue). Representative micrographs. Bar, 10 μm. (C) Quantification of aberrant nuclear morphology. Average of three independent experiments. Errors are SEM. Statistical significance compared with control values was determined by Student's t test; *p < 0.05; **p < 0.01; ***p < 0.001. (D) Assessment of the activation state of wild-type and catalytically inactive Net1. HeLa cells were transfected with HA-epitope-tagged wild-type Net1 or Net1 L321E. Two days later the cells were lysed and tested for interaction with GST or GST-A17RhoA in pull-down assays. Left, Western blots for HA-Net1 proteins and GST in the glutathione-agarose pull downs. Right, Western blots of HA-Net1 proteins and GAPDH in cell lysates. A representative experiment from three independent experiments. (E) Quantification of Net1 activity assays. Statistical significance was determined by Student's t test; ***p < 0.001.

Mentions: If our model is correct, then the catalytic activity of Net1 should be dispensable for mitotic progression. We therefore tested whether the mitotic defect resulting from Net1 knockdown could be equally rescued by expression of siRNA-resistant, wild-type, or catalytically inactive Net1. This approach required careful titration of Net1 expression, since overexpression of Net1 itself causes mitotic defects (Figure 1). HeLa cells were transfected with control or Net1 siRNAs and later retransfected with siRNA-resistant, wild-type mouse Net1, or Net1 L321E. Net1 L321E was been reported to be unable to activate RhoA or stimulate actin polymerization in cells (Alberts and Treisman, 1998; Schmidt and Hall, 2002). Two days later the cells were fixed and stained for Net1 expression, α-tubulin, and DNA, and then examined for aberrations in nuclear morphology. In these experiments, overexpression of wild-type Net1 or Net1 L321E in control siRNA–transfected cells caused >40% of cells to exhibit abnormally shaped nuclei, with the majority of these being micronucleated (Figure 7, A and C). These effects were slightly less robust than observed in earlier experiments (Figure 1), as less Net1 plasmid was transfected. Of importance, overexpression of Net1 L321E did not result in accumulation of multinucleated cells, which would have been expected if it were competent to stimulate RhoA activation. On the other hand, in Net1 siRNA–transfected cells, expression of wild-type Net1 or catalytically inactive Net1 L321E was equally efficient at rescuing nuclear morphology (Figure 7, B and C). To confirm that the Net1-L321E mutant is catalytically inactive, we performed GST-A17RhoA pull-down assays. A17RhoA is a nucleotide-free form of RhoA that binds tightly to active RhoGEFs and can be used as a probe to detect RhoGEF activation (Garcia-Mata et al., 2006; Carr et al., 2012). Wild-type Net1 bound strongly to GST-A17RhoA but Net1-L321E did not, confirming that this mutant is catalytically inactive (Figure 7, D and E). Taken together, these data indicate that Net1 does not mediate mitotic progression through the activation of Rho GTPases.


Rho GTPase-independent regulation of mitotic progression by the RhoGEF Net1.

Menon S, Oh W, Carr HS, Frost JA - Mol. Biol. Cell (2013)

Expression of wild-type or catalytically inactive Net1 rescues nuclear morphology in Net1-depleted cells. HeLa cells were transfected with control (A) or Net1-specific (B) siRNAs. One day later the cells were transfected with plasmids expressing siRNA-resistant, HA-epitope-tagged wild-type or catalytically inactive Net1 (L321E). Two days later the cells were fixed and stained for HA-epitope expression (green), α-tubulin (red), CREST (purple), and DNA (blue). Representative micrographs. Bar, 10 μm. (C) Quantification of aberrant nuclear morphology. Average of three independent experiments. Errors are SEM. Statistical significance compared with control values was determined by Student's t test; *p < 0.05; **p < 0.01; ***p < 0.001. (D) Assessment of the activation state of wild-type and catalytically inactive Net1. HeLa cells were transfected with HA-epitope-tagged wild-type Net1 or Net1 L321E. Two days later the cells were lysed and tested for interaction with GST or GST-A17RhoA in pull-down assays. Left, Western blots for HA-Net1 proteins and GST in the glutathione-agarose pull downs. Right, Western blots of HA-Net1 proteins and GAPDH in cell lysates. A representative experiment from three independent experiments. (E) Quantification of Net1 activity assays. Statistical significance was determined by Student's t test; ***p < 0.001.
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Figure 7: Expression of wild-type or catalytically inactive Net1 rescues nuclear morphology in Net1-depleted cells. HeLa cells were transfected with control (A) or Net1-specific (B) siRNAs. One day later the cells were transfected with plasmids expressing siRNA-resistant, HA-epitope-tagged wild-type or catalytically inactive Net1 (L321E). Two days later the cells were fixed and stained for HA-epitope expression (green), α-tubulin (red), CREST (purple), and DNA (blue). Representative micrographs. Bar, 10 μm. (C) Quantification of aberrant nuclear morphology. Average of three independent experiments. Errors are SEM. Statistical significance compared with control values was determined by Student's t test; *p < 0.05; **p < 0.01; ***p < 0.001. (D) Assessment of the activation state of wild-type and catalytically inactive Net1. HeLa cells were transfected with HA-epitope-tagged wild-type Net1 or Net1 L321E. Two days later the cells were lysed and tested for interaction with GST or GST-A17RhoA in pull-down assays. Left, Western blots for HA-Net1 proteins and GST in the glutathione-agarose pull downs. Right, Western blots of HA-Net1 proteins and GAPDH in cell lysates. A representative experiment from three independent experiments. (E) Quantification of Net1 activity assays. Statistical significance was determined by Student's t test; ***p < 0.001.
Mentions: If our model is correct, then the catalytic activity of Net1 should be dispensable for mitotic progression. We therefore tested whether the mitotic defect resulting from Net1 knockdown could be equally rescued by expression of siRNA-resistant, wild-type, or catalytically inactive Net1. This approach required careful titration of Net1 expression, since overexpression of Net1 itself causes mitotic defects (Figure 1). HeLa cells were transfected with control or Net1 siRNAs and later retransfected with siRNA-resistant, wild-type mouse Net1, or Net1 L321E. Net1 L321E was been reported to be unable to activate RhoA or stimulate actin polymerization in cells (Alberts and Treisman, 1998; Schmidt and Hall, 2002). Two days later the cells were fixed and stained for Net1 expression, α-tubulin, and DNA, and then examined for aberrations in nuclear morphology. In these experiments, overexpression of wild-type Net1 or Net1 L321E in control siRNA–transfected cells caused >40% of cells to exhibit abnormally shaped nuclei, with the majority of these being micronucleated (Figure 7, A and C). These effects were slightly less robust than observed in earlier experiments (Figure 1), as less Net1 plasmid was transfected. Of importance, overexpression of Net1 L321E did not result in accumulation of multinucleated cells, which would have been expected if it were competent to stimulate RhoA activation. On the other hand, in Net1 siRNA–transfected cells, expression of wild-type Net1 or catalytically inactive Net1 L321E was equally efficient at rescuing nuclear morphology (Figure 7, B and C). To confirm that the Net1-L321E mutant is catalytically inactive, we performed GST-A17RhoA pull-down assays. A17RhoA is a nucleotide-free form of RhoA that binds tightly to active RhoGEFs and can be used as a probe to detect RhoGEF activation (Garcia-Mata et al., 2006; Carr et al., 2012). Wild-type Net1 bound strongly to GST-A17RhoA but Net1-L321E did not, confirming that this mutant is catalytically inactive (Figure 7, D and E). Taken together, these data indicate that Net1 does not mediate mitotic progression through the activation of Rho GTPases.

Bottom Line: Accordingly, inhibition of Net1 expression results in spindle assembly checkpoint activation.The ability of Net1 to control mitosis is independent of RhoA or RhoB activation, as knockdown of either GTPase does not phenocopy effects of Net1 knockdown on nuclear morphology, and effects of Net1 knockdown are effectively rescued by expression of catalytically inactive Net1.These results identify Net1 as a novel regulator of mitosis and indicate that altered expression of Net1, as occurs in human cancers, may adversely affect genomic stability.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, TX 77008, USA.

ABSTRACT
Neuroepithelial transforming gene 1 (Net1) is a RhoA-subfamily-specific guanine nucleotide exchange factor that is overexpressed in multiple human cancers and is required for proliferation. Molecular mechanisms underlying its role in cell proliferation are unknown. Here we show that overexpression or knockdown of Net1 causes mitotic defects. Net1 is required for chromosome congression during metaphase and generation of stable kinetochore microtubule attachments. Accordingly, inhibition of Net1 expression results in spindle assembly checkpoint activation. The ability of Net1 to control mitosis is independent of RhoA or RhoB activation, as knockdown of either GTPase does not phenocopy effects of Net1 knockdown on nuclear morphology, and effects of Net1 knockdown are effectively rescued by expression of catalytically inactive Net1. We also observe that Net1 expression is required for centrosomal activation of p21-activated kinase and its downstream kinase Aurora A, which are critical regulators of centrosome maturation and spindle assembly. These results identify Net1 as a novel regulator of mitosis and indicate that altered expression of Net1, as occurs in human cancers, may adversely affect genomic stability.

Show MeSH
Related in: MedlinePlus