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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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Net1 knockdown inhibits Aurora A and Pak activation at centrosomes. (A) HeLa cells were transfected with control or Net1 siRNAs, fixed 72 h later, and stained for total Aurora A or phospho–threonine 288-Aurora A (red) and DNA (blue). Bar, 5 μm. (B) Quantification of total Aurora A or (C) phospho–Aurora A at the centrosome. Graphs represent average values from three independent experiments. Errors are SEM. Statistical significance was determined by Student's t test; *p < 0.05. (D) Distance between spindle poles in metaphase cells that were transfected with control or Net1 siRNAs. Values are from three independent experiments. Bars, median values. Statistical significance was determined by Student's t test; ***p < 0.001. (E) HeLa cells were transfected with control or Net1-specific siRNAs and then fixed and stained for phospho–T402-PAK (red) and DNA (blue). Bar, 5 μm. (F) Quantification of the phospho-Pak signal at the centrosome. Average of three independent experiments. Errors are SEM. Statistical significance was determined by Student's t test; **p < 0.01. (G) HeLa cells transfected with control and Net1 siRNAs were synchronized in prometaphase with nocodazole. Mitotic cells were collected by shake-off, and cell extracts were immunoprecipitated with control (IgG) or anti-PAK antibodies. Immunoprecipitates and total lysates were tested for the presence of GIT1, βPIX, PAK2, and Net1 by Western blotting. A representative experiment from three independent experiments. (H) Mitotic control or Net1 siRNA-transfected cells were collected as in G, and cell lysates were subject to immunoprecipitation with control IgG or Aurora A antibody. After washing, Aurora A kinase activity toward histone H3 serine 10 was assessed in vitro and visualized by Western blotting. A representative experiment from three independent experiments. (I) Mitotic control and Net1 siRNA–transfected cells were collected as in G, and cell lysates were tested by Western blotting with the indicated antibodies. A representative experiment from three independent experiments. (J) Control and Net1 siRNA–transfected cells were stained for TACC3 (red), α-tubulin (green), and DNA (blue). Bar, 5 μm. Representative maximum intensity z-planes from three independent experiments. (K) Western blot for phosphorylated (pTACC3) and total TACC3 from control and Net1 siRNA–transfected cells. A representative experiment from three independent experiments.
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Figure 8: Net1 knockdown inhibits Aurora A and Pak activation at centrosomes. (A) HeLa cells were transfected with control or Net1 siRNAs, fixed 72 h later, and stained for total Aurora A or phospho–threonine 288-Aurora A (red) and DNA (blue). Bar, 5 μm. (B) Quantification of total Aurora A or (C) phospho–Aurora A at the centrosome. Graphs represent average values from three independent experiments. Errors are SEM. Statistical significance was determined by Student's t test; *p < 0.05. (D) Distance between spindle poles in metaphase cells that were transfected with control or Net1 siRNAs. Values are from three independent experiments. Bars, median values. Statistical significance was determined by Student's t test; ***p < 0.001. (E) HeLa cells were transfected with control or Net1-specific siRNAs and then fixed and stained for phospho–T402-PAK (red) and DNA (blue). Bar, 5 μm. (F) Quantification of the phospho-Pak signal at the centrosome. Average of three independent experiments. Errors are SEM. Statistical significance was determined by Student's t test; **p < 0.01. (G) HeLa cells transfected with control and Net1 siRNAs were synchronized in prometaphase with nocodazole. Mitotic cells were collected by shake-off, and cell extracts were immunoprecipitated with control (IgG) or anti-PAK antibodies. Immunoprecipitates and total lysates were tested for the presence of GIT1, βPIX, PAK2, and Net1 by Western blotting. A representative experiment from three independent experiments. (H) Mitotic control or Net1 siRNA-transfected cells were collected as in G, and cell lysates were subject to immunoprecipitation with control IgG or Aurora A antibody. After washing, Aurora A kinase activity toward histone H3 serine 10 was assessed in vitro and visualized by Western blotting. A representative experiment from three independent experiments. (I) Mitotic control and Net1 siRNA–transfected cells were collected as in G, and cell lysates were tested by Western blotting with the indicated antibodies. A representative experiment from three independent experiments. (J) Control and Net1 siRNA–transfected cells were stained for TACC3 (red), α-tubulin (green), and DNA (blue). Bar, 5 μm. Representative maximum intensity z-planes from three independent experiments. (K) Western blot for phosphorylated (pTACC3) and total TACC3 from control and Net1 siRNA–transfected cells. A representative experiment from three independent experiments.

Mentions: Aurora A is a major regulator of mitotic progression. Its activity is required for initiation of mitosis, centrosomal maturation, and spindle assembly (Hirota et al., 2003; Dutertre et al., 2004; Kinoshita et al., 2005; Mori et al., 2007; Lu et al., 2008; Sasai et al., 2008; Venoux et al., 2008; Zhang et al., 2008; Carmena et al., 2009). Because cells lacking Net1 exhibited unstable mitotic spindles and failed to properly align their chromosomes during metaphase, we examined whether Aurora A activity was compromised by visualizing active Aurora A (pT288) or total Aurora A in mitotic cells. We observed that Net1 knockdown significantly inhibited endogenous Aurora A activation at the spindle poles and slightly inhibited overall spindle pole recruitment of Aurora A (Figure 8, A–C). This resulted in an increased distance between spindle poles (Figure 8D), which is consistent with aberrant mitotic microtubule motor activity caused by lack of Aurora A activation (Giet and Prigent, 2000; Bird and Hyman, 2008; Ma et al., 2011; Tanenbaum et al., 2011).


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

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

Net1 knockdown inhibits Aurora A and Pak activation at centrosomes. (A) HeLa cells were transfected with control or Net1 siRNAs, fixed 72 h later, and stained for total Aurora A or phospho–threonine 288-Aurora A (red) and DNA (blue). Bar, 5 μm. (B) Quantification of total Aurora A or (C) phospho–Aurora A at the centrosome. Graphs represent average values from three independent experiments. Errors are SEM. Statistical significance was determined by Student's t test; *p < 0.05. (D) Distance between spindle poles in metaphase cells that were transfected with control or Net1 siRNAs. Values are from three independent experiments. Bars, median values. Statistical significance was determined by Student's t test; ***p < 0.001. (E) HeLa cells were transfected with control or Net1-specific siRNAs and then fixed and stained for phospho–T402-PAK (red) and DNA (blue). Bar, 5 μm. (F) Quantification of the phospho-Pak signal at the centrosome. Average of three independent experiments. Errors are SEM. Statistical significance was determined by Student's t test; **p < 0.01. (G) HeLa cells transfected with control and Net1 siRNAs were synchronized in prometaphase with nocodazole. Mitotic cells were collected by shake-off, and cell extracts were immunoprecipitated with control (IgG) or anti-PAK antibodies. Immunoprecipitates and total lysates were tested for the presence of GIT1, βPIX, PAK2, and Net1 by Western blotting. A representative experiment from three independent experiments. (H) Mitotic control or Net1 siRNA-transfected cells were collected as in G, and cell lysates were subject to immunoprecipitation with control IgG or Aurora A antibody. After washing, Aurora A kinase activity toward histone H3 serine 10 was assessed in vitro and visualized by Western blotting. A representative experiment from three independent experiments. (I) Mitotic control and Net1 siRNA–transfected cells were collected as in G, and cell lysates were tested by Western blotting with the indicated antibodies. A representative experiment from three independent experiments. (J) Control and Net1 siRNA–transfected cells were stained for TACC3 (red), α-tubulin (green), and DNA (blue). Bar, 5 μm. Representative maximum intensity z-planes from three independent experiments. (K) Western blot for phosphorylated (pTACC3) and total TACC3 from control and Net1 siRNA–transfected cells. A representative experiment from three independent experiments.
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Figure 8: Net1 knockdown inhibits Aurora A and Pak activation at centrosomes. (A) HeLa cells were transfected with control or Net1 siRNAs, fixed 72 h later, and stained for total Aurora A or phospho–threonine 288-Aurora A (red) and DNA (blue). Bar, 5 μm. (B) Quantification of total Aurora A or (C) phospho–Aurora A at the centrosome. Graphs represent average values from three independent experiments. Errors are SEM. Statistical significance was determined by Student's t test; *p < 0.05. (D) Distance between spindle poles in metaphase cells that were transfected with control or Net1 siRNAs. Values are from three independent experiments. Bars, median values. Statistical significance was determined by Student's t test; ***p < 0.001. (E) HeLa cells were transfected with control or Net1-specific siRNAs and then fixed and stained for phospho–T402-PAK (red) and DNA (blue). Bar, 5 μm. (F) Quantification of the phospho-Pak signal at the centrosome. Average of three independent experiments. Errors are SEM. Statistical significance was determined by Student's t test; **p < 0.01. (G) HeLa cells transfected with control and Net1 siRNAs were synchronized in prometaphase with nocodazole. Mitotic cells were collected by shake-off, and cell extracts were immunoprecipitated with control (IgG) or anti-PAK antibodies. Immunoprecipitates and total lysates were tested for the presence of GIT1, βPIX, PAK2, and Net1 by Western blotting. A representative experiment from three independent experiments. (H) Mitotic control or Net1 siRNA-transfected cells were collected as in G, and cell lysates were subject to immunoprecipitation with control IgG or Aurora A antibody. After washing, Aurora A kinase activity toward histone H3 serine 10 was assessed in vitro and visualized by Western blotting. A representative experiment from three independent experiments. (I) Mitotic control and Net1 siRNA–transfected cells were collected as in G, and cell lysates were tested by Western blotting with the indicated antibodies. A representative experiment from three independent experiments. (J) Control and Net1 siRNA–transfected cells were stained for TACC3 (red), α-tubulin (green), and DNA (blue). Bar, 5 μm. Representative maximum intensity z-planes from three independent experiments. (K) Western blot for phosphorylated (pTACC3) and total TACC3 from control and Net1 siRNA–transfected cells. A representative experiment from three independent experiments.
Mentions: Aurora A is a major regulator of mitotic progression. Its activity is required for initiation of mitosis, centrosomal maturation, and spindle assembly (Hirota et al., 2003; Dutertre et al., 2004; Kinoshita et al., 2005; Mori et al., 2007; Lu et al., 2008; Sasai et al., 2008; Venoux et al., 2008; Zhang et al., 2008; Carmena et al., 2009). Because cells lacking Net1 exhibited unstable mitotic spindles and failed to properly align their chromosomes during metaphase, we examined whether Aurora A activity was compromised by visualizing active Aurora A (pT288) or total Aurora A in mitotic cells. We observed that Net1 knockdown significantly inhibited endogenous Aurora A activation at the spindle poles and slightly inhibited overall spindle pole recruitment of Aurora A (Figure 8, A–C). This resulted in an increased distance between spindle poles (Figure 8D), which is consistent with aberrant mitotic microtubule motor activity caused by lack of Aurora A activation (Giet and Prigent, 2000; Bird and Hyman, 2008; Ma et al., 2011; Tanenbaum et al., 2011).

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