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The Neuroepithelial transforming gene 1 (Net1) is a RhoA subfamily guanine nucleotide exchange factor that is overexpressed in a number of cancers and contributes to cancer cell motility and proliferation. Net1 also plays a Rho GTPase independent role in mitotic progression, where it promotes centrosomal activation of Aurora A and Pak2, and aids in chromosome alignment during prometaphase. To understand regulatory mechanisms controlling the mitotic function of Net1, we examined whether it was phosphorylated by the mitotic kinase Cdk1. We observed that Cdk1 phosphorylated Net1 on multiple sites in its N-terminal regulatory domain and C-terminus in vitro. By raising phospho-specific antibodies to two of these sites, we also demonstrated that both endogenous and transfected Net1 were phosphorylated by Cdk1 in cells. Substitution of the major Cdk1 phosphorylation sites with aliphatic or acidic residues inhibited the interaction of Net1 with RhoA, and treatment of metaphase cells with a Cdk1 inhibitor increased Net1 activity. Cdk1 inhibition also increased Net1 localization to the plasma membrane and stimulated cortical F-actin accumulation. Moreover, Net1 overexpression caused spindle polarity defects that were reduced in frequency by acidic substitution of the major Cdk1 phosphorylation sites. These data indicate that Cdk1 phosphorylates Net1 during mitosis and suggest that this negatively regulates its ability to signal to RhoA and alter actin cytoskeletal organization.

ATM activation following DNA damage is a critical event which is required for efficient DNA repair and cell survival, yet signaling mechanisms controlling its activation are incompletely understood. The RhoGEF Net1 has previously been reported to control Rho GTPase activation and downstream cell survival outcomes following double strand DNA damage. However the role of Net1 isoforms in controlling ATM-dependent cell signaling has not been assessed. In the present work we show that expression of the Net1A isoform is specifically required for efficient activation of ATM but not the related kinase DNA-PK after ionizing radiation. Surprisingly Net1A overexpression also potently suppresses ATM activation and phosphorylation of its substrate H2AX. This effect does not require catalytic activity towards RhoA or RhoB, and neither Rho GTPase affects ATM activation, on its own. Consistent with a role in controlling ATM activation, Net1A knockdown also impairs DNA repair and cell survival. Taken together these data indicate that Net1A plays a plays a previously unrecognized, Rho GTPase-independent role in controlling ATM activity and downstream signaling after DNA damage to impact cell survival.

Mechanisms accounting for the role of Net1 in cell proliferation have not been described. This study shows that Net1 plays a Rho GTPase–independent role in controlling mitotic spindle assembly and kinetochore attachment and is required for centrosome activation of Pak2 and Aurora A., 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.

Net1 is a RhoA guanine nucleotide exchange factor (GEF) that is overexpressed in a subset of human cancers and contributes to cancer cell motility and invasion in vitro. However, the molecular mechanism accounting for its role in cell motility and invasion has not been described. In the present work, we show that expression of both Net1 isoforms in breast cancer cells is required for efficient cell motility. Although loss of Net1 isoform expression only partially blocks RhoA activation, it inhibits lysophosphatidic acid (LPA)-stimulated migration as efficiently as knockdown of RhoA itself. However, we demonstrate that the Net1A isoform predominantly controls myosin light-chain phosphorylation and is required for trailing edge retraction during migration. Net1A interacts with focal adhesion kinase (FAK), localizes to focal adhesions, and is necessary for FAK activation and focal adhesion maturation during cell spreading. Net1A expression is also required for efficient invasion through a Matrigel matrix. Analysis of invading cells demonstrates that Net1A is required for amoeboid invasion, and loss of Net1A expression causes cells to shift to a mesenchymal phenotype characterized by high β1-integrin activity and membrane type 1 matrix metalloproteinase (MT1-MMP) expression. These results demonstrate a previously unrecognized role for the Net1A isoform in controlling FAK activation during planar cell movement and amoeboid motility during extracellular matrix (ECM) invasion.

The neuroepithelial cell transforming gene 1A (Net1A, an isoform of Net1) is a RhoA subfamily guanine nucleotide exchange factor (GEF) that localizes to the nucleus in the absence of stimulation, preventing it from activating RhoA. Once relocalized in the cytosol, Net1A stimulates cell motility and extracellular matrix invasion. In the present work, we investigated mechanisms responsible for the cytosolic relocalization of Net1A. We demonstrate that inhibition of MAPK pathways blocks Net1A relocalization, with cells being most sensitive to JNK pathway inhibition. Moreover, activation of the JNK or p38 MAPK family pathway is sufficient to elicit Net1A cytosolic localization. Net1A relocalization stimulated by EGF or JNK activation requires nuclear export mediated by CRM1. JNK1 (also known as MAPK8) phosphorylates Net1A on serine 52, and alanine substitution at this site prevents Net1A relocalization caused by EGF or JNK activation. Glutamic acid substitution at this site is sufficient for Net1A relocalization and results in elevated RhoA signaling to stimulate myosin light chain 2 (MLC2, also known as MYL2) phosphorylation and F-actin accumulation. Net1A S52E expression stimulates cell motility, enables Matrigel invasion and promotes invadopodia formation. These data highlight a novel mechanism for controlling the subcellular localization of Net1A to regulate RhoA activation, cell motility, and invasion.

Net1 isoform A (Net1A) is a RhoA GEF that is required for cell motility and invasion in multiple cancers. Nuclear localization of Net1A negatively regulates its activity, and we have recently shown that Rac1 stimulates Net1A relocalization to the plasma membrane to promote RhoA activation and cytoskeletal reorganization. However, mechanisms controlling the subcellular localization of Net1A are not well understood. Here, we show that Net1A contains two nuclear localization signal (NLS) sequences within its N-terminus and that residues surrounding the second NLS sequence are acetylated. Treatment of cells with deacetylase inhibitors or expression of active Rac1 promotes Net1A acetylation. Deacetylase inhibition is sufficient for Net1A relocalization outside the nucleus, and replacement of the N-terminal acetylation sites with arginine residues prevents cytoplasmic accumulation of Net1A caused by deacetylase inhibition or EGF stimulation. By contrast, replacement of these sites with glutamine residues is sufficient for Net1A relocalization, RhoA activation and downstream signaling. Moreover, the N-terminal acetylation sites are required for rescue of F-actin accumulation and focal adhesion maturation in Net1 knockout MEFs. These data indicate that Net1A acetylation regulates its subcellular localization to impact on RhoA activity and actin cytoskeletal organization.

The Neuroepithelioma transforming gene 1 (Net1) is a RhoA specific guanine nucleotide exchange factor (GEF) that is frequently overexpressed in human cancer, including breast cancer. We have previously reported that DNA damage activates Net1 to control RhoA and p38 MAPK mediated cell survival pathway in response to ionizing radiation (IR). However, others have shown that Net1 activation contributes to RhoB-mediated cell death after IR. Thus, the role of Net1 in controlling IR responses and cell survival is controversial. With the completion of the first year of this fellowship, we have found that the Net1A isoform is specifically required for DNA double-strand break (DSB)-induced signaling and DNA repair. Depletion of Net1A in human breast cancer cells reduced IR-stimulated ATM activation and signaling to its substrates Chk2 and H2AX. In addition, suppression of Net1A expression adversely affected cell survival after IR. Moreover, we observed that overexpression of the Net1A isoform significantly reduced yH2AX foci formation after IR, which required the unique N-terminal region of Net1A. Importantly, this effect did not require Rho GTPase activation by Net1A, and was not recapitulated by overexpression of RhoA or RhoB. Net1A was also found to co-immunoprecipitate with the DNA-PK complex in an IR-regulated manner. Taken together, our current data suggests a model in which Net1A functions as a non-catalytic binding protein to control DNA damage response signaling and DNA repair to affect cell survival after IR.

The Neuroepithelioma transforming gene 1 (Net1) is a RhoGEF that is frequently overexpressed in human cancer, including breast cancer. We have previously reported that DNA damage activates Net1 to control p38 MAPK signaling and protect cells from ionizing radiation (IR) induced apoptosis. However, others have shown that Net1 activation contributes to cell death by activating a RhoB-Bim signaling pathway. Thus, the role of Net1 in controlling IR responses and cell survival is controversial. Here we show that the Net1A isoform is uniquely important for DNA double-strand break (DSB)-induced signaling and DNA repair. SiRNA-mediated knockdown of Net1A in human breast cancer cells reduced IR-stimulated ATM activation and signaling to its substrates Chk2 and H2AX. In addition, suppression of Net1A expression adversely affected cell survival after IR. We also observed that Net1A overexpression significantly reduced γH2AX foci formation after IR, which required the unique N-terminal region of Net1A. Importantly, this effect did not require Rho GTPase activation by Net1A, and was not recapitulated by overexpression of RhoA or RhoB. Using comet assays, we found that Net1A depletion substantially slowed DNA DSB repair in response to IR. Intriguingly, knockdown of Net1A promoted non-homologous end joining (NHEJ) in the absence of DNA damage, and Net1A was found to co-immunoprecipitate with the DNA-PK complex in an IR-regulated manner. Taken together, these data suggest a model in which Net1A functions as a non-catalytic binding protein to control DNA damage response signaling and DNA repair to affect cell survival after IR. Citation Format: Wonkyung Oh, Jeffrey A. Frost. Regulation of ATM-dependent DNA damage signaling in human breast cancer cells by the RhoGEF Net1A . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1782. doi:10.1158/1538-7445.AM2013-1782