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• W2077856249 abstract "Rho family small G proteins are key regulators of cytoskeletal organization and oncogenic transformation whose activation is controlled by a family of proteins known as guanine nucleotide exchange factors (GEFs). In this work we have characterized the structural and biological determinants for cytoskeletal regulation and cell transformation by the neuroepithelioma transforming gene 1 (NET1), which is a GEF specific for RhoA, but not Cdc42 or Rac1. Previously it was shown that the biological activity and nuclear localization of NET1 is controlled by its amino terminus. Here we demonstrate that the amino terminus of NET1 does not function as cis-acting autoinhibitory domain, nor does it affect the ability of full-length NET1 to stimulate actin stress fiber formation. We also show that the nuclear localization of NET1 is controlled by two separate domains within its amino terminus, only one of which contains the previously identified NLS sequences. Importantly, we find that the ability of NET1 to stimulate actin stress fiber formation does not correlate with its transforming activity, because NET1 proteins that potently stimulate stress fiber formation do not transform cells. Furthermore, the presence of a potential PDZ binding site in the C terminus of NET1 is critical to its ability to transform cells, but is not required for enzymatic activity or for effects on the actin cytoskeleton. Thus, these data highlight a divergence between the ability of NET1 to stimulate cytoskeletal reorganization and to transform cells, and implicate the interaction with PDZ domain-containing proteins as critical to NET1-dependent transformation. Rho family small G proteins are key regulators of cytoskeletal organization and oncogenic transformation whose activation is controlled by a family of proteins known as guanine nucleotide exchange factors (GEFs). In this work we have characterized the structural and biological determinants for cytoskeletal regulation and cell transformation by the neuroepithelioma transforming gene 1 (NET1), which is a GEF specific for RhoA, but not Cdc42 or Rac1. Previously it was shown that the biological activity and nuclear localization of NET1 is controlled by its amino terminus. Here we demonstrate that the amino terminus of NET1 does not function as cis-acting autoinhibitory domain, nor does it affect the ability of full-length NET1 to stimulate actin stress fiber formation. We also show that the nuclear localization of NET1 is controlled by two separate domains within its amino terminus, only one of which contains the previously identified NLS sequences. Importantly, we find that the ability of NET1 to stimulate actin stress fiber formation does not correlate with its transforming activity, because NET1 proteins that potently stimulate stress fiber formation do not transform cells. Furthermore, the presence of a potential PDZ binding site in the C terminus of NET1 is critical to its ability to transform cells, but is not required for enzymatic activity or for effects on the actin cytoskeleton. Thus, these data highlight a divergence between the ability of NET1 to stimulate cytoskeletal reorganization and to transform cells, and implicate the interaction with PDZ domain-containing proteins as critical to NET1-dependent transformation. Rho family small G proteins play critical roles in regulating many aspects of cell physiology, including cytoskeletal organization, cell motility, vesicle trafficking, cell cycle progression, and neoplastic transformation (1Etienne-Manneville S. Hall A. Nature. 2002; 420: 629-635Crossref PubMed Scopus (3857) Google Scholar, 2Symons M. Rusk N. Curr. Biol. 2003; 13: R409-R418Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 3Pruitt K. Der C.J. Cancer Lett. 2001; 171: 1-10Crossref PubMed Scopus (250) Google Scholar, 4Jaffe A.B. Hall A. Adv. Cancer Res. 2002; 84: 57-80Crossref PubMed Scopus (255) Google Scholar). They do so by acting as molecular switches, cycling between their active, GTP-bound and inactive, GDP-bound states. Once activated, they stimulate signaling in multiple pathways by binding to downstream effector proteins and modulating their activities. Currently at least 21 mammalian Rho family GTPases have been identified, with the Rac1, Cdc42, and RhoA proteins being the most thoroughly characterized (5Wherlock M. Mellor H. J. Cell Sci. 2002; 115: 239-240Crossref PubMed Google Scholar). The activities of wild type Rho family proteins are controlled by three classes of enzymes known as Rho guanine nucleotide exchange factors (Rho GEFs 1The abbreviations used are: GEF, guanine nucleotide exchange factor; Dbl, diffuse B-cell lymphoma; DH, Dbl homology; PH, pleckstrin homology; NET1, neuroepithelioma transforming gene 1; wt, wild type; NLS, nuclear localization signal; NES, nuclear export signal; GST, glutathione S-transferase; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; GTPγS, guanosine 5′-3-O-(thio)triphosphate; PBS, phosphate-buffered saline; HA, hemagglutinin.), Rho GTPase-activating proteins, and Rho guanine nucleotide dissociation inhibitors (Rho GDIs) (6Symons M. Settleman J. Trends Cell Biol. 2000; 10: 415-419Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Rho GEFs stimulate the dissociation of GDP from inactive Rho proteins, thereby promoting their accumulation in the active, GTP-bound state. Rho GTPase-activating proteins stimulate the intrinsic GTPase activity of Rho proteins, thus causing their inactivation. Rho guanine nucleotide dissociation inhibitors are cytosolic proteins that bind to inactive, GDP-bound Rho proteins and localize them to the cytosol. Within this regulatory network, it is the Rho GEFs that mediate Rho protein activation in response to extracellular ligands. Thus, elucidating the regulatory mechanisms controlling Rho GEF activity is critical to understanding how growth factors control Rho-dependent signaling. The human Rho GEF family contains over 50 genes, all of which contain two conserved domains known as the Dbl (diffuse B-cell lymphoma) homology (DH) and pleckstrin homology (PH) domains (7Zheng Y. Trends Biochem. Sci. 2001; 26: 724-732Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar, 8Schmidt A. Hall A. Genes Dev. 2002; 16: 1587-1609Crossref PubMed Scopus (982) Google Scholar, 9Hoffman G.R. Cerione R.A. FEBS Lett. 2002; 513: 85-91Crossref PubMed Scopus (117) Google Scholar). The DH domain is named after the first mammalian Rho GEF identified and accounts for the enzymatic activity of these proteins. The function of the PH domain varies depending upon the Rho GEF being analyzed. Biochemical roles for the PH domain include subcellular targeting, stabilization of the DH domain and enhancement of its activity, and regulation of the interaction with substrate Rho proteins (10Zheng Y. Zangrilli D. Cerione R.A. Eva A. J. Biol. Chem. 1996; 271: 19017-19020Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 11Michiels F. Stam J.C. Hordijk P.L. van der Kammen R.A. Ruuls-Van Stalle L. Feltkamp C.A. Collard J.G. J. Cell Biol. 1997; 137: 387-398Crossref PubMed Scopus (211) Google Scholar, 12Han J. Luby-Phelps K. Das B. Shu X. Xia Y. Mosteller R.D. Krishna U.M. Falck J.R. White M.A. Broek D. Science. 1998; 279: 558-560Crossref PubMed Scopus (710) Google Scholar, 13Whitehead I.P. Lambert Q.T. Glaven J.A. Abe K. Rossman K.L. Mahon G.M. Trzaskos J.M. Kay R. Campbell S.L. Der C.J. Mol. Cell. Biol. 1999; 19: 7759-7770Crossref PubMed Google Scholar, 14Liu X. Wang H. Eberstadt M. Schnuchel A. Olejniczak E.T. Meadows R.P. Schkeryantz J.M. Janowick D.A. Harlan J.E. Harris E.A. Staunton D.E. Fesik S.W. Cell. 1998; 95: 269-277Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 15Worthylake D.K. Rossman K.L. Sondek J. Nature. 2000; 408: 682-688Crossref PubMed Scopus (305) Google Scholar, 16Das B. Shu X. Day G.J. Han J. Krishna U.M. Falck J.R. Broek D. J. Biol. Chem. 2000; 275: 15074-15081Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). All DH domain-containing Rho GEFs require the presence of a PH domain for activity in the cell. Rho GEFs exhibit distinct specificities for different Rho family small G proteins in vitro and in cells. For example, the Rho GEF Vav activates RhoA, Rac1, and Cdc42 equally well, whereas the Rho GEF Tiam1 is specific for Rac1 (7Zheng Y. Trends Biochem. Sci. 2001; 26: 724-732Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar, 8Schmidt A. Hall A. Genes Dev. 2002; 16: 1587-1609Crossref PubMed Scopus (982) Google Scholar). The biochemical mechanisms controlling the activities of most Rho GEFs have not been characterized. However, for those few that have been studied, two common regulatory themes have emerged. First, many Rho GEFs contain autoinhibitory domains that negatively regulate their enzymatic activities in the absence of an appropriate stimulus. When these domains are deleted, the Rho GEFs are constitutively activated in vitro and are oncogenic in cell transformation assays (8Schmidt A. Hall A. Genes Dev. 2002; 16: 1587-1609Crossref PubMed Scopus (982) Google Scholar, 17Whitehead I.P. Campbell S. Rossman K.L. Der C.J. Biochim. Biophys. Acta. 1997; 1332: F1-23Crossref PubMed Scopus (334) Google Scholar). Physiological mechanisms for release from autoinhibition include altered protein-protein interactions, binding to phosphatidylinositol phosphates, and site-specific phosphorylation. For example, Vav is activated by phosphorylation on tyrosine 174 and binding to phosphatidylinositol 3,4,5-trisphosphate, which together block the effects of an amino-terminal autoinhibitory domain (12Han J. Luby-Phelps K. Das B. Shu X. Xia Y. Mosteller R.D. Krishna U.M. Falck J.R. White M.A. Broek D. Science. 1998; 279: 558-560Crossref PubMed Scopus (710) Google Scholar, 18Han J. Das B. Wei W. Van Aelst L. Mosteller R.D. Khosravi-Far R. Westwick J.K. Der C.J. Broek D. Mol. Cell. Biol. 1997; 17: 1346-1353Crossref PubMed Scopus (276) Google Scholar, 19Teramoto H. Salem P. Robbins K.C. Bustelo X.R. Gutkind J.S. J. Biol. Chem. 1997; 272: 10751-10755Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 20Crespo P. Schuebel K.E. Ostrom A.A. Gutkind J.S. Bustelo X.R. Nature. 1997; 385: 169-172Crossref PubMed Scopus (680) Google Scholar, 21Bustelo X.R. Mol. Cell. Biol. 2000; 20: 1461-1477Crossref PubMed Scopus (448) Google Scholar). A second common mechanism for controlling Rho GEF activity is through the regulation of intracellular localization. This often entails translocation from the cytosol to receptor complexes at the plasma membrane. For example, the Rho GEF Tiam1 is recruited to the plasma membrane upon stimulation of cells with serum growth factors (11Michiels F. Stam J.C. Hordijk P.L. van der Kammen R.A. Ruuls-Van Stalle L. Feltkamp C.A. Collard J.G. J. Cell Biol. 1997; 137: 387-398Crossref PubMed Scopus (211) Google Scholar, 22Buchanan F.G. Elliot C.M. Gibbs M. Exton J.H. J. Biol. Chem. 2000; 275: 9742-9748Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The Rho GEF neuroepithelioma transforming gene 1 (NET1) codes for a 595-amino acid protein that consists of tandem DH and PH domains, flanked by amino-terminal and carboxyl-terminal extensions of 155 and 93 amino acids, respectively. It was originally cloned as a transforming gene in a screen for novel oncogenes using an NIH 3T3 cell focus formation assay (23Chan A.M. Takai S. Yamada K. Miki T. Oncogene. 1996; 12: 1259-1266PubMed Google Scholar). The oncogenic form of NET1 cloned from this screen lacked the first 145 amino acids of the wild type protein, which was critical to its transforming activity, because overexpression of wild type NET1 (wt NET1) was not transforming. Later it was shown that deletion of the first 121 amino acids of NET1 also produced a protein that was transforming in NIH 3T3 focus formation assays and that this form of NET1 (NET1ΔN) catalyzed GDP exchange on RhoA, but not Rac1 or Cdc42 in vitro (24Alberts A.S. Treisman R. EMBO J. 1998; 17: 4075-4085Crossref PubMed Scopus (97) Google Scholar). These authors also demonstrated that expression of NET1ΔN stimulated the formation of actin stress fibers, which is a hallmark of RhoA activation, and caused the phosphorylation of c-Jun by the JNK family of mitogen-activate protein kinases when co-expressed in cells. These data indicated that NET1 was a RhoA-specific GEF that was negatively regulated by its amino terminus. However, mechanisms controlling the activity of wt NET1 were not described. Recently it was shown that wt NET1 activity in the cell is controlled through subcellular localization, such that wt NET1 is localized to the nucleus when ectopically expressed in cells (25Schmidt A. Hall A. J. Biol. Chem. 2002; 277: 14581-14588Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). In this work it was demonstrated that the amino-terminal 121 residues of wt NET1 targets it to the nucleus through the actions of two nuclear localization signal (NLS) sequences contained within this region, and that mutation of these NLS sequences resulted in a partial redistribution of full-length NET1 to the cytosol (25Schmidt A. Hall A. J. Biol. Chem. 2002; 277: 14581-14588Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Furthermore, in this same study the PH domain of NET1 was shown to contain a nuclear export signal (NES) sequence. These data suggested that the ability of wt NET1 to regulate RhoA activation is negatively regulated by nuclear localization. However, despite considerable efforts by these authors, extracellular stimuli controlling the export of NET1 from the nucleus were not identified. To better understand the regulatory mechanisms controlling NET1 activity, we have characterized the requirement for distinct domains within NET1 in controlling its enzymatic activity, subcellular localization, and ability to transform cells. Our results demonstrate that two separate domains within the NET1 amino terminus target it to the nucleus, only one of which contains the previously identified NLS sequences. The contribution of this second domain to nuclear localization is critical, because the ability of NET1 to stimulate cytoskeletal rearrangement and cell transformation is dependent upon localization to the cytosol. In addition, we find that the amino terminus of NET1 does not negatively regulate its enzymatic activity in vitro, nor does it down-regulate the ability of NET1 to control actin cytoskeletal organization. The ability of NET1 proteins to stimulate actin stress fiber formation does not correlate with transforming activity, such that NET1 proteins that are extremely potent at stimulating stress fiber formation do not transform cells. Thus, these data highlight a divergence between the ability of NET1 to stimulate cytoskeletal reorganization and to transform cells. This point is emphasized by our finding that a potential PDZ binding site in the C terminus of NET1 must be present for efficient transformation of cells, but is not required for enzymatic activity or for effects on the actin cytoskeleton. Cell Culture and Transfections—HEK 293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen) plus 100 units/ml streptomycin/penicillin (Invitrogen). Swiss 3T3 and NIH 3T3 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% calf serum (Invitrogen) plus 100 units/ml streptomycin/penicillin (Invitrogen). HEK 293 and NIH 3T3 cells were transfected using Lipofectamine Plus (Invitrogen) with 2 μg of DNA, according to the manufacturer's instructions. Plasmids and Recombinant Proteins—For eukaryotic expression all cDNAs were contained in pEFHA (24Alberts A.S. Treisman R. EMBO J. 1998; 17: 4075-4085Crossref PubMed Scopus (97) Google Scholar). Wild type, mouse NET1, and NET1ΔN (amino acids 122–595) were as described previously (24Alberts A.S. Treisman R. EMBO J. 1998; 17: 4075-4085Crossref PubMed Scopus (97) Google Scholar). For NET1 plus NES, the nuclear export sequence (NES) from PKI (LALKLAGLDI) (26Wen W. Meinkoth J.L. Tsien R.Y. Taylor S.S. Cell. 1995; 82: 463-473Abstract Full Text PDF PubMed Scopus (1001) Google Scholar) was added onto the carboxyl terminus of wild type NET1 by PCR using Pfu polymerase (Stratagene). For NET1ΔN plus NLS, the nuclear localization signal (NLS) from the SV40 large T antigen (PKKKRKV) was added onto the carboxyl terminus of NET1ΔN by PCR (27Kalderon D. Roberts B.L. Richardson W.D. Smith A.E. Cell. 1984; 39: 499-509Abstract Full Text PDF PubMed Scopus (1866) Google Scholar). The mouse NET1A cDNA was obtained from the Mammalian Gene Collection (ATCC), and was subcloned into pEFHA by PCR. NET1 156–595, NET1 156–501, NET1-(122–501), NET1ΔNΔC4, and NET1ΔNAla were amplified by PCR and ligated into pEFHA. All cDNAs amplified by PCR were sequenced in their entirety to confirm correct amplification. Glutathione S-transferase (GST)-NET1 and GST-wild type RhoA fusion proteins were created by subcloning each cDNA into pGEXKG (Amersham Biosciences). BL21(DE3) Escherichia coli (Stratagene) were transformed with the plasmids, and the proteins were expressed by addition of 400 μm isopropyl 1-thio-β-d-galactopyranoside after the culture had reached an A600 = 0.8. Incubation with isopropyl 1-thio-β-d-galactopyranoside continued for 4 h at 37 °C. Cells were pelleted by centrifugation and stored at –80 °C. Cells were lysed by addition of lysozyme followed by sonication, insoluble proteins were pelleted by centrifugation (30 min at 35,000 × g, 4 °C), and GST fusion proteins were purified by glutathione-agarose affinity chromatography. Purified fusion proteins were dialyzed into dialysis buffer (20 mm Tris-HCl (pH 8.0), 100 mm NaCl, 1 mm dithiothreitol, 1 mm EDTA, 10% glycerol) and stored at –80 °C. For purification of GST-wt RhoA, 10 μm GDP was included in all purification and dialysis steps. Guanine Nucleotide Exchange Assays—GEF assays were performed essentially as described (28Zheng Y. Hart M.J. Cerione R.A. Methods Enzymol. 1995; 256: 77-84Crossref PubMed Scopus (70) Google Scholar), using equimolar amounts of GST-NET1 proteins. Briefly, GST-wt RhoA was preloaded for 5 min at room temperature with GDP loading buffer (20 mm Tris-HCl, pH 8.0, 100 mm NaCl, 1 mm EDTA, 100 μm AMP-PNP, 10 mm GDP, 1 mm dithiothreitol). Loading was terminated by the addition of MgCl2 to a final concentration of 10 mm. Approximately 700 nm GDP-wt RhoA was then incubated with 300 nm NET1 protein in GTPγS buffer (20 mm Tris-HCl, pH 8.0, 1 μCi of [35S]GTPγS, 5 μm GTPγS, 0.5 mg/ml bovine serum albumin, 100 mm NaCl, 10 mm MgCl2, 100 μm AMP-PNP, 0.2% Triton X-100) at 30 °C. At different times aliquots were removed, and the reaction was stopped by addition to a 10-ml volume of termination buffer (20 mm Tris-HCl, pH 8.0, 100 mm NaCl, 10 mm MgCl2). [35S]GTPγS bound to GST-wt RhoA was recovered by filtration through BA85 nitrocellulose filters (Schleicher and Schuell). The filters were washed with termination buffer, and bound nucleotide was quantified by liquid scintillation. Microinjection and Indirect Immunofluorescence—Swiss 3T3 cells were plated on glass coverslips and allowed to grow to 30% confluence. Prior to injection the cells were incubated for 24 h in Dulbecco's modified Eagle's medium plus 100 units/ml penicillin/streptomycin without calf serum. All plasmids were injected into the nuclei of cells at 0.2 mg/ml using an Eppendorf Injectman connected to an Eppendorf 5246 Transjector. Cells to be injected were visualized with a Zeiss Axiovert S100 microscope. Four hours after injection the cells were fixed with 3.7% formaldehyde in PBS at 37 °C for 5 min. Cells were then permeabilized with 0.2% Triton X-100 in PBS at room temperature for 5 min. To detect expressed, HA-tagged NET1 proteins the cells were incubated with 2 μg/ml mouse anti-HA (Santa Cruz Biotechnology) in PBS plus 0.2% Tween 20 and 1 mg/ml bovine serum albumin (PBST plus bovine serum albumin) for 1 h at 37 °C. After washing 3 × 5 min with PBST, the cells were incubated with Texas Red-conjugated donkey anti-mouse (Jackson Laboratories) and fluorescein isothiocyanate-phalloidin (Sigma) (to detect F-actin) diluted in PBST plus bovine serum albumin, for 1 h at 37 °C. The cells were washed 3 × 5 min with PBST and once with distilled water, and then mounted on glass microscope slides with FluorSave reagent (Calbiochem). Fluorescent cells were visualized with either a Zeiss Axioskop with a mounted Hamamatsu C4742-95 digital camera, or a Zeiss 510 Meta confocal microscope. Images were recorded using MetaVue or MetaMorph software (Universal Imaging). Subcellular Fractionation—NIH 3T3 cells in 10-cm dishes were transfected with HA epitope-tagged NET1 expression vectors using Lipofectamine Plus. Prior to harvest the cells were serum-starved in Dulbecco's modified Eagle's medium without calf serum for 24 h. Cells were then washed once with PBS, resuspended in 0.5 ml of hypotonic lysis buffer (50 mm Tris (pH 7.4), 1 mm EDTA, 1 mm dithiothreitol, 10 μg/ml pepstatin A, 10 μg/ml leupeptin, 2 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride, 50 mm NaF, 80 mm β-glycerophosphate, 1 mm sodium orthovanadate), and incubated on ice for 10 min. The cells were then lysed using 20 strokes in a Dounce homogenizer, and nuclei and unbroken cells were pelleted by centrifugation for 10 min at 1,500 × g, 4 °C. The supernatant was centrifuged for 30 min at 100,000 × g, 4 °C to isolate the cytosol. The nuclear pellets were washed once with 1 ml of hypotonic lysis buffer, and nuclei were pelleted again by centrifugation at 1,500 × g, 10 min at 4 °C. The nuclear pellets were then solubilized by resuspension in 2% SDS buffer (20 mm Tris (pH 8.0), 100 mm NaCl, 1 mm EDTA, 2.0% SDS, 50 mm NaF, 80 mm β-glycerophosphate, 1 mm sodium orthovanadate, 10 μg/ml aprotinin, 10 μg/ml pepstatin A, 2 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride), and DNA was sheared by drawing through a 20-gauge needle 10 times. Insoluble material was pelleted by centrifugation for 10 min at 16,000 × g at room temperature, and the supernatant was saved. The concentration of proteins present in the nuclear and cytoplasmic fractions was determined by bicinchoninic acid assay (Pierce), and equal amounts of each fraction were resolved by SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membrane (Amersham Biosciences), and HA-epitope-tagged NET1 proteins were detected by Western blotting using mouse or rabbit anti-HA (Santa Cruz Biotechnology), followed by HRP-conjugated donkey anti-mouse or donkey anti-rabbit (KPL). Western blots were developed using enhanced chemiluminescence and visualized with x-ray film (Kodak). The purity of the nuclear and cytoplasmic fractions was determined by Western blotting with rabbit anti-lamin B1 (Santa Cruz Biotechnology) and rabbit anti-superoxide dismutase-1 (Santa Cruz Biotechnology), which are localized to the nuclear and cytoplasmic compartments, respectively. Foci Formation Assays—Foci formation assays were performed essentially as described previously (29Solski P.A. Abe K. Der C.J. Methods Enzymol. 2000; 325: 425-441Crossref PubMed Google Scholar). Briefly, low passage NIH 3T3 cells were transfected in six centimeter dishes (BD Biosciences) with 2 μg of DNA using Lipofectamine Plus. All transfections were performed in duplicate. Cultures were refed every 3–4 days with growth media for a total of 16 days. Cells were then fixed with 10% acetic acid in water for 10 min at room temperature and then stained with 0.4% crystal violet (Sigma) in 10% ethanol in water for 10 min at room temperature. After staining, the cells were washed several times with water, allowed to air dry, and foci were counted. The Amino Terminus of NET1 Does Not Regulate Its Enzymatic Activity—The enzymatic activity of many Rho GEFs is regulated through the actions of a cis-acting autoinhibitory domain (7Zheng Y. Trends Biochem. Sci. 2001; 26: 724-732Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar, 8Schmidt A. Hall A. Genes Dev. 2002; 16: 1587-1609Crossref PubMed Scopus (982) Google Scholar). Because deletion of the amino terminus of NET1 creates a protein that is transforming, we examined whether the enzymatic activity of NET1 was also regulated by an amino-terminal autoinhibitory domain. Thus, we compared the relative abilities of the wild type and oncogenic forms of NET1 (wt NET1 and NET1ΔN, respectively) to stimulate GTPγS binding by RhoA in vitro. We also tested a splice variant of NET1 (NET1A) that lacks the first 85 amino acids of wild type NET1 and instead contains an unrelated 31-amino acid segment. This segment does not contain the two NLS sequences found in wild type NET1, and potentially replaces autoinhibitory sequences present in this portion of wt NET1. For these experiments wt NET1, NET1ΔN, and NET1A were expressed as glutathione S-transferase (GST) fusion proteins in E. coli and purified by glutathione-agarose affinity chromatography. Equimolar amounts of each protein were then tested for GEF activity toward purified GST-RhoA, which was measured through the binding of the nonhydrolyzable GTP analog GTPγS. As shown in Fig. 1, RhoA alone exhibited a modest ability to bind to GTPγS that was greatly enhanced by the addition of each NET1 protein. Furthermore, each NET1 protein exhibited a nearly equivalent activity toward RhoA in vitro. Thus, these data indicate that NET1 does not contain a negative regulatory domain in its amino terminus that directly controls its enzymatic activity, and sets NET1 apart from other Rho GEFs such as Vav, and Dbl, which do contain autoinhibitory domains (30Abe K. Whitehead I.P. O'Bryan J.P. Der C.J. J. Biol. Chem. 1999; 274: 30410-30418Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 31Bi F. Debreceni B. Zhu K. Salani B. Eva A. Zheng Y. Mol. Cell. Biol. 2001; 21: 1463-1474Crossref PubMed Scopus (68) Google Scholar). The Splice Variant NET1A Displays a Subcellular Localization and Cellular Activity That Is Distinct from wt NET1— Given that NET1 localization in the cytosol is critical to its ability to regulate the actin cytoskeleton (25Schmidt A. Hall A. J. Biol. Chem. 2002; 277: 14581-14588Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), we tested whether NET1A, which lacks the amino-terminal NLS sequences present in wt NET1, was cytosolic and thus behaved as a hyperactive form of NET1. To test this, we examined the ability of NET1A to elicit changes in the actin cytoskeleton as an indicator of NET1 activity. Thus, serum-starved Swiss 3T3 cells were microinjected with eukaryotic expression vectors for a control protein (β-galactosidase), wt NET1, NET1ΔN, or NET1A. Four hours later the cells were fixed and stained for expression of these proteins and for F-actin. As shown in Fig. 2A, serum-starved Swiss 3T3 cells are usually flat, stain positive for cortical F-actin, and exhibit few actin stress fibers. Injection of a control plasmid that drives the expression of β-galactosidase did not significantly affect this phenotype (panels a and b). Similarly, expression of wild type NET1 did not stimulate actin stress fiber formation (panels c and d). The wt NET1 was predominantly nuclear, although some cytoplasmic staining was observed in cells expressing very high amounts of NET1 (data not shown). In these cells some increase in F-actin staining was noted, as previously observed (25Schmidt A. Hall A. J. Biol. Chem. 2002; 277: 14581-14588Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). On the other hand, expression of NET1ΔN caused the formation of numerous actin stress fibers in every injected cell (panels e and f). These cells typically were less spread out than the surrounding cells, and the NETΔN protein was localized in both the nucleus as well as the cytoplasm. Importantly, when we tested for effects of NET1A expression, we found that it also potently stimulated actin stress fiber formation (panels g and h). In fact, NET1A was as efficient as NET1ΔN at stimulating the polymerization of actin stress fibers, although NET1A expression did not cause cells to round up in the way that NET1ΔN did. Staining for the expressed NET1A protein demonstrated that there were slightly higher levels of cytoplasmic expression as compared with wt NET1; however, the majority of the NET1A was localized to the nucleus. We confirmed the subcellular localization of these NET1 proteins by subcellular fractionation. For these experiments NIH 3T3 cells were transfected with the HA-epitope-tagged NET1 constructs shown. The cells were serum-starved, lysed in a hypotonic buffer, and then separated into nuclear and cytoplasmic fractions. Equal amounts of these fractions were resolved by SDS-PAGE, and the presence of NET1 proteins was detected by Western blotting using an antibody specific for the HA-epitope. We confirmed the validity of our fractionation procedure by blotting for lamin B1 and superoxide dismutase-1, which are localized to the nuclear and cytoplasmic compartments, respectively. This analysis confirmed that the majority of wt NET1 was localized to the nucleus (Fig. 2B). NET1A was also largely contained in the nuclear fraction, although there was slightly more NET1A in the cytosolic fraction as compared with wt NET1. The majority of NET1ΔN, on the other hand, was localized to the cytoplasm. Thus, taken together these data indicate that NET1A tends to localize to the cytoplasm to a slightly higher degree than wt NET1, and this leads to an increased ability of NET1A to stimulate actin polymerization in the cell. Because NET1A lacks the NLS sequences present in wt NET1 and does not contain an identifiable NLS in the unique portion of its amino terminus, it is unclear as to how NET1A localizes to the nucleus. The DH, PH, and C-terminal regions of wt NET1 do" @default.
• W2077856249 created "2016-06-24" @default.
• W2077856249 creator A5011850931 @default.
• W2077856249 creator A5020704363 @default.
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• W2077856249 date "2005-03-01" @default.
• W2077856249 modified "2023-10-14" @default.
• W2077856249 title "Characterization of the Biochemical and Transforming Properties of the Neuroepithelial Transforming Protein 1" @default.
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