Deniz Toksoz

Amino-acid substitutions at codon 13 of the N-ras oncogene in human acute myeloid leukaemia

DNAs from four out of five patients with acute myeloid leukaemia (AML) tested by an in vivo selection assay in nude mice using transfected mouse NIH 3T3 cells were found to contain an activated N-ras oncogene. Using a set of synthetic oligonucleotide probes, we have detected a mutation at codon 13 in all four genes. The same codon is mutated in an additional AML DNA that is positive in the focus-formation assay on 3T3 cells. DNA from the peripheral blood of one patient in remission does not contain a codon 13 mutation.

Transformation by Rho exchange factor oncogenes is mediated by activation of an integrin-dependent pathway.

Constitutive activation of growth factor receptor signaling pathways leads to uncontrolled growth, but why tumor cells become anchorage independent is less clear. The fact that integrins transmit signals required for cell growth suggests that constitutive activation of steps downstream from integrins mediates anchorage independence. Since the small GTPase Rho may mediate integrin signal transduction, the effects of serum and the Rho nucleotide exchange factor oncogenes dbl and lbc on cell growth and signaling pathways were examined. Our data show that these oncogenes induce anchorage‐independent but serum‐dependent growth and stimulation of signaling pathways. These results show, therefore, that anchorage‐independent growth results from constitutive activation of integrin‐dependent signaling events. They also support the view that Rho is a functionally important mediator of integrin signaling.

Activation of the Lbc Rho Exchange Factor Proto-Oncogene by Truncation of an Extended C Terminus That Regulates Transformation and Targeting

ABSTRACT The human lbc oncogene product is a guanine nucleotide exchange factor that specifically activates the Rho small GTP binding protein, thus resulting in biologically active, GTP-bound Rho, which in turn mediates actin cytoskeletal reorganization, gene transcription, and entry into the mitotic S phase. In order to elucidate the mechanism of onco-Lbc transformation, here we report that while proto- and onco-lbc cDNAs encode identical N-terminal dbloncogene homology (DH) and pleckstrin homology (PH) domains, proto-Lbc encodes a novel C terminus absent in the oncoprotein that includes a predicted α-helical region homologous to cyto-matrix proteins, followed by a proline-rich region. The lbc proto-oncogene maps to chromosome 15, and onco-lbc represents a fusion of the lbc proto-oncogene N terminus with a short, unrelated C-terminal sequence from chromosome 7. Both onco- and proto-Lbc can promote formation of GTP-bound Rho in vivo. Proto-Lbc transforming activity is much reduced compared to that of onco-Lbc, and a significant increase in transforming activity requires truncation of both the α-helical and proline-rich regions in the proto-Lbc C terminus. Deletion of the chromosome 7-derived C terminus of onco-Lbc does not destroy transforming activity, demonstrating that it is loss of the proto-Lbc C terminus, rather than gain of an unrelated C-terminus by onco-Lbc, that confers transforming activity. Mutations of onco-Lbc DH and PH domains demonstrate that both domains are necessary for full transforming activity. The proto-Lbc product localizes to the particulate (membrane) fraction, while the majority of the onco-Lbc product is cytosolic, and mutations of the PH domain do not affect this localization. The proto-Lbc C-terminus alone localizes predominantly to the particulate fraction, indicating that the C terminus may play a major role in the correct subcellular localization of proto-Lbc, thus providing a mechanism for regulating Lbc oncogenic potential.

Distinct roles for DH and PH domains in the Lbc oncogene

Members of the Dbl-homology (DH) family of proteins promote guanine nucleotide exchange on Rho GTPases. Lbc, which specifically acts on Rho but not Rac or Cdc42, was isolated as a transforming oncogene and is composed of a DH and a Pleckstrin-homology (PH) domain. We show here that the Lbc DH domain alone is capable of stimulating new DNA synthesis in quiescent fibroblasts and Rho-dependent actin stress fiber assembly. However, the PH domain is required for subcellular localization of Lbc along actin stress fibers and for efficient transformation of NIH3T3 cells. The results show that, in contrast to other Dbl-homology proteins, the PH domain of Lbc is dispensable for activation of Rho in vivo. The PH domain-dependent subcellular localization of Lbc may, however, be important for growth factor activation of endogenous Lbc and for oncogenic transformation.

Selective Inhibition of Growth of Tuberous Sclerosis Complex 2–Null Cells by Atorvastatin Is Associated with Impaired Rheb and Rho GTPase Function and Reduced mTOR/S6 Kinase Activity

Inactivating mutations in the tuberous sclerosis complex 2 (TSC2) gene, which encodes tuberin, result in the development of TSC and lymphangioleiomyomatosis (LAM). The tumor suppressor effect of tuberin lies in its GTPase-activating protein activity toward Ras homologue enriched in brain (Rheb), a Ras GTPase superfamily member. The statins, 3-hydroxy-3-methylglutaryl CoA reductase inhibitors, have pleiotropic effects which may involve interference with the isoprenylation of Ras and Rho GTPases. We show that atorvastatin selectively inhibits the proliferation of Tsc2-/- mouse embryo fibroblasts and ELT-3 smooth muscle cells in response to serum and estrogen, and under serum-free conditions. The isoprenoids farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP) significantly reverse atorvastatin-induced inhibition of Tsc2-/- cell growth, suggesting that atorvastatin dually targets a farnesylated protein, such as Rheb, and a geranylgeranylated protein, such as Rho, both of which have elevated activity in Tsc2-/- cells. Atorvastatin reduced Rheb isoprenylation, GTP loading, and membrane localization. Atorvastatin also inhibited the constitutive phosphorylation of mammalian target of rapamycin, S6 kinase, and S6 found in Tsc2-/- cells in an FPP-reversible manner and attenuated the high levels of phosphorylated S6 in Tsc2-heterozygous mice. Atorvastatin, but not rapamycin, attenuated the increased levels of activated RhoA in Tsc2-/- cells, and this was reversed by GGPP. These results suggest that atorvastatin may inhibit both rapamycin-sensitive and rapamycin-insensitive mechanisms of tuberin-null cell growth, likely via Rheb and Rho inhibition, respectively. Atorvastatin may have potential therapeutic benefit in TSC syndromes, including LAM.

Smooth Muscle α-Actin Expression and Myofibroblast Differentiation by TGFβ are Dependent Upon MK2

Fibroblasts play a major role in processes such as wound repair, scarring, and fibrosis. Differentiation into myofibroblasts, characterized by upregulation of smooth muscle α‐actin (smα) in response to profibrotic agents such as TGFβ is believed to be an important step in fibrosis. Therefore, elucidating mechanisms of myofibroblast differentiation might reveal novel targets in treating diseases such as idiopathic pulmonary fibrosis (IPF). MK2 is a kinase substrate of p38 MAP kinase that mediates some effects of p38 activation on the actin cytoskeleton. Using mouse embryonic fibroblasts (MEF) from MK2 knockout (MK2−/−) mice, we demonstrate that disrupting expression of MK2 expression reduces filamentous actin and stress fibers. It also causes MK2−/− MEF to express less smα than their corresponding wild‐type (WT) MEF at baseline and in response to TGFβ. Furthermore, TGFβ causes downregulation of smα in MK2−/− MEF, instead of upregulation observed in WT MEF. Expression of other fibroblast markers, such as collagen, is not altered in MK2−/− MEF. Our results further suggest that downregulation of smα in MK2−/− MEF is not due to lack of activation of serum responsive promoter elements, but probably due to reduced smα message stability in these cells. These results indicate that MK2 plays a key role in regulation of smα expression, and that targeting MK2 might present a therapeutic approach in managing conditions such as pulmonary fibrosis. J. Cell. Biochem. 100: 1581–1592, 2007.

Role of Lbc RhoGEF in Gα12/13-induced signals to Rho GTPase

Heterotrimeric Galpha12/13 signals induce cellular responses such as serum response element (SRE)-mediated gene transcription via Rho GTPase. Guanine nucleotide exchange factors (GEFs) are strong candidates for linking Galpha signals to Rho. For example, p115 RhoGEF transduces Galpha13 signals to Rho and inhibits Galpha12/13 signals via the RhoGEF LH domain which links to Galpha subunits. Here, we have evaluated the signaling capacity of Lbc RhoGEF in the context of Galpha12/13 signals. In vitro GEF assays indicate that baculoviral-expressed proto-Lbc has minimal exchange activity, implying that a stimulus is required for Lbc activity in vivo. Expression of a catalytically inactive proto-Lbc mutant in HEK293T cells attenuates Galpha12- and thrombin-induced activation of an SRE transcriptional reporter, and the levels of inhibition observed is similar to that obtained with an analogous p115 RhoGEF mutant. proto-Lbc mutant expression also led to decreased levels of Galpha12-induced RhoA activation in vivo. Complex formation between Galpha12 and Lbc forms was detected. Analysis of the Lbc peptide sequence reveals a previously undetected region which may link to Galpha subunit signals. These findings support a role for Lbc in Galpha12-induced signaling pathways to Rho.

Activated Gαq family members induce Rho GTPase activation and Rho‐dependent actin filament assembly

Rho GTPase is required for actin filament assembly and serum response element (SRE)‐dependent gene transcription. Certain G protein‐coupled receptors (GPCRs) induce Rho‐dependent responses, but the intermediary signaling steps are poorly understood. The heterotrimeric Gα12 family can induce Rho‐dependent responses. In contrast, there are conflicting reports on the role of the Gαq family in Rho signaling. We report that expression of activated Gαq members, or activation of endogenous Gαq via GPCR stimulation, induces SRE reporter activation via Rho, and increased GTP‐Rho levels. Moreover, microinjection of activated Gαq in fibroblasts induces actin stress fiber formation via Rho. Gαq functionally cooperates with Lbc Rho guanine nucleotide exchange factor. Overall, these findings indicate that Gαq family signals are sufficient to induce Rho‐dependent cellular responses.

Serotonin induces Rho/ROCK-dependent activation of Smads 1/5/8 in pulmonary artery smooth muscle cells

Serotonin (5‐HT) stimulates pulmonary artery smooth muscle cell proliferation and has been associated with pulmonary arterial hypertension (PAH). Bone morphogenetic protein receptor 2 (BMPR2) mutations similarly have been linked to PAH. However, possible crosstalk between 5‐HT and BMPR signaling remains poorly characterized. We report here that 5‐HT activates Smads 1/5/8 in bovine and human pulmonary artery smooth muscle cells (SMCs) and causes translocation of these Smads from cytoplasm to the nucleus. DN BMPR1A blocked 5‐HT activation of Smads 1/5/8 by 5‐HT and BMPR1A overexpression enhanced it. Activation of Smads by 5‐HT occurred through the 5‐HT 1B/1D receptor as it was blocked with the inhibitor GR 55562 but unaffected by inhibitors of the 5‐HT transporter and a variety of 5‐HT receptors. Activation of the Smads by 5‐HT depended on Rho/Rho kinase signaling as it was blocked by Y27632, but unaffected by inhibitors of PI3K or MAPK. Transfection of cells with BMPR1A and ligation of the BMP receptor with BMP‐2 also activated GTP‐Rho A of these SMCs, while DN BMPR1A blocked the activation. 5‐HT stimulated an increase in serine/threonine phosphorylation of BMPR1A, supporting the activation of BMPR1A by 5‐HT in SMCs. Infusion of 5‐HT into mice with miniosmotic infusion pumps caused activation of Smads 1/5/8 in lung tissue, demonstrating the effect in vivo. The studies support a unique concept that 5‐HT transactivates the serine kinase receptor, BMPR 1A, to activate Smads 1/5/8 via Rho and Rho kinase in pulmonary artery SMCs. Rho and Rho kinase also participate in the activation of Smads by BMP.—Liu, Y.,Ren, W., Warburton, R., Toksoz, D., Fanburg, B. L. Serotonin induces Rho/ROCK‐dependent activation of Smads 1/5/8 in pulmonary artery smooth muscle cells. FASEB J. 23, 2299–2306 (2009)

RAS GENES AND ACUTE MYELOID LEUKAEMIA

Acute myeloid leukaemia is an advantageous system to study genetic changes in human malignancy because of: (a) the existence of defined premalignant conditions. the myelodysplastic syndromes (MDS); (b) the existence of discrete stages of presentation, remission and relapse in the course of the malignancy: and (c) the ease of obtaining samples from patients a t all of these stages. The pattern of genetic change can therefore be monitored throughout the development and progression of the disease. A large body of evidence demonstrates that the blast cells which infiltrate the haemopoietic system in acute myeloid leukaemia (AML) are clonal, and that they derive from a multi-potent. haemopoietic stem cell (Fialkow et ul, 1979, 1981; Fearon et al, 1986). Isolated cases of AML have been reported where its occurrence is linked to a specific environmental agent such as X-rays (CourtBrown & Abbatt, 1955), occupational exposure to benzene (Aksoy et al. 1974) and therapy with alkylating agents (Rosner & Grunwald. 1975, 1980). However, most cases of AML have an unknown aetiology. Patients with myelodysplastic syndromes have chronic cytopenias and bone marrow dysplasia; about 20-30% of MDS progress to AML months or years later (Bennett rt al, 1982). However, the vast majority of dc novo cases of AML present without a previous history of myelodysplasia. The clonal origin of AML suggests that it is due to somatic heritable change(s). While there are non-random chromosomal abnormalities associated with probably all cases of AMI, (Yunis et a/, 1981), only one, t(15;17) (Golomb et 01, 1976). is tightly associated with an AML subtype (FAB M3) (Bennett et a!, 1976). Other non-random karyotypic abnormalities such as t(8,2 1) (Rowley, 1973) and inv(l6) (Arthur & Bloomfield, 1983) are linked with a particular FAB subtype, but not exclusively: while trisomy 8 and monosomy 7 can occur in any subtype (Rowley, 1978). Thus, in contrast to other leukaemias with a shared chromosomal alteration such as Burkitt’s lymphoma with t( 8;14) (Kaiser-McCaw rt a/, 1977). follicular lymphoma with t(14;18) (Yunis et al, 1982) and chronic myeloid leukaemia with t(9:22) (Nowell & Hungerford, 1987). there is no karyotypic change in AML which is common to the disease as a whole. This overall pattern of variable chromosomal abnormalities, coupled with the considerable morphological heterogeneity of AML. does not immediately point to a consistent genetic lesion on which to focus research efforts. In the last 5 years the use of gene transfer techniques to identify transforming genes in human malignancies has shown that a large fraction (25-90%) of some neo-