Mutations in LZTR1 drive human disease by dysregulating RAS ubiquitination.

The leucine zipper-like transcriptional regulator 1 (LZTR1) protein, an adaptor for cullin 3 (CUL3) ubiquitin ligase complex, is implicated in human disease, yet its mechanism of action remains unknown. We found that Lztr1 haploinsufficiency in mice recapitulates Noonan syndrome phenotypes, whereas LZTR1 loss in Schwann cells drives dedifferentiation and proliferation. By trapping LZTR1 complexes from intact mammalian cells, we identified the guanosine triphosphatase RAS as a substrate for the LZTR1-CUL3 complex. Ubiquitome analysis showed that loss of Lztr1 abrogated Ras ubiquitination at lysine-170. LZTR1-mediated ubiquitination inhibited RAS signaling by attenuating its association with the membrane. Disease-associated LZTR1 mutations disrupted either LZTR1-CUL3 complex formation or its interaction with RAS proteins. RAS regulation by LZTR1-mediated ubiquitination provides an explanation for the role of LZTR1 in human disease.

[Stress granules in the cells with intact and discrupted microtubules: analysis with new algorithm of image processing].

Stress granules--temporary RNP structures that are formed in cells under stress. They are studied mainly by means of fluorescence microscopy with the quantitative analysis of cell images. We have developed a new algorithm for automatic detection of stress granules in the cytoplasm of cultured animal cells having non-uniform cytoplasmic background. Using this approach, we have found that visible stress granules are formed in cells as "all or nothing", and their number in cells is rather constant. We also show that disruption of cellular microtubules lead to a decrease in the average size of stress granules and an increase in their number in the cell.

[Down-regulation of TRIP6 expression induces actin cytoskeleton rearrangements in human carcinoma cell lines].

Structure and dynamics of actin cytoskeleton play a role ih regulation of cell adhesion, spreading and migration. TRIP6 is a LIM domain-containing protein interacting with many actin-associated proteins and in addition modulating activity of certain transcription factors. To study functions of TRIP6 we inhibited its expression in A549 and A431 cells by short interfering RNAs (siRNAs). The TRIP6 knock-down lead to the increased number and length of stress fibers and to the induction of locomotive phenotype. There was observed decreased number and reorganization of focal adhesions revealed by staining for paxillin, and loss of cell to cell adhesions revealed by staining for E-cadherin. The above changes in cell morphology were accompanied by 2-fold increase in the cell motility rate assessed by the wound healing assay. Thus, down-regulation of TRIP6 in the cell lines used results in increase in the features characteristic to malignant transformation of epithelial cells. Possible mechanisms for the observed effects are discussed.

[The protective role of p53 in Ras-induced transformation of REF52 cells]. / Zashchitnaia funktsiia p53 pri ras-indutsirovannoi transformatsii kletok REF52.

A study was made of the effect of activated oncogene N-RAS on the function of tumor suppressor p53 and the proliferating ability of rat embryo fibroblasts REF52. The proliferation rate and the portion of S-phase cells increased in the first three days of N-RAS expression. After 5-7 days, the p53 function was enhanced, as manifest in increased p53 lifespan and nuclear content and induced transcription of p53-responsive genes. In particular, Cdk2 p21WAF1/CIP1, an inhibitor of cyclin-dependent kinase 2, was produced to a higher level and arrested the cell cycle in G1. Cells with abrogated or dramatically inhibited N-RAS expression were generated at this stage. Having a selective advantage, these cells gradually displaced N-RAS-expressing cells arrested in G1, so that one month after oncogene induction the culture mostly consisted of morphologically normal, actively proliferating Res-negative cells. Neither cell cycle arrest nor reversion to the normal phenotype were observed in N-RAS expressing cells devoid of the p53 function. Thus, p53 prevented stable N-RAS-induced transformation of REF52 cells, arresting the cell cycle and expediting revertant selection.

Dual-colour imaging of membrane protein targeting directed by poa semilatent virus movement protein TGBp3 in plant and mammalian cells.

The movement function of poa semilatent hordeivirus (PSLV) is mediated by the triple gene block (TGB) proteins, of which two, TGBp2 and TGBp3, are membrane proteins. TGBp3 is localized to peripheral bodies in the vicinity of the plasma membrane and is able to re-direct TGBp2 from the endoplasmic reticulum (ER) to the peripheral bodies. For imaging of TGBp3-mediated protein targeting, PSLV TGBp3 tagged with a red fluorescent protein (DsRed) was used. Coexpression of DsRed-TGBp3 with GFP targeted to the ER lumen (ER-GFP) demonstrated that ER-GFP was contained in typical ER structures and peripheral bodies formed by TGBp3 protein, suggesting an ER origin for these bodies. In transient coexpression with viral membrane proteins tagged with GFP, DsRed-TGBp3 directed to the peripheral bodies the homologous TGBp2 protein and two unrelated membrane proteins, the 6 kDa movement protein of beet yellows closterovirus and the putative movement protein encoded by the genome component 4 of faba bean necrotic yellows nanovirus. However, coexpression of TGBp3 with GFP derivatives targeted to the ER membranes by artificial hydrophobic tail sequences suggested that targeting to the ER membranes per se was not sufficient for TGBp3-directed protein trafficking to peripheral bodies. TGBp3-induced targeting of TGBp2 also occurred in mammalian cells, indicating the universal nature of the protein trafficking signals and the cotargeting mechanism.

[KAISO--a new member of the BTB/POZ family specifically bindsto methylated DNA sequences]. / KAISO, novyi chlen VTV/POZ semeistva, spetsifichno sviazyvaetsia s metilirovannymi posledovatel'nostiami DNK.

A protein specifically binding a symmetrically methylated DNA fragment of the first intron of the mts1 gene was studied. The protein was purified by gel filtration and affinity chromatography. Mass spectrometry showed that the protein is Kaiso, a new member of the BTB/POZ family. To study the association with methylated DNA sequences in vivo, the location of Kaiso in NIH 3T3 cells was analyzed. Immunofluorescent staining with polyclonal antibodies against Kaiso showed that the protein is predominantly associated with the nucleoli. The causes of its distribution awaits further investigation. The zinc-finger domains of Kaiso were for the first time demonstrated to specifically recognize symmetrically methylated DNA sequences in vitro.

p53 activation in response to microtubule disruption is mediated by integrin-Erk signaling.

The p53 tumor suppressor is activated in response to various stresses driving the cells into growth arrest or apoptosis. We have addressed the question of how disintegration of microtubule system induces activation of p53. Depolymerization of microtubules by colcemid in rat and human quiescent fibroblasts resulted in accumulation of transcriptionally active p53 that caused cell-cycle arrest at the G1/S boundary. The p53 activation correlated with prominent activation of Erk1/2 MAP kinases that resulted from colcemid-stimulated development of focal adhesions. Inhibition of focal contacts development by plating of cells onto poly-L-lysine abrogated both Erk1/2 and p53 activations in colcemid-treated cells, while plating of cells onto fibronectin caused transient up-regulation of p53 even in the absence of colcemid. Pre-treatment of cells with the specific MEK1 inhibitor PD098059 also attenuated colcemid-induced p53 activation and G1 cell cycle arrest. Cell types which either failed to develop focal adhesions in response to colcemid treatment (human MCF-7 epithelial cells), or lacked colcemid-induced sustained Erk activation (primary mouse embryo fibroblasts and 12(1) cells) showed virtually no p53 up-regulation in response to disruption of microtubules during G0/G1. Our results indicate that p53 activation is not triggered by disintegration of microtubule system by itself, but rather originates from some of the consequences of such disintegration, in particular, from the development of focal adhesions leading to activation of Erk signaling pathway.

p53 does not control the spindle assembly cell cycle checkpoint but mediates G1 arrest in response to disruption of microtubule system.

p53 plays a critical role as a tumour-suppressor in restricting the proliferation of damaged cells, thus preventing formation of genetically altered cell clones. Its inactivation leads, in particular, to accumulation of polyploid and aneuploid cells. To elucidate the role of p53 in control of chromosome number, we analysed its participation in the cell cycle checkpoints controlling: (1) spindle assembly; and (2) G1-to-S transitions in cells with disintegrated microtubule cytoskeleton. Treatment with 8-10 ng/ml of colcemid causing no visible destruction of the spindle leads to arrest of metaphase-to-anaphase transition in both p53-positive and p53-negative murine fibroblasts, as well as in p53-positive REF52 cells and their counterparts (where the p53 function was inactivated by transduction of dominant-negative p53 fragment). Furthermore, p53-positive and p53-defective rodent and human cells showed no significant difference in kinetics of metaphase-to-interphase transitions in cultures treated with high colcemid doses preventing spindle formation. These data argue against the hypothesis that p53 is a key component of the spindle-assembly checkpoint. However, p53 mediates activation of the G1 checkpoint in response to depolymerization of microtubules in interphase cells. Treatment of synchronized G0/G1 cells with colcemid causes arrest of G1-to-S transition. Inactivation of the p53 function by transduction of dominant-negative p53 fragment abolishes the G1 checkpoint that prevents entry into S phase of cells with disrupted microtubules. Transduction of kinase-defective dominant-negative c- raf mutant or application of PD 098059, a specific inhibitor of MEK1, also abrogates the G1 cell cycle arrest in cells with disintegrated microtubule system. It seems that Raf-MAP-kinase signalling pathways are responsible for p53 activation induced by depolymerization of microtubules.

P53-dependent effects of RAS oncogene on chromosome stability and cell cycle checkpoints.

Mutations activating the function of ras proto-oncogenes are often observed in human tumors. Their oncogenic potential is mainly due to permanent stimulation of cellular proliferation and dramatic changes in morphogenic reactions of the cell. To learn more on the role of ras activation in cancerogenesis we studied its effects on chromosome stability and cell cycle checkpoints. Since the ability of ras oncogenes to cause cell transformation may be dependent on activity of the p53 tumor-suppressor the cells with different p53 state were analysed. Ectopic expression of N-ras(asp12) caused in p53-deficient MDAH041 cell line an augmentation in the number of chromosome breaks in mitogenic cells, significant increase in the frequency of metaphases showing chromosome endoreduplication and accumulation of polyploid cells. Similar effects were induced by different exogenous ras genes (N-ras(asp12), H-ras(leu12), N-ras proto-oncogene) in Rat1 and Rat2 cells which have a defect in p53-upstream pathways. In contrast, in REF52 and human LIM1215 cells showing ras-induced p53 up-regulation, ras expression caused only slight increase in the number of chromosome breaks and did not enhance the frequency of endoreduplication and polyploidy. Inactivation in these cells of p53 function by transduction of dominant-negative C-terminal p53 fragment (genetic suppressor element #22, GSE22) or mutant p53s significantly increased the frequency of both spontaneous and ras-induced karyotypic changes. In concordance with these observations we have found that expression of ras oncogene caused in p53-defective cells further mitigation of ethyl-metansulphonate-induced G1 and G2 cell cycle arrest, but did not abrogate G1 and G2 cell cycle checkpoints in cells with normal p53 function. These data indicate that along with stimulation of cell proliferation and morphological transformation ras activation can contribute to cancerogenesis by increasing genetic instability.

Activation of p53-mediated cell cycle checkpoint in response to micronuclei formation.

Inactivation of p53 tumor-suppressor leads to genetic instability and, in particular, to accumulation of cells with abnormal numbers of chromosomes. In order to better define the role of p53 function in maintaining genome integrity we investigated the involvement of p53 in the control of proliferation of micronucleated cells resulting from abnormal chromosome segregation. Using cell lines expressing temperature-sensitive (ts) p53 or containing p53 genetic suppressor element (p53-GSE) we showed that inhibition of p53 function increases the frequency of cells with micronuclei. Immunofluorescence study revealed that in REF52 cell cultures with both spontaneous and colcemid-induced micronuclei the proportion of p53-positive cells is considerably higher among micronucleated variants as compared with their mononuclear counterparts. Analysis of 12(1)ConA cells expressing the beta-galactosidase reporter gene under the control of a p53-responsive promoter showed activation of p53-regulated transcription in the cells with micronuclei. Importantly, the percentage of cells manifesting specific p53 activity in colcemid-treated cultures increased with an augmentation of the number of micronuclei in the cell. Activation of p53 in micronucleated cells was accompanied by a decrease in their ability to enter S-phase as was determined by comparative analysis of 5-bromodeoxyuridine (5-BrdU) incorporation by the cells with micronuclei and their mononuclear counterparts. Inhibition of p53 function in the cells with tetracycline-regulated p53 gene expression, as well as in the cells expressing ts-p53 or p53-GSE, abolished cell cycle arrest in micronucleated cells. These results along with the data showing no increase in the frequency of chromosome breaks in REF52 cells after colcemid treatment suggest the existence of p53-mediated cell cycle checkpoint(s) preventing proliferation of micronucleated cells derived as a result of abnormal chromosome segregation during mitosis.