Cell cycle-dependent activation of Ras

Background Ras proteins play an essential role in the transduction of signals from a wide range of cell-surface receptors to the nucleus. These signals may promote cellular proliferation or differentiation, depending on the cell background. It is well established that Ras plays an important role in the transduction of mitogenic signals from activated growth-factor receptors, leading to cell-cycle entry. However, important questions remain as to whether Ras controls signalling events during cell-cycle progression and, if so, at which point in the cell-cycle it is activated.Results To address these questions we have developed a novel, functional assay for the detection of cellular activated Ras. Using this assay, we found that Ras was activated in HeLa cells, following release from mitosis, and in NIH 3T3 fibroblasts, following serum-stimulated cell-cycle entry. In each case, peak Ras activation occurred in mid-G1 phase. Ras activation in HeLa cells at mid-G1 phase was dependent on RNA and protein synthesis and was not associated with tyrosine phosphorylation of Shc proteins and their binding to Grb2. Significantly, activation of Ras and the extracellular-signal regulated (ERK) subgroup of mitogen-activated protein kinases were not temporally correlated during G1-phase progression.Conclusions Activation of Ras during mid-G1 phase appears to differ in many respects from its rapid activation by growth factors, suggesting a novel mechanism of regulation that may be intrinsic to cell-cycle progression. Furthermore, the temporal dissociation between Ras and ERK activation suggests that Ras targets alternate effector pathways during G1-phase progression.     

Cell cycle-dependent nuclear localization of yeast RNase III is required for efficient cell division

Members of the double-stranded RNA-specific ribonuclease III (RNase III) family were shown to affect cell division and chromosome segregation, presumably through an RNA interference-dependent mechanism. Here, we show that in Saccharomyces cerevisiae, where the RNA interference machinery is not conserved, an orthologue of RNase III (Rnt1p) is required for progression of the cell cycle and nuclear division. The deletion of Rnt1p delayed cells in both G1 and G2/M phases of the cell cycle. Nuclear division and positioning at the bud neck were also impaired in Deltarnt1 cells. The cell cycle defects were restored by the expression of catalytically inactive Rnt1p, indicating that RNA cleavage is not essential for cell cycle progression. Rnt1p was found to exit from the nucleolus to the nucleoplasm in the G2/M phase, and perturbation of its localization pattern delayed the progression of cell division. A single mutation in the Rnt1p N-terminal domain prevented its accumulation in the nucleoplasm and slowed exit from mitosis without any detectable effects on RNA processing. Together, the data reveal a new role for a class II RNase III in the cell cycle and suggest that at least some members of the RNase III family possess catalysis-independent functions.     

Characterization of a cell cycle-dependent nuclear autoantigen

Analysis of reactivity to nuclear antigens in autoimmune sera revealed a serum that produced a previously undescribed cell cycle-dependent immunofluorescence staining pattern. By indirect immunofluorescence using HEp-2 cells as substrate, the serum generated a speckled and nucleolar pleomorphic staining pattern. This characteristic immunofluorescence pattern was detected in different cell lines from various species indicating that the antigen was highly conserved. This serum immunoprecipitated a 85 kDa protein using an extract from [³⁵S]-labeled HeLa cells. Indirect immunofluorescence of proliferating mouse 3T3 cells displayed the characteristic pleomorphic staining which was not observed in serum-starved cells. Resting human and mouse peripheral blood lymphocytes were negative in immunofluorescence while mitogen-stimulated lymphocytes were positive. Germinal centers of mice two weeks after immunization with 2-phenyl-oxazolone showed speckled immunofluorescence staining in the dark zones whereas unimmunized mice were completely negative. Cell synchronization experiments showed a characteristic sequence of locations of the antigen during the cell cycle. In G₁, cells were completely negative. In late G₁, G₁/S and S phase, speckles were visible. In early G₂, speckles were visible, and later in G₂, the nucleoli were positive. During mitosis chromosomes were stained. Further characterization of this antibody specificity and cloning of cDNA are in progress.     

Pendulin, a Drosophila protein with cell cycle-dependent nuclear localization, is required for normal cell proliferation

We describe the dynamic intracellular localization of Drosophila Pendulin and its role in the control of cell proliferation. Pendulin is a new member of a superfamily of proteins which contains Armadillo (Arm) repeats and displays extensive sequence similarities with the Srp1 protein from yeast, with RAG-1 interacting proteins from humans, and with the importin protein from Xenopus. Almost the entire polypeptide chain of Pendulin is composed of degenerate tandem repeats of approximately 42 amino acids each. A short NH2-terminal domain contains adjacent consensus sequences for nuclear localization and cdc2 kinase phosphorylation. The subcellular distribution of Pendulin is dependent on the phase of cell cycle. During interphase, Pendulin protein is exclusively found in the cytoplasm of embryonic cells. At the transition between G2 and M-phase, Pendulin rapidly translocates into the nuclei where it is distributed throughout the nucleoplasm and the areas around the chromosomes. In the larval CNS, Pendulin is predominantly expressed in the dividing neuroblasts, where it undergoes the same cell cycle-dependent redistribution as in embryos. Pendulin is encoded by the oho31 locus and is expressed both maternally and zygotically. We describe the phenotypes of recessive lethal mutations in the oho31 gene that result in a massive decrease or loss of zygotic Pendulin expression. Hematopoietic cells of mutant larvae overproliferate and form melanotic tumors, suggesting that Pendulin normally acts as a blood cell tumor suppressor. In contrast, growth and proliferation in imaginal tissues are reduced and irregular, resulting in abnormal development of imaginal discs and the CNS of the larvae. This phenotype shows that Pendulin is required for normal growth regulation. Based on the structure of the protein, we propose that Pendulin may serve as an adaptor molecule to form complexes with other proteins. The sequence similarity with importin indicates that Pendulin may play a role in the nuclear import of karyophilic proteins and some of these may be required for the normal transmission and function of proliferative signals in the cells.     

Cell cycle-dependent phosphorylation, nuclear localization, and activation of human condensin

Condensin, one of the most abundant components of mitotic chromosomes, is a conserved protein complex composed of two structural maintenance of chromosomes (SMC) subunits (SMC2- and SMC4-type) and three non-SMC subunits, and it plays an essential role in mitotic chromosome condensation. Purified condensin reconfigures DNA structure using energy provided by ATP hydrolysis. To know the regulation of condensin in somatic cells, the expression level, subcellular localization, and phosphorylation status of human condensin were examined during the cell cycle. The levels of condensin subunits were almost constant throughout the cell cycle, and the three non-SMC subunits were phosphorylated at specific sites in mitosis and dephosphorylated upon the completion of mitosis. Subcellular fractionation studies revealed that a proportion of condensin was tightly bound to mitotic chromosomes and that this form was phosphorylated at specific sites. Condensin purified from mitotic cells had much stronger supercoiling activity than that purified from interphase cells. These results suggest that condensin functions in somatic cells are regulated by phosphorylation in two ways during the cell cycle; the phosphorylation of specific sites correlates with the chromosomal targeting of condensin, and its biochemical activity is stimulated by phosphorylation.     

Ran GTPase regulates Mad2 localization to the nuclear pore complex

In yeast and mammalian cells, the spindle assembly checkpoint proteins Mad1p and Mad2p localize to the nuclear pore complex (NPC) during interphase. Deletion of MAD1 or MAD2 did not affect steady-state nucleocytoplasmic distribution of a classical nuclear localization signal-containing reporter, a nuclear export signal-containing reporter, or Ran localization. We utilized cells with conditional mutations in the yeast Ran GTPase pathway to examine the relationship between Ran and targeting of checkpoint regulators to the NPC. Mutations that disrupt the concentration of Ran in the nucleus displaced Mad2p but not Mad1p from the NPC. The displacement of Mad2p in M-phase cells was correlated with activation of the spindle checkpoint. Our observations demonstrate that Mad2p localization at NPCs is sensitive to nuclear levels of Ran and suggest that release of Mad2p from NPCs is closely linked with spindle assembly checkpoint activation in yeast. This is the first evidence indicating that Ran affects the localization of Mad2p to the NPC.     

A permeable FGF-1 nuclear localization sequence peptide induces DNA synthesis independently of Ras activation

A 26-amino-acid peptide (designated PFNP) composed of the nuclear localization signal of fibroblast growth factor (FGF)-1 and a membrane-permeable peptide is known to mimic FGF-1's ability to stimulate DNA synthesis in various cell types at low cell densities. The underlying molecular mechanism is unknown, however. Here we show that PFNP activity is inhibited in murine fibroblasts by a tyrosine kinase inhibitor, that PFNP does not bind to the FGF receptor, and that PFNP does not induce phosphorylation of the FGF receptor substrate. In addition, expression of a dominant-negative form of Ras, which abolished the activities of epidermal growth factor (EGF) and heparin-binding EGF, had no affect on PFNP-induced DNA synthesis. Despite this apparent Ras independence, PFNP activity correlated with phosphorylation of ERK1/2 MAP kinases and was concentration dependently inhibited by inhibitors of ERK1/2 MAP kinase phosphorylation. These results indicate that whereas Ras activation is dispensable for PFNP-induced DNA synthesis, activation of tyrosine kinases and ERK1/2 kinases, albeit independently of the FGF receptor system, is crucial. Interestingly, FGF-1 signaling was predominantly Ras-independent when the cell density was optimum for PFNP, suggesting that PFNP and FGF-1 share the same signaling mechanism.     

Roles played by Ras subfamily proteins in the cell and developmental biology of microorganisms

The Ras subfamily proteins are monomeric GTPases that function as molecular switches in cellular signal transduction pathways. This review describes our current knowledge of the roles that these proteins play in the growth and differentiation of single celled microorganisms.     

The mechanism of Ras GTPase activation by neurofibromin

Individual rate constants have been determined for each step of the Ras.GTP hydrolysis mechanism, activated by neurofibromin. Fluorescence intensity and anisotropy stopped-flow measurements used the fluorescent GTP analogue, mantGTP (2'(3')-O-(N-methylanthraniloyl)GTP), to determine rate constants for binding and release of neurofibromin. Quenched flow measurements provided the kinetics of the hydrolytic cleavage step. The fluorescent phosphate sensor, MDCC-PBP was used to measure phosphate release kinetics. Phosphate-water oxygen exchange, using (18)O-substituted GTP and inorganic phosphate (P(i)), was used to determine the extent of reversal of the hydrolysis step and of P(i) binding. The data show that neurofibromin and P(i) dissociate from the NF1.Ras.GDP.P(i) complex with identical kinetics, which are 3-fold slower than the preceding cleavage step. A model is presented in which the P(i) release is associated with the change of Ras from "GTP" to "GDP" conformation. In this model, the conformation change on P(i) release causes the large change in affinity of neurofibromin, which then dissociates rapidly.     

Human cyclins A and B1 are differentially located in the cell and undergo cell cycle-dependent nuclear transport

We have used immunofluorescence staining to study the subcellular distribution of cyclin A and B1 during the somatic cell cycle. In both primary human fibroblasts and in epithelial tumor cells, we find that cyclin A is predominantly nuclear from S phase onwards. Cyclin A may associated with condensing chromosomes in prophase, but is not associated with condensed chromosomes in metaphase. By contrast, cyclin B1 accumulates in the cytoplasm of interphase cells and only enters the nucleus at the beginning of mitosis, before nuclear lamina breakdown. In mitotic cells, cyclin B1 associates with condensed chromosomes in prophase and metaphase, and with the mitotic apparatus. Cyclin A is degraded during metaphase and cyclin B1 is precipitously destroyed at the metaphase----anaphase transition. Cell fractionation and immunoprecipitation studies showed that both cyclin A and cyclin B1 are associated with PSTAIRE-containing proteins. The nuclear, but not the cytoplasmic form, of cyclin A is associated with a 33-kD PSTAIRE-containing protein. Cyclin B1 is associated with p34cdc2 in the cytoplasm. Thus we propose that the different localization of cyclin A and cyclin B1 in the cell cycle could be the means by which the two types of mitotic cyclin confer substrate specificity upon their associated PSTAIRE-containing protein kinase subunit.     

Calcium- and cell cycle-dependent association of annexin 11 with the nuclear envelope

Annexin 11 is a widely expressed calcium- and phospholipid-binding protein that resides in the nucleoplasm in many cultured cell lines. This is in contrast to its most extensively characterized in vitro ligand, the small calcium-binding protein S100A6 (calcyclin), which is concentrated in the nuclear envelope. Here we have examined the significance of the association of annexin 11 and S100A6 by asking whether circumstances exist in which the two proteins occupy the same subcellular localization. First, we show that in both A431 and vascular smooth muscle cells, elevation of intracellular Ca2+ leads to translocation of annexin 11 from the nucleus to the nuclear envelope where it co-localizes with S100A6. We also demonstrate, using fusions of annexin 11 with green fluorescent protein, that whereas the C-terminal core domain of annexin 11 is essential for Ca2+ sensitivity, the N-terminal domain is required for targeting to the nuclear envelope. Second, we show that annexin 11 relocalizes to the nuclear envelope as A431 cells transit from early to mid-prophase. In late prophase, at the time of nuclear envelope breakdown, annexin 11 and S100A6 become intensely localized with lamina-associated polypeptide 2 to folds in the nuclear envelope. From metaphase to telophase S100A6 is degraded, but in late telophase annexin 11 associates with the reforming nuclear envelope before resuming a nucleoplasmic location in interphase. These results show that co-localization of annexin 11 and S100A6 at the nuclear envelope may be regulated either by elevation of intracellular Ca2+ or by cell cycle progression and provide the first evidence that these proteins may associate in vivo.     

A cell cycle-dependent internal ribosome entry site

The eukaryotic mRNA 5' cap structure facilitates translation. However, cap-dependent translation is impaired at mitosis, suggesting a cap-independent mechanism for mRNAs translated during mitosis. Translation of ornithine decarboxylase (ODC), the rate-limiting enzyme in the biosynthesis of polyamines, peaks twice during the cell cycle, at the G1/S transition and at G2/M. Here, we describe a cap-independent internal ribosome entry site (IRES) in the ODC mRNA that functions exclusively at G2/M. This ensures elevated levels of polyamines, which are implicated in mitotic spindle formation and chromatin condensation. c-myc mRNA also contains an IRES that functions during mitosis. Thus, IRES-dependent translation is likely to be a general mechanism to synthesize short-lived proteins even at mitosis, when cap-dependent translation is interdicted.     

Dissection of the GTPase mechanism of Ras protein by MD analysis of Ras mutants

Controlling the hydrolysis rate of GTP bound to the p21ras protein is crucial for the delicate timing of many biological processes. A few mechanisms were suggested for the hydrolysis of GTP. To gain more insight into the individual elementary events of GTP hydrolysis, we carried out molecular dynamic analysis of wild-type p21ras and some of its mutants. It was recently shown that Ras-related proteins and mutants generally follow a linear free energy relationship (LFER) relating the rate of reaction to the pK(a) of the gamma-phosphate group of the bound GTP, indicating that proton transfer from the attacking water to the GTP is the first elementary event in the GTPase mechanism. However, some exceptions were observed. Thus, the Gly12 --> Aspartic p21ras (G12D) mutant had a very low GTPase activity although its pK(a) was very close to that of the wild-type ras. Here we compared the molecular dynamics (MD) of wild-type Ras and G12D, showing that in the mutant the catalytic water molecule is displaced to a position where proton transfer to GTP is unfavorable. These results suggest that the mechanism of GTPase is indeed composed of an initial proton abstraction from water by the GTP, followed by a nucleophilic attack of the hydroxide ion on the gamma-phosphorus of GTP.     

Characterization of A 54-kD protein of the inner nuclear membrane: evidence for cell cycle-dependent interaction with the nuclear lamina

Using a mAb (R-7), we have characterized a 54-kD protein of the chicken nuclear envelope. Based on its biochemical properties and subnuclear distribution p54 is likely to be an integral membrane component specific to the inner nuclear membrane. Fractionation experiments indicate that p54 interacts, directly or indirectly, with the nuclear lamina, and analysis of p54 in cultured cells suggests that this interaction is controlled by cell cycle-dependent posttranslational modification, most likely phosphorylation. Modification of p54 results in a slightly reduced electrophoretic mobility, and it converts the protein from a detergent-resistant to a detergent-extractable form. Detergent solubilization of p54 can be induced in vivo by treating isolated nuclei or nuclear envelopes with highly purified cdc2 kinase, one of the most prominent kinases active in mitotic cells. These results suggest that mitotic phosphorylation of p54 might contribute to control nuclear envelope dynamics during mitosis in vivo.     

Detection of Ras GTPase protein radicals through immuno-spin trapping

Over the past decade immuno-spin trapping (IST) has been used to detect and identify protein radical sites in numerous heme and metalloproteins. To date, however, the technique has had little application toward nonmetalloproteins. In this study, we demonstrate the successful application of IST in a system free of transition metals and present the first conclusive evidence of (?)NO-mediated protein radical formation in the HRas GTPase. HRas is a nonmetalloprotein that plays a critical role in regulating cell-growth control. Protein radical formation in Ras GTPases has long been suspected of initiating premature release of bound guanine nucleotide. This action results in altered Ras activity both in vitro and in vivo. As described herein, successful application of IST may provide a means for detecting and identifying radical-mediated Ras activation in many different cancers and disease states in which Ras GTPases play an important role.     

Expression of activated Ras during Dictyostelium development alters cell localization and changes cell fate

There is now a body of evidence to indicate that Ras proteins play important roles in development. Dictyostelium expresses several ras genes and each appears to perform a distinct function. Previous data had indicated that the overexpression of an activated form of the major developmentally regulated gene, rasD, caused a major aberration in morphogenesis and cell type determination. We now show that the developmental expression of an activated rasG gene under the control of the rasD promoter causes a similar defect. Our results indicate that the expression of activated rasG in prespore cells results in their transdifferentiation into prestalk cells, whereas activated rasG expression in prestalk causes gross mislocalization of the prestalk cell populations.