The Aurora/Ipl1p kinase family: regulators of chromosome segregation and cytokinesis.

Members of the Aurora/Ipl1p family of mitotically regulated serine/threonine kinases are emerging as key regulators of chromosome segregation and cytokinesis. Proper chromosome segregation and cytokinesis ensure that each daughter cell receives the full complement of genetic material. Defects in these processes can lead to aneuploidy and the propagation of genetic abnormalities. This review discusses the Aurora/Ipl1p kinases in terms of their protein structure and proposed function in mitotic cells and also the potential role of aurora2 in human cancer.

p34cdc2-mediated phosphorylation at T124 inhibits nuclear import of SV-40 T antigen proteins.

The nuclear import of transcription regulatory proteins appears to be used by the cell to trigger transitions in cell cycle, morphogenesis, and transformation. We have previously observed that the rate at which SV-40 T antigen fusion proteins containing a functional nuclear localization sequence (NLS; residues 126-132) are imported into the nucleus is enhanced in the presence of the casein kinase II (CK-II) site S111/112. In this study purified p34cdc2 kinase was used to phosphorylate T antigen proteins specifically at T124 and kinetic measurements at the single-cell level performed to assess its effect on nuclear protein import. T124 phosphorylation, which could be functionally simulated by a T-to-D124 substitution, was found to reduce the maximal extent of nuclear accumulation whilst negligibly affecting the import rate. The inhibition of nuclear import depended on the stoichiometry of phosphorylation. T124 and S111/112 could be phosphorylated independently of one another. Two alternative mechanisms were considered to explain the inhibition of nuclear import by T124 phosphorylation: inactivation of the NLS and cytoplasmic retention, respectively. Furthermore, we speculate that in vivo T124 phosphorylation may regulate the small but functionally significant amount of cytoplasmic SV-40 T antigen. A sequence comparison showed that many transcription regulatory proteins contain domains comprising potential CK-II-sites, cdc2-sites, and NLS. This raises the possibility that the three elements represent a functional unit regulating nuclear protein import.

A complex containing p34cdc2 and cyclin B phosphorylates the nuclear lamin and disassembles nuclei of clam oocytes in vitro.

Cell-free extracts prepared from activated clam oocytes contain factors which induce phosphorylation of the single 67-kD lamin (L67), disassemble clam oocyte nuclei, and cause chromosome condensation in vitro (Dessev, G., R. Palazzo, L. Rebhun, and R. Goldman. 1989. Dev. Biol. 131:469-504). To identify these factors, we have fractionated the oocyte extracts. The nuclear lamina disassembly (NLD) activity, together with a protein kinase activity specific for L67, appear as a single peak throughout a number of purification steps. This peak also contains p34cdc2, cyclin B, and histone H1-kinase activity, which are components of the M-phase promoting factor (MPF). The NLD/L67-kinase activity is depleted by exposure of this purified material to Sepharose conjugated to p13suc1, and is restored upon addition of a p34cdc2/p62 complex from HeLa cells. The latter complex phosphorylates L67 and induces NLD in the absence of other clam oocyte proteins. Our results suggest that a single protein kinase activity (p34cdc2-H1 kinase, identical with MPF) phosphorylates the lamin and is involved in the meiotic breakdown of the nuclear envelope in clam oocytes.

An adenovirus mutant that replicates selectively in p53-deficient human tumor cells.

The human adenovirus E1B gene encodes a 55-kilodalton protein that inactivates the cellular tumor suppressor protein p53. Here it is shown that a mutant adenovirus that does not express this viral protein can replicate in and lyse p53-deficient human tumor cells but not cells with functional p53. Ectopic expression of the 55-kilodalton EIB protein in the latter cells rendered them sensitive to infection with the mutant virus. Injection of the mutant virus into p53-deficient human cervical carcinomas grown in nude mice caused a significant reduction in tumor size and caused complete regression of 60 percent of the tumors. These data raise the possibility that mutant adenoviruses can be used to treat certain human tumors.

Human p53 inhibits growth in Schizosaccharomyces pombe.

Overexpression of wild-type p53 in mammalian cells blocks growth. We show here that the overexpression of wild-type human p53 in the fission yeast Schizosaccharomyces pombe also blocks growth, whereas the overexpression of mutant forms of p53 does not. The p53 polypeptide is located in the nucleus and is phosphorylated at both the cdc2 site and the casein kinase II site in S. pombe. A new dominant mutation of p53, resulting in the change of a cysteine to an arginine at amino acid residue 141, was identified. The results presented here demonstrate that S. pombe could provide a simple system for studying the mechanism of action of human p53.

Signal transduction by Ras-like GTPases: a potential target for anticancer drugs.

Members of the ras family of GTPases are involved in a wide variety of cellular processes including cell proliferation, differentiation, apoptosis, and transformation. The ras oncogene is one of the most frequently mutated genes in human cancer. In addition, other oncogene and tumor suppressor gene products are components of the signal transduction pathways in which Ras or other Ras-like GTPases play key regulatory functions. Current progress in the elucidation of these signal transduction pathways will be reviewed and the potential use of these insights for the development of novel therapeutic agents for the treatment of cancer will be discussed.

Identification of the guanine nucleotide dissociation stimulator for Ral as a putative effector molecule of R-ras, H-ras, K-ras, and Rap.

To identify proteins that bind to the Ras-related protein R-ras we performed a yeast two-hybrid cDNA library screen. Several clones were obtained encoding the C-terminal region of the guanine nucleotide dissociation stimulator for Ral (RalGDS). The R-ras-binding domain of RalGDS (RalGDS-RBD) is distinct from the conserved catalytic exchange factor regions. Using the two-hybrid system, we show that RalGDS-RBD interacts with H-ras, K-ras, and Rap, and with active but not with inactive point mutants of these Ras-like GTPases. Moreover, using purified proteins, we demonstrate the direct GTP-dependent interaction of the Ras-like GTPases with RalGDS-RBD and full-length RalGDS in vitro. Furthermore, we show that RalGDS-RBD and the Ras-binding domain of Raf-1 compete for binding to the Ras-like GTPases. These data indicate that RalGDS is a putative effector molecule for R-ras, H-ras, K-ras, and Rap.

Mechanism of interferon action. The interferon-induced phosphoprotein P1 possesses a double-stranded RNA-dependent ATP-binding site.

Protein P1, the interferon-induced protein phosphorylated in the presence of dsRNA in human amnion U-cells, was covalently labeled with [alpha-32P]ATP following ultraviolet irradiation. The photoaffinity labeling of protein P1 was dependent upon double-stranded RNA. Antibody prepared against phosphorylated protein P1 immunoprecipitated the double-stranded RNA-dependent photoaffinity-labeled product. The extent of photoaffinity labeling was significantly decreased by the addition of unlabeled ATP, GTP, or AMP; adenosine had little effect on the photoaffinity labeling of protein P1. These results suggest that protein P1 possesses a site capable of binding an adenine nucleotide in a double-stranded RNA-dependent manner.

Mechanism of interferon action. Activation of the human P1/eIF-2 alpha protein kinase by individual reovirus s-class mRNAs: s1 mRNA is a potent activator relative to s4 mRNA.

The ability of pure viral and cellular single-strand (ss) RNAs to activate the interferon-induced, double-stranded (ds) RNA-dependent P1/eIF-2 protein kinase purified from human amnion U cells was examined. In addition to the well-established activation of P1 kinase autophosphorylation in vitro by reovirus genome dsRNA, the P1 kinase was also efficiently activated by certain reovirus ssRNAs. The reovirus s1 mRNA was a potent activator of the kinase. By contrast, the reovirus s4 mRNA was a poor activator of the kinase. Likewise, adenovirus VAI RNA, transfer RNA, 5 S ribosomal RNA, and rabbit globin mRNA were not activators or were very poor activators of the purified P1/eIF-2 protein kinase. Analysis of hybrid ssRNAs produced between the reovirus s1 and s4 mRNAs revealed that both the 5' and the 3' portions of the s1 mRNA possessed nucleotide sequences capable of mediating kinase activation. Subsequent deletion analysis of the 5' portion of the s1 mRNA identified a 161-nucleotide region located between positions 416 and 576 which was sufficient for P1 kinase activation. Treatment of reovirus s1 mRNA transcripts with either ssRNA- or dsRNA-specific ribonucleases, but not with heat, destroyed the ability of s1 mRNA transcripts to activate the kinase. These results suggest that P1 kinase autophosphorylation in vitro may be selectively activated by individual ssRNAs in a differential manner, and that a secondary or higher-ordered ssRNA structure(s) may be important in mediating the activation.

Bcl-2 associates with the ras-related protein R-ras p23.

Apoptosis is an important but poorly understood mechanism of cell regulation. Growth factor deprivation can trigger apoptosis in a variety of cells, suggesting the existence of a signal transduction pathway responding to external signals and leading to apoptosis. Overexpression of the proto-oncogene bcl-2 can override these signals and block apoptosis, indicating that the bcl-2 protein (Bcl-2) is an important component of the apoptotic response. The identification of Bcl-2-binding proteins might help explain how Bcl-2 acts to regulate apoptosis. Here we use the yeast two-hybrid system to show that the human ras-related protein R-ras p23 (ref 16-18) binds to Bcl-2. This association is also detected in immunoprecipitates from human cell extracts. The association requires full-length Bcl-2 but the C-terminal 60 amino acids of R-ras p23 are sufficient for the interaction. These results provide evidence of a putative component of a signal transduction pathway involved in the regulation of apoptosis.

Transcriptional activation of the human proliferating-cell nuclear antigen promoter by p53.

Proliferating-cell nuclear antigen (PCNA) is a DNA damage-inducible protein that performs an essential function in DNA replication and repair as an auxiliary factor for DNA polymerases delta and epsilon. Examination of the human PCNA promoter DNA sequence revealed a site with homology to the consensus DNA sequence bound by p53. PCNA promoter fragments with this site intact bound p53 in vitro and were transcriptionally activated by wild-type p53 in transient expression assays in SAOS-2 cells. The resident p53-binding site could be functionally substituted by a previously described p53-binding site from the ribosomal gene cluster. A plasmid expressing a mutated version of p53 derived from a patient with Li-Fraumeni syndrome failed to activate the PCNA promoter in the cotransfection assay. In different cell types, activation of the PCNA promoter by the p53-binding sequence correlated with the status of p53. Activation of the PCNA promoter by wild-type p53 depends upon the level of p53 expression. This concentration dependence and cell type specificity reconciles the observations presented here with prior results indicating that wild-type p53 represses the PCNA promoter. These findings provide a mechanism whereby p53 modulates activation of PCNA expression as a cellular response to DNA damage.

Human p53 is phosphorylated by p60-cdc2 and cyclin B-cdc2.

The human anti-oncoprotein p53 is shown to be a substrate of cdc2. The primary site of phosphorylation is serine-315. Serine-315 is phosphorylated by both p60-cdc2 and cyclin B-cdc2 enzymes. The phosphorylation of p53 is cell cycle-dependent. The abundance of p53 also oscillates during the cell cycle. The protein is largely absent from cells that have just completed division but accumulates in cells during G1 phase. Phosphorylation by cdc2 might regulate the antiproliferative activity of p53.

Intermediate filament reorganization during mitosis is mediated by p34cdc2 phosphorylation of vimentin.

As cells enter mitosis, the intermediate filament (IF) networks of interphase BHK-21 cells are depolymerized to form cytoplasmic aggregates of disassembled IFs, and the constituent IF proteins, vimentin and desmin are hyperphosphorylated at several specific sites. We have characterized one of two endogenous vimentin kinases from a particulate fraction of mitotic cell lysates. Through several purification steps, vimentin kinase activity copurifies with histone H1 kinase and both activities bind to p13suc1-Sepharose. The final enriched kinase preparation consists primarily of p34cdc2 and polypeptides of 65 and 110 kd. The purified kinase complex phosphorylates vimentin in vitro at a subset of sites phosphorylated in vivo during mitosis. Furthermore, phosphorylation of in vitro polymerized vimentin IFs by the purified kinase causes their disassembly. Therefore, vimentin is a substrate of p34cdc2 and phosphorylation of vimentin contributes to M phase reorganization of the IF network.

The Ras-related protein R-ras interacts directly with Raf-1 in a GTP-dependent manner.

R-ras is a member of the ras family of small GTPases that associates with the apoptosis-suppressing proto-oncogene product Bcl-2. Using the yeast two-hybrid system we provide evidence for an interaction between R-ras and the Raf-1 kinase. This interaction requires only the N-terminal regulatory domain (amino acids 1-256) of Raf-1, and is observed with both the wild type and a constitutively active R-ras mutant, but not with a deletion mutant that lacks the potential effector domain or a mutant of R-ras impaired for GTP binding. Moreover, using an in vitro binding assay we show a direct GTP-dependent interaction of purified R-ras with a purified Raf-1 fragment corresponding to the proposed 81-amino-acid H-Ras-binding domain of Raf-1 (amino acids 51-131). Taken together, these data indicate that R-ras may exert its biological effect by means of modulating the activity of the Raf-1 kinase as its direct downstream effector.

A homologue of Drosophila aurora kinase is oncogenic and amplified in human colorectal cancers.

Genetic and biochemical studies in lower eukaryotes have identified several proteins that ensure accurate segregation of chromosomes. These include the Drosophila aurora and yeast Ipl1 kinases that are required for centrosome maturation and chromosome segregation. We have identified two human homologues of these genes, termed aurora1 and aurora2, that encode cell-cycle-regulated serine/threonine kinases. Here we demonstrate that the aurora2 gene maps to chromosome 20q13, a region amplified in a variety of human cancers, including a significant number of colorectal malignancies. We propose that aurora2 may be a target of this amplicon since its DNA is amplified and its RNA overexpressed, in more than 50% of primary colorectal cancers. Furthermore, overexpression of aurora2 transforms rodent fibroblasts. These observations implicate aurora2 as a potential oncogene in many colon, breast and other solid tumors, and identify centrosome-associated proteins as novel targets for cancer therapy.